Structural Cuticular Proteins Research Articles

Genome-Wide Identification and Transcriptome-Based Expression Profile of Cuticular Protein Genes in Antheraea pernyi.

Antheraea pernyi is one of the most famous edible and silk-producing wild silkworms of Saturniidae. Structural cuticular proteins (CPs) are the primary component of insect cuticle. In this paper, the CPs in the genome of A. pernyi were identified and compared with those of the lepidopteran model species Bombyx mori, and expression patterns were analyzed based on the transcriptomic data from the larval epidermis/integument (epidermis in the following) and some non-epidermis tissues/organs of two silkworm species. A total of 217 CPs was identified in the A. pernyi genome, a comparable number to B. mori (236 CPs), with CPLCP and CPG families being the main contribution to the number difference between two silkworm species. We found more RR-2 genes expressed in the larval epidermis of fifth instar of A. pernyi than B. mori, but less RR-2 genes expressed in the prothoracic gland of A. pernyi than B. mori, which suggests that the hardness difference in the larval epidermis and prothoracic gland between the two species may be caused by the number of RR-2 genes expressed. We also revealed that, in B. mori, the number of CP genes expressed in the corpus allatum and prothoracic gland of fifth instar was higher than that in the larval epidermis. Our work provided an overall framework for functional research into the CP genes of Saturniidae.

Antimicrobial Activity of Apidermin 2 from the Honeybee Apis mellifera.

Simple SummaryIn the honeybee Apis mellifera, apidermin 2 (APD 2) is known as a cuticular protein. However, the antimicrobial properties of A. mellifera APD 2 (AmAPD 2) have not been characterized. Herein, we provide the first demonstration that AmAPD 2 exhibits antibacterial and antifungal activities. We found that AmAPD 2 induced structural damage by binding to bacterial and fungal cell walls, indicating that AmAPD 2 has the antimicrobial action of an antimicrobial peptide. Our findings demonstrate a novel role of AmAPD 2 as an antimicrobial agent in honeybees.Apidermins (APDs) are known as structural cuticular proteins in insects, but their additional roles are poorly understood. In this study, we characterized the honeybee, Apis mellifera, APD 2 (AmAPD 2), which displays activity suggesting antimicrobial properties. In A. mellifera worker bees, the AmAPD 2 gene is transcribed in the epidermis, hypopharyngeal glands, and fat body, and induced upon microbial ingestion. Particularly in the epidermis of A. mellifera worker bees, the AmAPD 2 gene showed high expression and responded strongly to microbial challenge. Using a recombinant AmAPD 2 peptide, which was produced in baculovirus-infected insect cells, we showed that AmAPD 2 is heat-stable and binds to live bacteria and fungi as well as carbohydrates of microbial cell wall molecules. This binding action ultimately induced structural damage to microbial cell walls, which resulted in microbicidal activity. These findings demonstrate the antimicrobial role of AmAPD 2 in honeybees.

Antifungal roles of adult-specific cuticular protein genes of the red flour beetle, Tribolium castaneum

The insect cuticle is a composite structure that can further be divided into a few sub-structural layers. Its large moiety comprises a lattice of chitin fibrils and structural proteins, both of which are stabilized by covalent bonding among them. The cuticle covers the whole surface of insect body, and thus has long been suggested for the involvement in defense against entomopathogens, especially entomopathogenic fungi that infect percutaneously. We have been addressing this issue in the past few years and have so far demonstrated experimentally that chitin synthase 1, laccase2 as well as benzoquinone synthesis-related genes of Tribolium castaneum have indispensable roles in the antifungal host defense. In the present study we focused on another major component of the insect cuticular integument, structural cuticular proteins. We chose three genes coding for adult-specific cuticular proteins, namely CPR4, CPR18 and CPR27, and examined their roles in forming immunologically sound adult cuticular integuments. Analyses of developmental expression revealed that the three genes showed high level expression in the pupal stage. These results are consistent with their proposed roles in constructing cuticle of adult beetles. The RNA interference-mediated gene knockdown was employed to silence these genes, and the administration of double strand RNAs in pupae resulted in the adults with malformed elytra. The single knockdown of the three genes attenuated somewhat the defense of the resulting adult beetles against Beauveria bassiana and Metarhizium anisopliae, but statistical analyses indicated no significant differences from controls. In contrast, the double or triple knockdown mutant beetles displayed a drastic disruption of the host defense against the two entomopathogenic fungal species irrespective of the combination of targeted cuticular protein genes, demonstrating the important roles of the three cuticular protein genes in conferring robust antifungal properties on the adult cuticle. Scanning electron microscopic observation revealed that the germination of conidia attached on the adult body surface was still suppressed after the gene knockdown as in the case of wild-type beetles, suggesting that the weakened antifungal phenotypes resulted from the combined knockdown of the adult-specific cuticular protein genes could not be accounted for by the disfunction of secretion/retention of fungistatic benzoquinone derivatives.

Identification and expression analysis of cuticular protein genes in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

Structural cuticular proteins (CPs) are major components of the insect cuticle, and they play critical roles in insect development and insecticide resistance. Here, a total of 196 CP genes were successfully annotated in the Plutella xylostella genome. On the basis of motif analysis, these CPs were classified into 10 different families, including 122 CPR, 12 CPAP1, 8 CPAP3, 9 CPLCP, 2 Tweedle, 1 CPF, 1 CPFL, 1 CPCFC, 17 CPG and 2 18 aa proteins, and the remaining 21 unclassified CPs were classed as cuticular proteins hypothetical (CPH). A phylogenetic analysis of CPs from different insects revealed species-specific clades of RR-1 and RR-2 genes, suggesting that CP gene duplication might occur independently among insect taxa, while we also found that some other CPs (such as CPAP1 and CPAP3) had a closer relationship based on their conserved domain architecture. Using available RNAseq libraries, the expression profiles of the CPs were analyzed over the four developmental stages of the insect (i.e., egg, larva, pupa, and adult), revealing stage-specific expression patterns for the CPs. In a chlorpyrifos resistant strain, 18 CP genes were found to be more than two-fold upregulated compared to the susceptible control strain, and qRT-PCR analysis showed that these CP genes were overexpressed after exposure to chlorpyrifos, suggesting a potential role in the molecular mechanism of insecticide resistance in P. xylostella. This study provides the tools and molecular basis to study the role of CPs in the post-embryonal development and the mechanisms of insecticide resistance of P. xylostella.

Identification of Cuticular Protein Genes in the Colorado Potato Beetle Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

Structural cuticular proteins (CPs) are the primary components of insect cuticle, linings of salivary gland, foregut, hindgut and tracheae, and midgut peritrophic membrane. Variation of CPs in insect cuticle can cause penetration resistance to insecticides. Moreover, depletion of specific CP by RNA interference may be a suitable way for the development of potential pest control traits. Leptinotarsa decemlineata (Say) CPs are poorly characterized at present, and therefore, we mined the genome and transcriptome data to better annotate and classify L. decemlineata CPs in this study, by comparison with the annotated CPs of Tribolium castaneum Browse (Coleoptera: Tenebrionidae). We identified 175 CP genes. Except one miscellaneous CP with an 18-amino acid motif, these CPs were classified into 7 families based on motifs and phylogenetic analyses (CPs with a Rebers and Riddiford motif, CPR; CPs analogous to peritrophins, CPAP3 and CPAP1; CPs with a tweedle motif, TWDL; CPs with a 44-amino acid motif, CPF; CPs that are CPF-like, CPFL; and CPs with two to three copies of C-X5-C motif, CPCFC). Leptinotarsa decemlineata CPRs could be categorized into three subfamilies: RR-1 (50), RR-2 (85), and RR-3 (2). The RR-1 proteins had an additional motif with a conserved YTADENGF sequence. The RR-2 members possessed a conserved RDGDVVKG region and three copes of G-x(3)-VV. Few genes were found in TWDL (9), CPAP1 (9), CPAP3 (8), CPF (5), CPFL (4), and CPCFC (2) families. The findings provide valuable information to explore molecular modes of penetration resistance to insecticides and to develop dsRNA-based control method in L. decemlineata.

Structural glycoprotein LmAbd-9 is required for the formation of the endocuticle during locust molting

Insect cuticle is a composite made of chitin filaments embedded in a proteinaceous matrix, consisting mainly of structural cuticular proteins. In the present study, an endocuticle structural glycoprotein gene, LmAbd-9, was characterized based on the Locusta migratoria transcriptome. LmAbd-9 encodes a glycoprotein with a chitin binding domain 4, belonging to RR-1 subclass of the CPR family, which has two potential O-linked glycosylation sites (S115 and T137) at which glycosylation modification may occur. LmAbd-9 was highly expressed in the integument and showed periodic expression during molting. The expression levels of LmAbd-9 were significantly down-regulated after injection with 20-hydroxyecdysone (20E) for 6, 12, and 24 h, whereas it was upregulated after double-stranded RNA-mediated RNA interference of the 20E receptor gene LmEcR and LmFTZ-F1beta at day 2 of fifth instar nymphs for 48 h. After injection of dsLmAbd-9 on day 2 of fifth instar nymphs, the insects could normally molt to adults and showed no macroscopic phenotype; however, the cuticle of the adults was thinner, and there were significantly fewer endocuticular lamellae than in the control. Thus, LmAbd-9 that negatively regulated by the 20E signaling pathway was involved in the formation of the endocuticle in L. migratoria.

Proteomics reveals localization of cuticular proteins in Anopheles gambiae

Anopheles gambiae devotes over 2% of its protein coding genes to its 298 structural cuticular proteins (CPs). This paper provides new LC-MS/MS data on two adult structures, proboscises and palps, as well as three larval samples – 4th instar larvae, just their terminal segment, and a preparation enriched in their tracheae. These data were combined with our previously published results of proteins from five other adult structures, whole adults, and two preparations chosen for their relatively clean cuticle, the larval head capsules left behind after ecdysis and the pupal cuticles left behind after adult eclosion. Peptides from 28 CPs were recovered in all adult structures; 24 CPs were identified for the first time, 6 of these were members of the TWDL family. Most newly identified proteins came from the larval sources. Based solely on peptide recovery, from our data and from other investigators, most available on VectorBase, there were only 4 CPs that were restricted to a single adult structure. More were restricted to a single metamorphic stage, 14 in larvae, 0 in pupae and 32 in adults. Expression data from our earlier RT-qPCR studies reduces these numbers. Charting restriction of CPs to stage or structure is a step forward in establishing their specific roles.

Cuticular protein defective Bamboo mutant of Bombyx mori is sensitive to environmental stresses

Insect cuticle acts as a primary protective barrier against environment stresses that may directly impact the insect body. Here, we report the mechanical defense function of a structural cuticular protein, BmorCPH24, to environmental stresses using a silkworm Bamboo (Bo) mutant with this gene mutation. Ultraviolet (UV) irradiation and topical application of an acetone insecticide were used as environmental stresses to determine the differences in susceptibility between Bo and wild-type larvae. UV irradiation resulted in a sunburn phenotype in the Bo strains earlier than the wild-type indicating the sensitivity of Bo. Higher malondialdehyde (MDA) content and a lower survival ratio were also observed in the Bo strains. Treatment with deltamethrin revealed that Bo larvae were more sensitive to insecticides than the wild-type. Furthermore, cuticle analysis by microsection revealed thinner cuticle and a significant decrease in the endocuticle layer (∼64.0%) in Bo. These results suggest that BmorCPH24 mutation can lead to deficiency in resources required to construct the cuticle in Bo resulting in thin cuticle and reduced resistance to UV and insecticides. These results provide us new insight into the role of structural cuticular proteins in insect cuticle against environment stresses.

Microscopic cuticle structure comparison of pupal melanic and wild strain of Spodoptera exigua and their gene expression profiles in three time points

The beet armyworm, Spodoptera exigua (Hubner), is one of the major crop pests and is a target for current pest control approaches using insecticides. S. exigua melanic mutants (SEM) spontaneously occurred in the S. exigua wild type (SEW) strain and have been maintained under laboratory conditions on an artificial diet. Scanning electron microscopy showed that the inner cuticle of the SEM had a denser and less orderly structure. We investigated the cuticle protein genes using RNA-seq at three different developmental stages of both SEM and SEW. Comparison of cDNA libraries showed that 7257 CPs were significantly up-regulated and 664 genes were significantly downregulated in SEM at the developmental stage of 46-h in the fifth instar. In addition, 460 genes were significantly up-regulated and 439 genes were significantly down-regulated in the SEM at the development stage of 4-h before pupation. Moreover, 162 genes were significantly up-regulated and 293 genes were significantly downregulated in the SEM, just after pupation. Two genes CPR63 and CPR97 were identified from RNA sequences to verify the differentially expressed gene (DEG) results through quantitative real-time PCR (qRT-PCR). The results show that expression of both CPR63 and CPR97 structural cuticular proteins were significantly different between SEM and SEW. This functional analysis may help in understanding the role that these genes play in the cuticle pattern of the SEM.

Preliminary characterization of putative structural cuticular proteins in the malaria vector Anopheles sinensis.

The insect cuticle protects against environmental stresses such as insecticides, physical injury, dehydration and pathogenic microorganisms. Structural cuticular proteins (CPs) are the primary components of the cuticle, and are of interest for physiology-based pest management methods. Anopheles sinensis CPs are poorly characterised at present, and therefore we performed whole-genome sequence analysis and re-analysed available transcriptome data to identify potential insecticide resistance-associated CPs. Genome analysis revealed 238 putative CPs belonging to 11 different families; 136 proteins possessing a Rebers and Riddiford motif (CPR), 12 CPs analogous to peritrophins with a single chitin-binding domain (CPAP1), eight CPs analogous to peritrophins with three chitin-binding domains (CPAP3), four proteins with a 44 amino acid motif (CPF), five CPF-like (CPFL) proteins, nine Tweedle proteins, three proteins of low complexity with alanine residues (CPLCA), 26 proteins of low complexity with conserved glycine residues (CPLCG), seven proteins of low complexity with invariant W residues (CPLCW), 27 proteins of low complexity with proline residues (CPLCP), and one protein with two or three copies of a C-X -C motif (CPCFC). Two CPs belonging to the CPR and CPAP3 families were over-expressed in a deltamethrin-resistant strain. These results establish an information framework for An. sinensis CPs that provides a basis for further molecular research. © 2017 Society of Chemical Industry.

RNAi-Mediated Functional Analysis of Bursicon Genes Related to Adult Cuticle Formation and Tanning in the Honeybee, Apis mellifera.

Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk), thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, Apis mellifera. RNAi-mediated knockdown of AmBurs α and AmBurs β bursicon genes prevented the complete formation and tanning (melanization/sclerotization) of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of AmBurs α, AmBurs β and AmRk is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of AmBurs α, AmBurs β and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.

An RNAi Screen for Genes Involved in Nanoscale Protrusion Formation on Corneal Lens in Drosophila melanogaster

The "moth-eye" structure, which is observed on the surface of corneal lens in several insects, supports anti-reflective and self-cleaning functions due to nanoscale protrusions known as corneal nipples. Although the morphology and function of the "moth-eye" structure, are relatively well studied, the mechanism of protrusion formation from cell-secreted substances is unknown. In Drosophila melanogaster, a compound eye consists of approximately 800 facets, the surface of which is formed by the corneal lens with nanoscale protrusions. In the present study, we sought to identify genes involved in "moth-eye" structure, formation in order to elucidate the developmental mechanism of the protrusions in Drosophila. We re-examined the aberrant patterns in classical glossy-eye mutants by scanning electron microscope and classified the aberrant patterns into groups. Next, we screened genes encoding putative structural cuticular proteins and genes involved in cuticular formation using eye specific RNAi silencing methods combined with the Gal4/UAS expression system. We identified 12 of 100 candidate genes, such as cuticular proteins family genes (Cuticular protein 23B and Cuticular protein 49Ah), cuticle secretion-related genes (Syntaxin 1A and Sec61 ββ subunit), ecdysone signaling and biosynthesis-related genes (Ecdysone receptor, Blimp-1, and shroud), and genes involved in cell polarity/cell architecture (Actin 5C, shotgun, armadillo, discs large1, and coracle). Although some of the genes we identified may affect corneal protrusion formation indirectly through general patterning defects in eye formation, these initial findings have encouraged us to more systematically explore the precise mechanisms underlying the formation of nanoscale protrusions in Drosophila.

Distribution of cuticular proteins in different structures of adult Anopheles gambiae

Anopheles gambiae devotes over 2% (295) of its protein coding genes to structural cuticular proteins (CPs) that have been classified into 13 different families plus ten low complexity proteins not assigned to families. Small groups of genes code for identical proteins reducing the total number of unique cuticular proteins to 282. Is the large number because different structures utilize different CPs, or are all of the genes widely expressed? We used LC-MS/MS to learn how many products of these genes were found in five adult structures: Johnston's organs, the remainder of the male antennae, eye lenses, legs, and wings. Data were analyzed against both the entire proteome and a smaller database of just CPs. We recovered unique peptides for 97 CPs and shared peptides for another 35. Members of 11 of the 13 families were recovered as well as some unclassified. Only 11 CPs were present exclusively in only one structure while 43 CPs were recovered from all five structures. A quantitative analysis, using normalized spectral counts, revealed that only a few CPs were abundant in each structure. When the MS/MS data were run against the entire proteome, the majority of the top hits were to CPs, but peptides were recovered from an additional 467 proteins. CP peptides were frequently recovered from chitin-binding domains, confirming that protein-chitin interactions are not mediated by covalent bonds. Comparison with three other MS/MS analyses of cuticles or cuticle-rich structures augmented the current analysis. Our findings provide new insights into the composition of different mosquito structures and reveal the complexity of selection and utilization of genes coding for structural cuticular proteins.

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

The insect exoskeleton is composed of cuticle primarily formed from structural cuticular proteins (CPs) and the polysaccharide chitin. Two CPs, TcCPR27 and TcCPR18, are major proteins present in the elytron (highly sclerotized and pigmented modified forewing) as well as the pronotum (dorsal sclerite of the prothorax) and ventral abdominal cuticle of the red flour beetle, Tribolium castaneum. Both CPs belong to the CPR family, which includes proteins that have an amino acid sequence motif known as the Rebers & Riddiford (R&R) consensus sequence. Injection of double-stranded RNA (dsRNA) for TcCPR27 and TcCPR18 resulted in insects with shorter, wrinkled, warped and less rigid elytra than those from control insects. To gain a more comprehensive understanding of the roles of CPs in cuticle assembly, we analyzed for the precise localization of TcCPR27 and the ultrastructural architecture of cuticle in TcCPR27- and TcCPR18-deficient elytra. Transmission electron microscopic analysis combined with immunodetection using gold-labeled secondary antibody revealed that TcCPR27 is present in dorsal elytral procuticle both in the horizontal laminae and in vertical pore canals. dsRNA-mediated RNA interference (RNAi) of TcCPR27 resulted in abnormal electron-lucent laminae and pore canals in elytra except for the boundary between these two structures in which electron-dense molecule(s) apparently accumulated. Insects subjected to RNAi for TcCPR18 also had disorganized laminae and pore canals in the procuticle of elytra. Similar ultrastructural defects were also observed in other body wall regions with rigid cuticle such as the thorax and legs of adult T. castaneum. TcCPR27 and TcCPR18 are required for proper formation of the horizontal chitinous laminae and vertical pore canals that are critical for formation and stabilization of rigid adult cuticle.

CutProtFam-Pred: Detection and classification of putative structural cuticular proteins from sequence alone, based on profile Hidden Markov Models

The arthropod cuticle is a composite, bipartite system, made of chitin filaments embedded in a proteinaceous matrix. The physical properties of cuticle are determined by the structure and the interactions of its two major components, cuticular proteins (CPs) and chitin. The proteinaceous matrix consists mainly of structural cuticular proteins. The majority of the structural proteins that have been described to date belong to the CPR family, and they are identified by the conserved R&R region (Rebers and Riddiford Consensus). Two major subfamilies of the CPR family RR-1 and RR-2, have also been identified from conservation at sequence level and some correlation with the cuticle type. Recently, several novel families, also containing characteristic conserved regions, have been described. The package HMMER v3.0 (http://hmmer.janelia.org/) was used to build characteristic profile Hidden Markov Models based on the characteristic regions for 8 of these families, (CPF, CPAP3, CPAP1, CPCFC, CPLCA, CPLCG, CPLCW, Tweedle). In brief, these families can be described as having: CPF (a conserved region with 44 amino acids); CPAP1 and CPAP-3 (analogous to peritrophins, with 1 and 3 chitin-binding domains, respectively); CPCFC (2 or 3 C-x(5)-C repeats); and four of five low complexity (LC) families, each with characteristic domains. Using these models, as well as the models previously created for the two major subfamilies of the CPR family, RR-1 and RR-2 (Karouzou et al., 2007), we developed CutProtFam-Pred, an on-line tool (http://bioinformatics.biol.uoa.gr/CutProtFam-Pred) that allows one to query sequences from proteomes or translated transcriptomes, for the accurate detection and classification of putative structural cuticular proteins. The tool has been applied successfully to diverse arthropod proteomes including a crustacean (Daphnia pulex) and a chelicerate (Tetranychus urticae), but at this taxonomic distance only CPRs and CPAPs were recovered.

Temporal and spatial expression of cuticular proteins of Anopheles gambiae implicated in insecticide resistance or differentiation of M/S incipient species

BackgroundPublished data revealed that two of the 243 structural cuticular proteins of Anopheles gambiae, CPLCG3 and CPLCG4, are implicated in insecticide resistance and a third, CPF3, has far higher transcript levels in M than in S incipient species. We studied the distribution of transcripts for these three genes in the tissues of An. gambiae and the location of the proteins in the cuticle itself to gain information about how these cuticular proteins contribute to their important roles. Our data are consistent with CPLCG3/4 contributing to a thicker cuticle thus slowing penetration of insecticides and CPF3 possibly having a role in the greater desiccation tolerance of the M form.MethodsUsing RT-qPCR, we established the temporal expression of the genes and by in situ hybridization we revealed the main tissues where their mRNAs are found. Electron microscopy immunolocalization, using secondary antibodies labeled with colloidal gold, allowed us to localize these proteins within different regions of the cuticle.ResultsThe temporal expression of these genes overlaps, albeit with higher levels of transcripts from CPF3 in pharate adults and both CPLCG3 and CPLCG4 are higher in animals immediately after adult eclosion. The main location of mRNAs for all three genes is in appendages and genitalia. In contrast, the location of their proteins within the cuticle is completely different. CPF3 is found exclusively in exocuticle and CPLCG3/4 is restricted to the endocuticle. The other CPF gene expressed at the same times, CPF4, in addition to appendages, has message in pharate adult sclerites.ConclusionsThe temporal and spatial differences in transcript abundance and protein localization help to account for An. gambiae devoting about 2% of its protein coding genes to structural cuticular proteins. The location of CPLCG3/4 in the endocuticle may contribute to the thickness of the cuticle, one of the recently appreciated components of insecticide resistance, while the location of CPF3 might be related to the greater desiccation resistance of the M form.

Changes in transcript abundance for cuticular proteins and other genes three hours after a blood meal in Anopheles gambiae

Numerous studies have examined changes in transcript levels after Anopheles gambiae takes a blood meal. Marinotti et al. (2006) used microarrays and reported massive changes in transcript levels 3 h after feeding (BF3h) compared to non-blood fed (NBF). We were intrigued by the number of transcripts for structural cuticular proteins (CPs) that showed such major differences in levels and employed paired-end (50 bp) RNA-seq technology to compare whole body transcriptomes from 5-day-old females NBF and BF3h. We detected transcripts for the majority of CPs (164/243) but levels of only 12 were significantly altered by the blood meal. While relative transcript levels of NBF females were somewhat similar to the microarray data, there were major differences in BF3h animals, resulting in levels of many transcripts, both for CPs and other genes changing in the opposite direction. We compared our data also to other studies done with both microarrays and RNA-seq. Findings were consistent that a small number of CP genes have transcripts that persist even in 5-day-old adults. Some of these transcripts showed diurnal rhythms (Rund et al., 2013; Rinker et al., 2013). In situ hybridization revealed that transcripts for several of these CP genes were found exclusively or predominantly in the eye. Transcripts other than for CPs that changed in response to blood-feeding were predominantly expressed in midgut and Malpighian tubules. Even in these tissues, genes responsible for proteins with similar functions, such as immunity or digestion, responded differently, with transcript levels for some rising and others falling. These data demonstrate that genes coding for some CPs are dynamic in expression even in adults and that the response to a blood meal is rapid and precisely orchestrated.

Formation of Rigid, Non-Flight Forewings (Elytra) of a Beetle Requires Two Major Cuticular Proteins

Insect cuticle is composed primarily of chitin and structural proteins. To study the function of structural cuticular proteins, we focused on the proteins present in elytra (modified forewings that become highly sclerotized and pigmented covers for the hindwings) of the red flour beetle, Tribolium castaneum. We identified two highly abundant proteins, TcCPR27 (10 kDa) and TcCPR18 (20 kDa), which are also present in pronotum and ventral abdominal cuticles. Both are members of the Rebers and Riddiford family of cuticular proteins and contain RR2 motifs. Transcripts for both genes dramatically increase in abundance at the pharate adult stage and then decline quickly thereafter. Injection of specific double-stranded RNAs for each gene into penultimate or last instar larvae had no effect on larval–larval, larval–pupal, or pupal–adult molting. The elytra of the resulting adults, however, were shorter, wrinkled, warped, fenestrated, and less rigid than those from control insects. TcCPR27-deficient insects could not fold their hindwings properly and died prematurely approximately one week after eclosion, probably because of dehydration. TcCPR18-deficient insects exhibited a similar but less dramatic phenotype. Immunolocalization studies confirmed the presence of TcCPR27 in the elytral cuticle. These results demonstrate that TcCPR27 and TcCPR18 are major structural proteins in the rigid elytral, dorsal thoracic, and ventral abdominal cuticles of the red flour beetle, and that both proteins are required for morphogenesis of the beetle's elytra.

The Distribution of GYR- and YLP-Like Motifs in Drosophila Suggests a General Role in Cuticle Assembly and Other Protein-Protein Interactions

BackgroundArthropod cuticle is composed predominantly of a self-assembling matrix of chitin and protein. Genes encoding structural cuticular proteins are remarkably abundant in arthropod genomes, yet there has been no systematic survey of conserved motifs across cuticular protein families.Methodology/Principal FindingsTwo short sequence motifs with conserved tyrosines were identified in Drosophila cuticular proteins that were similar to the GYR and YLP Interpro domains. These motifs were found in members of the CPR, Tweedle, CPF/CPFL, and (in Anopheles gambiae) CPLCG cuticular protein families, and the Dusky/Miniature family of cuticle-associated proteins. Tweedle proteins have a characteristic motif architecture that is shared with the Drosophila protein GCR1 and its orthologs in other species, suggesting that GCR1 is also cuticular. A resilin repeat, which has been shown to confer elasticity, matched one of the motifs; a number of other Drosophila proteins of unknown function exhibit a motif architecture similar to that of resilin. The motifs were also present in some proteins of the peritrophic matrix and the eggshell, suggesting molecular convergence among distinct extracellular matrices. More surprisingly, gene regulation, development, and proteolysis were statistically over-represented ontology terms for all non-cuticular matches in Drosophila. Searches against other arthropod genomes indicate that the motifs are taxonomically widespread.ConclusionsThis survey suggests a more general definition for GYR and YLP motifs and reveals their contribution to several types of extracellular matrix. They may define sites of protein interaction with DNA or other proteins, based on ontology analysis. These results can help guide experimental studies on the biochemistry of cuticle assembly.

Molecular Evolution of Drosophila Cuticular Protein Genes

Several multigene families have been described that together encode scores of structural cuticular proteins in Drosophila, although the functional significance of this diversity remains to be explored. Here I investigate the evolutionary histories of several multigene families (CPR, Tweedle, CPLCG, and CPF/CPFL) that vary in age, size, and sequence complexity, using sequenced Drosophila genomes and mosquito outgroups. My objective is to describe the rates and mechanisms of ‘cuticle-ome’ divergence, in order to identify conserved and rapidly evolving elements. I also investigate potential examples of interlocus gene conversion and concerted evolution within these families during Drosophila evolution. The absolute rate of change in gene number (per million years) is an order of magnitude lower for cuticular protein families within Drosophila than it is among Drosophila and the two mosquito taxa, implying that major transitions in the cuticle proteome have occurred at higher taxonomic levels. Several hotspots of intergenic conversion and/or gene turnover were identified, e.g. some gene pairs have independently undergone intergenic conversion within different lineages. Some gene conversion hotspots were characterized by conversion tracts initiating near nucleotide repeats within coding regions, and similar repeats were found within concertedly evolving cuticular protein genes in Anopheles gambiae. Rates of amino-acid substitution were generally severalfold higher along the branch connecting the Sophophora and Drosophila species groups, and 13 genes have Ka/Ks significantly greater than one along this branch, indicating adaptive divergence. Insect cuticular proteins appear to be a source of adaptive evolution within genera and, at higher taxonomic levels, subject to periods of gene-family expansion and contraction followed by quiescence. However, this relative stasis is belied by hotspots of molecular evolution, particularly concerted evolution, during the diversification of Drosophila. The prominent association between interlocus gene conversion and repeats within the coding sequence of interacting genes suggests that the latter promote strand exchange.