Beetle Forewing Formation Requires Two Major Proteins (2012) PDF

Formation of Rigid, Non-Flight Forewings (Elytra) of a Beetle Requires Two Major Cuticular Proteins Yasuyuki Arakane1,2*, Joseph Lomakin3, Stevin H. Gehrke3, Yasuaki Hiromasa1, John M. Tomich1, Subbaratnam Muthukrishnan1, Richard W. Beeman4, Karl J. Kramer1,4, Michael R. Kanost1 1 Department of Bioc...

Formation of Rigid, Non-Flight Forewings (Elytra) of a Beetle Requires Two Major Cuticular Proteins Yasuyuki Arakane1,2*, Joseph Lomakin3, Stevin H. Gehrke3, Yasuaki Hiromasa1, John M. Tomich1, Subbaratnam Muthukrishnan1, Richard W. Beeman4, Karl J. Kramer1,4, Michael R. Kanost1 1 Department of Biochemistry, Kansas State University, Manhattan, Kansas, United States of America, 2 Division of Plant Biotechnology, College of Agriculture and Life Science, Chonnam National University, Gwangju, Korea, 3 Department of Chemical and Petroleum Engineering, University of Kansas, Lawrence, Kansas, United States of America, 4 Center for Grain and Animal Health Research, Agricultural Research Service, United States Department of Agriculture, Manhattan, Kansas, United States of America Abstract 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. Citation: Arakane Y, Lomakin J, Gehrke SH, Hiromasa Y, Tomich JM, et al. (2012) Formation of Rigid, Non-Flight Forewings (Elytra) of a Beetle Requires Two Major Cuticular Proteins. PLoS Genet 8(4): e1002682. doi:10.1371/journal.pgen.1002682 Editor: David L. Stern, Janelia Farm Research Campus, Howard Hughes Medical Institute, United States of America Received December 13, 2011; Accepted March 12, 2012; Published April 26, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by National Science Foundation grants IOS0726425 and MRI0521587 (http://www.nsf.gov/index.jsp). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction number present in the red flour beetle, Tribolium castaneum. Anopheles gambiae (malaria mosquito) and Bombyx mori (oriental How arthropods manufacture exoskeletons with a wide array of silkworm) have an even larger number of genes encoding CP-like mechanical properties, ranging from hard and rigid to soft and proteins, each species harboring more than 200 putative CP genes flexible, is an important question in developmental biology. The [6–10]. Expression of specific CPs may be required to produce insect exoskeleton, or cuticle, covers the entire body wall and cuticles with a range of morphological and mechanical properties attached appendages as well as the foregut, hindgut and tracheae. in different regions of the insect body and at different It is a complex extracellular biocomposite, secreted by the developmental stages. epidermis and consisting of several functional layers including Insect CPs are classified into several distinct families defined by the waterproofing envelope, the protein-rich epicuticle and the the presence of specific sequence motifs [7,10]. The largest of these chitin-rich procuticle. Cuticular proteins (CPs) and the is the CPR family, which includes proteins that have a conserved polysaccharide chitin are the primary structural components of amino acid sequence known as the Rebers & Riddiford (R&R) the exo- and endocuticular layers that comprise the procuticle. motif. The R&R motif contains a putative chitin-binding During cuticle maturation and tanning (sclerotization and domain that may help to coordinate the interactions between pigmentation), some of the CPs are post-translationally modified chitin fibers and the proteinaceous matrix [12,13]. and cross-linked by quinones or quinone methides produced by A major event in the evolution and diversification of beetles was the laccase-mediated oxidation of N-acylcatechols [2,3]. This the transformation of the membranous forewings into thickened, process stabilizes and hardens the exoskeleton, protecting insects hardened, non-flight covers (elytra) for protection of the delicate from microbial, physical and other environmental stresses. hindwings and dorsal abdomen [14–16]. The elytron is composed However, little is known about the functional importance of of ventral and dorsal layers of epidermal cells that secrete thin individual insect cuticular proteins in the morphogenesis and lower and thicker upper cuticular laminae [17,18]. In the mechanical properties of the exoskeleton. developing elytron, the space between these two layers is filled More than 100 genes encoding CP-like proteins have been with hemolymph and supporting structures known as trabeculae identified in the fruit fly, Drosophila melanogaster , with a similar that function as mechanical struts, connecting and fortifying the PLoS Genetics | www.plosgenetics.org 1 April 2012 | Volume 8 | Issue 4 | e1002682  Rigid Cuticle Proteins in Tribolium Author Summary and membranous in comparison to the dorsal layer. The surface of the ventral elytral cuticle is relatively smooth and makes close Primitive insects have two pairs of membranous flight contact with the underlying and folded hindwings. The surface of wings, but during the evolution of the beetle lineage the the dorsal elytral cuticle, on the other hand, contains numerous forewings lost their flight function and became modified sensory setae and rib-like structures (striae) that extend longitudi- as hard, rigid covers called elytra for protection of soft nally, apparently adding rigidity to the structure. Initially, the body parts of the abdomen and also the delicate flexible elytra are short, colorless and soft, but they expand in both length hindwings, which retained their flight function. This and width shortly after eclosion, and subsequently harden and transformation is manifested by a greatly thickened and darken. A similar cuticle tanning process occurs in most of the rigid (sclerotized) exoskeletal cuticle secreted by the adult body wall. forewing epidermis. We demonstrate that this evolutionary modification is accompanied by the incorporation of two In this study we have identified two highly abundant proteins highly abundant structural proteins into the elytral cuticle, that are present in rigid cuticle of the elytron, pronotum and namely TcCPR18 and TcCPR27. Depletion of these proteins ventral abdomen but not in the flexible cuticle of the dorsal by RNA interference results in malformation and weaken- abdomen and hindwing of T. castaneum adults, characterized their ing of the elytra, culminating in insect death. These genes and expression profiles, and analyzed their roles in adult proteins are also abundant in hard cuticle from other cuticle formation and stabilization. We have also determined that regions such as the pronotum and ventral abdomen, but these two CPR proteins are essential structural components in the are absent in soft cuticles, and therefore may function as sclerotized dorsal cuticle of the elytron and are required for key determinants of rigid cuticle. Expression of such normal morphological, functional and mechanical properties. proteins at high levels in the modified forewing appears to have been a fundamental evolutionary step in the Results transformation of the membranous wing into a thickened and rigid elytron in the Coleoptera. Elytra from T. castaneum contain two predominant cuticular proteins Extracts of untanned elytra dissected from newly emerged dorsal and ventral cuticular layers. As the elytron matures, the adults contained two highly abundant proteins with apparent sizes epidermal layers are reduced in size, possibly dying or fusing of 10 and 20 kDa based on their electrophoretic mobilities together, and the hemolymph is resorbed, leaving a cavernous (Figure 1). To characterize these major proteins further, each was interior. The dorsal layer of the elytron becomes highly tanned digested with trypsin, and the resulting peptides were analyzed by and rigid as a result of both pigmentation and sclerotization. The MALDI-TOF/TOF mass spectrometry. Comparison of these ventral layer also exhibits some pigmentation, but it remains thin results with conceptual trypsinization of the computed proteome of Figure 1. Identification of two major elytral cuticle proteins from T. castaneum. Extracts of SDS-soluble proteins from elytra dissected from newly emerged adults were analyzed by SDS-PAGE. The two major proteins (subsequently named TcCPR27 and TcCPR18), with apparent sizes of 10 and 20 kDa, respectively, were digested with trypsin, and the resulting peptides were analyzed by MALDI-TOF mass spectrometry (see Figure S1 and Table S1). Amino acid sequences deduced from cloned cDNA sequences for each protein are shown on the left. Both proteins contain an RR-2 motif (highlighted in gray). Predicted secretion signal peptides are underlined. In both TcCPR27 and TcCPR18, the amino-terminal residue of the mature forms after secretion (Gln 20) is apparently modified to pyroglutamic acid (Table S1). doi:10.1371/journal.pgen.1002682.g001 PLoS Genetics | www.plosgenetics.org 2 April 2012 | Volume 8 | Issue 4 | e1002682  Rigid Cuticle Proteins in Tribolium T. castaneum revealed two candidate genes, denoted as TcCPR27 TcCPR27 and TcCPR18 are abundantly expressed in rigid (XP_971678) and TcCPR18 (XP_967633), which are members of cuticle but not in flexible cuticle the Rebers and Riddiford family of cuticular proteins (Figure 1, To investigate whether TcCPR27 and TcCPR18 are present in Table S1 and cuticle DB: http://biophysics.biol.uoa.gr/ other regions of the adult cuticle, we extracted proteins from cuticleDB). Peptide coverages for TcCPR27 and TcCPR18 were cuticular samples dissected from the pronotum and the ventral 68.2 and 88.7%, respectively (Figure S1). abdomen just after adult eclosion. As in the case of the elytra, We cloned cDNAs corresponding to these cuticular protein TcCPR27 and TcCPR18 proteins were also the predominant genes (accession numbers HQ634478 and HQ634479). The protein constituents of the pronotum, although their yields were cDNA sequence of TcCPR27 was identical to that of the NCBI low relative to those obtained from the elytra (Figure 2). We RefSeq gene prediction, whereas the RefSeq prediction for hypothesized that because the extent of tanning of the pronotum TcCPR18 had one in-frame deletion of three consecutive just after eclosion is substantially greater than that of the elytron, nucleotides and a single nucleotide mismatch compared to the which tans at a later time (Figure 2), pronotum cuticular proteins cDNA, resulting in a deletion of one amino acid (one of the twelve were already cross-linked and much less extractable at the time of consecutive glycines at amino acid positions 65–76 in the RefSeq adult eclosion. To delay pronotum cuticle tanning, dsRNA for prediction) and a phenylalanine-to-leucine substitution at amino laccase-2 (dsTcLac2) was injected into 0–1 d-old pupae , and the acid position 85 in the RefSeq prediction. TcCPR27 and TcCPR18 pronotum cuticular proteins were subsequently extracted from encode proteins containing putative secretion signal peptide samples obtained soon after adult eclosion. The yields of sequences, with theoretical molecular masses for the mature TcCPR27 and TcCPR18 were much higher in those extracts, proteins of 11.4 and 16.4 kDa, respectively. indicating that the two proteins had not undergone substantial Each mature protein contains a single RR-2 cuticular protein cross-linking in the absence of laccase and therefore were more motif. Nearly all RR-2 proteins have a consensus region as follows: readily extractable (Figure 2). G-X(8)-G-X(6)-Y-X(6)-GF. Both of these Tribolium proteins, Like the elytron, the adult ventral abdominal cuticle undergoes however, have a slightly different RR2 motif. TcCPR27 contains tanning and becomes hardened 3–5 d post eclosion, whereas the G-X(8)-G-X(6)-Y-X(5)-GA, whereas TcCPR18 has G-X(8)-H- dorsal abdominal cuticle in the adult remains relatively untanned, X(7)-Y-X(6)-GF. The former has a GA rather than the almost flexible and transparent like the hindwing. TcCPR27 and universal GF or GY at the end of the consensus, and the conserved TcCPR18 were abundant in extracts recovered from ventral Y and the third G are interrupted by five amino acids instead of abdominal cuticle of newly emerged adults, but very little or no the typical six. In the case of TcCPR18, the second conserved G is TcCPR27 or TcCPR18 was present in extracts of the dorsal replaced by an H with no G residue located nearby. abdominal cuticle (Figure S4A). Similarly, the levels of TcCPR27 TcCPR18 is rich in glycine (21.6%), whereas TcCPR27 has a and TcCPR18 in the flexible hindwing were very low or high content of both glycine (16.5%) and histidine (15.5%). undetectable (Figure S4A). These results show that TcCPR27 TcCPR18 is an apparent ortholog of the ecdysteroid-regulated, and TcCPR18 are major proteins in cuticles that become highly adult-specific cuticle protein acp22 of Tenebrio molitor (yellow sclerotized and rigid, but they are absent or only very minor mealworm), with 67% sequence identity (Figure S2). Both components of cuticles that are more flexible and membranous. TcCPR27 and TcCPR18 map to linkage group 3 of the T. castaneum genome, but they are not tightly linked (BeetleBase: http:// TcCPR27 and TcCPR18 genes are expressed at high levels beetlebase.org). Elytra of three other Tribolium species, T. in body regions with rigid adult cuticle brevicornis, T. confusum and T. freemani, also contain predominant Few or no transcripts for TcCPR27 or TcCPR18 were detected cuticular proteins with high amino acid sequence similarities to during the egg, larval or early pupal stages of development. TcCPR27 and TcCPR18 (Figure S3 and Table S2). However, the transcript levels of these genes dramatically Figure 2. TcCPR27 and TcCPR18 are abundant in cuticle of the pronotum. To suppress cuticle tanning, dsRNA for TcLac2 (2 ng per insect) was injected into day 0 pupae. Proteins from the pronotum and elytra were extracted from five adults at 0–30 min after eclosion, and the extracts were analyzed by SDS-PAGE (left panel). In the dsVer-treated control animals, the extent of tanning of the pronotum was substantially greater than that of the elytron (right panel). S: PBS homogenate supernatant, P: PBS homogenate pellet. doi:10.1371/journal.pgen.1002682.g002 PLoS Genetics | www.plosgenetics.org 3 April 2012 | Volume 8 | Issue 4 | e1002682  Rigid Cuticle Proteins in Tribolium increased at the pharate adult stage 0–1 d before eclosion, declining antibody specifically detected CPR27 but not CPR18 (Figure 4A). soon thereafter (Figure 3A, 3B). Transcript levels of TcCPR27 and In pharate adults, TcCRP27 was co-localized with chitin in the TcCPR18 in the elytron were approximately 1,700- and 55-fold dorsal elytral cuticle as well as in the ventral abdominal cuticle, both higher, respectively, than those in the membranous hindwing of which become more rigid and darker as the adult matures (Figure S4B). Both genes were also expressed in the pronotum and (Figure 4B, panels 1, 3). Little or no TcCRP27 immunoreactivity ventral abdomen, whose cuticles become highly sclerotized and was detected in the pupal, hindwing, or ventral elytral cuticles. hardened in mature adults. Transcript levels of TcCPR27 and TcCPR18 in the ventral abdomen were approximately 3,000 and RNAi–mediated knockdown of TcCPR27 and TcCPR18 770 times higher, respectively, than the levels in the transparent, expression leads to malformed and weakened elytra flexible and membranous dorsal abdomen (Figure S4B). RNA interference (RNAi) was used to investigate the functions of TcCPR27 and TcCPR18. As a negative control we injected TcCPR27 is localized in rigid cuticle of the dorsal elytra dsRNA for T. castaneum tryptophan oxygenase (the vermilion gene, and ventral abdomen abbreviated Ver), a gene required for normal eye pigmentation The high histidine content of TcCPR27 and TcCPR18 (15.5%. Following dsRNA injections into last instar larvae, mRNA and 10.1%, respectively) allowed us to purify these proteins from and protein levels of TcCPR27 and TcCPR18 were analyzed by elytra dissected from newly molted adults by utilizing nickel-affinity real-time PCR and SDS-PAGE. Injection of these dsRNAs led to chromatography (Figure 4A). A polyclonal antibody directed substantial and specific down-regulation of each gene at the against purified TcCPR27 was then generated. The CPR27 mRNA (Figure 5A) and protein (Figure 5B) levels. TcCPR27 Figure 3. Expression profiles of TcCPR27 and TcCPR18 genes during development. (A) To analyze the expression profiles of TcCPR27 and TcCPR18 during development, real-time PCR experiments were done using total RNA extracted from five whole insects at different developmental stages (embryo to adult). Both genes were highly expressed at the pupal stage. (B) To analyze more rigorously the expression patterns of these genes, the stages analyzed were expanded between the early pharate pupal and young adult stages. The transcript levels of both genes dramatically increased at the pharate adult stage and declined rapidly thereafter. E, embryos; YL, young larvae; ML, mature larvae; PP, pharate pupae; P, pupae; A, adults; PP0, day 0–1 pharate pupae; PP1, day 1–2 pharate pupae; P0, day 0 pupae; P1, day 1 pupae, P2, day 2 pupae; P3, day 3 pupae; P4, day 4 pupae (pharate adults); P5, day 5 pupae (pharate adults); A0, day 0 adults; A1, day 7 adults. Expression levels for TcCPR27 and TcCPR18 are presented relative to the levels of expression in embryos (E) or 0–1 d old pharate pupae (PP0). The transcript levels of the T. castaneum ribosomal protein S6 (rpS6) were measured to normalize for differences between samples in the concentrations of cDNA templates. doi:10.1371/journal.pgen.1002682.g003 PLoS Genetics | www.plosgenetics.org 4 April 2012 | Volume 8 | Issue 4 | e1002682  Rigid Cuticle Proteins in Tribolium Figure 4. Immunolocalization of TcCPR27 in elytral cuticle. (A) Coomassie staining and immunoblot analyses of purified TcCPR27 and TcCRP18. (B) Immunolocalization of TcCPR27 in pharate adults. Cryosections (5–10 mm) of 5 d old pupae that had been injected previously with dsRNA for TcCPR27 or TcVer (T. castaneum Vermilion) in the last larval instar were incubated with the anti-TcCPR27 antibody. Anti-TcCPR27 antibody was detected by Alexa Fluor 488-conjugated anti-rabbit IgG antibody (green arrows in panels 1 and 2). The same sections were also stained with a rhodamine-conjugated chitin-binding probe (red arrows in panels 3 and 4). Nuclei were stained with DAPI (blue). E = elytron, H = hindwing, TC = thoracic cuticle, PC = pupal cuticle, D = elytral dorsal cuticle, V = elytral ventral cuticle. Scale bar = 10 mm. doi:10.1371/journal.pgen.1002682.g004 immunostaining also was strongly reduced after injection of unable to fold normally. Such insects died approximately one week dsTcCPR27 (Figure 4B, panel 2). Chitin staining in TcCPR27- after eclosion, apparently from dehydration that resulted from deficient insects, however, was detected at approximately the same failure of the misshapen elytra to properly cover the membranous level as in dsVer-treated control animals (Figure 4B, panel 4). These dorsal abdomen and to thereby seal it against trans-cuticular water results were further supported by staining of elytral chitin with a loss. The shape of a normal elytron is ‘‘inverted boat-like’’ to fit fluorescent chitin-labeling reagent, FITC-CBD. There was no snugly on top of the hindwings and abdomen in order to protect difference in chitin staining among elytra collected from the latter structures (Figure S6). In contrast, elytra from dsTcCPR27-, dsTcCPR18- and dsVer-treated insects (Figure S5). dsTcCPR27-treated insects were flatter and/or warped and did Injection of dsTcCPR27 or dsTcCPR18 into larvae had no not cover the entire abdomen. Manual excision of the distal half of apparent effect on larval-pupal or pupal-adult molting or on the the elytron from a mature adult also led to high mortality, whereas morphology of the pupal cuticle, as expected from the observed removing an entire hindwing did not cause significant mortality, as late pupa-specific expression of these genes. However, the elytra of long as the elytra could adopt their normal juxtaposition (Figure the resulting adults were malformed (Figure 6 and Figure S6). The S7), consistent with our observation that properly formed elytra surface of the elytra of dsTcCPR18-treated adults was irregular and are essential to prevent desiccation of the adult, in addition to rough compared to those of control insects. Adults derived from other potentially protective functions. These results support the dsTcCPR27-injected larvae exhibited even more severe morpho- hypothesis that TcCPR27 and TcCPR18 are major structural logical defects. Their elytra were very short, wrinkled, warped and proteins in rigid elytral cuticle, and are required for normal elytral fenestrated (Figure 6 and Figure S6) and their hindwings were morphogenesis, hindwing folding and body hydration. PLoS Genetics | www.plosgenetics.org 5 April 2012 | Volume 8 | Issue 4 | e1002682  Rigid Cuticle Proteins in Tribolium Figure 5. Knockdown of TcCPR27 and TcCPR18 transcripts by RNAi. Late instar T. castaneum larvae were injected with 0.2 mg of dsRNA for TcCPR27 or TcCPR18. Following dsRNA injections, expression of TcCPR27 and TcCPR18 genes was analyzed by real-time PCR (A) and SDS-PAGE (B) to evaluate transcript and protein levels. cDNAs were prepared from total RNA isolated from five whole insects at pupal day 5 (10 d post-injection). For real-time PCR, expression levels of TcCPR27 and TcCPR18 are presented relative to the levels in Ver control insects (injected with dsRNA for T. castaneum Vermilion gene). The transcript levels of the T. castaneum ribosomal protein S6 (rpS6) were measured to normalize for differences between samples in the concentrations of cDNA templates. Proteins were extracted from elytra from five newly emerged adults for each treatment. A faint band (red arrow) with a mobility similar to that of TcCPR18 observed in extracts of whole insects injected with dsRNA for TcCPR18 was identified by mapping of tryptic peptides by MALDI-TOF to be a different CPR RR2 protein, TcCPR33, with a theoretical mass of 19.1 kDa. These data indicate that both TcCPR27, TcCPR18 were specifically down-regulated at both the mRNA and protein levels after dsRNA injections. doi:10.1371/journal.pgen.1002682.g005 Effect of dsRNA for TcCPR27 and TcCPR18 on the had intermediate values of strength, consistent with the less severe mechanical properties of elytra visible phenotype observed with TcCPR18 knockdown (Figure 6). We also analyzed the effects of depletion of TcCPR27 and In addition, the dsTcCPR27 elytra had reduced values for tan d, an TcCPR18 on the mechanical properties of elytra. Dynamic indication that they experienced a higher degree of cross-linking mechanical experiments were carried out to determine the storage than the control. Lower E0 or tan d in polymeric networks is modulus E9 and the loss modulus E0 of elytra as a function of typically associated with a reduction in the network of uncross- oscillation frequency and strain. E9 is a measure of the elastically linked material, dangling chains (chains linked to the network at recoverable deformation energy, whereas E0 is a measure of only one end), loops and other network imperfections. It is a viscous energy dissipation (dampening) and hence is also known as well-established principle in the synthesis of gels or networks by the viscous modulus. The ratio E0/E9 is known as the ‘‘loss cross-linking polymers, that increasing the ratio of cross-linker tangent’’ or simply tan d, where d is the phase angle between molecules to polymer molecules will typically increase the overall sinusoidally applied stress and strain. For materials such as the cross-link density of a network and reduce the fraction that is not elytra where E9..E0, E9 is approximately equal to the Young’s cross-linked. Thus, for elytral cuticle, the deficiency of a modulus obtained from the slope of simple stress-strain measure- major structural cuticular protein such as TcCPR27, while ments at the same strain rate [23,24]. Hence, E9 is a measure of maintaining a constant concentration of quinone cross-linking the stiffness of the elytra. Elytra from animals injected with molecules, would be expected to lead to a greater average number dsTcCPR27 were significantly less rigid (lower E9) than the dsVer- of cross-links per protein molecule. Thus, for elytral cuticle, treated controls (Figure 7). Elytra from dsTcCPR18-injected beetles reducing the expression level of a major structural cuticular PLoS Genetics | www.plosgenetics.org 6 April 2012 | Volume 8 | Issue 4 | e1002682  Rigid Cuticle Proteins in Tribolium Figure 6. T. castaneum elytral defects produced by injection of dsRNAs for TcCPR27 and TcCPR18. To investigate the functions of TcCPR27 and TcCPR18, specific dsRNAs for TcCPR27 (dsCPR27) or TcCPR18 (dsCPR18) (0.2 mg per insect) were injected into late instar larvae. Dorsal views of the resulting adults (1-d old) (left panel) and elytra (right panel) are shown. dsRNA for Ver (dsVer) was injected to serve as a negative control. doi:10.1371/journal.pgen.1002682.g006 protein such as TcCPR27 or TcCPR18, while maintaining a immature stages. Highly abundant cuticular proteins related to constant concentration of quinone cross-linking molecules, would TcCPR27 and TcCPR18 were also present in protein extracts of be expected to lead to a greater average number of cross-links per elytra dissected from three other Tribolium species including T. protein molecule. The more severe phenotype for knockdown of brevicornis, T. confusum and T. freemani (Figure S3 and Table S2). TcCPR27, relative to TcCPR18 might be due to differences in the TcCPR27 and TcCPR18 were also identified in extracts of degree of knockdown of their expression in the RNAi experiments, pronotum cuticle, in which tanning had been initiated before adult or perhaps could be due to differences in their structural properties eclosion. The protein yields, however, were much lower than those or cross-linking chemistry. A greater reduction in protein levels for obtained from elytral extracts unless cuticle tanning was suppressed TcCPR27 than TcCPR18 in the knockdown animals might have by injection of dsRNA for the tanning enzyme TcLac2 (Figure 2). led to relatively more cross-linking, thus reducing tan d, and a These results support the hypothesis that TcCPR27 and TcCPR18 greater reduction in TcCPR27 protein expression would lead to a are cross-linked by highly reactive quinones and quinone methides lower storage modulus. Similar observations were previously that are produced by the cuticle tanning phenoloxidase laccase-2, reported for elytra from insects subjected to Lac2 knockdown and that these proteins become inextractable after tanning has (reduced tan d combined with reduced E9). occurred. Previously, Missios et al. extracted two major cuticular proteins of 10 and 20 kDa, consistent with the apparent Discussion masses of TcCPR27 and TcCPR18, respectively, from extracts of cuticle from whole bodies of newly eclosed T. castaneum adults. The basic genetic patterning mechanism for dorsal appendages Neither of these proteins was extractable from 7 day-old adults, such as wings has been described for both D. melanogaster and T. consistent with an interpretation that these proteins become cross- castaneum [27–29]. Similar gene networks are used to pattern linked during maturation of the cuticle. wings, elytra and halteres, despite the profound morphological and To study the functions of TcCPR27 and TcCPR18, we functional divergence of these appendages during insect evolution. performed RNAi and successfully down-regulated levels of Individual structural proteins are likely to substantially affect the TcCPR27 and TcCPR18 mRNAs and proteins (Figure 5). These physical properties of elytra. However, to date, there have been no deficiencies caused several elytral defects. Although TcCPR27 and detailed reports about the contributions of individual structural TcCPR18 are also present in other body regions such as the proteins to elytral morphogenesis. cuticles of the pronotum and ventral abdomen, which are heavily Like other beetle species, T. castaneum adults possess elytra, tanned in the mature adult, we did not observe visible phenotypic modified forewings with a highly sclerotized and pigmented dorsal changes in those cuticles after injection of dsTcCPR27 and cuticle. Immediately after eclosion, untanned elytra have a soft dsTcCPR18. The size and shape of these body regions do not white cuticle. Elytra expand shortly thereafter and then become change much after adult eclosion, in contrast to the elytra that are rigid and darker during cuticle maturation. The role of structural greatly expanded shortly after eclosion. The elytra of both proteins in this developmental process is the focus of this study. We TcCPR27- and TcCPR18-deficient insects failed to fully expand, identified two proteins from the RR2 family of cuticular proteins, and their dorsal surfaces were not smooth (Figure S6). The elytra TcCPR27 and TcCPR18, which are highly abundant in protein of TcCPR27-deficient insects, particularly, were very short, extracts of elytra dissected from newly emerged adults. TcCPR27 wrinkled and fragile. Elytra from dsTcCPR27- and dsTcCPR18- and TcCPR18 transcripts were strongly up-regulated at the treated insects appear to contain more cross-linked proteins than developmental stage when adult cuticular proteins are expected to the elytra from dsVer-treated control insects (Figure 6). Lacking be synthesized (pupal day 4), and were nearly absent at earlier these major cuticular proteins apparently increases the effective PLoS Genetics | www.plosgenetics.org 7 April 2012 | Volume 8 | Issue 4 | e1002682  Rigid Cuticle Proteins in Tribolium Figure 7. Mechanical properties of Tribolium dsTcCPR27 and dsTcCPR18 elytra. Elytra from beetles on day 2 after adult eclosion were examined by dynamic mechanical analysis over a frequency range of 0.1 to 600 rad/s. Typical scans are shown in the top panels, and mean values at 1 rad/s are presented in the lower panels. Error bars represent standard deviation (n = 3–4). The asterisk indicates significant difference from the dsVer control (p,0.05) as determined by analysis of variance and Tukey’s Multiple Comparison Test. Reduced expression of the abundant CPR proteins resulted in weaker (smaller E9) yet more cross-linked (smaller tan d) elytra. doi:10.1371/journal.pgen.1002682.g007 concentration of cross-linking agents (NADA and NBAD qui- TcCPR27 and TcCPR18 are required not only for rigid cuticle nones), resulting in aberrant cross-linking among the remaining development, but also for morphogenesis, elytral mechanical proteins and shortened warped elytra. The effect was seen most properties, and survival of the red flour beetle. In contrast, these clearly in dsTcCPR27-treated insects, in which the modulus and proteins are essentially undetectable in soft cuticles. Expression of tan d were significantly reduced relative to control insects. The such cuticular proteins in the modified forewings appears to be a dsTcCPR18- treated insects had a smaller decrease in modulus and fundamental evolutionary step in transforming the flexible and tan d, perhaps because of a smaller degree of protein reduction. thin membranous wing into a thickened and rigid elytron in the The difference could also arise from structural differences between Coleoptera. In the case of TcCPR18, an orthologous gene is found TcCPR27 and TcCPR18, which could have different propensities in the only other beetle species examined, the lesser grain borer, for forming intermolecular vs. intramolecular cross-links. Howev- Rhyzopertha dominca, in the family Bostrichidae (Schlipalius, D. and er, the present data cannot draw that level of distinction. All of Beeman, R. W., unpublished observations) but not in any of the these results suggest that TcCPR27 and TcCPR18 are critical for other sequenced arthropod genomes, including representatives of normal elytral morphogenesis and are required to prevent the Diptera, Hymenoptera and Lepidoptera. These structural dehydration and death of the adult. proteins are probably cross-linked during sclerotization, via In summary, we have identified in beetles two major structural formation of histidyl-catechol adducts [32,33]. Rigidification of proteins, TcCPR27 and TcCPR18, which account for approxi- the beetle forewing has likely been achieved in part through both mately half of the extractable cuticular proteins in the elytra and structural protein incorporation and multiple co-options of the also are major components of other hard cuticular structures. It is sclerotization pathway acting downstream of conserved wing gene interesting to note that the proteins utilized for hard cuticles of network components, with the final product being primarily a rigid other body regions of the beetle were apparently used to build the interpenetrating network of chitin embedded in a cross-linked elytron’s hard cuticle. In some saturniid moth species, proteins protein matrix [3,29,34,35]. To gain a more comprehensive from the same CPR family are also used to form rigid structures understanding of the roles of cuticular proteins in defining the such as tubercles, head capsules and hard pupal cuticle. We morphology and properties of the beetle elytron and rigid body now have biomechanical evidence on just how important these wall cuticle, future studies are required to determine, at the kinds of proteins are. ultrastructural level, the precise localization of TcCPR27, PLoS Genetics | www.plosgenetics.org 8 April 2012 | Volume 8 | Issue 4 | e1002682  Rigid Cuticle Proteins in Tribolium TcCPR18 and other structural proteins, and to assess the nature oxidation of methionine as a variable modification. Sequence and extent of their covalent cross-linking during sclerotization. motif analysis of the predicted protein sequence was searched in motifs database including PROSITE profiles and Pfam HMMs. Materials and Methods TcCPR27 and TcCPR18 cDNAs Insects The full-length coding sequences for TcCPR27 and TcCPR18 The GA-1 strain of T. castaneum was used in this study. Beetles (351 bp and 504 bp, respectively) were amplified from total RNA were reared at 30uC under standard conditions. extracted from pupae (mixture of 0 d- to 5 d-old pupae) by RT- PCR. The cDNAs for TcCPR27 and TcCPR18 were amplified Protein extraction and identification using the following gene specific primers, which included predicted Elytra of newly emerged adults (n = 10) were homogenized in start and stop codons: 59 ATG CAC GGT GGA GCA GTT C 39 100 ml of cold PBS containing protease inhibitor cocktail (Roche). and 59 TCA GTT GCC TCC AAT CCC G 39 for TcCPR27, and The homogenate was centrifuged for 2 min at 4uC. The 59 ATG AGA TTA TTT ATT ACA TTG GCC 39 and 59 CTA supernatant was collected as PBS soluble fraction. The pellet GAT TAA TAA TGT GGT TTG TAA G 39 for TcCRP18. PCR was homogenized in 100 ml of SDS-PAGE sample buffer, heated products were cloned into pGEMT (Promega) and sequenced. at 95uC for 10 min, centrifuged for 2 min. The supernatant was collected as PBS pellet fraction. Protein extracts were analyzed by Real-time PCR 15% SDS-PAGE or 4–12% Bis-Tris gradient gel (Invitrogen). Total RNA isolation, cDNA synthesis and real-time PCR were Proteins were digested with trypsin, and the resulting fragments done as described previously using the following primer sets: were analyzed by MALDI-TOF mass spectrometry. 59AGG TTA CGG CCA TCA TCA CTT GGA 39 and 59ATT GGT GGT GGA AGT CAT GGG TGT 39 for TcCPR27, 59 GAA Identification of proteins by mass spectrometry TAC CGC ATC CGT GAC CAC AAA 39 and 59CAG GTT CCA After staining gels with Coomassie G-250, the selected gel band ACA AAC TGT AGG TTC CC 39 for TcCPR18. Total RNA was was excised as 1–2 mm diameter pieces and transferred to a isolated from whole insects (n = 5) to analyze developmental 1.5 mL Eppendorf tube. A protein-free region of the gel was also expression patterns and knock-down levels after RNAi of TcCPR27 excised as background control. The control and test gel sections and TcCPR18. Total RNA also was isolated from elytra, hindwings, were destained using three 30 min washes of 60 mL 1:1 ventral abdomens and dorsal abdomens of pharate adults (5 d-old acetonitrile: water at 30uC. Gel pieces were then dried for pupae) (n = 10). The transcript levels of the T. castaneum ribosomal 10 min under vacuum. The gel sections were subjected to protein S6 (rpS6) were measured to normalize for differences reduction and alkylation using 50 mM Tris (2-carboxyethyl) between samples in the concentrations of cDNA templates. phosphine (TCEP) at 55uC for 10 min followed by 100 mM iodoacetamide in the dark at 30uC for 60 min. The carbox- Double-stranded RNA synthesis and injection ymethylated gels were thoroughly washed and re-dried in vacuo, dsRNA for TcCPR27 and TcCPR18 was synthesized as then incubated with sequencing grade trypsin (Trypsin Gold, described previously using the primers 59-(T7)-GAC CAC Promega, Madison, WI), 20 ng/mL in 40 mM ammonium CAC ACC CAT G-39 and 59-(T7)-TCA GTT GCC TCC AAT bicarbonate, in 20 mL. Upon rehydration of the gels, an additional C-39 for TcCPR27, and 59-(T7)-GGA AGA GTA CGG TCA TC - 15 mL of 40 mM ammonium bicarbonate and 10% acetonitrile 39 and 59-(T7)-GGT TCC CTT TAC TTT G-39 for TcCPR18, was added, and gel sections were incubated at 30uC for 17 h in where T7 indicates the T7 RNA polymerase recognition sequence. sealed Eppendorf tubes. The aqueous digestion solutions were The sizes of dsRNAs for TcCPR27 and TcCPR18 were 204 bp and transferred to clean 1.5 mL Eppendorf tubes, and tryptic 325 bp, respectively. dsRNAs were injected into last instar larvae fragments remaining within the gel sections were recovered by a. dsRNA for the T. castaneum vermilion gene (dsVer) was used as a single extraction with 50 ml of 50% acetonitrile and 2% negative control. trifluoracetic acid (TFA) at 30uC for 1 h. The acetonitrile fractions were combined with previous aqueous fractions and the liquid was Purification of TcCPR27 and TcCPR18 from extracts of removed by speed vacuum concentration. The dried samples were resuspended in 10 mL of 30 mg/mL 2,5-dihydroxylbenzonic acid elytra (DHB) (Sigma, St. Louis, MO) dissolved in 33% acetonitrile/0.1% Proteins were extracted from 200 pairs of elytra of 5 d-old TFA and 2 mL of peptide/matrix solution was applied on a Bruker pupae as described in Materials and Methods. The homogenate Massive Aluminum plate for MALDI-TOF and TOF/TOF was centrifuged for 2 min at 4uC. The supernatant was applied to analysis. Mass spectra and tandem mass spectra were obtained a Ni-NTA column equilibrated with 50 mM Tris-HCl, pH 7.5 on a Bruker Ultraflex II TOF/TOF mass spectrometer. Positively containing 0.2 M NaCl and 20 mM imidazole and washed with charged ions were analyzed in the reflector mode. MS and MS/ the same buffer. Bound proteins were eluted with a 20 to 200 mM MS spectra were analyzed with Flex analysis 3.0 and Bio Tools 3.0 imidazole gradient. The fractions were analyzed for protein software (Bruker Daltonics). Measurements were externally content by SDS-PAGE. Purified TcCPR27 was used as antigen to calibrated with 8 different peptides ranging from 757.39 to generate rabbit antiserum by Cocalico Biologicals, Inc., PA, USA. 3147.47 (Peptide Calibration Standard I, Bruker Daltonics) and internally recalibrated with peptides from the autoproteolysis of Mechanical analysis of elytra trypsin. Peptide ion searches were performed with Beetlebase Mechanical analysis of elytra was carried out using a TA (http://www.bioinformatics.ksu.edu/BeetleBase/) (as well as Instruments RSAIII dynamic mechanical analyzer, by methods Metazoa domain_201000104 in NCBInr database) using MAS- described previously. COT software (Matrix Science). The following parameters were used for the database search: MS and MS/MS accuracies were set Accession numbers to ,0.5 Da, trypsin/P as an enzyme, missed cleavages 1, cDNA sequences are deposited at NCBI with accession carbamidomethylation of cysteine as fixed modification, and numbers HQ634478 (TcCPR27) and HQ634479 (TcCPR18). PLoS Genetics | www.plosgenetics.org 9 April 2012 | Volume 8 | Issue 4 | e1002682  Rigid Cuticle Proteins in Tribolium Supporting Information dsTcCPR27- and dsTcCPR18-elytra were remarkably soft and fragile compared with dsVer-elytra. The fluorescence was observed Figure S1 Trypsinization and peptide mass fingerprinting (PMF) using a Leica MZ FLIII fluorescence stereomicroscope equipped by TOF-MS. Two major elytral cuticular proteins were digested with the following filter set: excitation = 480/40 nm, bar- with trypsin and the resulting peptides were analyzed by MALDI- rier = 510 nm. TOF mass spectrometry. Results were compared with conceptual (TIF) trypsinization products of the computed proteome of T. castaneum. Matched peptides are shown in red. Coverage for TcCPR27 and Figure S6 Scanning electron micrographs of TcCPR27- and TcCPR18 was 88.7 and 68.2%, respectively. Underlined residues TcCPR18-deficient elytra. Elytra were dissected from 1 d-old are predicted signal peptides, which are not included in the adults that had been injected with dsRNA for TcCPR27, TcCPR18 theoretical molecular mass calculations. or TcVer (200 ng per insect) as last instar larvae. The dorsal view of (TIF) elytra is shown. dsRNA for Ver was injected to serve as a negative control. Figure S2 Amino acid sequence alignment of TcCPR18 and (TIF) Tenebrio molitor adult-specific protein, Tmacp22. Alignment of deduced amino acid sequences was made using ClustalW software. Figure S7 Survival rate after removing elytra or hindwings from The symbols below the aligned amino acid sequences indicate mature T. castaneum adults. Elytra or hindwings were removed identical (*), highly conserved (:) and conserved (.) amino acids. from mature adults (n = 20), and viability was monitored (insects TcCPR18 is a putative ortholog of the T. molitor ecdysteroid- were reared at 30uC and 50% humidity). A: whole hindwings regulated adult-specific cuticle protein, TmACP22, with 67% removed. B: half of distal part of elytra removed. C: whole elytra sequence identity and 74% similarity. removed. Loss of an entire hindwing did not affect adult survival, (TIF) whereas removing elytra resulted in high mortality, probably Figure S3 Highly abundant proteins similar to TcCRP27 and because of dehydration. Thus, the elytra but not hindwings are TcCPR18 are predominant cuticular proteins in elytra of other essential for T. castaneum adult viability. Yellow and red lines Tribolium species. Extracts of elytra from newly emerged adults of indicate moribund and dead adults, respectively. T. castaneum, T. brevicornis, T. confusum and T. freemani as well as (TIF) Tenebrio monitor were analyzed by 4–12% Bis-Tris gel (Invitrogen). Table S1 Major proteins identified in extracts of unsclerotized Like T. castaneum, two to three abundant proteins with the elytra. Based on the cDNA sequence, the amino-terminal amino apparent masses of approximately 10 and 20 kDa were obtained acid residue for both proteins is predicted to be a glutamine from each species. These major proteins were digested with (Figure 1). The observed mass of native 10 kDa band was trypsin, and the resulting peptides were analyzed by MALDI-TOF 11,467 Da as determined by MALDI-linear-TOF MS, suggesting mass spectrometry (see Table S2). The green and orange arrows that amino-terminus of mature protein of TcCPR27 might be a indicate bands that exhibited high scores for similarity to modified glutamine. Further analysis of MS and MS/MS profiles TcCRT27 and TcCRT18, respectively. T. monitor adult cuticle revealed that the most likely candidate for the amino-terminal proteins, TmACP20 and TmACP22 , were also identified. S: residue is pyroglutamic acid. The tryptic peptide of 2143.1 Da PBS homogenate supernatant, P: PBS homogenate pellet, M: from TcCPR27 corresponds to pyro-E20GGEGYGHHHLEEYI- protein size markers. DYR37. A similar amino-terminal modification was confirmed in (TIF) the 20 kDa band identified as TcCPR18 (1451.5 Da peptide, Figure S4 Expression patterns of TcCPR27 and TcCPR18 in the pyro-E20GGGGGEEEYGHHR33). These results suggest that for adult ventral vs. dorsal abdominal cuticles and elytra vs. both proteins, Gln 20 is both deamidated and then dehydrated to hindwings. (A) The ventral and dorsal abdominal cuticles were form the observed modification. dissected from five 0–30 min old adults. TcCRP27 and TcCRP18 (DOC) were identified by peptide mapping in the ventral abdominal Table S2 Identification of major proteins extracted from elytra cuticle but not in the dorsal abdominal cuticle. S: PBS of three Tribolium species. homogenate supernatant, P: PBS homogenate pellet. (B) To (DOC) analyze the transcript levels of TcCPR27 and TcCPR18 in the ventral and dorsal abdomen as well as in the elytra and hindwings, real-time PCR was done using total RNA extracted from tissues of Acknowledgments ten pharate adults (5 d-old pupae). Expression levels for TcCPR27 We thank Neal Dittmer, Kansas State University; Haobo Jiang, Oklahoma and TcCPR18 are presented relative to the levels of expression in State University; Yoshinori Tomoyasu, Miami University; and Patricia A. elytra (E) or ventral abdomen (V). The transcript levels of the T. (Huber) Sprouse, University of Kansas, for helpful discussions and castaneum ribosomal protein S6 (rpS6) were measured to normalize comments on the manuscript. This is publication 11-035-J from the for differences between samples in the concentrations of cDNA Kansas Agricultural Experiment Station. Mention of trade names or templates. H: hindwings, D: dorsal abdominal cuticle. commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or (TIF) endorsement by the U.S. Department of Agriculture. USDA is an equal Figure S5 Elytral chitin staining with FITC-CBD. Elytra were opportunity provider and employer. removed from pharate adults (5 d-old pupae) that had been injected dsRNA for TcCPR27, TcCPR18 or TcVer (200 ng per Author Contributions insect) at the late larval instar stage. The elytra were incubated Conceived and designed the experiments: YA SHG RWB SM KJK MRK. with 10 N NaOH at 95uC for 5 h to remove protein, followed by Analyzed the data: YA SHG RWB SM KJK MRK. Wrote the paper: YA staining with the fluorescein-conjugated chitin-binding domain SHG RWB SM KJK MRK. Performed gene cloning, protein purification, probe (FITC-CBD, New England BioLabs). The appearance immunohistochemistry, and RNAi: YA. Performed MALDI-TOF and of the elytra did not differ until after adult eclosion, although TOF/TOF: YH JMT. Performed mechanical analysis: JL. PLoS Genetics | www.plosgenetics.org 10 April 2012 | Volume 8 | Issue 4 | e1002682  Rigid Cuticle Proteins in Tribolium References 1. Moussian B (2010) Recent advances in understanding mechanisms of insect epidermis from the elytra and hindwings of the red flour beetle, Tribolium cuticle differentiation. Insect Biochem Mol Biol 40: 363–375. castaneum. J Proteome ResIn press. 2. 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(2011) sclerotization and pigmentation in Tribolium castaneum. Insect Biochem Mol Biol Proteomic and transcriptomic analyses of rigid and membranous cuticles and 40: 267–273. PLoS Genetics | www.plosgenetics.org 11 April 2012 | Volume 8 | Issue 4 | e1002682