Expression profiles of cuticular protein genes in wing tissues during pupal to adult stages and the deduced adult cuticular structure of Bombyx mori

Accepted Manuscript Expression profiles of cuticular protein genes in wing tissues during pupal to adult stages and the deduced adult cuticular structure of Bombyx mori

Rima Shahin, Masashi Iwanaga, Hideki Kawasaki PII: DOI: Reference:

S0378-1119(17)31042-9 https://doi.org/10.1016/j.gene.2017.11.076 GENE 42384

To appear in:

Gene

Received date: Revised date: Accepted date:

27 September 2017 6 November 2017 30 November 2017

Please cite this article as: Rima Shahin, Masashi Iwanaga, Hideki Kawasaki , Expression profiles of cuticular protein genes in wing tissues during pupal to adult stages and the deduced adult cuticular structure of Bombyx mori. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), https://doi.org/10.1016/j.gene.2017.11.076

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ACCEPTED MANUSCRIPT Expression profiles of cuticular protein genes in wing tissues during pupal to adult stages and the deduced adult cuticular structure of Bombyx mori

Rima Shahin,a Masashi Iwanaga,a Hideki Kawasakia, *

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a Faculty of Agriculture, Utsunomiya University, 350 Mine, Utsunomiya, Tochigi 321-8505, Japan

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* Corresponding author Fax: +81-28-649-5401

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E-mail: [email protected]

Abstract

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We aimed to clarify the regulation of cuticular protein (CP) gene expression and the resulting

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insect cuticular layers by comparing the expression pattern of CP genes and related ecdysoneresponsive transcription factor (ERTF) genes, the coding amino acid sequences of CP genes, and

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histological observation. The expression of CP and ERTF genes during pupal and adult stages was examined via qPCR. The number of CP genes expressed during pupal and adult stages

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decreased as compared to that during prepupal to pupation stages, particularly in CPRs. The peaks of transcripts were observed at P5, P6, P9, A0, and A1. The order of the ERTF and CP genes expression resembled that at prepupal and pupation stages, suggesting the relatedness of

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ERTFs with the same CP genes at both stages. Moreover, the order of expression of CP genes resembled that in prepupal to pupation stages, by which we presumed the spaces of CPs in the epicuticle, outer-exocuticle, inner-exocuticle, endocuticle layer. Key words: Bombyx mori, ecdysone, cuticular protein, BHR4, FTZ-F1, E74A

1. Introduction 1

ACCEPTED MANUSCRIPT Ecdysone pulses trigger the major developmental transition during the life cycle of insects. The most dramatic transition is larval to adult metamorphosis in holometabolous insects. Ecdysone signaling regulates insect metamorphosis through successively expressed ecdysone-responsive transcription factors (ERTFs; Fletcher and Thummel, 1995; White et al., 1997; Lam et al., 1997). The expression profiles of several ERTFs during insect development have been reported (Sullivan and Thummel, 2003; Riddiford et al., 2003; Shahin et al., 2016). The results of their

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interactions triggered the successive activation of metamorphosis-related genes, including CP

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genes. Shahin et al. (2016) suggested that successively expressed CP genes were induced by

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successively expressed ERTFs, where different ERTFs regulated different target genes, resulting in successful metamorphosis.

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Recent genomic analysis has revealed the existence of a number of cuticle protein genes in D. melanogaster (Karouzou et al., 2007), the honey bee Apis mellifera (Honeybee Genome

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Sequencing Consortium, 2006), A. gambiae (Cornman et al., 2008), and B. mori (Futahashi et al., 2008). Most of them have been transcribed and cataloged in EST databases. More than 200 CP

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genes were identified in B. mori, and 52 were found in Bombyx EST libraries of wing discs (Futahashi et al., 2008). Genomic analyses clarified that the regulatory regions of cuticle protein

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genes and ERTF binding sites, which were predicted from the genomic information, appeared to be functional (Nita et al., 2009; Wang et al., 2009a; Wang et al., 2009b; Wang et al., 2010).

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These regulatory sequences concerned ecdysone responsiveness and the developmental expression patterns of CP genes. Direct regulation by EcR/USP was observed in the expression

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of the CP gene, BmorCPR21 (BMWCP10), where the Broad-Complex functioned together with EcR/USP (Wang et al., 2010). Transcripts of BmorCPR99 (BMWCP2) and BmorCPR92

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(BMWCP5) were induced by an ecdysone pulse through FTZ-F1 that bound to the upstream region of these CP genes and increased their promoter activity (Nita et al., 2009; Wang et al., 2009b). We followed the nomenclature of Willis (2010) and Futahashi et al. (2008), except we deleted ‘Bmor’ before the gene name. Numerous studies on cuticular proteins have been conducted, in which amino acid compositions have been given (Sridhara, 1981). The mechanical properties of the cuticle are influenced not only by the chitin architecture and the degree of sclerotization and hydration but also by the precise combination of proteins in the cuticular matrix, which plays a role in the determination of its properties. The most pronounced differences are often observed between the 2

ACCEPTED MANUSCRIPT outer layer (pre-ecdysial, exocuticle) and the inner layer (post-ecdysial, endocuticle), which were observed both in the nymphal cuticle (Nohr and Andersen, 1993) and in the adult cuticle of Locusta migratoria as well as Schistocerca gregaria (Andersen and Hojrup, 1987; Andersen, 1988). The pre-ecdysial proteins in locusts are predominantly hydrophobic and rich in alanine (Ala), valine (Val), and proline (Pro), while the post-ecdysial proteins are acidic and hydrophilic (Andersen and Hojrup, 1987; Andersen, 1988; Cox and Willis, 1987).

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The cuticle shows drastic difference in appearance, architecture, and composition at

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metamorphosis. Synthesis and deposition of the cuticular proteins are governed by the changing

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titers of ecdysone. We offer here the systematic structure of the insect cuticular layer and its regulation by ERTF through the CP gene expression profiles and amino acid sequences of their

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coding proteins. Thus, many CP genes are identified, and the present findings help to clarify the involvement of a large number of CP genes and the structure of cuticle layers. By comparing the

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expression profiles of CP genes and their amino acid coding sequences, we speculate on the

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constructed cuticular layers. In doing so, we offer new information to this field.

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2. Results

2.1. Histological differentiation in the wing tissue

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We observed histological wing development to compare with the expression profile of CP genes during pupal and adult stages. Wing tissues became flattened bilayers by P3, and veins and

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intervein regions became distinguished. The epidermis detached from the old cuticle, and the number of epidermal cells increased. Adult cuticle deposition was not yet observed. The wing bilayers were separated by P4, and the nuclei of epidermal cells enlarged. Envelope was observed over the upper face of the wing epidermis (Fig. 1A). By P5, 6, and 7, the epidermal cells became visibly differentiated and lobulated (Fig. 1B, C). The epidermal cells were arranged in rows and were highly vacuolated. Scales became more developed by P7. From P5, a cuticular layer was observed. A thick cuticular layer was observed at P9 around the veins and margins (Fig. 1D). By A0, very thin cuticular layers were formed in inter-vein after molting, and the epidermal cells were small and shrunken especially in the intervein region. In contrast, the 3

ACCEPTED MANUSCRIPT integument showed distinct formation, and the tracheae became larger in the vein. By A1, A2, A3, A4, and A5, no striking histological change was observed, and the cells became very sparse and much attenuated. The exo- and endocuticle became more distinguished only at the veins and margins of the wing (Fig. 1E). Hardening and sclerotization were in progress at the epicuticle and exocuticle with a deep and dark color.

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2.2. Expression profile of CP genes in pupal and adult stages

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Fifty-two CP genes that were identified in wing disc EST libraries and 24 CP genes in the

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compound eye EST library were examined, since the compound eye is an imaginal organ as well as wing disc, and its EST library was constructed during pupal stage. Moreover, adult cuticular

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proteins were reported to show similar distribution between wing and eye lens of Anopheles gambiae (Zhou et al., 2016).

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A distinct change was observed in the number of expressed CP genes between the pupal to adult (P-A) stages and the prepupal to pupation (PP-P) stages (Shahin et al., 2016), especially a

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decrease in the number of transcribed RR-1 and RR-2 CP genes. Moreover, the average strength of expression of P-A stages was weaker than that of PP-P stages, which reflects the thickness of

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the pupal cuticular layer. CP genes that showed the low level expression were ignored in the present study except for the CPH33 expressed at P4, since CPH33 was only one CP gene that

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showed a peak at P4. Expression profiles are shown in Fig. 2. The transcripts of CP genes during P-A stages was observed from P4 until A3, which

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occurred after the beginning of the hemolymph ecdysteroid began to decline and took 10 days. In contrast, it required 3 days for pupal cuticle layers (Shahin et al., 2016). At P4, only CPH33

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showed a sharp peak (G1). Nine genes showed peaks at P5. We divided these genes into three subgroups according to their expressions as follows: Subtype 1 peaked at P5 and showed sharp peaks (G2-1); CPG11 and CPR34 belong to this group. Subtype 2 peaked at P5 and showed high expression at P6; CPR78, CPG9, CPG24, and CPH28 belong to this group (G2-2). Subtype 3 peaked at P5 and decreased gradually, expressing until P8 (G2-3); CPT2, CPG12, and CPG13 belong to this group, except that CPG13 showed a peak at A0. Five CP genes belong to G3; four RR-2 genes and one CPH gene belong to this group. CPR107 and CPR109 showed broad expression from P4 until A1 and peaked at P6 (G3-1). The peak of CPR75 was not clear, but the expression resembled these two; therefore, we assigned it to G3-1. CPR93 and CPH1 peaked at 4

ACCEPTED MANUSCRIPT P6 but showed different expression patterns, so we assigned them to different groups, G3-2 and G3-3, respectively. We assigned six CP genes that showed their transcripts peak around the late pupal stage to G4. Two CP genes of G4 showed a similar profile; transcripts of CPR67 and CPR71 were from P7 to P9 and peaked at P9 (G4-1). Transcripts of CPR15 and CPT3 were slightly different from them: transcripts of CPR15 continued after adult eclosion, indicating construction of an

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endocuticle. CPT3 transcripts showed a small peak at P5, indicating involvement in the outer

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exocuticle. CPG14 showed high transcription from P5 and peaked at P9, indicating involvement

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in inner and outer exocuticle construction. CPFL3 transcripts showed strongest among the all of the CP genes of P-A stages, peaking at P7 and P9. Six genes showed peaks at A0 (G5).

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Transcripts of CPR54, CPFL1, CPH2, and CPH31 showed dramatic peaks at A0, and then declined, indicating the construction of adult endocuticle layers (G5-1). CPR 10 transcripts

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started to be observed from P5, declined at P9, and peaked at A0 (G5-2). CPH30 showed transcription similar to that of CPR10, starting from P6 and peaking at A0 (G5-2). The

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transcripts of three CPH genes and one RR-1 CP gene showed peaks at A1 (G6), and their transcripts increased from A0 and peaked at A1. CPH3 showed expression at P2, indicating

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construction of the pupal and adult endocuticle.

2.3. Developmental profiles of ERTFs during pupal and adult stages

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Nine ERTFs were examined in the present experiment. Shahin et al. (2016) suggested the relatedness of BHR3, BHR4 (registered as BmGRF; Charles et al., 1999), βFTZ-F1, and E74A

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with CP genes expressed in PP-P stages. Therefore, we first examined the expression profiles of these four ERTF genes (Fig. 3). Transcripts of BHR3 increased from P3 and peaked at P5, while those of BHR4 showed a broad peak from P5 to P7 and gradually decreased. βFTZF1 transcripts increased incrementally from P5 then declined sharply at adult eclosion. E74A transcripts showed a sharp peak at A0. The order of the expression of these ERTFs is the same as that in PPP stages (Shahin et al., 2016). BHR38 relatedness with the adult cuticle has been reported (Bruey-Sedano et al., 2005; Kozlova et al., 2009); therefore, we examined the BHR38 expression profile during pupal and adult stages. BHR38 transcripts showed expression at A0.

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ACCEPTED MANUSCRIPT The transcripts of E74B fluctuated without distinctive peaks during the pharate adult stage, showing a slight increase after eclosion (Supplementary Figure 1). E75A transcripts were observed at the early pupal stage and then declined from P5, with a small increase only at A0 (Supplementary Figure 1). In contrast, those of E75B showed a broad peak, sharply decreased at P9, and had a small increase at A0 (Supplementary Figure 1), while BR-C Z4 transcription was

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not expressed at P-A stages.

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2.4. Amino acid sequences of CPs

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We divided CPs into six groups, and each group was divided into several subgroups according to the expression peaks of their genes as described above: G1, G2-1, G2-2, G2-3, G3-1, G3-2, G3-3,

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G4-1, G4-2, G5-1, G5-2, G6-1, and G6-2 (Fig. 2, Table 1). We calculated percentages of the characteristic amino acids of CPs that determine the nature of cuticular layers (Table 1). CPH33

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in G1 and CPGs in G2 (CPG11, 9, 24, 12, 13) contained high percentages of histidine and lysine (His&Lys). CPH33 in G1 and CPGs in G2 (CPG11, 24, 12, 13) contained high percentages of Val and Pro. G3-1 RR-2 CPs, CPR107, and CPR109 contained high percentages of His&Lys. In

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contrast, G4-1 RR-2 CPs, CPR67, and CPR71 contained low percentages of His&Lys but high

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percentages of Ala. Most of the CPs in G5 and G6 also contained high percentages of Ala. Thus, most CPs in Groups 1, 2, and 3 showed high percentages of His&Lys, together with high

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percentages of Pro and Val and a low percentage of Ala (Table 1). Thus, CPs that are coded by CP genes expressed at earlier stages have high percentages of His&Lys, Pro, and Val. In contrast,

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CPs belonging to Groups 4, 5, and 6 contained low percentages of His&Lys, Pro, and Val but

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high percentages of Ala. The amino acid sequences of characteristic CPs are shown in Fig. 4.

3. Discussion

3.1. Developmental expression of CP genes The composition and function of the cuticle depend on the stages: the pupal cuticle is thicker and more elastic than that of adult wings that are thin and stiff and covered with scales (Sridhara, 1981; Vincent, 2001). Of the 52 CP genes expressed during PP-P stages only 31 were expressed during the P-A stages. Most of the CP genes that became undetectable during P-A stages were 6

ACCEPTED MANUSCRIPT RR-1 and RR-2 CP genes. R&R consensus was demonstrated to bind to chitin (Rebers and Willis, 2001; Togawa et al., 2004). The result reflects the developed lamellar structure of the pupal cuticle. Six peak stages were observed, at P4, P5, P6, P9, A0, and A1. All genes were induced after the hemolymph ecdysteroid titer began to decline, when BHR3 peaked as observed at PP-P stages (Shahin et al., 2016). Most genes expressed during P-A stages were also expressed during PP-P stages (Shahin et

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al., 2016); therefore, we compared the groups from P-A stages with those from PP-P stages. The

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data of Table 2 depend on Shahin et al. (2016). Most of the G1 and G2 genes belong to G1 of the

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PP-P stages (Tables 1 and 2). Most of the G3 genes belong to G1-3 of the PP-P stages. The genes in G4-1 belong to G3 of the PP-P stages. The genes of G5-1 belong to G3-3 of the PP-P stages.

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The order resembles that of the PP-P stages; therefore, we presumed the relatedness of the

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expression of CP genes with ERTFs, as described below.

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3.2. Expression profiles of ERTFs during P-A stages

We examined the expression of nine ERTF genes during P-A stages. The expression profiles of

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eight ERTF genes were observed during the prepupal stage (Shahin et al., 2016), and a similar expression profile of the ERTF genes was exhibited during adult wing formation. BHR3

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increased from P3 and peaked at P5. BHR4 increased from P5 and decreased after P7. βFTZ-F1 increased from P5 until P9, then rapidly decreased. E75A and E74A showed expression at A0

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with a slight increase and a sharp peak, respectively. The order of expression of BHR3, BHR4, βFTZ-F1, and E74A resembled that of PP-P stages; therefore, it is suggested that the same

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regulation of CP gene expression exists in P-A stages. DHR3 has been reported as a transition switch, repressing early genes, E74A and E75A, and inducing βFTZ-F1 (Lam et al., 1997, 1999; Sun et al., 1994), and βFTZ-F1 has been reported to re-induce early genes E75A and E74A (Thummel, 2001), with which the present results agree. We also examined BHR38 expression, since DHR38 has been reported to influence the cuticle formation and be required for adult cuticle gene ACP65A (Bruey-Sedano et al., 2005; Kozlova et al., 1998, 2009). Therefore, we expected that BHR38 would be expressed during the pupal stage instead of BR-C Z4. However, it showed a sharp peak at A0. The reason for this and the function of BHR38 remain elusive. Four early genes, E75A, E75B, E74B, and BR-C Z4, showed expression at the early molted pupa 7

ACCEPTED MANUSCRIPT (Supplementary Fig. 2), when the ecdysteroid titer in the hemolymph is low (Kawasaki et al., 1986).

3.3. Four ERTFs suggested to regulate different groups of CP genes We observed five peaks of CP genes in PP-P stages (Shahin et al., 2016), where we presumed the relatedness of ERTFs and CP genes (Fig. 5), depending on the expression profiles of ERTFs and

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CP genes and previous reports (Wang et al., 2009; Nita et al., 2009; Shahin et al., 2016). By

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comparing the expression profiles of ERTF and CP genes in P-A stages with those of PP-P

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stages, we assumed that the G1, G2, and G3 genes of the pupal stage are related with BHR3 and BHR4, those of G4 are with βFTZ-F1, and those of G5 and G6 are with E74A and/or BHR38

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(Fig. 6).

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3.4. Construction of cuticular layers

The insect cuticle is made up of two major layers, the epicuticle and procuticle. The epicuticle

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consists of the cement, wax, and cuticulin layers (Locke, 1961; Wigglesworth, 1972). The procuticle is divided into the upper exocuticle and lower endocuticle. The procuticle is secreted

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before and after ecdysis in response to the rise and decline of hemolymph ecdysteroids (Wigglesworth, 1972; Andersen, 2000). The exocuticle and endocuticle consist of chitin, CPRs,

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and other types of CPs whose disordered and low-complexity (LCP; Willis, 2010) sequences of amino acids are suggested to construct or fill the spaces in the exocuticle and endocuticle

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(Andersen, 2002).

Based on the above studies and the comparison of the successive expression of CP genes at

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P-A and PP-P stages (Tables 1 and 2), we offer the following structure of insect cuticular layer. CPH33 and CPG11 belong to G1-1 of the PP-P stages and G1 and G2-1 of the P-A stages, respectively. They contain high percentages of His&Lys, Pro, and Val. Williamson (1994) reported that Pro residues acted as binding sites for polyphenols. CPH33, CPG11, CPG12, and CPG13 contain high percentages of Pro, which suggests that they bind to polyphenols in the epicuticle; therefore, these CPs are suggested to construct the epicuticle layer or exist close to the epicuticle layer. CPH33, CPG11, CPG24, CPG12, and CPG13 contain high percentages of Val; therefore, they are very hydrophobic and are fit for the sclerotizing exocuticle. The epicuticle and exocuticle 8

ACCEPTED MANUSCRIPT become sclerotized after ecdysis (Hopkins et al., 2000), when phenolic compounds incorporate into the CPs (Andersen, 2010). RR-2 CPs contain high percentages of His&Lys, and these amino acids react with sclerotizing reagents (Iconomidou, 2005), which suggests that RR-2 CPs construct the exocuticle. RR-2 CPs construct the hard cuticle, such as larval tubercles and pupal sclerites (Gu and Willis, 2003), adult scales (Fu et al., 2011), the elytra of beetles (Dittmer et al., 2012; Arakane et al., 2012), and pupal wings (Shahin et al., 2016). Vannini and Willis (2017)

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showed that RR-2 CPs were in the exocuticle layer using antibody and transmission electron

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microscopy. Therefore, G1 RR-2 CPs of the PP-P stages and G3 CPs of P-A stages are believed

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to construct the exocuticle. Mun et al. (2015) recently reported that the CP of T. castaneum, TcCP30, having a low-complexity sequence, cross-linked with TcCPR18 and TcCPR27 by

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laccases but did not with TcCPR4 that belongs to RR-1 CP. TcCP30, TcCPR18, and TcCPR27 contained high percentages of His that functions for cross-linkage. As well as TcCP30, CPH33,

are suggested to cross-link with RR-2 CPs.

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CPG11, CPG24, CPG9, and CPG12 in G1 and G2 contain high percentages of His&Lys. They

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The His&Lys residues in CP are used for sclerotization of the cuticle (Schaefer et al., 1987; Hopkins et al., 2000; Andersen, 2010). Wigglesworth (1948) observed outer and inner exocuticle

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layers. A CP that contains high percentages of His&Lys is suggested to construct the outer exocuticle, such as CPR107 and CPR109 in G1-3 of PP-P and G3-1 of P-A stages. The large

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numbers and the strong expression of the His&Lys RR-2 CP genes of PP-P stages are suggested to bring out the hard and thick exocuticle. The hydrophobic nature of this region represents the

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first barrier of the insect surface that protects the insects against hydration and swelling (Moussian, 2010). Thus, CPs that are coded by CP genes expressed at earlier stages have a high

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percentage of His&Lys among RR-2 CPs and are suggested to construct outer exocuticle layer. In contrast, G4-1 in P-A stages (Table 1) and G2 and G3 RR-2 CPs in PP-P stages (Table 2), such as CPR 67 and CPR 71, contained less than 20% His&Lys, even though these percentages are higher than those of RR-1 CPs. From this, G4 of P-A stages and G3-1 and G3-2 of PP-P stages are suggested to construct the inner exocuticle layer (Wigglesworth, 1948). Thus, CPs in the lower region showed a low percentage of His&Lys, which suggests less involvement of these CPs in sclerotization (Andersen et al., 1995). Expression peaks at P5–P6 are used for production of the outer exocuticle layer, and those at P9 are used for production of the inner exocuticle.

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ACCEPTED MANUSCRIPT Thus, CP genes used for outer and inner exocuticles were expressed at different stages and are suggested to be regulated by different ERTFs. G5 CPs in the P-A stages, such as CPH2, CPH30, CPH31, CPR10, and CPFL1, are in G3-3 and G4 of the PP-P stages, containing high percentages of Ala; the expression of their transcription is at A0 and P0. Also, G6 CPs in the P-A stages contain high percentages of Ala. The predicted lower region of the cuticular layer in the present study contains high percentages

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of Ala, suggesting the functional difference of PVPV and AAPA, which are used for protein

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folding (Andersen et al., 1995). Cox and Willis (1987) reported that acidic amino acids were

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hydrophilic and involved in the soft cuticle, such as the intersegmental membrane or larval cuticle (Missios et al., 2000). Therefore, we suggest that acidic amino acid residues are

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frequently involved in the CP in the endocuticle, which is a hydrophilic region. However, our results did not support this; the percentages of Asp&Glu in RR-1 CPs were not high (Table 1).

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Additionally, the percentages of His&Lys in RR-1 CPs are lower than those in RR-2 CPs (Tables 1 and 2), which indicates the low involvement of RR-1 CPs in sclerotization of the exocuticle

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and endocuticle as described by Andersen et al. (1995). The present results correspond with reports that RR-1 CPs constructed a flexible cuticle, such as the larval integument (Rebers et al.,

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1997; Gu and Willis, 2003; Okamoto et al., 2008; Fu et al., 2011), the intersegmental region of pupa (Rebers et al., 1997), and the endocuticle region (Andersen et al., 1998). Developmental

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order of CPs and estimated cuticular layers are shown in Fig. 7. Thus, by comparing successively expressed CP genes and their amino acid sequences, we

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assumed the construction of cuticular layers as follows. There are RR-2 CPs with high His&Lys content and RR-2 CPs with low His&Lys content, followed by RR-1 CPs; low-complexity CPs

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are abundant in all of the layers, with high percentages of His&Lys and Pro in the upper layers and high percentages of Ala in the lower layers. Successively expressed CP genes that are regulated by ERTFs construct thick cuticular layers. Although, not all the CP genes were examined and several reports showed that different CPs were used for different regions (Gu and Willis, 2003; Vannini et al., 2014), the present data can offer new information in this field. Distinctive motifs found in LCPs are PVPV, PVPY, PYPV, AAPA, APAA, GGG, and GGY, which are considered to enable protein folding (Andersen et al., 1995). Disordered regions have no fixed structure, are highly flexible and extensible, and change forms in accordance with their neighbor molecules (Andersen, 2011). The present results agreed with this; the function of 10

ACCEPTED MANUSCRIPT LCPs is to fill the interspace of cuticular layers and interact with CPRs, depending on their region. From this, we speculated the cuticular structure of B. mori as shown in Fig. 8.

3.5. Possible regulation of cuticular layers by ERTF From the evidence we obtained in the present results, the regulation and construction of the adult cuticular layers of B. mori are presumed as follows. The epicuticle layer is suggested to be

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induced by BHR3, and CPH33 and CPG11 are involved in the construction of this layer. The

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outer exocuticle layer is suggested to be induced by BHR4, and CPT2, CPG12, CPR107, and

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CPR109 are involved in the construction of this layer. The third layer is an inner exocuticle. This layer is suggested to be induced by βFTZ-F1, and CPR67 and CPR 71 are involved in the

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construction of this layer. The most inner layer is the endocuticle, suggested to be induced by E74A, and CPH2 and CPH31 are involved in the construction of this layer. Based on the present

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results together with pupal cuticle production (Shahin et al., 2016), the epicuticle is suggested to be induced by BHR3, the outer exocuticle by BHR4, the inner exocuticle by βFTZ-F1, and the

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endocuticle by E74A, respectively. The CP genes that construct each peak are suggested to

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encode CPs for the outer exocuticle, inner exocuticle, and endocuticle.

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4. Experimental procedures

4.1. Insects

A hybrid strain of B. mori (N124 and C124) was used in the present study. Insects were reared on mulberry leaves at 25 °C. Larvae began wandering after the sixth day of the fifth larval instar, and pupation occurred 3 days later; adults eclosed 10 days after pupation. The periods (in days) correspond to the developmental stages, and the days of pupation and eclosion were designated as P0 and A0, respectively.

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ACCEPTED MANUSCRIPT 4.2. Quantitative PCR Wing tissues were dissected during the pupal and adult stages of B. mori. Total RNA was isolated from wing tissues by using ISOGEN (Nippon Gene, Japan). Wing tissues were homogenized by repeated forcing through a 23-gauge (0.60 x32mm) needle attached to a sterile plastic syringe at least 30 times, in case of the tissue samples were soft (from P2 to P4). In case of hard tissues from P5 to A5, homogeneous lysate was achieved effectively using IKA T10

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basic ULTRA-TURRAX Homogenizer system. First-strand cDNA was synthesized from 1 µg of

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total RNA in a 10 µl reaction mixture using ReverTra Ace (Toyobo, Japan). Quantitative PCR

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(quantitative RT-PCR: qRT-PCR, here we use as qPCR) was conducted on LightCycler 96 (Roche) using FastStart Universal SYBR Green Master (Roche) according to the manufacturer’s

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protocol. The primer sets of CP and ERTF genes are listed in Supplementary Table 1. Each amplification reaction was performed in a 15 µl qPCR reaction mixture under the following

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conditions: denaturation at 95 °C for 10 min followed by 40 cycles of treatment at 95 °C for 10 s and at 60 °C for 1 min. Ribosomal protein S4 (Bmrpl: GenBank accession no. NM_001043792) was used as a control gene. The data were normalized by the determination of the amount of

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Bmrpl in each sample to eliminate variations in mRNA and cDNA quality and quantity. ∆Ct

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method was used for quantifying the qRT-PCR data. The transcript abundance value of each datum was the mean and S.E.M. of two to five biological replicates. Each pair of primers was

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designed using Primer3 software (http://frodo.wi.mit.edu/).

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4.3. Mallory's triple stain

Wing tissues were fixed with Carnoyʼs solution (ethyl alcohol: chloroform: acetic acid; 6:3:1)

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and cut into sections of seven microns after dewaxing and rehydrating through descending series of xylene, ethanol, and rinsing under running water for 15 minutes. All samples were stained in two solutions: (A) Samples were immersed in acid azocarmine (0.1 g of Azocarmine G and 1 ml of acetic acid in 100 ml distilled water) and incubated at 37 °C for 20 minutes, then washed in distilled water. The sections were then treated with 5% phosphotungstic acid for 20 minutes. After being washed in distilled water, the sections were stained for 30 minutes in the second solution, (B) Orange G 0.4 g, aniline blue 0.2 g, acetic acid 1 ml in 100 ml distilled water. The sections were then dehydrated through progressive series of ethanol and xylene and mounted

12

ACCEPTED MANUSCRIPT using Canada balsam with a cover slip. The sections were visualized under a light microscope (Olympus BX50) and documented using DP-BSW software.

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Acknowledgements

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This work was supported by the Ministry of Education, Science, and Culture of Japan. We thank Dr. F. Yoshizawa for his advices about amino acid characterization.

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75, 45-57.

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Figure legends

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Fig. 1 Development of wing tissue during pupal to adult stages. A broken line shows the ecdysteroid titer during the pupal stage (Kawasaki et al., 1986). Microphotograph of a wing tissue at P4 (A), P6 (B), P7 (C), P9 (D), and A3 (E). Bars equal 20µm. Env: envelope, Ep: epidermis, S: Scale, Cu: cuticle, SC: scale cell, Ex: exocuticle, En: endocuticle.

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Fig. 2 Developmental profile of CP genes. Each datum was calculated from two to five independent experiments. Results are expressed as the mean ± S.E.M. RNA was extracted from wing discs at the indicated stages and reverse-transcribed to cDNA for use in qRT-PCR. Values are the ratio to the mRNA level of the ribosomal protein S4. A Group 1 gene shows a peak at P4. Group 2, Group 3, Group 4, Group 5, and Group 6 genes show peaks at P5, P6, P9, A0, and A1, respectively.

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Fig. 3 Developmental profile of ERTF genes. BHR3, BHR4, FTZ-F1, E74A, and BHR38 expressions are shown. Treatment of samples and data analysis are the same as in Fig. 2. Fig. 4 Amino acid sequences of selected CPs from epicuticle (CPG11), outer exocuticle (CPR109), inner exocuticle (CPR67), and endocuticle (CPH3) layers. Lines indicate R&R consensus; distinctive amino acids are marked with red or blue. K: Lys; H: His; P: Pro; A: Ala.

Fig. 5 Schematic presentation of the regulation of cuticular protein expression by different ecdysone-responsive transcription factors (ERTFs) during the prepupal to pupation (PP-P) stages.

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ACCEPTED MANUSCRIPT BHR3, BHR4, FTZ-F1, and E74A regulate G1, G2, G3, G4 and G5 groups of cuticular protein genes, respectively.

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Fig. 6 Schematic presentation of the regulation of cuticular protein expression by different ecdysone-responsive transcription factors (ERTFs) during the pupal to adult (P-A) stages. BHR3, BHR4, FTZ-F1, and E74A regulate G1, G2, G3, G4, G5 and G6 groups of cuticular protein genes, respectively.

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Fig. 7 Developmental profile of CP gene groups and presumed their production. P3; three days after pupation, A0; the day of eclosion, Epi; epicuticle, O-exo; outer-exocuticle, I-exo; innerexocuticle, Endo; Endocuticle.

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Fig. 8 Schematic presentation of cuticular layers of pupae. RR-2 cuticular proteins (CPs) are deposited in the exocuticular layer. RR-1 CPs and other types of CPs are in the exo- and endocuticular layers. CPG, CPT, CPH, and CPFL are low-complexity CPs, as described in the text.

19

ACCEPTED MANUSCRIPT Table 1 Groups of CPs of P-A stages and their amino acid percentages Group 1

CP Name

Accession No.

Percentage of amino acid

Amino acid Numbers

D+E

H+K

Q

V

G

P

A

BR000500

128

0.10

0.21

0.03

0.23

0.03

0.19

0.02

CPG11

BR000432

252

0.10

0.19

0.01

0.24

0.02

0.21

0.02

CPR34

BR000535

191

0.15

0.07

0.14

0.05

0.06

0.07

0.08

CPR78

BR000579

222

0.09

0.08

0.19

0.04

0.05

0.13

0.06

CPG9

BR000430

194

0.18

0.19

0.05

0.03

0.15

0.04

0.04

CPG24

BR000445

373

0.14

0.21

0.02

0.20

0.08

0.14

0.01

CPH28

BR000493

0.03

0.13

0.05

0.05

0.13

0.10

CPT2

BR000651

294

0.07

0.07

0.03

0.04

0.27

0.11

0.06

CPG12

BR000433

D E

0.11

335

0.11

0.19

0.01

0.23

0.06

0.21

0.03

CPG13

BR000434

351

0.09

0.15

0.02

0.23

0.06

0.19

0.03

BR000576

156

0.08

0.10

0.05

0.10

0.04

0.08

0.20

BR000608

161

0.14

0.30

0.04

0.08

0.09

0.04

0.07

CPR109

BR000610

160

0.14

0.29

0.04

0.08

0.10

0.04

0.06

CPR93

BR000594

226

0.09

0.17

0.04

0.08

0.04

0.09

0.18

2-2

CPR75

3-1

3-2

CPR107

496

E C

T P

C A

U N

M

A

I R

C S

2-1

2-3

T P

CPH33

20

ACCEPTED MANUSCRIPT 3-3

CPH1

BR000451

72

0.07

0.04

0.01

0.03

0.03

0.19

0.17

CPR67

BR000568

162

0.09

0.10

0.04

0.14

0.06

0.11

0.19

CPR 71

BR000572

167

0.08

0.11

0.02

0.14

0.04

0.11

0.24

CPT3

BR000652

302

0.04

0.07

0.03

0.03

0.34

0.08

0.06

CPR15

BR000516

265

0.08

0.03

0.05

0.01

0.24

0.05

0.15

CPG14

BR00435

183

0.06

0.10

0.05

0.08

0.16

0.08

0.08

CPFL3

BR000420

220

0.00

0.06

0.00

0.07

0.04

0.10

0.32

CPR54

BR000555

297

0.10

0.05

0.13

0.02

0.06

0.18

0.10

CPFL1

BR000418

242

0.06

0.14

0.03

0.08

0.16

0.06

0.27

CPH2

BR000452

199

0.07

0.11

0.04

0.11

0.11

0.06

0.25

CPH31

BR000496

235

0.08

0.07

0.08

0.02

0.05

0.11

0.28

CPR10

BR000511

266

0.09

0.05

0.14

0.04

0.07

0.12

0.10

CPH30

BR000495

E C

194

0.06

0.14

0.04

0.04

0.11

0.08

0.28

97

0.17

0.06

0.02

0.09

0.07

0.07

0.10

BR000466

121

0.03

0.07

0.01

0.07

0.07

0.11

0.20

BR000469

154

0.02

0.06

0.02

0.19

0.04

0.12

0.18

BR000453

265

0.11

0.07

0.06

0.13

0.05

0.08

0.22

4-1

4-2

5-1

5-2 CPR46

6-1

CPH11 CPH14

6-2

CPH3

BR000547

C A

T P

D E

M

A

I R

C S

U N

T P

CPs coded by low expression genes are not cited. CPRs in red chracter are RR2. CPs in yellow columns are identified in both PP-P

21

ACCEPTED MANUSCRIPT and P-A stages. Amino acid numbers do not include signal peptids. Percentages over 12% are shown in red.

T P

I R

C S

U N

A

D E

M

T P

E C

C A

22

ACCEPTED MANUSCRIPT Table 2 Groups of CPs of PP-P stages and their amino acid percentages Group

Percentage of amino acid

Accession No.

Amino acid Numbers

CPH33

BR000500

128

0.10

CPG11

BR000432

252

CPR124

BR000625

CPG24

CP Name

D+E H+K

Q

V

G

P

A

0.21

0.03

0.23

0.03

0.19

0.02

0.10

0.19

0.01

0.24

0.02

0.21

0.02

161

0.12

0.24

BR000445

373

0.14

CPR122

BR000623

221

0.14

CPR93

BR000594

226

0.09

CPR107

BR000608

161

CPR109

BR000610

CPR111

BR000612

CPG14

BR00435

CPT2

C S 0.04

0.11

0.08

0.06

0.09

U N

0.02

0.20

0.08

0.14

0.01

0.24

0.06

0.09

0.04

0.06

0.09

0.17

0.04

0.05

0.04

0.09

0.18

0.14

0.30

0.04

0.08

0.09

0.04

0.07

160

0.14

0.29

0.04

0.08

0.10

0.04

0.06

178

0.14

0.28

0.08

0.08

0.11

0.03

0.06

183

0.06

0.10

0.05

0.08

0.16

0.08

0.08

BR000651

294

0.07

0.07

0.03

0.04

0.27

0.11

0.06

BR000652

302

0.04

0.07

0.03

0.03

0.34

0.08

0.06

BR000535

191

0.15

0.07

0.14

0.05

0.06

0.07

0.08

CPR75

BR000576

156

0.08

0.10

0.05

0.10

0.04

0.08

0.20

CPR92

BR000593

280

0.08

0.17

0.03

0.14

0.04

0.11

0.16

1-2

CPT3 CPR34

E C

C A

D E

T P

1-3

1-4

T P

I R

1-1

A

M

0.21

2-1

23

ACCEPTED MANUSCRIPT CPR95

BR000596

215

0.08

0.12

0.04

0.11

0.04

0.07

0.22

CPR97

BR000598

164

0.09

0.12

0.05

0.11

0.04

0.08

0.20

CPR98

BR000599

231

0.07

0.11

0.06

0.11

0.03

0.09

0.21

CPR 99

BR000600

231

0.07

0.11

0.06

0.11

0.03

CPR104

BR000104

205

0.09

0.16

0.03

0.10

CPR105

BR000606

207

0.09

0.15

0.03

CPR138

BR000639

165

0.08

0.10

CPG12

BR000433

335

0.11

CPH18

BR000473

93

0.00

CPR4

BR000505

105

CPR45

BR000546

CPR2

BR000503

CPR3

BR000504

CPR23

BR000524

0.22

0.05

0.07

0.20

0.10

0.07

0.22

0.05

0.10

0.04

0.09

0.20

0.19

C S

0.05

0.01

0.23

0.06

0.21

0.03

0.02

0.00

0.12

0.01

0.16

0.31

0.15

0.05

0.05

0.09

0.14

0.07

0.11

0.10

0.06

0.18

0.05

0.06

0.11

0.08

127

0.13

0.09

0.04

0.09

0.07

0.10

0.12

118

0.13

0.03

0.09

0.07

0.09

0.10

0.11

87

0.13

0.06

0.10

0.06

0.09

0.08

0.08

BR000625

460

0.07

0.06

0.06

0.07

0.10

0.08

0.04

BR000421

288

0.00

0.07

0.00

0.10

0.06

0.09

0.28

CPR 71

BR000572

167

0.08

0.11

0.02

0.14

0.04

0.11

0.24

CPR103

BR000604

205

0.09

0.16

0.03

0.10

0.05

0.07

0.20

2-2

CPT1

T P

CPFL4

E C

C A

D E 158

2-3

2-4

T P

0.09

A

U N

M

I R

3-1

24

ACCEPTED MANUSCRIPT CPR67

BR000568

162

0.09

0.10

0.04

0.14

0.06

0.11

0.19

CPG13

BR000434

351

0.09

0.15

0.02

0.23

0.06

0.19

0.03

CPR10

BR000511

266

0.09

0.05

0.14

0.04

0.07

0.12

0.10

CPH2

BR000452

199

0.07

0.11

0.04

0.11

0.11

CPH31

BR000496

235

0.08

0.07

0.08

0.02

CPFL1

BR000418

242

0.06

0.14

0.03

CPR55

BR000556

267

0.08

0.04

CPG4

BR000425

393

0.02

CPH30

BR000495

194

0.06

CPG17

BR000438

76

CPH1

BR000451

3-2

4

5

D E 72

T P

0.06

0.25

0.05

0.11

0.28

0.08

C S

0.16

0.06

0.27

0.20

0.02

0.07

0.12

0.07

0.01

0.10

0.10

0.09

0.18

0.14

0.04

0.04

0.11

0.08

0.28

0.08

0.24

0.08

0.03

0.32

0.01

0.00

0.07

0.04

0.01

0.03

0.03

0.19

0.17

3-3

A

U N

M

0.03

I R

T P

CPs coded by showed uniqu expression are not cited. CPRs in red chracter are RR2. CPs in yellow columns are identified in both PP-P and P-A stages. Percentages over 12% are shown in red.

E C

C A

25

ACCEPTED MANUSCRIPT Abbreviations CP, cuticular protein; ERTF, ecdysone-responsive transcription factor; qPCR, quantitative real-time PCR; BM, basement membrane; W3, three days after the beginning of wandering; 20E, 20-hydroxyecdysone; BR-C, Broad Complex; CPR, cuticular protein with R&R consensus, CPG, glycine rich cuticular protein; CPH, cuticular protein homolog, CPT, cuticular protein with Tweedle motif; CPFL, cuticular protein with 51 amino acid motif Like

IP

T

Highlights

AC

CE

PT

ED

M

AN

US

CR

1. We examined the expression pattern of cuticular protein (CP) and ecdysone-responsive transcription factor genes during pupal and adult stages. 2. We grouped CP genes to six groups (G1-G6) according to their peak expression stages. 3. BHR3, BHR4, FTZ-F1 and E74A are suggested to regulate CP genes of G1, G2, G3, G4, and G5&G6, respectively. 4. Depending on the amino acid sequences coded by CP genes, CP gene expression order, we speculated the cuticular layer structure. 5. BHR3, BHR4, FTZ-F1 and E74A are presumed to regulate epicuticle, outer exocuticle, inner exocuticle, endocuticle, respectively.

26

Graphics Abstract

Env Ep

A

S Cu S

C SC

E

S Cu Cu

B Ep

Ep

S

D

P3

P5

P7

Developmental Figure 1

P9

stage

A0

Ex En Ep

G1

0.4

mRNA level

0.3

CPH33 0.2

0.1

0.0

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

A5

Developmental stage

G2-1

G2-1

12

6

10

5

CPR34 4

8

mRNA level

mRNA level

CPG11 6

4

3

2

1

2

0

0

P2 P3 P4 P5 P6 P7 P8 P9 A0 A1 A2 A3 A4 A5

P2 P3 P4 P5 P6 P7 P8 P9 A0 A1 A2 A3 A4 A5

Developmental stage

Developmental stage

Figure 2r1

G2-2 16

8

30

25 7

14 25

6

20

12

CPH28

CPG24

4

10

15

mRNA level

mRNA level

6

15

mRNA level

CPG9

CPR78 8

10

5 2

4 3 2 1

5

0

0

0

0

-1

-2

P2

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

A5

P2

P3

Developmental stage

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

P3

P4

P5

P6

P2 P3 P4 P5 P6 P7 P8 P9 A0 A1 A2 A3 A4 A5

A5

Developmental stage

P7

P8

P9

A0

A1

A2

A3

A4

A5

Developmental stage

Developmental stage

G2-3 60

24 4.8 50

CPG12

18

40

mRNA level

CPT2

2.4

mRNA level

3.6

mRNA level

mRNA level

5

20

10

30

20

CPG13 12

6

1.2 10

0.0

0

0 P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

A2

A3

A4

A5

P2 P3 P4 P5 P6 P7 P8 P9 A0 A1 A2 A3 A4 A5

Developmental stage

Figure 2r2

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

A2

A3

A4

A5

G3-1 5.2

5.2

3.6

3.9

3.9

2.7

CPR109

1.3

CPR75

mRNA level

mRNA level

2.6

2.6

1.3

1.8

0.9

0.0

0.0

0.0

P3

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

A5

P2

Developmental stage

P3

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

A5

P2

P3

P4

P5

Developmental stage

P6

P7

G3-3 14.4

3.2

10.8

CPR93

2.4

CPH1 1.6

7.2

0.8

3.6

0.0

0.0

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

P2

A5

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

Developmental stage

Figure 2r3

P8

P9

A0

A1

Developmental stage

G3-2

mRNA level

P2

mRNA level

mRNA level

CPR107

A2

A3

A4

A5

A2

A3

A4

A5

G4-1 50 36 48

40 27

CPR67

CPR15

24

CPR71

mRNA level

mRNA level

mRNA level

36

18

30

20

9

12

10

0

0

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

A5

0

P2

P3

P4

P5

Developmental stage

P6

P7

P8

P9

A0

A1

A2

A3

A4

P2

A5

P3

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

Developmental stage

Developmental stage

G4-2 16.8

200

20

CPG14 12.6

150

15

CPFL3

10

mRNA level

mRNA level

mRNA level

CPT3 8.4

4.2

5

0.0

0

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

A2

A3

A4

A5

100

50

0

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

Figure 2r4

A2

A3

A4

A5

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

A2

A3

A4

A5

A5

G5-1 72

10

8

50

40

54

CPH2

6

4

mRNA level

CPFL1 mRNA level

mRNA level

CPR54

36

30

20

18 10

2

0

0

0

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

A5

P2

P3

P4

P5

Developmental stage

P6

P7

P8

P9

A0

A1

A2

A3

A4

P2

A5

P3

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

A5

Developmental stage

Developmental stage

G5-2

G5-1 56

48

48

CPR10 CPH31

36

42

36

24

12

mRNA level

mRNA level

mRNA level

CPH30 28

12

14

0

0

0 P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

A5

P2

Developmental stage

24

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

Figure 2r5

A2

A3

A4

A5

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

A2

A3

A4

A5

G6-1 14 60

40 50

12

CPR46 10 30

30

20

20

CPH14

8

mRNA level

mRNA level

CPH11

6 4

10 2 10

0

0 0

-2 P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

P2

A5

P3

P4

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

Developmental stage

Developmental stage

40 35 30

CPH3 25 20 15 10 5 0 -5 P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

Figure 2r6

A2

A5

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

G6-2

mRNA level

mRNA level

40

A3

A4

A5

A2

A3

A4

A5

1.4

3.6 1.2 1.0

2.7

BHR3

mRNA level

mRNA level

0.8 0.6 0.4

BHR4 1.8

0.9 0.2 0.0

0.0

-0.2 P2 P3 P4 P5 P6 P7 P8 P9 A0 A1 A2 A3 A4 A5

P2

P3

P4

Developmental stage

P5

P6

P7

P8

P9

A0

A1

A2

A3

A4

A5

Developmental stage

2.0 0.8 1.28

0.7

1.5

BHR38

1.0

0.5

mRNA level

0.5

mRNA level

mRNA level

0.6

E74A

0.96

Bm-FTZF1

0.64

0.32

0.4 0.3 0.2 0.1

0.0

0.00

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

A2

A3

A4

A5

0.0 -0.1 P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

Figure 3

A2

A3

A4

A5

P2

P3

P4

P5

P6

P7

P8

P9

A0

A1

Developmental stage

A2

A3

A4

A5

CPG11 MRLMRSSLLLAVVIGLAAAEEKAAKAEEQLEPKKQDKRGLSEYYGSYDEHGGGHEEHVKTITVVKKV PVPYPVEKHIPVPVEKNVPYPVKVPVPHPYPVIKTVHFPVKEYIKVPEYIPKPYPVTKHVPVPVKVHVDN PVPVRVYEHVPVPVEKAVPVPVKVPVPHPYPVEKKVPFPVKVPVKVHVPYPVEKIIHYPVKVPVDNPIP VHVDKPVPVHIEKPVPYPVEKPVPYPVKVPVDRPVPVHVEKPVPYPVKVPGTRTIPCRKSNTVSR CPR109 MYSKVLLVATILAAATARPQEGHGHGHDHGHAVSSQSIILHTSHGHEHQPHHAPAHHQILLTQHAGHHD HEELHHGVHLVQHHGHEHHHGHDDHHVDYHAHPKYAFEYKVEDPHTGDNKYQHETRDGDVVKGVY SLHEADGTIRTVEYSADKHSGFNAVVRREGHARHVVPEHHHHH CPR67 MAFKFVVLACLVAVASAGVVPVAQYGYAAPALHAAPVSYSAPIAKVAVEEYDAHPQYSFAYDVQDGVT GDSKSQHETRDGDVVQGSYSVVDPDGIKRTVEYTADPHNGFNAVVHREPLGHAAKVAYAAPVAKIAAP VTYAASPVVHSAPIVHSAPVAYSSPIAKYPAPFTYSAPIYHH CPH3 MRFLIVSALVACVAAAPSHLVPFPAVAYHAVAIPAVVPTLSPGDIQAAAIDAQVKAADLAQAAADKAIAIN EQNAENYNVKAVVNTNLAQEQAVDGVWAVEDKKWQALDALKTAEAQLDGAVASQAVQLAKSAVGA APYVVAPVFPVVYPGIASPAIKSIATQPPVEEVKTVADVEASAKAEEGPAELEVGKVEGNTDSVAVEAKS ASEAAESSAIQSAAKTSAVESDAQTSGVLGAGHISTIQGAIATKTNYPTIPLVGPAFLAHPQVPLVFAVASP S Figure 4

CP genes G1

G1

G3, G4, G5

G2

Ecdysteroid BHR3

βFTZ-F1

E74A

BHR4

W2

W3E

W3M

Developmental Figure 5

W3L

stage

P0

CP genes G1

G2, G3

G5, G6

G4

Ecdysteroid

E74A BHR3 BHR4

βFTZ-F1

BHR38

P0

P3

P5

P7

Developmental Figure 6

stage

A0

Developmental stage

P3

G1 G2

A0

G4

G3

G5 G6

I-exo

Epi

O-exo

Endo Figure 7

V

P

H V H P

H

P H HH

H

V H H

H H V H H

H H

V

H

HV P H H H H H H VH H V

P P H H V H V H PH H H P H PH H H H H H H H HH H H V VHH H PV H HV P VH H PV P V P H P H V V V V

H

H

A A

A

A

A

A

A

A

A A

A

A G

A

A

V

G

A

A

G

G

A

A A

V

A

A

A

A

A A

A V A A

A

A V A

V V A

A

V

A

Epicuticle

A

A

A A A

A A A

Adult cuticle

V A

A

A

A A

A

A

Outer-exocuticle Inner-exocuticle

A

Endocuticle

Pupal Cuticle RR2

RR1

Presumed Cuticular Layer Figure 8

LCP