Wing Discs Of Bombyx Mori Research Articles

DIA-based proteome reveals the involvement of cuticular proteins and lipids in the wing structure construction in the silkworm

Wing discs of Bombyx mori (B. mori) are transformed into wings during metamorphosis via dramatic morphological and structural changes. Mutations in genes related to the wings cause the adults to have altered wing shapes or abnormal wing colour. At present, there are more than 20 wing mutants recorded in the silkworm. However, the key factors that influence B. mori wing development are still unclear. Here, we used the strains +Wes/+Wes and Wes/+Wes that are typical for the normal wing and shriveled wing phenotypes, respectively, to identify differentially expressed proteins by label-free data-independent acquisition (DIA). Ten enriched GO terms and 9 KEGG pathways were identified based on the 3993 proteins in the wings. Among the identified and quantified proteins, 370 differentially expressed proteins (DEPs) were detected (P-value <0.01, |log2FC| > 0.58). Mapping of the DEPs to the reference canonical pathways in KEGG showed that the top 20% of the pathways were related to fatty acid, cutin, suberin and wax biosynthesis, protein processing in endoplasmic reticulum, protein export, etc. Of the 370 DEPs, 238 were down-regulated, and 132 were up-regulated of Wes/+Wes compared with +Wes/+Wes. Numerous cuticular proteins were down-regulated, and fatty metabolism enzymes were up-regulated, in Wes/+Wes compared with +Wes/+Wes. SignificanceThe comparative analysis of proteomes suggested that cuticular proteins and fatty metabolism enzymes are the main abnormally expressed proteins in the pupal wings of Wes/+Wes, leading to curly and shrunken wings after moth transformation. Our results also identify the substances affecting the development of silkworm wings from the perspective of proteins. The information from this study is important for further research on the molecular mechanisms of wing development in lepidopteran insects, and these differentially expressed genes may be targets for Lepidoptera pest control.

Comparative transcriptome analysis of wing discs from Bombyx mori and Bombyx mandarina

Abstract The insect wing is developed from the wing imaginal disc which is designed from the embryonic ectoderm. To get insight into gene expression profiles in wing discs of Bombyx mori during metamorphosis, we compared the gene expression in the wing between B. mori and B. mandarina moth through RNA-seq. Out of total valid reads identified from the 5th day of 5th instar larvae of silkworm (L5), 7th day of pupae (P7), 1st day of moth (M1) and 1st day of wild silkworm moth (WM1), 20,092,004, 29,251,647, 24,654,695 and 19,753,089 reads were mapped to the mRNA reference sequences of silkworm, respectively. 9229, 7048, 9268 and 6701 differentially expressed genes (DEGs) were respectively recorded in P7 vs L5, M1 vs P7, M1 vs L5 and WM1 vs M1. Further, the peroxisome, ribosome, endocytosis and oxidative phosphorylation pathways were significantly regulated in the metamorphosis of the silkworm. Our study identified 16 orthologous genes with a positive selection from M1, which might be subjected to artificial selection in the domestication of B. mori and would play vital roles in the flight of B. mandarina.

Successively expressed cuticular protein genes at the prepupal stage in wing discs of Bombyx mori

BackgroundWe classified cuticular protein genes expressed at prepupal stage in wing discs of Bombyx mori into six groups according to their developmental expression and ecdysone responsiveness. Their expression pattern is suggested to be regulated by ecdysone-responsive transcription factors, whose transcripts showed resemblance with those of cuticular protein gene expression.ResultGroup1 and Group2 CP genes showed peak expression at stage W2. Group3 CP genes showed high expression at stage W3E and W3L and were upregulated by 20E addition, showing a peak 12 h after 20E pulse treatment. Group4 CP gene transcripts started expression from stage V5 and peaked at stage W3L. Some genes showed significant increase 4 or 6 h after 20E addition and were induced 6 h and showed a peak 18 h after the 20E pulse treatment. Group5 CP gene transcripts peaked at the same stage W3L. Some Group5 genes showed significant increase 6 h after the 20E addition, while others were not induced by the 20E addition. These different sub-groups showed different expression profiles in the feeding stage. Transcripts of this group genes were induced 12 h and showed a peak 18 h after the 20E pulse treatment. Group6 CP genes peaked at the stage P0, were not induced by the 20E addition, and showed a peak 24 h after the 20E pulse treatment. Group3, 4, 5, and 6 CP genes are suggested to be regulated by BHR4, BR-C, E74A, and βFTZ-F1, respectively. ERTFs showed different responsiveness to 20E concentration. BR-C was most and E74A was least insensitive. The addition of cycloheximide inhibited BR-C, E74A, and βFTZ-F1 expression depending on the length of treatment after ecdysone pulse treatment, which suggests that BHR4 induced BR-C, E74A, and βFTZ-F1.ConclusionExpression patterns of CPs were determined by the ecdysone-responsiveness and the related ERTFs expressed in the prepupal stage in B. mori wing discs.

Transcriptional regulation of cuticular protein glycine-rich13 gene expression in wing disc of Bombyx mori, Lepidoptera.

Cuticular protein genes are good models to study the molecular mechanisms of signaling by ecdysteroids, which regulate molting and metamorphosis in insects. The present research demonstrates on hormonal regulation and analysis of the regulatory sequences and transcription factors important for Bombyx mori cuticular protein glycine-rich13 (CPG13) gene expression. Expression of CPG13 was strong at prepupal stage in wing tissues of B. mori. CPG13 expression was induced by the addition of 20E, which was inhibited by cycloheximide in the wing disc. The upstream region of the CPG13 gene was analyzed using a transient reporter assay with a gene gun system and identified two BR-Z2 binding sites to be important cis-acting elements for the transcription activation of the luciferase reporter gene by an ecdysone pulse. Site-directed mutagenesis of these sites, followed by introduction into wing discs, significantly decreased the reporter activity. It was found that the regions carrying the binding sites for the ecdysone-responsive transcription factor BR-Z2 were responsible for the hormonal enhancement of the reporter gene activity in wing discs. Mutation of the BR-Z2 binding sites decreased the reporter activity suggesting that the BR-Z2 isoform can bind to the upstream region of the cuticle protein gene, CPG13 and activates its expression.

Stage-specific activation of the E74B promoter by low ecdysone concentrations in the wing disc of Bombyx mori

To understand the transcriptional regulation of E74B by low concentrations of ecdysone, the promoter activity of Bombyx mori E74B was assessed in the B. mori wing disc using a transient reporter assay. We identified the transcription start sites of BmE74B and found that the core promoter region consists of initiator (Inr) and downstream promoter elements (DPE). The 3.6-kb upstream promoter region of BmE74B was responsive to 20-hydroxyecdysone (20E) in a dose-dependent manner, and the highest luciferase activity was observed in the presence of 0.2μg/ml 20E. Moreover, the upstream BmE74B promoter activity was induced by 20E in a stage-specific and time-dependent manner, and the 3.6-kb promoter contained essential elements for the temporal regulation of BmE74B. Furthermore, we found a set of putative ecdysone response elements (EcREs). Five of these elements are highly conserved, capable of binding to the ecdysone receptor. Mutation of more than three putative EcREs, followed by introduction into the wing discs, abolished the activation of the BmE74B promoter by a low concentration of ecdysone. The results confirmed the role of ecdysone response elements in the transcription regulation of BmE74B and demonstrated that multiple putative EcREs were involved in the maximum response of BmE74B to low concentrations of ecdysone.

Ecdysone-responsive transcriptional regulation determines the temporal expression of cuticular protein genes in wing discs of Bombyx mori

The present study was conducted to clarify the regulatory mechanism of cuticular protein genes of Bombyx mori expressed in wing discs in the prepupal stage. BHR3, BHR4, E74A, and ßFTZ-F1 were successively expressed in wing discs at the pre-pupal stage. BHR3 showed different ecdysone responsiveness from other ecdysone-responsive transcription factors (ERTFs) and was induced by ecdysone addition but showed decrease by ecdysone removal after treatment (ecdysone pulse). BHR4 and E74A were induced by the ecdysone addition and by the ecdysone pulse. ßFTZ-F1 was not induced by the ecdysone addition but was induced by the ecdysone pulse. Thus, ERTFs showed different hormone responsiveness, resulting in the different expression timing. We selected cuticular protein genes that showed the same expression stage with each transcription factor and examined their ecdysone responsiveness. The developmental expression and ecdysone responsiveness of BmorCPH5, BmorCPR34, BmorCPR23, and BmorCPR99 resembled those of BHR3, BHR4, E74A, and ßFTZ-F1, respectively. The results of the transient reporter assay strongly suggested the regulation of each cuticular protein promoter by ERTF. These ERTFs regulated different cuticular protein genes and determined their expression timing and probably the nature of the cuticle layer of insects.

Existence of prophenoloxidase in wing discs: a source of plasma prophenoloxidase in the silkworm, Bombyx mori.

In insects, hemocytes are considered as the only source of plasma prophenoloxidase (PPO). PPO also exists in the hemocytes of the hematopoietic organ that is connected to the wing disc of Bombyx mori. It is unknown whether there are other cells or tissues that can produce PPO and release it into the hemolymph besides circulating hemocytes. In this study, we use the silkworm as a model to explore this possibility. Through tissue staining and biochemical assays, we found that wing discs contain PPO that can be released into the culture medium in vitro. An in situ assay showed that some cells in the cavity of wing discs have PPO1 and PPO2 mRNA. We conclude that the hematopoietic organ may wrongly release hemocytes into wing discs since they are connected through many tubes as repost in previous paper. In wing discs, the infiltrating hemocytes produce and release PPO probably through cell lysis and the PPO is later transported into hemolymph. Therefore, this might be another source of plasma PPO in the silkworm: some infiltrated hemocytes sourced from the hematopoietic organ release PPO via wing discs.

Expression of cuticular protein genes, BmorCPG11 and BMWCP5 is differently regulated at the pre-pupal stage in wing discs of Bombyx mori

Through BLAST search of the genomic database of Bombyx mori, we found a clone, BmorCPG11, which has only putative BR-C binding sites in the 2 kb upstream region. Both BmorCPG11 and BR-Z2 were expressed in the cephalic region of the epidermis, differently from BR-Z1 and BR-Z4. BR-Z2 transcripts increased by the addition of 20-hydroxyl-ecdysone (20E), which was slightly inhibited by cycloheximide. BmorCPG11 expression was also induced by the addition of 20E, which was inhibited by cycloheximide. Both BmorCPG11 and BR-Z2 were induced by the 20E pulse treatment, but they were inhibited by the addition of cycloheximide. Both genes showed similar expression pattern in wing discs during the developmental stage and in vitro ecdysone responsiveness, and both showed relatedness. The result of a reporter assay demonstrated the strong relatedness of BmorCPG11 and BR-Z2. The expression profiles of BmorCPG11 and BR-Z2 are different from those of BMWCP5 and βFTZ-F1. The present findings showed different regulation of cuticular protein genes by ecdysone-responsive transcription factors at the pre-pupal stage of wing discs of B. mori.

Ecdysone directly and indirectly regulates a cuticle protein gene, BMWCP10, in the wing disc of Bombyx mori

The hormonal regulation of cuticle protein genes is a good model to study the molecular mechanism of signaling by ecdysteroids, which initiates each of the major developmental transitions in insects. This study was conducted to clarify the regulation of the expression of an ecdysone-inducible cuticle protein gene, BMWCP10. Induction of the BMWCP10 transcript by ecdysone was partly inhibited in the presence of cycloheximide, which implies that the BMWCP10 promoter is directly and indirectly activated by ecdysone. Using electrophoretic mobility shift analysis and a competition experiment, we identified a putative ecdysone response element (EcRE1) located at positions −93 to −81 relative to the transcription start site. Site-directed mutagenesis of this site, followed by introduction into wing discs, dramatically abolished the reporter activity. This EcRE1 is necessary for the activation of the promoter by 20-hydroxyecdysone (20E) in the wing disc, since the mutation of EcRE1 caused loss of responsiveness to 20E. Collectively, the data obtained in our current and previous work indicate that ecdysone receptor and Broad-Complex Z2 (BR-Z2) are required for maximal BMWCP10 expression in wing disc.

βFTZ-F1 and Broad-Complex positively regulate the transcription of the wing cuticle protein gene, BMWCP5, in wing discs of Bombyx mori

The present study was undertaken to clarify the mechanism regulating cuticle protein gene expression. Expression of BMWCP5 was strong at around pupation and weak at the mid-pupal stage in wing tissues of Bombyx mori. We analyzed the upstream region of the BMWCP5 gene using a transient reporter assay with a gene gun system to identify the regulatory elements responsible for its unique expression pattern. We identified two βFTZ-F1 binding sites to be important cis-acting elements for the transcription activation of the luciferase reporter gene by an ecdysone pulse. Site-directed mutagenesis of these sites, followed by introduction into wing discs, significantly decreased the reporter activity. We also found that the regions carrying the binding sites for the ecdysone-responsive factor BR-C Z4 (BR-Z4) were responsible for the hormonal enhancement of the reporter gene activity in wing discs. Mutation of the BR-Z4 binding sites decreased the reporter activity. The nuclear proteins that bound to these βFTZ-F1 and BR-Z4 sites were identified by an electrophoretic mobility shift assay (EMSA). The results demonstrate for the first time that the BR-Z4 isoform can bind to the upstream region of the cuticle protein gene, BMWCP5, and activate its expression. The results also suggest that the BMWCP5 transcription is primarily regulated by the ecdysone pulse through βFTZ-F1, and the stage-specific enhancement is brought about through BR-Z4.

Activation of BMWCP10 promoter and regulation by BR-C Z2 in wing disc of Bombyx mori

The cuticle protein gene BMWCP10 is transcriptionally upregulated by ecdysone during development. In the present study, using a transient reporter assay, the activity of various genomic segments at the 5′-flanking region of the BMWCP10 gene in driving gene expression and their involvement in ecdysone-mediated activation were assessed in the Bombyx wing disc. The promoter activity of BMWCP10 was responsive to 20-hydroxyecdysone (20E) in a dose-dependent manner, and the highest luciferase activity was observed in the presence of 2 μg/ml 20E. Furthermore, the upstream BMWCP10 promoter was activated by 20E in a stage-specific manner, and the 2.9-kb promoter contained essential elements for the temporal regulation of BMWCP10 in the Bombyx wing disc. Deletion studies revealed that the -598/-387 bp region was required for high-level transcription. In this region, a BR-C Z2 binding element was identified by electrophoretic mobility shift assay (EMSA). Site-directed mutagenesis of this element in the context of the 598-bp promoter fragment significantly decreased the reporter activity in response to ecdysone treatment. The results confirmed the role of BmBR-C Z2 in the transcription regulation of BMWCP10 and suggested the contribution of BmBR-C Z2 to BMWCP10 induction by 20E.

Analysis of ecdysone-pulse responsive region of BMWCP2 in wing disc of Bombyx mori

The present study was undertaken to clarify the regulation of cuticle-protein gene expression. Bombyx BAC library was screened to obtain the sequences of regulatory regions of cuticle protein genes isolated from wing discs of Bombyx mori. Two kb upstream of BMWCP2 was cloned into a reporter plasmid, and a reporter assay was operated. Plasmids were introduced into wing discs and wing tissues using a gene gun. DNA introduction into wing discs was confirmed with plasmid pA3GFP. The upstream region of BMWCP2 showed stage-specific activity: strongest at P0. EMSA analysis indicated the binding of BmbetaFTZ-F1. Ecdysone pulse-responsive sequences were examined in vitro. A luciferase assay was performed using reporter plasmids that contained different length upstream-regions of BMWCP2. With this method, we identified the ecdysone-responsive region. With deletion of the BMWCP2 upstream region, mutagenesis of the BmbetaFTZ-F1 binding site and EMSA analysis, it was confirmed that the BMWCP2 expression was regulated by BmbetaFTZ-F1 through the ecdysone pulse. This is first to apply the introduction of reporter plasmids into small organs to examine the developmental and hormonal regulation of the cuticle protein gene expression. We demonstrated that the binding of BmbetaFTZ-F1 facilitated the promoter activity of the BMWCP2 cuticle protein gene in vitro.

Identification of molting fluid carboxypeptidase A (MF-CPA) in Bombyx mori

Using microarray analyses, we identified carboxypeptidase A (MF-CPA), which was induced during pupal ecdysis in the wing discs of Bombyx mori. Here, we report the functional characterization of MF-CPA. MF-CPA has amino acid sequence similarities with the proteins in the carboxypeptidase A/B subfamily, from human to nematode. The MF-CPA gene is expressed during the molting periods in the epithelial tissues. MF-CPA is detected in the molting fluid, which fills the space between the old and new cuticle during molting. By Western blot analysis, we show that MF-CPA is secreted as a zymogen and processed in the molting fluid. Recombinant MF-CPA expressed in the insect cells has carboxypeptidase A activity. We propose that MF-CPA degrades the proteins from the old cuticle during the molting periods and contributes to recycling of the amino acids.

Characteristics of two genes encoding proteins with an ADAM-type metalloprotease domain, which are induced during the molting periods inBombyx mori

By microarray analyses, we identified two genes (BmADAMTS-1 and BmADAMTS-like) encoding a protein, which are induced during the pupal ecdysis in the wing discs of Bombyx mori; these genes are homologous to ADAMTS family members (a disintegrin and metalloproteinase domain, with thrombospondin type-1 repeats). A complete metal-binding motif of the ADAM-type metalloprotease domain (HEXXHXXGXXHD) was contained in both amino acid sequences. However, thrombospondin type 1 (TSP-1) repeats were observed only in BmADAMTS-1. The BmADAMTS-1 gene was expressed in the hemocyte and midgut of the larvae at day 2 of wandering stage (W2), and strongly induced during the pupal ecdysis in the hemolymph. The BmADAMTS-like gene was expressed in the epithelial tissues of the larvae at W2, and had expression peaks slightly later than the BmADAMTS-1 gene. Our results indicate that BmADAMTS-1 and BmADAMTS-like may cleave the extracellular matrix (ECM) in the degenerating and remodeling tissues during the molting periods.

Change in the expressed gene patterns of the wing disc during the metamorphosis of Bombyx mori

The present study was conducted to clarify what occurs during the metamorphosis of the imaginal disc in insects. To understand the metamorphosis on a molecular level, the changes in expression profiles in the imaginal disc during metamorphosis were investigated. For this purpose, we constructed cDNA libraries from four different stages of wing discs of Bombyx mori, sequenced about 1000 cDNAs randomly collected from each library, and constructed a database of expressed sequence tags (EST). The morphological changes and expression profiles from EST were compared during those four stages. Microarray analysis was applied to quantify the expression of each gene in each stage in order to confirm whether the expression of the genes identified from EST was induced by 20-hydroxyecdysone (20E) in a stage-specific manner. Wing discs showed dynamic morphogenesis in 4–5 days during the preparatory stage of metamorphosis, which was under the control of an ecdysteroid. Different expressed profiles were observed in each of the four different stages by comparison of each EST clone. These profiles reflected the morphological changes of the Bombyx wing disc during metamorphosis. The results of expression profiles from the four stages suggested that the V4 stage was cell proliferating; W0, proliferating and the beginning of differentiation; W2, morphologically changing; W3, cuticle secreting. Microarray analysis showed the effectiveness of its application on 20E induction of genes in wing discs. The wing disc of B. mori is an exceptionally suitable system for understanding the relationship between morphological changes and the distribution of mRNA.

Microarray analysis of gene expression profiles in wing discs of Bombyx mori during pupal ecdysis

Wing discs of holometabolous insects undergo dramatic morphological changes during metamorphosis, a process that is controlled by the actions of hundreds of gene products. Using cDNA microarrays constructed from 5086 ESTs, we monitored the gene expression profiles in wing discs of Bombyx mori at 13 time points during pupal ecdysis (day-4 fifth instar larvae to day-0 pupae). Of the 5086 ESTs on the microarrays, 2998 ESTs had significant signals in more than half of the experiments. Of the 2998 ESTs, genes represented by 683 ESTs showed significant perturbations during pupal ecdysis. Genes previously known to be induced during metamorphosis were identified, including E75, Urbain, Chitinases, and cuticle proteins. The expressions of genes represented by 59 ESTs induced at the beginning of wandering contained genes predicted to be involved in protein degradation, amino acid metabolism, and amino acid transport. The expressions of genes represented by 147 ESTs induced after the ecdysteroid peak had a role in cuticle synthesis, pigmentation, ion transport, protein transport, and transcription regulation. The expressions of genes represented by 85 ESTs repressed after the ecdysteroid peak were predicted to be involved in nucleotide and nucleic acid metabolism and cell cycle. This indicates the involvement of several biological processes in wing disc development during metamorphosis.

Isolation and comparison of different ecdysone-responsive cuticle protein genes in wing discs of Bombyx mori

Microarray analysis was used to isolate an ecdysone up-regulated cuticle protein gene from wing discs of Bombyx mori. Transcripts of isolated cDNAs were identified by Northern blot analysis. Expression of the BMWCP10 gene was observed during the W0-W3 stages with the strongest signal being at the W2 stage. In contrast, expression of the BMWCP2 gene was observed at the W3and P0 stages. Expression of BMWCP10 was identified after exposure to 20E in vitro, while that of BMWCP2 was identified after exposure to 20E followed by its removal. Induction of BMWCP10 by 20E was observed in 30 min and was not inhibited by cycloheximide. Expression of BMWCP2 was observed in wing discs cultured for more than 18 h in a hormone-free medium after 20E removal. At least 4 h exposure to 20E was required before removal for induction of BMWCP2. Induction of BMWCP2 required protein synthesis. Thus, different ecdysone-responsive cuticle protein genes in wing discs of Bombyx mori were isolated.

Isolation and expression of an ecdysteroid-inducible neutral endopeptidase 24.11-like gene in wing discs of Bombyx mori

In the process of comparison of two cDNA libraries (W0, W2), we isolated a clone from the wing discs of Bombyx mori encoding a putative neutral endopeptidase 24.11-like gene. The predicted open reading frame encoded 772 amino acid residues, having about 53% identity with Drosophila GH07643, 36% with rat NEP, and 34% with rat ECE. This is the first NEP gene isolated in invertebrate. A 3.6-kb transcript was found to accumulate in the wing disc according to the increase of ecdysteroid titer during metamorphosis. Accumulation of the transcript was induced in wing discs with 20-hydroxyecdysone about 20 h after incubation, which was inhibited by cycloheximide. This gene is ecdysone-inducible, appears to encode a functional protein, and may function during wing metamorphosis.

Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations

Multiple cloning of cuticle protein genes was performed by sequencing of cDNAs randomly selected from a cDNA library of wing discs just before pupation, and nine different cuticular protein genes were identified. Thirty-one clones of a cuticle protein gene were identified from the 1050 randomly sequenced clones; about 3% were cuticle protein genes in the W3-stage wing disc cDNA library. The sequence diversity of the deduced amino acid sequences of isolated Bombyx cuticle genes was examined along with the expression profiles. The deduced amino acid sequences of the nine cuticle protein genes contained a putative signal peptide at the N-terminal region and a very conserved hydrophilic region known as the R and R motif. The developmental expression of cuticle genes was classified into two types: pupation (five clones were expressed only around pupation) and pupation and mid-pupal (four clones were expressed around this stage). All the isolated genes were expressed in the head, thoracic, and abdominal regions of the epidermis at different levels around pupation, but no expression was observed in the epidermis at the fourth molting stage.

Isolation and expression of the ecdysteroid-inducible angiotensin-converting enzyme-related gene in wing discs of Bombyx mori

We isolated a clone encoding a putative angiotensin-converting enzyme-related gene from the wing disc cDNA library of the silkworm, Bombyx mori (refer to as BmAcer). The predicted open reading frame encoded 648 amino acids with about 50% identities with the Drosophila melanogaster angiotensin-converting enzyme Ance and Acer. Northern analysis identified a 2.2-kilobase mRNA which was abundant in wing discs two days after the beginning of wandering. An accumulation of the transcript was observed approximately 2 h after 20–hydroxyecdysone (20E) exposure in vitro and was blocked slightly by a protein synthetic inhibitor. These data suggest that the transcription of the BmAcer gene is directly 20E-inducible.