Developmental proteome dynamics of silk glands in the 5th instar larval stage of Bombyx mori L (CSR2 × CSR4)

Biochimica et Biophysica Acta 1864 (2016) 860–868

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Developmental proteome dynamics of silk glands in the 5th instar larval stage of Bombyx mori L (CSR2 × CSR4) Venugopal Reddy Bovilla a, Mahesh Kumar Padwal b, Prasanthi Siripurapu a, Bhakti Basu b,⁎, Anitha Mamillapalli a,⁎ a b

Department of Biotechnology, GITAM Institute of Science, GITAM University, Visakhapatnam 530045, India Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400085, India

a r t i c l e

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Article history: Received 18 December 2015 Received in revised form 23 February 2016 Accepted 15 March 2016 Available online 23 March 2016 Keywords: Bombyx mori CSR2 × CSR4 Silk gland Proteome Developmental proteomics

a b s t r a c t Bivoltine breed of Bombyx mori (B. mori), CSR2 × CSR4 is an Indian high yielding silkworm strain. Silk gland proteome of this strain was not studied till now. Methods of improving silk production by chemical approaches have reached saturation and transgenic methods are needed in further to boost silk production. An understanding of proteomic changes during silk gland development helps in designing experiments to enhance silk production by transgenic approaches. The present study reports comprehensive developmental proteomic analysis of CSR2 × CSR4, 5th instar whole silk glands. Eighty six unique protein IDs were obtained from the analysis of one hundred and twenty protein spots. Among the identified proteins, majority of the proteins were involved in metabolism (41%) followed by proteins involved in protein homeostasis (30%). Sixty percent of the identified proteins showed dynamic nature by expression analysis from day 1, day 3, day 5 and day 7 gels. In comparison to the published data till now on silk gland proteomics this study reports identification of 20 new proteins from the silk glands for the first time. Significance: The paper reports for the first time proteomic analysis of high yielding silkworm strain of India. The study analyzes whole silk glands to understand the tissue in total during 5th instar development. Lowering fibroin content made us to identify a large number of new proteins which were not reported till now in the silk gland proteome. Proteins which are involved in silk synthesis and release were found to be developmentally regulated. The study identified alanine, serine and glycine tRNA ligases for the first time and also showed their upregulation on day 7 of 5th instar larval stage. The amino acid repeat of fibroin protein is enriched with the three amino acids, glycine, serine and alanine. The identified proteins could be studied further to understand their functional role in-depth. © 2016 Elsevier B.V. All rights reserved.

1. Introduction In India primarily four varieties of silk are produced, tussar, eri, mulberry and muga. Mulberry silk is produced by Bombyx mori (B. mori) which is essentially monophagous and survives solely on mulberry leaves. Silkworm rearing is almost exclusively dependent on the mulberry leaf quantity and quality and also profoundly influenced by the climate and hence there is a demand for region and season specific silkworm races [1,2]. In India, the silkworm hybrids which have been exploited for commercial silk production are either multivoltine × multivoltine, multivoltine × bivoltine or bivoltine × bivoltine combination. Works by Basavaraja et al. [3]; Datta et al. [4–7]; Suresh Kumar et al. [8,9]; Mal Reddy et al. [10]; Dandin et al. [11], led to the evolution of highly productive CSR bivoltine ⁎ Corresponding authors. E-mail addresses: [email protected] (B. Basu), [email protected] (A. Mamillapalli).

http://dx.doi.org/10.1016/j.bbapap.2016.03.013 1570-9639/© 2016 Elsevier B.V. All rights reserved.

breeds which have potential to produce international grade silk. These CSR breeds in various combinations viz., CSR2 × CSR4, CSR2 × CSR5, CSR3 × CSR6, CSR13 × CSR5, CSR12 × CSR6, CSR16 × CSR17, CSR18 × CSR19, CSR20 × CSR29, CSR46 × CSR47, CSR50 × CSR51 etc. have been developed for commercial exploitation to meet the demand of quality silk. As per the statistics available with Central silk board, more than 90% of the silk produced in the country is of multivoltines × bivoltine hybrid cocoons [12]. Bivoltine varieties were introduced in India with the help of Japanese hybrids. The bivoltine breeds thus obtained were tested for pupation percentage, cocoon yield, cocoon weight, shell weight, silk filament length etc. One of the bivoltine breed, CSR2 × CSR4 was found to be suitable to rear in the Southern Indian states like, Andhra Pradesh, Karnataka and Tamil Nadu [13]. The larvae of this strain has bluish white body color with intermediate shape cocoons of medium size. This variety is suitable to grow in the months of September to February. Productive Bivoltine hybrid, CSR2 × CSR4, has all important economic

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parameters like shell ratio N 23.0%; raw silk 19–20%; average cocoon yield 70–80 kg/100 dfls; renditta 5.2 to 5.5 and silk grade — 3 A to 4 A [14]. The silk gland is an organ specialized for the synthesis and secretion of silk proteins. Silk glands of B. mori are of ectodermal origin and are divided into three anatomically and physiologically distinct regions, anterior silk gland (ASG), middle silk gland (MSG) and posterior silk gland (PSG). Specialized cells present at the PSG devote 85% of their protein synthesis activity to silk production [15]. Silk glands appear very early in the embryonic development and increase in size and volume due to endoreduplication of its genome [16,17]. Studies related to silk gland development received very early attention. Changes in the tRNA species during silk gland development was reported forty years back [18]. Proteomic studies were performed largely with Dazhao and Qiufeng, Chinese strains of B. mori. Proteomic studies of PSG and MSG of the two strains showed significant differences in spot distribution and expression during development [19–21]. Spinning mechanism was understood by ASG proteomics and transcriptome analysis [22]. Proteomic studies of silk glands during larval-pupal metamorphosis identified programmed cell death proteins, proteasome subunit, caspases, elongation factor and HSPs [23]. However, proteome based studies on the high yielding variety B. mori CSR2 × CSR4 is not investigated as yet. The present study demonstrates comparative proteomic analysis of the day 1, day 3, day 5 and day 7 of the 5th instar larvae of B. mori CSR2 × CSR4 strain. We have specifically selected these days of 5th instar as, worms resume feeding on day 1 after fourth molt; actively feed up to day 3, show maximum gain in protein concentration on day 5 and maximum protein quantity is observed on day 7. The worms stop feeding and go to spinning stage after day 7. We identified 86 proteins from CSR2 × CSR4 proteome, of which 20 proteins were novel. The data records both quantitative and qualitative changes in the silk gland proteome during 5th instar larval development. 2. Materials and methods

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the standard Lowry method using bovine serum albumin as standard. The protein extracts were subjected to enzymatic treatments with DNase I and RNase I (10 μg/ml each) (Himedia, India) for 2 h on ice, to digest DNA and RNA, respectively. 300 μg of protein was further concentrated to 5 μl using lyophilizer (LARK, India) and then solubilized in 30 μl of rehydration buffer (8 M urea, 1 M thiourea, 4% CHAPS, 150 mM DTT, 2% IPG buffer, traces of Bromophenol Blue for 1 h at room temperature. The solubilized protein extracts were either used immediately or stored at −70 °C until use. 2.4. 2D gel electrophoresis and analysis of protein spots The silk gland proteins (300 μg) were resolved by 2D gel electrophoresis as described earlier [26]. In brief, the proteins were resolved in the first dimension by iso-electric focusing (pH 3–10 NL, 11 cm IPG strips) using PROTEAN® i12™ IEF System (Bio-Rad, India) by cup loading method as per the manufacturer's instructions. Second dimensional resolution was achieved by 12% SDS-PAGE. The gels were stained with Coomassie Brilliant Blue R250 (CBB) stain for 45 min followed by destaining overnight. Three biological replicates were generated at various stages of development of 5th instar larva by repeating the experiment at least three times and analyzing at least one 2D gel in each experiment and at each developmental stage. Dyversity-6 gel imager and GeneSnap software [Syngene, UK] were used to digitize the gel images. PDQuest (version 8.1.0, Bio-Rad) was used to match three biological replicates of 2D gels, at each developmental stage, with a minimum correlation coefficient value of 0.7. Spot detection and matching was initially done in automatic detection mode. The spots which were not present within three replicates were removed from analysis by manual editing. The spots were normalized using local regression method. One way global ANOVA were applied to find the statistically significant differentially expressed protein on different day of development in spread sheet program “MS Excel 2013”. Tukey's HSD post hoc test (p value b 0.05) was applied to diagnose the days among which proteins expression were changed significantly.

2.1. Rearing of silk worms B. mori silkworms, bivoltine breed (CSR2 × CSR4), in 3rd molt stage were procured from the department of sericulture, Government of Andhra Pradesh. The larvae were acclimatized to the lab rearing conditions in the 4th instar stage. The larvae were reared in trays at 23° ± 1 °C and 65–70% relative humidity. The worms were fed three times a day with fresh mulberry leaves till the end of the 5th instar stage. 2.2. Extraction and estimation of total protein content of the silk glands The whole silk glands, dissected from 5th instar larvae in different stages of development (day 1–7), were homogenized using mortar and pestle. 1X Laemmli sample buffer was then added to the homogenate (10 times the wet weight of the tissue), vortexed for 1 min and followed by boiling for 10 min. Cell debris was removed by centrifugation at 10,000 rpm for 10 min. Protein estimation was carried out by modified Lowry protocol with DOC–TCA precipitation [24]. 2.3. Protein sample preparation for 2D electrophoresis The silk glands were dissected from 5th instar larvae at different stages of development (day 1–7) and were resuspended in the insect ringer solution (0.68% NaCl). Immediately after isolation, the glands were frozen for a period of 6 h at −86 °C to eliminate fibroin interference [25]. The glands were brought to 4 °C and homogenized in prechilled lysis buffer (1 mM Tris–HCl, pH 8 containing 1 mM phenyl methyl sulphonyl fluoride). Cell debris was removed by centrifugation at 2000 rpm for 2 min, at 4 °C. The supernatant was sonicated for 1 min, with pulse time 2 s on, 5 s off (PCI Analytics, India) and again centrifuged at 21,000 g for 50 min at 4 °C. Protein estimation was carried by

2.5. Protein identification by Matrix Assisted Laser Desorption Ionization mass spectrometry One hundred and eighteen protein spots were manually excised from the 2D gels and were processed for in-gel trypsin digestion. Various steps such as destaining, reduction, alkylation, in-gel trypsin digestion and elution of oligopeptides were carried out exactly as described earlier [26]. All the chemicals used were LC–MS grade, (Chromasolv, Sigma, India) unless stated otherwise. The eluted oligopeptides were stored at −70 °C, if necessary. The oligopeptides were co-crystallized with CHCA matrix (5 mg/ml in 0.1% TFA with 50% ACN) and spotted onto a 384 well ground steel target plate (Bruker Daltonics, Germany). The Matrix Assisted Laser Desorption/Ionization–Time of Flight/Time of Flight (MALDI ToF/ToF, UltraFlex III (Bruker Daltonics, Germany) mass spectrometer was externally calibrated using Peptide calibration mix I (Bruker Daltonics, Germany) as per the manufacturer's instructions. The spectra were acquired in positive ion reflector mode, using a standard ToF–MS protocol and in the mass range of 600–4500 Da. Peak list was generated using FlexAnalysis software 3.0 (Bruker Daltonics, Germany) and the mass spectra were imported into the MASCOT database search engine (BioTools v3.1 connected to Mascot, Version 2.2.04, Matrix Science). Mascot searches were conducted using the NCBI non-redundant database (release March 2014 or later with minimum of 38,032,689 sequences actually searched) with the following settings: Number of miss cleavages permitted was 1 (or 2 for spot no. 97); variable modification of oxidation on methionine residue; fixed modifications of carbamidomethyl on cysteine residue; peptide tolerance of 100 ppm; enzyme used as trypsin and a peptide charge setting as + 1. A match with B. mori protein with the best score in each Mascot search was accepted as successful identification. Protein

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identification was considered significant when Mascot score was ≥ 75 (p b 0.05). For very low molecular weight proteins (b12 kDa), protein identification was considered significant if the score was ≥50 (p b 0.05).

exported and visualized in the Java Treeview. For clustering the Euclidean distance and complete linkage method were used. 3. Results

2.6. Hierarchical cluster analysis The proteins spots were analyzed on the basis of their abundance on different days of development. The densitometric value of 15,000 was considered as 0 and protein abundance above or below 15,000 was expressed in terms of fold difference (log2 values). Hierarchical clustering of all the identified proteins spots normalized quantities were done using “GeneCluster” toolbox for gene cluster [27]. Cluster tree were

3.1. Proteome map of silk glands in the 5th instar larval stage of Bombyx mori L (CSR2 × CSR4) Silk glands were isolated on different days of 5th instar larval and prepupal stages. Both length and weights of the silk glands increased from day 1 to day 7 of the 5th instar larval stage and decreased from day 9 to day 11 (Supplementary Fig. 1A). The protein content of the

Fig. 1. Representative 2D gel images of cellular proteins (300 μg) from 5th instar larval silk glands, resolved by iso-electric focusing (pH 3–10NL, 11 cm) followed by 12% SDS-PAGE and CBB R250 staining. Different days indicate different developmental stages of 5th instar larva. The pH range was given at the top of each gel and molecular weight markers are indicted at right hand side. Numbers indicate protein spots that were identified in this study.

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Fig. 2. Pie diagram displays distribution of (A) all identified proteins and (B) differentially expressed proteins during silk gland development of 5th instar larva, belonging to various functional categories such as metabolism (M), miscellaneous enzymes (ME), protein homeostasis (PH), transport protein (TP), structural proteins (StP), storage proteins (SP), cellular detoxification (CD) proteins with no known function (PUF). (C) Hierarchical clustering of all identified proteins according to their expression levels. The densitometric value of 15,000 was considered as 0 and protein abundance above or below 15,000 was expressed in terms of fold difference (log2 values). The dendrogram depicts the relationships among the expression patterns of high or low abundance proteins. Set 1–3, marked at the top of the dendrogram, represent three replicates respectively, on indicated stages of development. Annotated dendrogram is provided as Supplementary Fig. 2.

silk glands increased till the end of the larval stage and decreased later during prepupal stage (Supplementary Fig. 1). Proteome complement of silk glands, during the development of 5th instar larva, was studied by resolution of proteins by two dimensional gel electrophoresis and identification of proteins by mass spectrometry. On an average, 202, 218, 243 and 242 protein spots were observed on day 1, day 3, day 5 and day 7 silk gland 2D proteome profiles, respectively (Fig. 1 and Supplementary Table 1). In all, we identified 118 protein spots by MALDI ToF/ToF mass spectrometry (Fig. 1, Supplementary Tables 1 and 2, and Supplementary mass spectrometry data). The identified protein spots were expression products of 86 unique genes and were classified based on their function, as per KEGG database [28,29]. They belonged to the major functional categories of metabolism (M) (34 proteins, 40%), protein homeostasis (PH) (25 proteins, 29%), miscellaneous enzymes (ME) (11 proteins, 13%), transport protein (TP) (1 protein, 1%), structural proteins (StP) (2 proteins, 2%), storage proteins (SP) (4 proteins, 5%), and cellular detoxification (CD) (1 protein, 1%) (Fig. 2A and Supplementary Table 2). Seven identified proteins (9%) have no known function (PUF) (Fig. 2A and Supplementary Table 2). Among the identified proteins, 64 protein spots (belonging to 51 proteins) were differentially expressed (p b 0.05) throughout the

development of 5th instar larva. Among the differentially expressed proteins (DEPs), 47% were metabolic proteins, 23% were related to protein homeostasis, 16% were miscellaneous enzymes, 6% were structural proteins, 2% belonged to cellular detoxification, 2% were structural proteins and 6% had no known function (Fig. 2B and Supplementary Table 2). 3.2. Expression levels of cellular proteins of silk glands vary widely We observed a large variation in the expression levels of silk gland proteins (Fig. 2C, Supplementary Tables 2 and 3). In general, the proteins expressed in high abundance (densitometric values N 20,000) on all the days were protein homeostasis related [HSP90 (spot no. 42), heat shock protein 20.4 (spot no. 32), protein disulfide-isomerase like protein ERp57 (spot no. 5, 6); metabolic enzymes [aldose reductase-like isoform X1 (spot no. 1), arginine kinase (spot no. 2), actin-4 (spot no. 3), alaninetRNA ligase, cytoplasmic (spot no. 11), transketolase (spot no. 17), glyceraldehyde-3-phosphate dehydrogenase (spot no. 23, 28), NADPHspecific isocitrate dehydrogenase (spot no. 26), cytosolic malate dehydrogenase (spot no. 29), Phosphoribosylformylglycinamidine synthase-like (spot no. 47), cytosolic10-formyltetrahydrofolate dehydrogenase-like

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(spot no. 49, 50); miscellaneous enzymes [carboxylesterase CarE-7 (spot no. 14) and aminoacylase-1-like (spot no. 20); transport protein such as cellular retinoic acid binding protein (spot no. 35) and storage lipoprotein 30 K protein 1 (spot no. 31) (Fig. 2C and Supplementary Fig. 2). Proteins expressed in low abundance (densitometric values b 10,000) on all the days were metabolic enzymes [arginine kinase (spot no. 88), and fructose

1,6-bisphosphate aldolase (spot no. 116); miscellaneous enzyme aminoacylase-1-like (spot no. 110); and protein homeostasis related proteasome subunit beta type-5-like (spot no. 100) and proteasome subunit beta type-6-like (spot no. 115). Expression levels of all other proteins were either medium (between 10,001–19,999) or varied on different days.

Fig. 3. Hierarchical clustering of DEPs according to their expression levels on different days of development. Two main clusters are further divided into sub-clusters according to expression pattern of their constituent proteins. The numbers in parenthesis indicate spot nos. as given in Fig. 1 and Supplementary Table 2.

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3.3. Several silk glands proteins were differentially expressed during 5th instar larval development The DEPs were further subjected to hierarchical cluster analysis to reveal development specific expression pattern of silk gland cellular proteins. They displayed 2 main clusters and 4 sub-clusters each (Fig. 3). Cluster 1 A: Proteins which were abundantly expressed throughout the development of 5th instar larval silk glands were clustered in the cluster 1 A (Fig. 3). These were cyclophilin like protein (spot no. 33), putative deoxynucleoside kinase (spot no. 85), 40s ribosomal protein S4 (spot no. 22), abnormal wing disc like protein (spot no. 34) and DNA supercoiling factor (spot no. 40). DNA supercoiling factor induces negative supercoils in the DNA which is important before transcription and replication. This protein was identified from the posterior silk gland of B. mori [30]. It plays a role in transcriptional activation via alteration of chromatin structure [31]. These proteins are mainly involved in metabolism, replication, transcription and translation. As silk glands in the initial days of 5th instar development prepare themselves for growth, endoreduplication and active protein synthesis, abundant expression of these proteins in the silk glands throughout the 5th instar larval stage is logical. Cluster 1B.1: Cluster 1B.1 had proteins whose expression increased throughout the development of 5th instar larval silk glands (Fig. 3). These include storage proteins 30 K protein 1 (spot no. 31), low molecular mass 30 kDa lipoprotein 21G1 (spot no. 38) and low molecular 30 kDa lipoprotein PBMHPC-23 (spot no. 98); protein homeostasis related heat shock protein 20.4 (spot no. 37) and protein disulfideisomerase like protein ERp57 (spot no. 5), elongation factor 1 gamma (spot no. 74) and glycine-tRNA ligase (spot no. 52); and metabolic enzymes like aldose reductase-like isoform X1 (spot no. 1), transketolase (spot no. 16), aminoacylase-1-like (spot no. 20). Along with constitutively expressed metabolic enzymes, enzymes belonging to this cluster may help in central metabolic pathways. Cluster 1B.2: Cluster 1B.2 had cluster of proteins with medium level of protein expression and with minor but significant modulations in the early or late phase of development (Fig. 3). Metabolic enzymes arginine kinase (spot no. 2), glyceraldehyde-3-phosphate dehydrogenase (spot no. 28), cytosolic malate dehydrogenase (spot no. 29) displayed medium level expression in the first 3 days but their expression increased at the later phase of development. On the other hand, protein homeostasis related elongation factor 1 gamma (spot no. 72); alphatocopherol transfer protein-like (spot no. 119); metabolic enzymes glycine-tRNA ligase (spot no. 13), S-adenosyl-L-homocysteine hydrolase (spot no. 70), NADPH-specific isocitrate dehydrogenase (spot no. 25), serine-tRNA ligase, cytoplasmic (spot no. 64); and structural protein actin-depolymerizing factor 1 (spot no. 36) had higher expression levels in the early phase of development and their expression decreased in the later phase. The proteins belonging to this cluster may play important role in necessary housekeeping functions. Cluster 1B.3: Proteins which were abundantly expressed in the early phase (1st–3rd day) of development but expression declined in the later phase of development (5th–7th day) of 5th instar larval silk glands were clustered in the cluster 1B.3 (Fig. 3). This cluster had oxidative stress defense protein thiol peroxiredoxin (spot no. 84); protein homeostasis related translation elongation factor 2 isoform 2 (spot no. 53), ribosomal protein L13 (spot no. 101) and serine protease inhibitor 18 precursor (spot no. 111); and metabolic enzymes probable bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase 2-like isoform X2 (spot no. 75) and putative acetyl transferase (spot no. 96). Other proteins belonging to this cluster are involved in housekeeping functions like protein translation, metabolism, and maintain cellular homeostasis. Cluster 2 A: In the second cluster, a small cluster 2 A comprised of 3 proteins that displayed low expression on 5th day of development (Fig. 3). These were delta-1-pyrroline-5-carboxylate dehydrogenase, mitochondrial-like (spot no. 56), heat shock protein 20.4 (spot no. 32)

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and cytosolic 10-formyltetrahydrofolate dehydrogenase-like (spot no. 49). These proteins are involved in metabolism and protein folding. Physiological relevance of their low expression on 5th day of development is not currently clear. Cluster 2B.1: Cluster 2B.1 comprised of protein that displayed low expression till 5th day of development and their expression increased on 7th day (Fig. 3). These were metabolic enzymes like cytosolic 10-formyltetrahydrofolate dehydrogenase-like (spot nos. 48, 50), integument esterase 1 precursor (spot no. 15), phosphoserine aminotransferase 1 (spot no. 27, 82) and carboxylesterase CarE-7 (spot no. 14). We speculate that silk-gland specific carboxylesterases, (COEs) may participate in synthesis of silk proteins and detoxify the xenobiotics entered into silk gland. Cluster 2B.2: Cluster 2B.2 proteins showed low expression in the early phase of development (1st–3rd day) and medium level expression in the later phase (5th–7th day). It comprised of protein homeostasis related transitional endoplasmic reticulum ATPase, TER94 (spot no. 9); and other metabolic enzymes like vacuolar ATP synthase catalytic subunit A (spot no. 44), arginine kinase (spot no. 88), enolase (spot no. 73) and alanine-tRNA ligase, cytoplasmic (spot nos. 89, 90). Metabolic enzymes belonging to this cluster may help maintain cellular homeostasis. Cluster 2C: A large cluster 2C constituted proteins whose early medium level expression declined throughout the development of the silk glands (Fig. 3). It indicates that the proteins belonging to this group might have functional significance in the initial phase of silk gland development. These include metabolic enzymes like glucose-6-phosphate 1-dehydrogenase-like (spot no. 95), fructose 1,6bisphosphate aldolase (spot no. 116), pyruvate kinase-like isoform X3 (spot no. 93), isocitrate dehydrogenase (spot no. 68), bifunctional purine biosynthesis protein PURH-like (spot no. 57), probable medium-chain specific acyl-CoA dehydrogenase, mitochondrial-like isoform X1 (spot no. 62). Miscellaneous enzymes like aminoacylase-1like (spot no. 110), S-formylglutathione hydrolase-like isoform X1 (spot no. 78), glutathione S-transferase sigma 2 (spot no. 114), receptor for activated protein kinase C (spot nos. 117, 118) were also included in this group. Protein homeostasis related proteasome subunit alpha type1-like (spot no. 113), Translation elongation factor 2 isoform 2 (spot no. 102), proteasome subunit beta type-4-like (spot no. 113), chaperonin (spot no. 106) belonged to cluster 2C. It also included La protein homolog isoform X1 (spot no. 105). La proteins were initially identified in vertebrates and later identified in invertebrates. The function of these proteins is to make multiple rounds of transcription by RNA Pol III and is reported to be very essential for growth and development. The expression of La protein homolog isoform X1 is coinciding with the growth of the silk gland [32]. 3.4. Identification of novel proteins that have not been earlier detected in silk glands of B. mori Supplementary Table 4 shows the comparison between the proteins identified in this study and reported in the published literature on B. mori silk gland proteomics [20,21,23,33–36]. Twenty proteins, identified in this study from 5th instar silk gland of B. mori, have not been reported earlier. Of these, 12 proteins were found to be developmentally regulated. They are cyclophilin like peptidyl-prolyl cis-trans isomerase (spot no. 33); pyruvate kinase-like isoform X3 (spot no. 93); alanine–tRNA ligase, cytoplasmic (spot nos. 11, 12, 89, 90); glycine–tRNA ligases (spot nos. 52, 13), serine–tRNA ligase, cytoplasmic-like (spot nos. 64, 67); probable bifunctional methylene tetrahydrofolate dehydrogenase/cyclohydrolase 2-like isoform X2 (spot no. 75); cytosolic 10-formyltetrahydrofolate dehydrogenase-like (spot nos. 48, 49, 50); putative deoxynucleoside kinase (spot no. 85); glutathione S-transferase sigma 2 (spot no. 114); integument esterase 1 precursor (spot no. 15); aldose reductase-like isoform X1 (spot nos. 1, 77) (Fig. 1 and Supplementary Tables 2, 3). Remaining proteins are

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proteasome subunit beta type-5-like (spot no. 100); dipeptidyl peptidase 3-like isoformX2 (spot no. 7); uncharacterized protein C05D11.1-like isoform X2 (spot no. 41); triosephosphate isomerase (spot no. 80); aspartate aminotransferase, cytoplasmic-like isoform X1 (spot no. 63); oxysterol-binding protein-related protein 11-like isoform X1 (spot no. 107); uncharacterized protein LOC101743840 isoform X3 (spot no. 30); uncharacterized protein LOC101743815 (spot no. 97) (Fig. 1 and Supplementary Tables 2, 3). 4. Discussion In all, we identified 118 protein spots which were expression products of 86 unique genes. Two thirds of the proteins were involved in metabolic and protein homeostatic function. Proteins involved in metabolism, replication, transcription and translation were found to be expressed on all days of development. Importantly, about 60% of the proteins were found to be differentially expressed showing the dynamic nature of silk glands during 5th instar larval development. Proteins involved in metabolism represent maximum number of identified proteins. Glucose 6 phosphate dehydrogenase (G6PD) plays an important role in lipid metabolism [37], was identified earlier in mid gut [38], and prothoracic glands [37], of B. mori. Fructose 1, 6-bisphosphate aldolase was earlier identified in hemolymph and anterior silk gland of B. mori, and plays a role in central carbon metabolism [39]. Expressions of G6PD and fructose 1, 6-bisphosphate aldolase indicate that pentose phosphate pathway is the most predominant pathway of carbohydrate metabolism during early development of silk gland. Glyceraldehyde-3-phosphate dehydrogenase was identified earlier in hemolymph [39], silk gland [33], fat body of pupae [40], and fertilized eggs [41], and is involved in the glucose metabolism. NADPH-specific isocitrate dehydrogenase (Q1HQ47), identified in domesticated silkworm [42], wing disc [43], fat body of pupae [40], posterior silk gland, where it plays a vital role in dehydrogenase activity and oxidoreductase activity of carbohydrate metabolism [34]. Isocitrate dehydrogenase (Q2F681) was identified earlier in fat body of pupae [40], and hemolymph [39], and plays enzymatic role in glyoxylate cycle. Cytosolic malate dehydrogenase, was earlier identified in hemolymph and functions in cellular carbohydrate metabolic processes [39]. Medium level expression of these enzymes throughout silk gland development suggests active glycolysis and TCA cycles to boost generation of molecular energy [ATP and NAD(P)H]. Among other differentially expressed metabolic enzymes, abnormal wing disc like protein has nucleoside diphosphate kinase activity and is involved in purine and pyrimidine metabolism in the baculovirus infected cell lines of B. mori [31]. Expression of this protein was earlier reported in larval mid gut [44], and fertilized eggs of Dazao strain [41]. Arginine kinase has been earlier detected in anterior silk gland [35], fat body [42], mid gut [45,38], fertilized egg [41], and hemolymph [39]. It catalyzes the transfer of phosphate from ATP to arginine [31]. While arginine kinase 1 is a key enzyme of energy metabolism [42], expression of arginine kinase 2 was specific to tissue or developmental stage [46]. Phosphoserine aminotransferase 1, reported in this study is a crucial enzyme in silk production and cell proliferation. Its expression was low during initial days of 5th instar larva of B. mori and increased on day 7. Phosphoserine aminotransferase 1 was earlier found in hemolymph [39], fat body [40], and wing disc [43], of B. mori and is implicated in catabolism, catalyzing the second step in the biosynthesis of the amino acid serine. The serine synthesis is crucial since silk is enriched in serine [47], and serine is also a precursor for the synthesis of purine nucleotides [48,49]. This enzyme was found to be upregulated on the day 7 of silk gland development which correlates well with the synthesis of silk. Pyruvate kinase is involved in energy metabolism, which regulates the glycolytic pathway when the oxygen supply is low or when the body cannot meet energy requirements [50–52]. This protein was earlier identified in prothoracic glands of B. mori and is developmentally regulated during 5th instar larval development [37]. It was predicted to

have a role in ecdysteroidogenesis during the fifth instar larval development. Probable medium-chain specific acyl-CoA dehydrogenase has been identified in silk gland earlier [35]. Vacuolar ATP synthase catalytic subunit A was identified earlier in silk glands [20], fat body [42], and is reported to be involved in maintenance of pH in the anterior region of silk gland. This helps in maintaining silk in liquid state at the anterior region of silk gland [42,53]. Its increased expression may be very crucial on day 7 as the silk secretion reaches to the maximum levels and the gland needs to store them properly before releasing it at the end of the 5th instar stage. Proteins belonging to protein homeostasis group constituted the second major component of the proteins identified in the present study. We demonstrate for the first time that the alanine, serine and glycine tRNA ligases were developmentally regulated in the silk glands. They were up-regulated on day 7 of 5th instar larval stage. The amino acid repeat of fibroin protein is enriched with the three amino acids, glycine, serine and alanine. Transcriptome analysis of Bombyx mandarina showed enrichment of cytoplasmic alanine tRNA ligase in the PSG which is important for efficient synthesis of fibroin [54]. 40S ribosomal protein S4, involved in protein translation was identified in the mid gut of B. mori [45]. Translation elongation factor 2 is earlier identified in silk glands [35]. In hemolymph, it is involved in mitotic spindle elongation and translocation of ribosomes during translation elongation [39]. Elongation factor 1 gamma is a functional component of protein translation machinery. Transitional endoplasmic reticulum ATPase, TER94, was identified earlier in fat body [40]. It is involved in protein folding. EST profile showed the expression of transitional endoplasmic reticulum ATPase, TER94, as maximum in spinning stage and decreases drastically in pupal stage. Similar result was obtained in our study at the protein level. Expression of heat shock protein 20.4 has been observed in silk gland [33], embryos [55], fertilized egg [41], pupae [40], and fat body [56], of B. mori. They possess chaperone activity to refold polypeptides trapped in protein aggregates and to degrade the misfolded proteins in endoplasmic reticulum [36,57]. These proteins are also involved in the regulation of programmed cell death [58–62], and play a role under stress condition [63]. Protein disulfide-isomerase like protein ERp57 was earlier found in anterior silk gland [35], where it plays a major role in protein disulfide isomerization and cell redox homeostasis. It interacts with two lectin chaperones and thereby promotes the oxidative folding of newly synthesized glycoproteins [31]. Cyclophilin protein displays protein folding function in B. mori eggs [41]. These were identified in the silk gland of Antheraea mylitta and predicted to play a role in the protection of the cocoon [64]. Serine protease inhibitor 18 precursor, shown to be present in hemolymph [65], helps in the regulation of protease activity in silk gland [34]. Proteasome subunit alpha type-1-like was earlier identified in wing disc [43], silk gland [20], and in PSG it plays a major role in proteolysis and peptydolysis [34]. Apart from the above two major categories of identified proteins, we demonstrate that the expression of 30 K proteins was increased from day 1 to day 7 of 5th instar larval silk gland development. 30 K proteins are a group of lipoproteins with a molecular mass of approximately 30 kDa. The 30 K proteins are of special interest as they are involved in storage [66], self-defense system [67], and possess anti-apoptotic functions [68]. Apart from silk glands [20], they have been detected in various organs such as fat body [40,42], wing disc [43] and endocrine organs [69]. They accumulate in a stage-dependent fashion in the larval hemolymph of the silkworm, B. mori [70]. The expression of these proteins increased gradually as the silkworm grew, and became a major component of hemolymph in pupation [65] and disappear during eclosion. Thus, increased expression of 30 K proteins in the silk glands shows correlation with their function. A small group of enzymes with varied functions were identified which includes esterases and glutathione S transferase (GST). Esterases (integument esterase precursor 1 and carboxylesterase), play an important role in xenobiotic detoxification, pheromone degradation, neurogenesis and regulation of development. They show tissue specific

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expression [71]. Removal of pheromones from integuments helps in the identification and control of signals in insects [72]. During signaling, integument esterase plays important role in inactivation of pheromones [72,73]. Carboxylesterase CarE-7 has been identified in the integument [74], of B. mori. COEs have the activities of hydrolase and ethyl ester synthase [75]. GST is a redox related protein identified in fertilized egg [41], head [76], and scales [74], and helps in detoxification [31]. It belongs to a family of phase II detoxification proteins which protect the cellular biomolecules from degradation. GSTs identified in B. mori fall into six classes [77]. Functional elucidation of this category of GST enzyme in Drosophila showed their role in lipid peroxidation [78]. The lowering of its expression in silk glands on day 7 of 5th instar could have helped in the gland degradation during the pupal stages. Present analysis showed for the first time enrichment of its protein on day 7 in B. mori silk gland. Cellular detoxification plays an important role in silk gland development. Thiol peroxiredoxin, earlier identified in fat body of pupa [40]and silk gland [33], acts as a physiological anti-oxidant [34], and helps in stress and immune response [35]. Peroxiredoxins are proteins which protect tissues/cells from oxidative damage. This protein was shown to be important in preserving homeostasis and extending life span in Drosophila [79]. Present study showed it to be abundantly expressed in the early phase (1st–3rd day) of development and declined in the later phase of development (5th–7th day) of 5th instar larval silk glands. The expression pattern of thiol peroxiredoxin may help in the degeneration of silk glands in the spinning stages (day 9 to day 11). Taken together, the present study found interesting correlation between protein expression and their function in silk glands development of B. mori. 5. Conclusion The present work reports for the first time B. mori total silk gland proteome analysis of the high yielding strain from India. Analysis of development associated gene expression showed interesting results and the findings of this study are summarized in Fig. 4. The proteins which were highly expressed on all days were involved in nucleotide metabolism, transcription and replication, while low abundance proteins were restricted to metabolism and proteolysis categories. Proteins involved in amino acid biosynthesis, protein translation and folding, storage and energy metabolism displayed increasing expression while proteins metabolism, proteolysis and anti-apoptotic functions demonstrated decreasing expression during development of silk glands. This work

Fig. 4. Schematic representation depicting salient proteomic modulations during silk gland development in 5th instar larva of Bombyx mori.

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throws new light on the development of silk glands of CSR2 × CSR4 strain and the newly identified proteins can be further studied for their role in silk production. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2016.03.013. Conflict of interest The authors declare no conflict of interest. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgment This work was supported by the Department of Atomic Energy as Board of Research in Nuclear Sciences (BRNS) grant to Anitha Mamillapalli (Sanction No. 2012/37B/29/BRNS). Venugopal Reddy acknowledges BRNS for providing research fellowship. The authors thank the Department of Sericulture, Government of Andhra Pradesh for providing the silkworms. References [1] J. Nagaraju, Application of genetic principles in improving silk production, Curr. Sci. 83 (2002) 409–414. [2] K. Thangavelu, Silkworm Breeding: Priorities for India, Advances in Mulberry Sericulture, C.V.G. Publications, Bangalore 1999, p. 187207. [3] H.K. Basavaraja, S. Nirmal Kumar, N. Suresh Kumar, N. Mal Reddy, K. Giridhar, M.M. Ahsan, R.K. Datta, New productive bivoltine hybrids, Indian Silk 34 (1995) 5–9. [4] R.K. Datta, H.K. Basavaraja, N. Mal Reddy, S. Nirmal Kumar, M.M. Ahsan, N. Suresh Kumar, M. Ramesh Babu, Evolution of new productive bivoltine hybrids, CSR2 × CSR4 and CSR2 × CSR5, Sericologia 40 (2000) 151–167. [5] R.K. Datta, H.K. Basavaraja, N. Mal Reddy, S. Nirmal Kumar, M.M. Ahsan, N. Suresh Kumar, M. Ramesh Babu, Evolution of new productive bivoltine hybrid CSR3 × CSR6, Sericologia 40 (2000) 407–416. [6] R.K. Datta, H.K. Basavaraja, N. Mal Reddy, S. Nirmal Kumar, N. Suresh Kumar, M. Ramesh Babu, M.M. Ahsan, K.P. Jayaswal, Breeding of new productive hybrid CSR12 × CSR6 of silkworm Bombyx mori L, Int. J. Indust. Entomol. 3 (2001) 127–133. [7] R.K. Datta, N. Suresh Kumar, H.K. Basavaraja, C.M. Kishore Kumar, N. Mal Reddy, CSR18 × CSR19 — a robust bivoltine hybrid for all season rearing in the tropics, Indian Silk 39 (2001) 5–7. [8] N. Suresh Kumar, H.K. Basavaraja, C.M. Kishore Kumar, N. Mal Reddy, R.K. Datta, On the breeding of CSR18 × CSR19— a robust bivoltine hybrid silkworm, Bombyx mori L. for the tropics, Int. J. Indust. Entomol. 5 (2002) 155–162. [9] N. Suresh Kumar, H.K. Basavaraja, P.G. Joge, N. Mal Reddy, G.V. Kalpana, S.B. Dandin, Development of new robust bivoltine hybrid (CSR46 × CSR47) of Bombyx mori L. for the tropics, Indian J Seric. 45 (2006) 21–29. [10] N. Mal Reddy, H.K. Basavaraja, N. Suresh Kumar, P.G. Joge, G.V. Kalpana, S.B. Dandin, R.K. Datta, Breeding of productive bivoltine hybrid, CSR16 × CSR17 of silkworm Bombyx mori L, Int. J. Indust. Entomol. 8 (2003) 129–133. [11] S.B. Dandin, N. Suresh Kumar, H.K. Basavaraja, N. Mal Reddy, G.V. Kalpana, P.G. Joge, B. Nataraju, M. Balavenkatasubbaiah, B. Nanjegowda, Development of new silkworm hybrid, Chamaraja (CSR50 × CSR51) of Bombyx mori L. for tropics, Indian J. Seric. 45 (2006) 35–44. [12] Dayananda, S.B. Kulkarni, P. Rama Mohana Rao, O.K. Gopinath, S. Nirmal Kumar, Evaluation and selection of superior bivoltine hybrids of the silkworm Bombyx mori L. for tropics through large scale in-house testing, Int. J. Plant Anim. Environ. Sci. 1 (2011) 16–22. [13] N. Mal Reddy, A. Naseema Begum, B.B. Bindroo, CSR2 × CSR4 Productive bivoltine hybrid, Technical bulletin No. 13 [Eng], Silkworm breeding laboratory. @ Copyright CSR&TI, Mysore, May 2014. [14] D. Gangopadhyay, Sericulture industry in India—a review, India, Science and Technology, S&T for Rural India and Inclusive Growth, 2008 (http://www.nistads.res. in/indiasnt2008/t6rural/t6rur16.htm). [15] F.M. Wurm, Human therapeutic proteins from silkworms, Nat. Biotechnol. 21 (2003) 34–35. [16] S.P. Gillot, DNA synthesis and endomitosis in the giant nuclei of the silk gland of Bombyx mori, Biochimie 61 (1979) 171–204. [17] S. Dhawan, K.P. Gopinathan, Cell cycle events during the development of the silk glands in the mulberry silkworm Bombyx mori, Dev. Genes Evol. 213 (2003) 435–444. [18] P. Delaney, M.A. Siddiqui, Changes in the in vivo levels of charged transfer RNA species during development of the posterior silk gland of Bombyx mori, Dev. Biol. 44 (1975) 54–62.

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V.R. Bovilla et al. / Biochimica et Biophysica Acta 1864 (2016) 860–868

[19] J.Y. Li, L.P. Ye, J.Q. Che, J. Song, Z.Y. You, S.H. Wang, B.X. Zhong, Proteome identification of the silkworm middle silk gland, Data in Brief 6 (2016) 903–907. [20] P. Zhang, Y. Aso, K. Yamamoto, Y. Banno, Y. Wang, K. Tsuchida, Y. Kawaguchi, H. Fujii, Proteome analysis of silk gland proteins from the silkworm, Bombyx mori, Proteomics 6 (2006) 2586–2599. [21] J.Y. Li, L.P. Ye, J.Q. Che, J. Song, Z.Y. You, Comparative proteomic analysis of the silkworm middle silk gland reveals the importance of ribosome biogenesis in silk protein production, J. Proteome 126 (2015) 109–120. [22] H. Chang, T. Cheng, Y. Wu, W. Hu, R. Long, C. Liu, P. Zhao, Q. Xia, Transcriptomic analysis of the anterior silk gland in the domestic silkworm (Bombyx mori) – insight into the mechanism of silk formation and spinning, PLoS ONE 10 (2015), e0139424. [23] S.H. Jia, M.W. Li, B. Zhou, W.B. Liu, Y. Zhang, X.X. Miao, R. Zeng, Y.P. Huang, Proteomic analysis of silk gland programmed cell death during metamorphosis of the silkworm Bombyx mori, J. Proteome Res. 6 (2007) 3003–3010. [24] G.L. Peterson, Determination of total protein, Methods Enzymol. 91 (1983) 95–119. [25] P. Agrawal, K.P. Gopinathan, Analysis of nuclear proteins from silk glands of Bombyx mori, J. Biosci. 13 (1988) 379–391. [26] N. Anaganti, B. Basu, A. Gupta, D. Joseph, S.K. Apte, Depletion of reduction potential and key energy generation metabolic enzymes underlies tellurite toxicity in Deinococcus radiodurans, Proteomics 15 (2015) 89–97. [27] M.B. Eisen, P.T. Spellman, P.O. Brown, D. Botstein, Cluster analysis and display of genome-wide expression patterns, Proc. Natl. Acad. Sci. 25 (1998) 14863–14868. [28] M. Kanehisa, S. Goto, KEGG: Kyoto Encyclopedia of Genes and Genomes, Nucleic Acids Res. 28 (2000) 27–30. [29] M. Kanehisa, S. Goto, Y. Sato, M. Kawashima, M. Furumichi, M. Tanabe, Data, information, knowledge and principle: back to metabolism in KEGG, Nucleic Acids Res. 42 (2014) 199–205. [30] T. Ohta, S. Hirose, Purification of a DNA supercoiling factor from the posterior silk gland of Bombyx mori, Proc. Natl. Acad. Sci. 87 (1990) 5307–5311. [31] H.J.R. Popham, J.J. Grasela, C.L. Goodman, A.H. McIntosh, Baculovirus infection influences host protein expression in two established insect cell lines, J. Insect Physiol. 56 (2010) 1237–1245. [32] C.J. Yoo, S.L. Wolin, La proteins from Drosophila melanogaster and Saccharomyces cerevisiae: a yeast homolog of the La autoantigen is dispensable for growth, Mol. Cell. Biol. 14 (1994) 5412–5424. [33] J.Y. Li, H.J. Yang, T.Y. Lan, H. Wei, H.R. Zhang, M. Chen, W. Fan, Y.Y. Ma, B.X. Zhong, Expression profiling and regulation of genes related to silkworm posterior silk gland development and fibroin synthesis, J. Proteome Res. 10 (2011) 3551–3564. [34] Y. Hou, Q.Y. Xia, P. Zhao, Y. Zou, H. Liu, J. Guan, J. Gong, Z. Xiang, Studies on middle and posterior silk glands of silkworm (Bombyx mori) using two-dimensional electrophoresis and mass spectrometry, Insect Biochem. Mol. Biol. 37 (2007) 486–496. [35] Q. Yi, P. Zhao, X. Wang, Y. Zou, X. Zhong, C. Wang, Z. Xiang, Q.Y. Xia, Shotgun proteomic analysis of the Bombyx mori anterior silk gland: an insight into the biosynthetic fiber spinning process, Proteomics 13 (2013) 2657–2663. [36] J. Li, L. Ye, T. Lan, M. Yu, J. Liang, B. Zhong, Comparative proteomic and phosphoproteomic analysis of the silkworm (Bombyx mori) posterior silk gland under high temperature treatment, Mol. Biol. Rep. 39 (2012) 8447–8456. [37] X.F. Wu, X.H. Li, W.F. Yue, B. Roy, G.L. Li, J.M. Liu, B.X. Zhong, Q.K. Gao, W.C. David, Y.G. Miao, Proteomic identification of the silkworm (Bombyx mori L) prothoracic glands during the fifth instar stage, Biosci. Rep. 29 (2009) 121–129. [38] L. Qin, H. Xia, H. Shi, Y. Zhou, L. Chen, Q. Yao, X. Liu, F. Feng, Y. Yuan, K. Chen, Comparative proteomic analysis reveals that caspase-1 and serine protease may be involved in silkworm resistance to Bombyx mori nuclear polyhedrosis virus, J. Proteome 75 (2012) 3630–3638. [39] J.Y. Li, J.S. Li, B.X. Zhong, Proteomic profiling of the hemolymph at the fifth instar of the silkworm Bombyx mori, Insect Sci. 19 (2012) 441–454. [40] H. Yang, Z. Zhou, H. Zhang, M. Chen, J. Li, Y. Ma, B. Zhong, Shotgun proteomic analysis of the fat body during metamorphosis of domesticated silkworm (Bombyx mori), Amino Acids 38 (2010) 1333–1342. [41] Y. Xu, Q. Fu, S. Li, N. He, Silkworm egg proteins at the germ-band formation stage and a functional analysis of BmEP80 protein, Insect Biochem. Mol. Biol. 41 (2011) 572–581. [42] Z.H. Zhou, H.J. Yang, M. Chen, C.F. Lou, Y.Z. Zhang, K.P. Chen, Y. Wang, M.L. Yu, F. Yu, J.Y. Li, B.X. Zhong, Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and on artificial diet, J. Proteome Res. 7 (2008) 5103–5111. [43] Y.L. Zhang, R.Y. Xue, G.L. Cao, Y.X. Zhu, Z.H. Pan, C.L. Gong, Shotgun proteomic analysis of wing discs from the domesticated silkworm (Bombyx mori) during metamorphosis, Amino Acids 45 (2013) 1231–1241. [44] S. Zhang, Y. Xu, Q. Fu, L. Jia, Z. Xiang, N. He, Proteomic analysis of larval midgut from the silkworm (Bombyx mori), Comp. Funct. Genomics 2011 (2011), e876064. [45] H.P. Yao, X.W. Xiang, L. Chen, A.Q. Guo, F.Q. He, L.P. Lan, X.M. Lu, X.F. Wu, Identification of the proteome of the midgut of silkworm, Bombyx mori L, by multidimensional liquid chromatography (MDLC) LTQ-Orbitrap MS, Biosci. Rep. 29 (2009) 363–373. [46] L. Kang, H. Shi, X. Liu, C. Zhang, Q. Yao, Y. Wang, C. Chang, J. Shi, J. Cao, J. Kong, K. Chen, Arginine kinase is highly expressed in a resistant strain of silkworm (Bombyx mori, Lepidoptera): Implication of its role in resistance to Bombyx mori nucleopolyhedrovirus, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 158 (2011) 230–234. [47] Z.Z. Cong, F. Confalonieri, N. Medina, Y. Zivanovi, C. Esnault, T. Yang, M. Jacquet, J. Janin, M. Duguet, R. Perasso, Z.G. Li, Fine organization of Bombyx mori fibroin heavy chain gene, Nucleic Acids Res. 28 (2000) 2413–2419. [48] J.Y. Baek, D.Y. Jun, D. Taub, Y.H. Kim, Characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of l-serine biosynthesis, Biochem. J. 373 (2003) 191–200. [49] T.J. de Koning, K. Snell, M. Duran, R. Berger, B.T. Poll-The, R. Surtees, L-serine in disease and development, Biochem. J. 371 (2003) 653–661.

[50] V.A. Zammit, I. Beis, E.A. Newsholme, Maximum activities and effects of fructose bisphosphate on pyruvate kinase from muscles of vertebrates and invertebrates in relation to the control of glycolysis, Biochem. J. 174 (1978) 989–998. [51] M.E. Munoz, E. Ponce, Pyruvate kinase: current status of regulatory and functional properties, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 135 (2003) 197–218. [52] A. Dabrowska, Erythrocyte pyruvate kinase–an enzyme that may have an influence on oxygen transport to tissues, Postepy Hig. Med. Dosw. 51 (1997) 305–318. [53] M. Azuma, Y. Ohta, Changes in H+-translocating vacuolar-type ATPase in the anterior silk gland cell of Bombyx mori during metamorphosis, J. Exp. Biol. 201 (1998) 479–486. [54] T. Cheng, B. Fu, Y. Wu, R. Long, C. Liu, Q. Xia, Transcriptome sequencing and positive selected genes analysis of B. mandarina, PLoS One 10:3 (2015), e0122837. [55] J.Y. Li, S.H. Hosseini Moghaddam, J.E. Chen, M. Chen, B.X. Zhong, Shotgun proteomic analysis on the embryos of silkworm Bombyx mori at the end of organogenesis, Insect Biochem. Mol. Biol. 40 (2010) 293–302. [56] S.H. Hosseini Moghaddam, X. Du, J. Li, J. Cao, B. Zhong, Y.Y. Chen, Proteome analysis on differentially expressed proteins of the fat body of two silkworm breeds, Bombyx mori, exposed to heat shock exposure, Biotechnol. Bioprocess Eng. 13 (2008) 624–631. [57] J.P. Hendrick, F.U. Hartl, The role of molecular chaperones in protein folding, FASEB J. 9 (1995) 1559–1569. [58] C.Y. Li, J.S. Lee, Y.G. Ko, J.I. Kim, J.S. Seo, Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation, J. Biol. Chem. 275 (2000) 25665–25671. [59] D.D. Mosser, A.W. Caron, L. Bourget, C.D. Larose, B. Massie, Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis, Mol. Cell. Biol. 17 (1997) 5317–5327. [60] S.R. Kirchhoff, S. Gupta, A.A. Knowlton, Cytosolic heat shock protein 60, apoptosis, and myocardial injury, Circulation 105 (2002) 2899–2904. [61] S. Xanthoudakis, R. Sophie, D. Rasper, T. Hennessey, Y. Aubin, R. Cassady, P. Tawa, R. Ruel, A. Rosen, D.W. Nicholson, Hsp60 accelerates the maturation of pro-caspase-3 by upstream activator proteases during apoptosis, EMBO J. 18 (1999) 2049–2056. [62] R. Ran, A. Lu, L. Zhang, Y. Tang, H. Zhu, H. Xu, Y. Feng, C. Han, G. Zhou, A.C. Rigby, F.R. Sharp, Hsp70 promotes TNF-mediated apoptosis by binding IKKγ and impairing NFκB survival signaling, Genes Dev. 18 (2004) 1466–1481. [63] Z.W. Li, X. Li, Q.Y. Yu, Z.H. Xiang, H. Kishino, Z. Zhang, The small heat shock protein (sHSP) genes in the silkworm, Bombyx mori, and comparative analysis with other insect sHSP genes, BMC Evol. Biol. 9 (2009) 215. [64] M. Samita, I.G. Sagar, S. Roy, S. Ghorai, S. Bhattacharyya, A. Venugopalan, A.K. Ghosh, Analysis of transcripts expressed in one-day-old larvae and fifth instar silk glands of tasar silkworm, Antheraea mylitta, Comp. Funct. Genomics 2010 (2010), e246738. [65] Y. Zhang, Z. Dong, D. Wang, Y. Wu, Q. Song, P. Gu, P. Zhao, Q. Xia, Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins, Amino Acids 46 (2014) 1021–1031. [66] E. Mine, S. Izumi, M. Katsuki, S. Tomino, Developmental and sex-dependent regulation of storage protein synthesis in the silkworm, B. mori, Dev. Biol. 97 (1983) 329–337. [67] M. Ujita, Y. Katsuno, I. Kawachi, Y. Ueno, Y. Banno, H. Fujii, A. Hara, Glucan-binding activity of silkworm 30-kDa apolipoprotein and its involvement in defense against fungal infection, Biosci. Biotechnol. Biochem. 69 (2005) 1178–1185. [68] E.J. Kim, W.J. Rhee, T.H. Park, Isolation and characterization of an apoptosisinhibiting component from the hemolymph of Bombyx mori, Biochem. Biophys. Res. Commun. 285 (2001) 224–228. [69] J.Y. Li, X. Chen, W. Fan, S.H. Hosseini Moghaddam, M. Chen, Z.H. Zhou, H.J. Yang, J.E. Chen, B.X. Zhong, Proteomic and Bioinformatic Analysis on Endocrine Organs of Domesticated Silkworm, Bombyx mori L. for a comprehensive understanding of their roles and relations, J. Proteome Res. 8 (2009) 2620–2632. [70] Y. Hou, Y. Zou, F. Wang, J. Gong, X. Zhong, Q. Xia, P. Zhao, Comparative analysis of proteome maps of silkworm hemolymph during different developmental stage, Proteome Sci. 8 (45) (2010) 1–10. [71] Q.Y. Yu, C. Lu, W.L. Li, Z.H. Xiang, Z. Zhang, Annotation and expression of carboxylesterases in the silkworm, Bombyx mori, BMC Genomics 10 (2009) 553. [72] M. Maibeche-Coisne, C. Merlin, M.C. Francois, I. Queguiner, P. Porcheron, E. JacquinJoly, Putative odorant-degrading esterase cDNA from the moth Mamestra brassicae: cloning and expression patterns in male and female antennae, Chem. Senses 29 (2004) 381–390. [73] R.G. Vogt, L.M. Riddiford, Pheromone binding and inactivation by moth antennae, Nature 293 (1981) 161–163. [74] Q. Fu, P. Li, Y. Xu, S. Zhang, L. Jia, X. Zha, Z. Xiang, N. He, Proteomic analysis of larval integument, trachea and adult scale from the silkworm, Bombyx mori, Proteomics 11 (2011) 3761–3767. [75] R.S. Holmes, J. Chan, L.A. Cox, W.J. Murphy, J.L. Vande Berg, Opossum carboxylesterases: sequences, phylogeny and evidence for CES gene duplication events predating the marsupial–eutherian common ancestor, BMC Evol. Biol. 8 (54) (2008) 1–13. [76] J.Y. Li, S.H. Hosseini Moghaddam, X. Chen, M. Chen, B. Zhong, Shotgun strategybased proteome profiling analysis on the head of silkworm Bombyx mori, Amino Acids 39 (2010) 751–761. [77] Q. Yu, C. Lu, B. Li, S. Fang, W. Zuo, F. Dai, Z. Zhang, Z. Xiang, Identification, genomic organization and expression pattern of glutathione S transferase in the silkworm, Bombyx mori, Insect Biochem. Mol. Biol. 38 (2008) 1158–1164. [78] B. Agianian, P.A. Tucker, A. Schouten, K. Leonard, B. Bullard, P. Gros, Structure of a Drosophila sigma class glutathione S­transferase reveals a novel active site topography suited for lipid peroxidation products, J. Mol. Biol. 326 (2003) 151–165. [79] B. Benoit, J. Karpac, S. Supoyo, M.D. Gennaro, R. Lehmann, H. Jasper, Lifespan extension by preserving proliferative homeostasis in Drosophila, PLoS Genet. 6 (2010), e1001159.