MATERIALS AND METHODS

Rearing: A hybrid strain (Tamil Nadu white×NB4D2, Bombyx mori) of the silkworm was used for this experiment, which was maintained in our Insect Molecular Biology Laboratory at Bharathidasan University, Tamil Nadu, India. Disease free eggs of B. mori were kept at ambient temperature (27±2°C) and a relative humidity (75±5%) until hatching of larvae as reported previously³¹. The hatched larvae fed with chopped tender leaves of the mulberry variety (MR2) until III instar and with coarse leaves provided until the end of the last instar.

Protein extraction: The heads were collected from 20 individuals of 5th instar larvae and freshly emerged adults of B. mori, washed twice with milli-Q water taken in a centrifuge tube and stored at -85°C for further use. The heads were grounded in 200 μL of extraction buffer containing 7 M urea, 2 M thio urea, 4% CHAPS, 20 mM DTT, 5 mM EDTA, 2 mM PMSF, 1.2% biolyte, 10% glycerol, RNase (1 mg mL^–1) and DNAse (1 mg mL^–1). The mixture was centrifuged at 15,000 rpm for 20 min at 4°C. The supernatant was collected for further centrifugation at 15,000 rpm for 20 min at 4°C. The supernatant was prepared as aliquots and stored at -85°C until electrophoresis performed. The protein concentrations were determined according to Bradford method³².

02D-elecrophoresis: A 300 μg of protein taken from the each head sample and mixed in 350 μL of rehydration buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 65 mM DTT 0.8% biolytes, 0.002% bromophenol blue. Each sample was loaded onto 17 cm immobiline dry strips (17 cm, pH 3-10 Linear, Bio-rad) to passive rehydration for overnight. Iso Electric Focusing (IEF) was performed using Ettan IPGphor 3 U (GE healthcare) at 20°C. The IEF program was set as: Step 500 V for 1 h, gradient 3000 V for 1 h, gradient 8000 V for 3 h and step 8000 V for 7 h (a total of 55 028 Vh). The isoelectric focused strips were incubated for 15 min in an equilibration buffer containing 50 mM tris-HCl buffer pH 8.8, 6 M urea, 2% SDS and 30% glycerol and 1% DTT (1%) and the strip was again equilibrated for another 15 min with same buffer except that DTT was replaced with 2.5% iodoacetamide. The equilibrated strips were transferred to 12% SDS-PAGE³³ and sealed with 0.5% agarose to prevent the shaking of the strip on the top of the PAGE. The SDS-PAGE was performed at 15°C using protean II Xi cell unit (Bio-rad) at constant power of 2 W gel^–1 for 1 h followed by 6 W gel^–1 until the bromophenol blue frontier reached the bottom of the gel. The gel was stained for 2 h or overnight in 0.1% of coomassie brilliant blue R-250 solution (Sigma) in 40% methanol and 10% glacial acetic acid. Then the gel was rinsed thrice with milli-Q for a minute and the remain stain was removed using destaining solution containing 10% glacial acetic acid and 40% methanol in milli-Q until clear protein spots were visualized. Then the gel was stored in 10% ethanol at 4°C if for long times. Molecular mass and pI were calculated from digitalized 2D images using protein ladder (Bench Mark^TM protein ladder, life technologies, Cat No: 10747-012). Triplicate analysis was performed for each sample and a comparison of relative intensity of protein spots between the larvae and adult heads were conducted.

2D-PAGE spot analysis: Two-dimensional spot images were scanned at an optical resolution of 400 dpi with gray scale using high-resolution image scanner. The scanned gels were analyzed using Image Master^TM 2D platinum 7.0 software according to the instruction manual given by GE healthcare. The triplicate images were merged and subjected to analysis including background subtraction, spot detection, spot matching and quantitative intensity normalization analysis, etc. All the statistical analysis was done with PAST version 2.08.

In-gel trypsin digestion: Based on the comparative and the differential expression, 30 protein spots were manually excised from the Coomassie blue stained gel and subjected to in-gel trypsin digestion³⁴. Briefly, the gels were washed twice in milli-Q water. The gel pieces were placed in a solution containing 50% of methanol and then dehydrated by acetonitrile (ACN) and dried in a SpeedVac (Thermo Savant). The dried gel pieces were rehydrated using 0.02 μg μL^–1 trypsin and 25 mM ammonium bicarbonate containing a 10% ACN solution on ice and digested at 37°C for 16 h. The enzymatic reactions were stopped using 50 μL of a solution including 5% trifluoroacetic acid (TFA) and 67% ACN. After centrifugation, the peptide mixture was transferred into a centrifuge tube and sonicated for 15 min and then concentrated in a SpeedVac before being analyzed in a mass spectrometer. These MS/MS analysis were replicated twice for each protein sample in this experiment.

MALDI-TOF/TOF MS analysis and database searching: Prepared peptide samples w e re resolved in 0.1% TFA, mixed with an equal volume of matrix solution α-cyano-4-hydroxy-cinnamic acid (CHCA, Sigma, St., Louis, MO) and spotted on the target plate. The samples were allowed to air-dry and analyzed UltrafleXtreme MALDI-TOF/TOF-MS/MS (Bruker Daltonik, UK). Protein identification using peptide mass fingerprinting was performed using the Mascot search engine version 2 (Matrix Science, London, UK) against the NCBI and Swiss-prot protein database. The following search parameters were used: Taxonomy: Bombyx mori, Database: NCBI nr database or Swiss-prot database, enzyme: trypsin with one max missed cleavage, monoisotopic masses, fixed modifications of carbamidomethyl, variable modifications of oxidation on methionine, peptide charge state of 1+ and peptide MS/MS tolerance of ±0.7. Protein identification with significant hits as defined by a mascot probability analysis (Protein score greater than 95%, p<0.05) was accepted. The identified proteins were further classified based on their biological, cellular and molecular functions based on the functional annotations in the software tool for rapid identification of proteins (STRAP online tool, version 0.15)³⁵.

RESULTS

2D-PAGE pattern of proteins from larvae and adult head of B. mori: The greatest difference between the 2D gel patterns of head proteins from larval and adult of B. mori was showed in (Fig. 1, 2). The triplicates of each head proteins patterns were highly reproducible and acceptable. In total, ~315±3 and 296±3 protein spots were detected from larvae and adult heads, respectively in the pI range of 3-10 and molecular masses of ~5-120 kDa (Table 1). In this 315±3 spots on the larvae head, around 65±1 spots are present at >50 kDa and the spots present below 50 kDa are nearly 250±5, whereas 55±1 spots are observed as a major spot in larvae. In the adults head, 296±3 protein spots were observed of which 48±2 proteins are present above 50 kDa and 248±4 spots below 50 kDa and 38±2 spots are observed to be the major spots in adult head out of 224. The variation of proteins spots was measured using one-way ANOVA.

Table 1:	Univariate statistics of proteins spots in heads from larva and adult of Bombyx mori obtained by 2-DE

Fig. 1:	2D-PAGE analysis of proteins from larvae head of Bombyx mori

Fig. 2:	2D-PAGE analysis of proteins from adult head of Bombyx mori

Table 2:	Distributional variation of protein spots obtained from the head of larva and adult of Bombyx mori using 2-DE

Intra and inter variation of protein spots distributed in 2-DE in the heads of larva and adult were not significantly varied and this result was tested with Levene’s test based on mean (Table 2). The variation of protein spots of 2-DE between the larva and adult was tested with Mann-Whitney and Kruskal-Wallis analyses (Table 2). The larval head protein spots were more significant than the adult proteins spots revealed by these analyses and it was further tested with Monte Carlo (0.8076). The theoretical isolelectric point (pI) and Molecular Weight (MW) of the identified proteins were calculated using the compute pI/MW tool (http://web.expasy.org/compute_pi/) according to the predicted amino acid sequence. The results showed that distribution of major protein spots in the larvae within the range of pI 5-7 and in adult within the range of pI 4-5 and 7-9 (Fig. 3). The spots present in between each pI is calculated to be 11±1, 64±1, 132±3, 77 ±1, 20±1, 9±1 and 3±1 for 3-4, 4-5, 5-6 , 6-7, 7-8, 8-9 and 9-10 pI, respectively. The spots present in each pI is calculated to be 9±2 spots in 3-4, 66±1 spots in 4-5, 117±1 spots in 5-6, 62±2 spots in 6-7, 31±1 spots in 7-8, 10±1 spots in 8-9 and 2 ±1 spots in 9-10.

Qualitative comparisons of 2-DE protein patterns: The enlarged view of comparatively analysed spots and its expression in 3D view are represented in (Table 3) through the analysis of Image Master^TM 2D platinum 7.0 (GE healthcare). The results revealed that the majority of proteins were expressed at different level in both heads. Of 10 proteins (Spot 1-9, 16) were found to be a two fold higher expression in the larval head and 6 proteins (Spot 10-15) were two fold expression in the adult head. In addition, 6 proteins (Spot 17-22) were unique in the larval stage and whereas 8 were unique in the adult stage (Spot 23-30).

Mass spectrometric analysis of protein spots: The comparative and differentially expressed proteins from the head of larvae and adult were carefully analysed and subjected to mass spectrometry analysis. In total 30 proteins were identified from both larvae and adult heads using mass spectrometric approach and the results were shown on Table 4.

Fig. 3:	Distribution of proteins based on the isoelectric point in both larvae and adult head of Bombyx mori

The list of comparatively identified proteins between the head of larvae and adult as follows cytoskeleton proteins (Spot 1), structural constituent and chitin based cuticle protein (Spots 2, 3, 6, 7), odorant binding proteins (Spot 4), three metabolic enzymes identified as characterized proteins such as glycerophosphodiester, enolase and monooxygenase/reductase (Spot 8, 10 and 13) and two uncharacterized proteins are also associated with enzymatic activity includes Phosphatase (Spot 9) and N-acetyltransferase activity (Spot 12), insect neuro hormone (Spot 11) and few uncharacterized proteins (5, 14, 15, 16) are associated with molecular binding activities.

Six proteins were identified uniquely in the larval head as follow: Actin-4 (Spot 17), apolipophorin-III (Spot 18), centromere-associated protein E like (Spot 19), iron-ion binding (Spot 20), cuticular protein RR2 motif 59 precursor (21) and one uncharacterized protein with phosphatase activity (Spot 22). Eight proteins from adult head were found to be unique namely, metabolic activity (23), uncharacterized protein with no function (Spot 24, 25, 29), glycoprotein hormone alpha2 (Spot 26), DNA binding activity, ALY protein (Spot 27, 30), clathrin coat assembly protein (Spot 28). In the present study, all spots had significant peptide hits but their pI/MW was not consistent with comparative theoretical and computational/values due to database limit and protein mass variation for homologous protein in different organisms. The identified proteins were classified based on the molecular, cellular and biological functions (Fig. 4).

Table 3:	Selected 2D-PAGE gel areas, corresponding 3D profile of selected proteins identified in larvae and adult of B. mori
Spot volumes were generated from Image MasterTM 2D platinum 7.0 (GE Lifescience, USA). Protein identifications related to spots numbers are presented in this table

Fig. 4(a-c):	Gene Ontology (GO) categories of the common and unique expressed proteins from larval and adult heads of Bombyx mori, (a) Biological process (b) Cellular component and (c) Molecular function

DISCUSSION

In the present study, the total protein isolated from larval as well as adult head of Bombyx mori was subjected to 2D-PAGE analysis. The 2-DE results clearly showed 315±3 protein spots from adult head and 296±3 spots in the larval head of B. mori. Most of the protein spots in the larval and adult head are observed in the pI range of 4-10 and 3.5-8, respectively. Like other kinds of organs/tissues of B. mori exhibited a huge variation in the protein spots through proteomics analysis. For instance, 400 protein spots were identified in silk glands¹², 128 spots in hemolymph⁹, 566 spots in male midgut and 547 spots in female midgut²⁴. The results of the present study suggests that enormous numbers of protein are synthesized during the larval stage (active feeding stage) for the growth and development whereas in adult showed less number of protein in the narrow range of pI due to changes in the adult feeding trait and non-synthesizing of major proteins during the adult development other than the ecolsion and reproduction.

When we carefully analysed the protein spots of larvae and adult head, a total of 30 similar and dissimilar proteins were observed, out of this results in more than 99% contrast with the study of Li et al.¹⁸. However, these proteins were individually observed from different tissues of B. mori for example collateral glands⁸, silk glands, haemolymph and fat body^10,12, midgut²³ and newly hatched larva³⁰.

Interestingly, we observed that the expression of Odorant Binding Proteins (OBPs), apolipophorin III precursor (Apo-Lp III) and iron-ion transport protein (Spot 4, 18, 20) were higher in the larval head than the adult head. This result indicates that larval feeding activity requires OBPs, Apo-Lp III and iron-ion transport protein. In the larval stage, the OBPs help to transport of hydrophobic odorant from mulberry leaves to olfactory receptors^2,36,37, Apo-Lp III and iron-ion transport protein may have the role to provide energy for feeding of larva. However, Apo-Lp III transports of diacylglycerol (DAG) from the fat body lipid storage deport to flight muscles in the adult^38,39 and Iron-Ion transport protein is regulated to electron transport, neurotransmitter and synaptic-vesicle exocytosis⁴⁰.

The RNA silencing process in B. mori is mediated by argonaute protein⁴¹. In the present study, the argonaute protein (Spot 1) expression was found to be two fold higher in the larval head when compared to the adult head, which reveal that agronaute protein occupy the major role to regulate the translation of proteins during larval developmental stage^42,43. In addition, we identified the Centromere-associated protein-E (CENP-E) (Spot 19) from the larval head. The CEBP-E may conduct to mitotic kinesin, required for efficient and stable microtubule capture at kinetochores. The CEBP-E also directly binds to a kinetochore-associated kinase (BubR1) implicated in the mitotic checkpoint, the major cell cycle control pathway in which unattached kinetochores prevent anaphase onset⁴⁴.

Most of the cuticular proteins are synthesized during the larval stage by dermal cells and secreted to its surface after each moulting in B. mori³⁰. Similarly the foremost expressions of cuticular proteins (Spot 2- 3, 6-7, 21) were discovered in the larval and adult head of our study. In addition, the abundance of Cuticular protein RR-2 motif 59 precursors (Spot 21) and the uncharacterized protein (Spot 22) which are found to be involved in Cuticular development and metabolic process in the larval stage only.

Table 4:	List of identified proteins from larval and adult heads of silkworm, B. mori
ASpot ID is the symbol for every identified protein, which also noted on the 2-DE patterns (Fig. 1),BAccession number is the protein identifier in the NCBI or uniprot database (http://www.ncbi.nlm.nih.gov/), CSpecies indicate the organism that the matched proteins identified from NCBI or uniprot database, DMatched peptides are the number of paring experimental spectrums to a known protein and protein score is the results from mascot searching and is the main parameter for identification confidence,EScore value, FThe theoretical pI/MW represents the values of isoelectric point (pI) and Molecular Weight (MW) retrieved from protein databases of NCBI, GBiological functional annotation of the identified success protein and HMolecular function of the identified success proteins in each heads

These cuticular proteins are found to be playing a significant role in protecting the insect’s body from dehydration, physical injury, infection and penetration of environmental factors^45-47,18. The actin-4 was identified in spot 17 of our 2-DE of larval head, which is playing an important role in Synaptogenesis, for example cell motility, cytokinesis and morphogenesis^48-52.

In total, five enzymes were identified in the study. Of these, three characterized enzymes are as follows: Enolase, cytochrome P450 and glycerol phosphodiesterase and the other two are uncharacterized enzymes. Enolase is a glycolytic enzyme (spot 10), involved in the regulation and control of transcription, apoptosis and cell differentiation⁵³ and feeding activity of the larva^54,55. The cytochrome P450 enzyme (Spot 8) involved metabolism/detoxification of drugs, environmental pollutants and pesticides¹⁸ and glycerolphosphodiesterase (Spot 13) for glycerol metabolism⁷. The other two uncharacterized enzymes are involved in phosphatase and N-acetyl transferase activity (Spot 9 and 12) in both larvae and adult heads. Similarly, the N-acetyl transferase activity found in the silkworm head and presumed the function as a neurochemical mediator of photoperiodic control and event such as moulting and ecolsion^56,57.

In the present study, expression of a glyco-protein hormone alpha 2 subunit observed as heterodimer in the head of the larva (Spot 11) and monomer in the adult head (Spot 26). It is evidenced to the study of Sudo et al.⁵⁸ that the glyco-protein hormone is an insect neurohormone, it is a heterodimer and participate in developmental regulation includes cuticle formation, testis and ovary differentiation in B. mori. Also this glyco-protein hormone which regulates ion transport in hindgut of adult Aedes aegypti⁵⁹. Further a unique expression of adult specific uncharacterized protein (Spot 23) which is involved in carboxlic acid metabolic process observed in the present study.

CONCLUSION

We speculate that all these adult unique protein and other highly expressed proteins may have a potential role in adult development and reproduction. The uncharacterized proteins (Spot 5, 24, 25, 29) from adult head observed with no functions. Further study need to be carried out for better understanding of the molecular network involves in biological functions of Bombyx mori. This study might lay a strong foundation to understand the feeding behaviour of larvae and adult development in B. mori and also in other lepidopteron insect pests.

ACKNOWLEDGMENT

We thank the Central Silk Board, Bangalore, India (CSB-31/2(PROJECT)/2010-11-RCS dated 16.09.2011) for financial support. V. Arun Prasanna is grateful to the Central Silk Board, Bangalore, India for the award of Junior Research Fellowship. We also thank to Dr. L. Uma, Director, National Facility for Marine Cyanobacteria (NFMC), Bharathidasan University, Tiruchirappalli, India for permitting to analyse the 2D-PAGE image. We sincerely thank Mr. A Jaleel, Mass Spectrometry and Proteomics Core Facility, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram-695 014, Kerala, India.