1. Introduction

The accessory glands arise from a special set of cells in the male primordium of the genital disc whose developmental fate is determined by the male sex determination pathway during the third instar larvae. It plays an essential role in insect reproduction. Insect have yielded a variety of basic information about ultrastuctural, physiological and biochemical analysis. In many insects the main function of accessory gland is the production of spermatophore for sperm transfer from male to female genital tract during mating ^{[1, 2]}.

Accessory glands secretory protein (ACP) plays an important role in fertile reproduction. ACP’s are major components of Drosophila seminal fluid. The seminal fluid of Drosophila melanogaster contains over 80 proteins and peptides, which are transferred along with sperm from male to female during mating ^[3]. Seminal proteins induce morphological and behavioural changes in mated females.

Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis (SDS-PAGE) is a relatively simple rapid low cost and high sensitive tool used for the separation of proteins and to determine their molecular weight. The separation of proteins by electrophoresis is based on the fact that charged molecules will migrate through a matrix upon application of an electrical field. It is a well known fact that proteins are dissociated into their constituent polypeptide chains by detergents like SDS. SDS is a denaturing and solubilizing agent; induce conformational changes in the proteins ^[4].

The SDS-PAGE analysis and quantity of proteins ejected during mating has been analyzed in D. melanogaster, D. n. nasuta, D. s. neonasuta, D. rubida, D. pararubida, D. varians and D. ananassae ^{[5, 6]}) and also qualitative and quantitative correlations of male accessory gland secretions has been analysed in D. nasuta sub group ^[7].

Periodic Acid Schiff (PAS) staining is widely used in the detection of glycoprotein. Glycoprotein plays an important role in numerous developmental and physiological processes, especially in the nervous system. The glycosylated protein fraction varies in different species of Drosophila ^{[5, 8]}.

In spite of more than 110 species in melanogaster species group, which consists of sibling, sympatric and closely related species, the information about ACP is available only of D. melanogaster. Therefore to know the nature and characteristics of ACP in different species, species belonging to various groups/ subgroups of Drosophila were taken for the present study.

2. Materials and Methods

D. melanogaster, D. simulans, D. mauritiana, D. ananassae, D. varians, D. bipectinata, D. malerkotliana and D. rajasekari, belonging to melanogaster species group, D. virilis and D. texana, belonging to virilis species group and D. hydei belonging to repleta species group were obtained from Drosophila stock centre, Department of Zoology, University of Mysore, Mysore, India for the present work. The stocks were maintained using wheat cream agar medium and prepared as per the procedure described by Shivanna et al., ^[9]. The cultures of Drosophila were maintained at a constant temperature of 22 ± 1ºC and humidity of 70-80%.

The virgin male and female flies were separated within 1 hour of their eclosion from the cultures and they were kept in a separate vial and aged for 7 days. After 7 days the flies were etherized and accessory glands from adult male were dissected in physiological saline (0.7% of NaCl). Glands were isolated and fixed in 95% ethanol. The ethanol fixed glands were washed in a mixture of methanol and chloroform (1:1) and dried at 37ºC for 15 minutes. The sample buffer about 40 µl (0.623 M Tris-HCl, pH 6.8, 1% sodium dodecyl sulphate (SDS), 1% β- mercaptoethanol, and 10% glycerol) was added to each sample to dissolve the glands. The eppendorf tube containing the sample was boiled for about 10 minutes in boiling water bath and centrifuged at 1000rpm for 10 minutes.

SDS polyacrylamide gel of (T=13.7% C=3.5%) 1.5mm thickness was prepared. 40µl of protein sample was loaded separately into each slot of the stocking gel (3.8 ml of distilled water, 3.8ml of 0.5M Tris HCl, pH 6.8, 118µl of 20% SDS, 3.8ml 0f 22% acryl amide, 100µl of 10% APS and 8µl of TEMED). Electrophoresis was performed at room temperature with 60V for 1 hour. Later it was increased to 80V until the dye migrated to 7cm in the running gel (16.8ml of distilled water, 6.9ml of 1.5M Tris-HCl pH 8.8, 208µl of 20% SDS, 16.8ml of 22% acryl amide, 184µl of 10% APS, 18.4µl of TEMED). The gel was then removed and stained with Coomassie Brilliant Blue R-250 for three hours and destained in a mixture of 25% methanol and 7.5% acetic acid.

To identify the glycosylated protein fractions of the ACPs, the gel was stained employing the procedure described by Segrest and Jackson ^[10]. The gel was fixed overnight in PAS fixative solution (40% ethanol, 5% glacial acetic acid and 55% distilled water). The gels were then treated with periodic acid (0.7%) solution covering the gels for 2-3 hours. This was followed by treatment with sodium metabisulfate (0.2%) with one solution change after 30 minutes, and then the gels were transferred to Schiff’s reagent. Colour develops in 12-18 hours at room temperature and thereafter the gels were stored at 4ºC and photographed.

Molecular weight of each protein fractions was calculated using the marker proteins (phosphorylase-97,400 Da, Bouines serum albumin-66,000 Da, ovalbumin-43,000 Da, Carbonic anhydrase-29,000 Da, Soybean Trypsin Inhibitor-20,100 Da, Lysozyme-14, 300 Da) with the help of log graph.

3. Results

Figure 1 shows the SDS-PAGE patterns of accessory gland protein in different species of Drosophila. Table 1 embodies the number of major and minor accessory gland protein fractions, less than and more than 50 kD fractions. The molecular weight of each accessory gland protein fractions in different species of Drosophila were recorded along with glycosylated fractions (Table 2).

Table 1. Major and minor accessory gland protein fractions in different species of Drosophila

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Table 2. Molecular weight of accessory gland protein fractions in different species of Drosophila

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Figure 1. Coomassie Brilliant Blue stained SDS-PAGE patterns of accessory glands secretory proteins in different species of Drosophila

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Among the sibling species, the ACP in D. melanogaster has composed of highest number of major protein fractions (7) with molecular weight of 134, 69, 68, 62, 39, 20 and 18 kD. In D. simulans, the secretions are composed of 3 major fractions having molecular weight 68, 59 and 19 Kd, and D. mauritiana is composed of three major protein fractions with molecular weight of 200, 71 and 58 kD.

Among sympatric species (D. ananassae, D. bipectinata, D. malerkotliana and D. rajasekari), it was found that the proteins of D. bipectinata composed of 2 major protein fractions with molecular weight of 62 and 21 kD. D. ananassae, consists of 4 major ACP fractions with molecular weights of 52, 39, 27 and 21 kD and D. malerkotliana is composed of 3 major protein fractions with molecular weight of 61, 59 and 22 kD and D. rajasekari has 2 major fractions with molecular weight of 63 and 22 kD. ananassae subgroup species, D. varians contains 7 major ACP fractions with molecular weights of 68, 59, 40, 32, 28, 24 and 22kD.

Among the closely related species D. virilis is composed of 6 major protein fractions with molecular weight of 66, 60, 29, 28, 22 and 20 kD. D. texana has 4 major protein fractions with molecular weight of 39, 27, 25 and 12 kD. D. hydei is composed of 8 major protein fractions with molecular weight of 126, 89, 84, 72, 64, 58, 40 and 38 kD.

Glycoprotein stains pink with fuchsin on a clear background. Out of 11 fractions 5, 2 and 1 protein fractions are glycosylated in D. melanogaster, D. simulans and D. ananassae respectively. In D. mauritiana out of 10 only 3 fractions are glycosylated. In D. bipectinata and D. malerkotlina only one fraction is glycosylated out of 7 and 8 fractions. Out of 14, 5 are glycosylated in D. virilis. In D. texana and D. varians 3 fractions, in D. rajasekari 2 protein fractions are glycosylated whereas in D. hydei none of the major protein fractions are glycosylated (Table 2 and Figure 2).

Figure 2. Periodic acid-Schiff stained SDS-PAGE patterns of accessory glands secretory proteins in different species of Drosophila

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4. Discussion

Drosophila has been used as a model organism for over a century. It is the most valuable organism in biological research, particularly in genetics, developmental biology and reproductive biology. Reproductive system of male Drosophila accessory gland is a bilobed structure composed of a single layer of cell surrounding a lumen ^[11]. Newly emerged male requires 12 hours to synthesize their glandular secretions. In D. melanogaster the accessory gland secretory proteins have been found in glandular lumen even in the pupae ^[12]. The reproductive tract tissue of male produces seminal fluid and it is transferred to female during mating, it induces numerous physiological and behavioral changes like, decreased receptivity to remating, affecting sperm storage parameters etc., ^[13]. For maximum synthesis and accumulation of secretion the accessory gland requires 7-15 days depending on the type of species, therefore for the present investigations 7 days aged flies were used. Whereas for D. virilis, D. hydei and D. texana 15 days old flies were used ^[14]. Accessory gland secretion is generally composed of carbohydrates, lipids, amino acids and amines. The major components of accessory gland secretion are proteins ^[15].

The secretory proteins of D. melanogaster on 7.5 % and 10% SDS polyacrylamide gel yielded a minimum of 12 and more than 40 fractions with molecular weight ranging from 12 to 175 kD respectively ^[1]. In immigrans species group, D. n. nasuta consists of 10 fractions with molecular weight ranges from 14 to 120 kD, in D. s. neonasuta one more fraction with molecular weight of 92 kD, D. rubida and D. pararubida accessory gland secretion comprises of about 8 and 9 fractions with molecular weight ranges from 12 to 90 kD ^[5]. D. nasuta sub group species consists of 4 to 18 major protein fractions. Based on their mobility, the major protein fractions are categorized into 3 groups and their molecular weights ranges from 14 to 92 kD ^[16]. D. ananassae accessory gland secretory protein consists of 12 fractions with molecular weight ranges from 15 to 200 kD. In D. n. nasuta, D. s. neonasuta, D. rubida and D. pararubida the low molecular weight fractions are not glycosylated ^[5].

Among the melanogaster species group D. rajasekari has lowest (5) whereas D. varians has highest (13) number of protein fractions. D. virilis has the highest (14) and D. texana has lowest (9) protein fractions which belonging to virilis species group. D. hydei has highest (14) number of fractions. Species like D. melanogaster, D. simulans and D. ananassae has similar (11) number of protein fractions which belongs to same group, whereas D. virilis and D. hydei belongs to different group has similar (14) number of protein fraction, this indicates that the number of fractions is not similar in species belonging to same group or different group. Though the number of fractions is similar in species belonging to same or different group their molecular weights are different. Thus there is no uniformity in number of fractions and their molecular weights in different species. The glycosylated fractions among all the species is also not similar it ranges from 0 to 5 in different species. Earlier reports and the present study on 11 species of Drosophila revealed that the variations in molecular weight, number of fractions and their glycosylation are unique to the species and this shows that, they are species specific.

5. Acknowledgement

Thanks are due to Drosophila Stock Centre Department of Zoology, of , ‘Manasagangotri’ , for Drosophila stocks, UGC- SAP and UGC- MRP F No.37-241/2009 for financial assistance to Dr. N. Shivanna.

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