Identification of pheromone-binding proteins of the maize stem borer, Chilo partellus (Swinhoe, 1885) (Lepidoptera: Crambidae)

Sample collection

Chilo partellus male moths were collected from the maize fields by using pheromone traps in the farmer fields in Vaddeswaram, Guntur, India.

Sample protein preparation

Male C. partellus moth antenna tissue (100 mg) was taken and homogenized with the help of mortar and pestle using 500 μl of 1 M Tris buffer (pH 7.5) containing 10% SDS and 0.02 M EDTA. The homogenate was centrifuged at 14,000 rpm for 12 min at 4 °C. Supernatant fraction was carefully collected in fresh autoclaved tubes; the sample was stored at − 20 °C until further use. The protein was estimated according to standard protocol suggested by Lowry et al. (1951) using bovine serum albumin (BSA) as standard.

Preparation of sample protein for SDS-PAGE

A ratio of 1:1 protein sample and 1.0 M Tris buffer (pH 6.8) containing 10% SDS, 2% β-mercaptoethanol, 10% glycerol, and 0.002% bromophenol blue was added and kept at 90 °C for 5 min (Kasim et al. 2015).

Separation of PBPs by SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the molecular weight distribution of insect proteins as described by Nagnan-Le et al. (1996). To study the SDS-PAGE protein profile, one-dimensional SDS-PAGE (12% separating gel and 4% stacking gel) was carried out in a small vertical system. To 10 μg of protein, 10 μl of lamellae buffer (loading dye) was mixed and samples were incubated at 95 °C for 5 min in water bath and then immediately load the protein sample into the wells. The loading buffer containing bromophenol blue acts as a tracking dye. A medium range marker (NEX-GEN blue spray prestain protein ladder) was also loaded into the well for the determination of molecular weight through protein bands. The gel was run at constant voltage of 80 V for 3–4 h followed by staining with Coomassie brilliant blue R-250 overnight. The relative mobility of protein bands was determined and the zymogram was constructed.

Extraction and purification of proteins from SDS-PAGE

After SDS-PAGE electrophoresis, proteins were extracted and purified according to Nagnan-Le et al. (1996). The visualized bands are removed from the gel using a surgical blade and washed using 2 ml of 250 mM Tris buffer/250 mM EDTA, pH of 7.4, thoroughly for three times then finally washed with autoclaved distilled water. The water was completely removed from the gel slices and homogenized with 1 ml of Tris buffer (pH 7.4). The samples were subjected to sonication then centrifuged at 12,000 rpm for 10 min at 4 °C. The protein sample was equilibrated with 20 mM Tris buffer, pH 7.4, containing 0.1% SDS. The partially purified protein aliquots were run in the native PAGE; it is used for the densitometric analysis and determination of the recovery percentage.

Densitometry analysis

To identify the universal expression pattern of existing protein spots on gel, densitometry analysis was employed. It is majorly used to measure protein expression levels and the intensities of specific bands by using an online software tool (Gelquant.NET). In this study, the relative amounts of protein were quantified by using a computational program from a TIFF image assisted with a commercial scanner. Relative mobility (Rf) of each stained band, its concentration, and area occupied by it with reference to total protein present in the lane can be calculated by gel perfect program explained by Bozzo and Retamal (1991). SDS-PAGE gels of purified proteins were digitized, and peak areas of both purified and crude protein bands were compared. The relative concentration of areas provides us the percentage recovery.

Separation of PBPs by SDS-PAGE

Extracted proteins from the sensory organs were quantified by using Lowry’s method and the protein concentration was found to be 0.77 μg/mg. Molecular weight of the extracted proteins was determined using SDS-PAGE (12% resolving gel and 4% stacking gel). Usually, molecular weight of PBP was of 17 kDa and found to be the same in our results (de Santis et al. 2006) using a protein marker (NEX-GEN blue spray pre-stain protein ladder) (Fig. 1).

Extraction and purification of proteins from SDS-PAGE

The identified proteins were further purified through gel extraction and recovery of protein varied from 80 to 95%. Purified samples were run on native PAGE gel with negative control (Fig. 2). Few samples are homogenized and purified using Tris buffer (20 mM, pH 7.4) without SDS, while recovery percentage was observed to be slightly lower when compared to SDS. Our results illustrate that it was not critical to use a large quantity of starting material to achieve an adequate amount of purified protein either to improve an immunoassay or to characterize a protein chemically.

Densitometry analysis

Through densitometry analysis, the electrophoretic mobility was calculated, using molecular weights of proteins with reference to protein marker standards (Priya et al. 2013). In this analysis, the perfect gel picture on relative mobility of protein was observed to be 0.574 nm (Fig. 3a) and the band intensity was approximately 250 units as shown in (Fig. 3b).

Results of this study may serve as preliminary information to identify olfaction-based approaches which interfere with insect host location and mating behavior through a strategy called push-pull pest control. PBPs are the ideal targets for understanding the olfactory code of insects as well as for interfering with their communication system. These proteins are specifically expressed in the antennae of male moths. Insects survive by possessing a specialized sensory system to monitor various odors present in the environment. Thus, sense of smell often plays a crucial role for detecting food, mating partner, and choosing for oviposition site or even protecting themselves from predators and pathogens to fit among various existing ecological lifestyles (Cande et al. 2013). Considering the importance of PBPs of male moths, they play a vital role in the detection of sex pheromones. Leal (2013) stated that male moths detect sex pheromones with tuned odorant receptors, which confer specificity to peripheral odorant receptor neurons, with the assistance of other olfactory proteins.