Genelute Mammalian Genomic DNA extraction kit was purchased from Sigma, the reagents used for PCR amplification were procured from GeNei, and the polymerase chain reaction (PCR) amplification was performed using Biometra thermal cycler (T-Personal 48). All other reagents were procured from Hi-Media, Mumbai.
Collection and identification of spiders
The spiders were collected early in the morning from soybean and mango fields of Bhada village, Tq. Ausa, Dist. Latur as per the procedure described by Coddington, Griswold, Silva, Penaranda, and Larcher (1991) and Toti, Coyle, and Miller (2000). The identification of spiders was carried on the basis of morphological characteristics. The species level identification was further confirmed by the 18S rRNA gene sequencing.
18S rRNA gene sequencing
DNA Extraction was carried out using Genelute Mammalian Genomic DNA extraction kit (Sigma, G1 N70-1KT). The DNA isolated from spider was subjected to polymerase chain reaction (PCR) amplification using Biometra thermal cycler (T-Personal 48). The PCR reaction mix contained 2.5 μl of 10X buffer, 1 μl of each forward and reverse primers with base sequence: 18s5F (Forward)-CTGGTTGATYCTGCCAGT and 18s1100R (Reverse)-CTTCGAACCTCTGACTTTCG, 2.5 μl of 2.5 mM of each dNTP, 2.5 Units of Taq DNA polymerase, and 1 μl Template DNA and 8.5 μl nuclease-free water. The PCR amplification cycle consist of a cycle of 5 min at 94 °C; 35 cycles of 1 min at 94 °C, 1 min at 55 °C, 2 min at 72 °C; and additionally 1 cycle of 7 min at 72 °C. Gel electrophoresis was performed using 1.0% agarose to analyze the size of amplified PCR product. The size obtained was approx. 1000 bp for partial 18s rRNA region. The PCR product was purified using AxyPrep PCR Clean up kit (Axygen, AP-PCR-50). It was further sequenced using Applied Biosystems 3730xl DNA Analyzer USA and chromatogram was obtained. The DNA sequences were analyzed using online Nucleotide Basic Local Alignment Search Tool (BLASTn) facility of National Center for Biotechnology Information (NCBI). The BLAST results were used to find out evolutionary relationship of spider. Altogether, 20 sequences, including sample sequence, were used to generate phylogenetic tree. The tree was constructed in NCBI using neighbor joining method.
Antimicrobial activity of silk
Collection of spider silk
Spider silk was collected by running the sterile pipette through the web from soybean and mango fields of Bhada village, Tq. Ausa, Dist. Latur. This silk was employed for the assessment of antimicrobial activity.
The silk of P. brevivulva (1.0 mg) was placed in sterile glass borosilicate test tubes and 10 ml of different solvents like chloroform, formic acid, ethanol and methanol, water, and 1 N HCl were added separately to test the solubility of silk.
Both gram-positive as well as gram-negative bacterial strains were used for the assessment of antibacterial activity. Bacillus megaterium (MTCC 2444) and Staphylococcus aureus (MTCC 96) were the gram-positive bacterial strains while Klebsiella pneumoniae (ATCC 15380), Pseudomonas aeruginosa (MTCC 2488), Proteus vulgaris (MTCC 1771), and Salmonella typhi (ATCC 23564) were the gram-negative bacterial strains used in the study. The selected test fungi were Aspergillus niger (MTCC 1781), Aspergillus flavus (MTCC 873), Candida albicans (MTCC 227), Ustilago maydis (MCIM 983), Alternaria solani (MCIM 887), and Mucor hiemalis (MCIM 873). Each bacterial species was inoculated in nutrient broth and fungal species in potato dextrose broth separately on orbital shaking incubator (REMI-24 BL) for 24 to 48 h.
Silk extract of P. brevivulva was tested for antibacterial activity against six bacterial species such as B. megaterium, K. pneumoniae, P. aeruginosa, P. vulgaris, S. typhi, and S. aureus. The antibacterial activity was carried out by disc diffusion method. Nutrient agar plates were prepared and 100 μl of the test microbe was pipetted onto the center of the plate and then spread around using the glass spreader. Disc loaded with silk sample was placed onto the surface of the agar. Then, the plates were placed in an incubator at 37 ± 1 °C and left for around 24 h. The result was recorded by measuring the diameter of zone of inhibition (mm).
Silk extract of P. brevivulva was tested against six fungal species viz. A. niger, A. flavus, C. albicans, U. maydis, A. solani, and M. hiemalis. The antifungal activity was carried out by agar well diffusion method. Potato dextrose agar plates were prepared and after solidification, 100 μl of the spore suspension was pipetted on to the center of the plate and then spread around using the glass spreader. The wells were made on agar plate by using cork borer. Then, 30 μl of silk extract was introduced in the well. The plates were incubated at 28 ± 1 °C for 24–48 h. The antifungal activity was evaluated by measuring the zone of inhibition (mm).
Dialysis of silk samples
The membrane dialysis of silk extract was performed as per the procedure described by Lombardi and Kaplan (1990). The silk extract was dialyzed against 1000 ml of 10 mM Tris-HCl buffer (pH 7.0) for 24 h and the samples were re-dissolved in dimethyl sulfoxide (DMSO) for further analysis.
Antimicrobial activity of DMSO fraction of silk samples
The antibacterial activity of P. brevivulva silk was confirmed by testing DMSO fraction of silk against B. megaterium, S. typhi, and K. pneumoniae by disc diffusion method. Streptomycin (50 μg/ml) was used as positive control and DMSO was used as negative control.
The DMSO fraction of P. brevivulva silk was also tested for the confirmation of its antifungal potential against A. niger, A. flavus, C. albicans, U. maydis, and A. solani by agar well diffusion method. Nystatin (50 μg/ml) was used as positive control and DMSO was used as negative control.
Estimation of protein content of DMSO fraction of silk samples
The protein content of DMSO fraction of P. brevivulva silk was determined by the method as described by Bradford (1976) with Coomassie Brilliant Blue (G-250) dye using bovine serum albumin as the standard.
Determination of minimum inhibitory concentration
Minimum inhibitory concentration of DMSO fraction of Pardosa brevivulva silk was determined using Alamer Blue Assay (Rampersad, 2012; Yajko et al., 1995) for three different bacterial species (B. megaterium, S. typhi, and K. pneumoniae). The DMSO fraction of Pardosa brevivulva silk was also was tested for the determination of minimum inhibitory concentration against C. albicans and Aspergillus flavus. Each well of microtitre plate was initially added with 100 μl of sterile broth. Then 100 μl of sample was added in the first well and twofold serial dilutions of silk sample were made. At last, 20 μl of bacterial/fungal cell suspension was added to each well. Microtitre plate was incubated for 24 h at 37 ± 1 °C temperature for bacteria and 24–48 h at 28 ± 1 °C for fungus. After incubation, 30 μl of Alamer blue dye was added to each well and re-incubated for 4 h at room temperature.
Characterization of antimicrobial compounds from bioactive fraction of spider silk
The silk sample was lyophilized by first freezing at − 22 ± 1 °C in a deep freezer for 8 h. The frozen material was then employed for freeze drying in freeze-dryer at − 40 ± 1 °C for 12 h at 0.01 MPa pressure. This material was subsequently used for the Fourier-transform infrared spectroscopy (FT-IR), Carbon-13 nuclear magnetic resonance (13C NMR) & Proton nuclear magnetic resonance (1H NMR), and C18 column reversed-phase high-performance liquid chromatography (RP-HPLC) analysis.
The FT-IR spectrum of lyophilized silk sample was recorded on a Shimadzu FT-IR Spectrophotometer. The lyophilized silk sample was mixed with KBr and pellet technique was adopted to record the spectra in cm−1. The spectrum was recorded at room temperature with the resolution of 2(1/cm) for 45 scans in the range from 4000 to 500 cm−1.
13C and 1H NMR
13C NMR and spectrum was recorded at room temperature on Bruker AC-250 spectrometer using DMSO as solvent. The 1H NMR spectrum was also recorded at room temperature on Bruker AC-250 spectrometer using CDCl3 as solvent and TMB (tetramethyl saline) as standard.
C18 column RP-HPLC analysis
The HPLC analysis of silk sample was carried out using C18 reverse phase colunm with pyridine/acetate buffer (pH 4.0) and 1-propanol as the organic modifier. Silkworm Sericin was used as standard.