Isolation and characterization of S. vinaceusdrappus (S12-4)

Soil samples were collected from the Venganayakkan region of Western Ghats of Tamil Nadu, India, and transported aseptically to the laboratory using sterile plastic containers. Streptomyces vinaceusdrappus (S12-4) which, through preliminary screening was shown to be biologically active against mosquito larvae, was isolated from the soil samples by drop-plating serial dilutions of the samples onto Actinomycetes Isolation Agar in petri dishes. The dishes were incubated at 28°C for 10–14 d (Valanarasu et al. 2009). The pure isolate was characterized morphologically and physiologically as per Bergey's Manual of Systematic Bacteriology (Locci 1989) at various pH levels, sodium chloride (NaCl) concentrations, temperatures, and on various growth media for differing lengths of incubation for optimal production. The more biologically active isolates were selected for biochemical characterization using a biochemical kit (KB014 HiAcinetobacter Identification Kit, HiMedia Laboratories, Mumbai, India) according to the manufacturer's protocol.

Secondary metabolite extraction

Five liters of the International Streptomyces Project No. 2 (ISP-2) broth in an Erlenmeyer flask were inoculated with an active isolate of S12-4 and incubated at 28°C for 10–14 d. The culture was then centrifuged at 3,293 × g for 15 min to separate the biomass and supernatant. The biomass was immersed in methanol and the supernatant was mixed in an equal volume of ethyl acetate. The solvent was separated using a separating funnel and concentrated with a vacuum rotary evaporator at 50°C. The resultant extract was transferred to a sterile vial at −20°C (Saravanakumar et al. 2012).

Antibiotic sensitivity

A slightly modified disc diffusion method as described by Yao (2002) was used to assess antibiotic sensitivity of isolate S12-4. The isolate culture was grown on slants of ISP-2 agar at 28°C for 2–4 d. Aliquots of these cultures were mixed in 5 ml sterile water (Waksmans 1961) and, within 1 h, the suspension was drop-plated and uniformly spread on the surface of ISP-2 agar in petri dishes and allowed to air-dry for 15 min. Commercially prepared discs of 37 antibiotics were placed on the surface of the agar and removed after 30 min at room temperature allowing for diffusion into the medium. These plates were incubated at 28°C for 2–3 d. Zones of inhibition were measured and recorded in millimeters.

Insects and bioassays

Culex quinquefasciatus, An. stephensi, and Ae. aegypti larvae were obtained from colonies maintained at the Entomology Research Institute, Loyola College, Chennai. The colonies were maintained at 75–85% relative humidity and 27 ± 1°C and on a light phase of 11 ± 0.5 h. Larvae were fed with dog food and brewer's yeast in a 3:2 ratio.

Larvicidal activity of the crude extract of isolate S12-4 was evaluated using a slightly modified method of the World Health Organization (2005). Concentrations of 62.5, 125, 250, and 500 ppm were prepared using 0.5% dimethyl sulfoxide (DMSO). Third-instar larvae were placed in each suspension, with 20 larvae per replicate and 5 replicates per concentration and mosquito species. DMSO and water alone, each without the extract, served as controls. Numbers of dead larvae were counted 24 h after exposure. Mortality data were corrected using Abbott's (1925) and were subjected to probit analysis (US EPA Probit Analysis software, version 1.5) to estimate concentration–mortality response to the S12-4 extract.

Ovicidal activity was evaluated using modified methods of Elango et al. (2009). The concentrations were prepared as previously described with the same treatments and numbers of replicates. Twenty newly deposited eggs of three mosquito species were placed in the treatment suspensions. Eggs were monitored using stereomicroscope (Wild M7S TYP 308700, Switzerland). The number of unhatched versus hatched eggs was tabulated at 120 h after initial exposure to the treatments. Unhatched eggs were identified as those with unopened opercula.

Molecular analyses

DNA of the S12-4 isolate was extracted using the HipurA Streptomyces DNA purification kit-MB 527-50 pr (HiMedia) according to the manufacturer's guidelines. The primers 27F-5′AGAGTTTGATCMTGGCTCAG3′ and 1492R-5′TACGGYTACCTTGTTACGACTT3′ were used to amplify the 16S ribosomal sequence from the genomic DNA in a thermal cycler as follows: initial denaturation for 3 min at 94°C, 35 cycles of denaturation 1 min at 94°C, annealing at 54°C for 1 min, extension at 72°C for 2 min, and final extension at 72°C for 7 min and then held at 4°C. The polymerase chain reaction (PCR) products were confirmed with 1% agarose gel electrophoresis stained by ethidium bromide (Farris et al. 2007, Valanarasu et al. 2008). The confirmed PCR product was sequenced using the di-deoxy chain termination method in an Applied Biosystems Automated Sequencer (Synergy Scientific Services, Chennai, India). The sequences were identified and compared to the reference species of actinomycetes closely related to the genomes in the database using the National Center for Biotechnical Information (NCBI) Basic Local Alignment Search Tool (BLAST) (http:www.ncbi.nlm.nih.gov/BLAST). A phylogenetic tree for isolate S12-4 was constructed with the neighbor-joining method using MEGA4 software. The sequence was submitted to the GenBank, NCBI (Saltau and Nei 1987).

Gas chromatography–mass spectrometry analysis

The S12-4 isolate extract was subjected to gas chromatography–mass spectrometry (GC-MS) analysis on GC-MS-5975 (Agilent, Palo Alto, CA), column DB 5 ms Agilent, dimension length = 30.0 m, internal diameter = 0.2 mm, film thickness = 0.25 μm, with a temperature program of 70–300°C, 10°C /min, injection temperature = 240°C, carrier gas = helium, flow rate =1.51 ml/minute, equipped with GC-MS NIST-II library.