In each case 50 larvae were used in 300 ml of a previously autoclaved medium (water plus food at the concentration of 0,4 g l-1). selleck Each day a count was realized. The monitoring
of the experiment was carried for 18 days. When reaching the pupal stage, half of the pupae were sampled and conserved for further analysis. The second half of the pupae was let to molt; after emergence, adults were immediately sampled and conserved. Statistical analysis The results were analyzed to assess if there is a statistically significant difference between the treated larvae and the controls, in terms of mortality and development. The statistical analyses were carried out using the non parametric test U of Mann-Whitney under the SPSS software (ver.17, SPSS inc, USA). Values of P < 0.05 were considered as statistically significant. Analysis of the bacterial community of An. stephensi Total DNA was extracted from pupae and adults of An. stephensi using the CTAB method with a prior cell lysis ARN-509 by enzymatic method and followed by an isopropanol precipitation of the DNA, as described by Jara et al. . PCR
amplification for DGGE was carried out using primers 357f (5′-CCTACGGGAGGCAGCAG-3′) and 907r (5′-CCGTCAATTCCTTTRAGTTT-3′) with a GC clamp, as described by Sanchez et al. . DGGE (Denaturant Gradient Gel Electrophoresis) analysis was carried out on each PCR amplicon using a DCodeTM Universal Mutation Detection System (BioRad, Hercules, USA), following the procedure described previously . Electrophoresis was performed in 0.5-mm polyacrylamide gel (7% (w/v) acrylamide–bisacrylamide 37.5:1) containing a 35–55% urea–formamide denaturing gradient (100% Cisplatin supplier corresponds to 7M urea and 40% (v/v) formamide) according to the method of Muyzer et al. , increasing in the electrophoretic run direction.
The gel was subjected to a constant voltage of 90 V for 15 h at 60 °C in TAE Buffer 1X (50X TAE stock solution consisting in 2 M Tris base, 1 M glacial acetic acid, 50 mM EDTA). After electrophoresis, the DGGE gels were stained in 1X TAE solution containing SYBR Green (Molecular Probes, Leiden, The Netherlands) for 45 min and photographed under a UV illumination using a GelDoc 2000 apparatus (BioRad, Hercules, USA). For the sequencing of DGGE bands, bands of interest were excised from the gels with a sterile blade, mixed with 50 μl of sterile water, and incubated overnight at 4°C to allow the DNA of the bands to diffuse out of the polyacrylamide gel blocks. Two microliters of this aqueous solution was used to reamplify the PCR products with the same primers described above, excluding the GC clamp. Reamplified bands were then sequenced using ABI technology .