Molecular analyses
DNA Extraction and Polymerase Chain Reaction (PCR). Total DNA from individual mosquitoes was extracted following a modified method proposed by Collins et al. (1987). The DNA were pelleted, dissolved in water, and subjected to a phenol chloroform isoamyl extraction followed by chloroform. DNA were precipitated using NaAc/ethanol and dissolved in 50 µl of deionized water (Sigma-Aldrich, St. Louis, MO). The extracted and purified DNA were screened initially for Wolbachia infection by PCR using wsp gene, forward primer (81F, 5′-TGGTCCAATAAGTGATGAAGAAAC-3′ and reverse primer 691R, 5′- AAAATTAAACGCTACTCCA-3′), which amplified about 600-700 bp product. This served as positive control for Wolbachia genotyping.
PCR conditions were as follows: An initial denaturation of 5 min (950C) was followed by Þve cycles of 940C for 40 s (denaturation), 450C for 1 min (annealing), and 72 0C for 1 min (extension) and 35 cycles of 940C for 40 s (denaturation), 510C for 1 min (annealing), 720C for 1 min (extension), and a final extension at 720C for 10 min following a modified protocol of Hasebe et al. (2002). The 500C reaction included 1.5 U of Thermus aquaticus polymerase, 5 µl of 10 × PCR buffer, 2.5m Mmagnesium chloride, 2.5 µl of Q solution (QIAGEN GmbH, Hilden, Germany), and 0.5 µl of 10 pmol each of forward and reverse primers, along with the DNA of the mosquito species.
Wolbachia genotyping among the positive samples was performed by a multiplex PCR using Wolbachia strain specific primers; 328F/691R for wAlb A and 183F/691R for wAlb B. The PCR products obtained using general primers were diluted; 1µL in 99µL of sterilized sigma water prior to the PCR reaction. Similar to general PCR reaction, the volume/ reaction was calculated according to the number of samples to be processed, and once the MM is prepared, 48µL of it is added to tubes followed by 2µL of template DNA. A negative control was placed to determine whether the amplification. PCR cycling conditions Wolbachia genotyping are 95°C for 2 min , 35 cycles at 94°C for 1min, 55°C for 1min 30 sec, 72°C for 2mins, 1 cycle at, 72°C for 7 min, 4°C hold.
DNA Sequencing and Analysis. The amplified fragments were run on a 1% agarose gel to check the integrity of the fragments and the PCR product was purified by QIAGEN GmbH PCR purification kit. The purified products were eluted to 20 µl of deionized water, and a portion of it was lyophilized in a Speed Vac concentrator (Thermo Electron Corporation, Waltham, MA) and was shipped to MWG (Lederberg, Germany/Microsynth, Balgach, Switzerland) for custom sequencing. Both reads (from forward primer as well as reverse primer) were done, and the sequences were analyzed as follows. The DNA sequences were subjected to alignment using ClustalW. Sequence divergences among individuals were quantified by using the Kimura two-parameter distance model (Kimura 1980). The phytogenic tree was constructed and genetic distance was calculated using MEGA 5.0. The neighbour-joining (NJ) method was followed using Kimura two-parameter (K2P) genetic distances (Kimura, 1980) to deduce the phylogenetic tree.