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.