Results
All DNA extractions were done using adults 105, of which 69 were males
and 36 were female. Among Wolbachia infection positive samples,
12 of them including 6 samples with general WSP primers and 6
samples with strain specific primers were then sequenced for
confirmation of the strains amplified.
Among the positive samples 22 amplified products were sequenced
(Table 1 ). The sequences were then aligned and subjected to
phylogenetic analysis and further analyzed between and among the groups
formed within the phylogenetic trees. The evolutionary history was
inferred by using the Maximum Likelihood method based on the Tamura-Nei
model ( Tamura and Nei, 1993). The two trees with the highest
log likelihood were constructed using Bootstrap method with 1000
replicates. The trees are drawn to scale, with branch lengths measured
in the number of substitutions per site (next to the branches). The
analysis involved 7 nucleotide sequences. Evolutionary analyses were
conducted in MEGA 5.
The phylogenetic tree based on general amplified products as apparent,
shown in figure 1 , showed two major division designated A and
B. The samples sequenced after general amplification from
Thiruvananthapuram when analyzed with strain specific primers showed
single infection with walbA strain as similar to the phylogenetic
tree observed. As well as the sample from Kottayam showed single
infection with B strains.
The strain specific samples showed two distinct groups showingwAlbA and wAlbB strain shown in figure 2 . The
genetic difference between the groups was found to be 0.301 (Kimura
2-parameter model. Kimura’s two parameter model (1980) corrects for
multiple hits, taking into account transitional and transversional
substitution rates, while assuming that the four nucleotide frequencies
are the same and that rates of substitution do not vary among sites.
There was no gene flow between group A and group B (Nm = 0.00. (P value=
0.0011) A variable site contains at least two types of nucleotides or
amino acids. Some variable sites can be singleton or
parsimony-informative. The sequence analysis showed 4 singleton variable
sites within the groups, with no parsimony informative sites. Also 1
segregating site was observed causing to have 2 haplotypes within each
group. The nucleotide diversity was found to be similar in both A and B
(0.00099).
The Wolbachia infection was detected in St.albopicta in all the 3 study sites having homology of
superinfection with A and B groups. Then the selected samples
representing the study sites were subjected to phylogenetic analysis.
However the second and third study areas showed infection with single
strains; Thiruvananthapuram, showed 5 samples with single infection ofwAlbA strain and 3 samples in Kottayam with wAlbB strains.
A total of 7 samples were found to be uninfected. The table 2shows the details of the Wolbachia infection status,
table 3 shows infection status among males and females.