Journal of Mosquito Research, 2013, Vol.3, No.8, 58-64
ISSN 1927-646X
http://jmr.sophiapublisher.com
59
implementation of appropriate vector control
measures and the development of strategies for
monitoring the spatial-temporal fluctuations of vector
species that are needed to assess the potential risk of
malaria outbreaks. In the present study the attempts
have been made to generate the genetic profile of the
laboratory populations of four different mosquito
species
Anopheles stephensi
(
An. Stephensi
)
,
Aedes
aegypti
(
Ae. aegypti
),
Aedes albopictus
(
Ae. Albopictus
)
and
Culex quinquefasciatus
(
Cx. quinquefasciatus
)
by
using polymerase chain reaction-restriction fragment
length polymorphism (PCR-RFLP) of cytochrome
oxidase I (
COI
)
gene and to understand the
phylogenetic relationship among them.
COI
gene is
the mitochondrial marker often used for evolutionary
study since it is the largest among the three subunits
and its protein sequence contains highly conserved
functional domains and variable regions. The
amplified
COI
gene was subjected for restriction
digestion by
Mbo
I,
Dra
I,
Taq
I,
Bgl
II,
Eco
RI and
Msp
I
as single and double digestions. The results obtained
from double digestions with the combination of
Mbo
I
and
Dra
I were able to differentiate among the four
mosquito species. The banding patterns were analyzed
using POPGENE 1.31 software and phylogenetic tree
was constructed. The
Aedes
and
Culex
belong to the
Culicinae family were separated from the
Anopheles
species and among themselves based on their
phylogeny and distance.
2
Results
2.1
Genomic DNA extraction and PCR amplification
of
COI
gene
The genomic DNA from single mosquito was isolated
as described in our previous work Sharma et al. 2009
and analyzed on 0.8% agarose gel for purity. PCR
amplification of
COI
gene led to an approximately
580
bp fragment. PCR product of the
COI
gene was
subjected to PEG-NaCl purification for digestion
purpose. The purified product represented by a single
band of 580 bp with no impurities of primer-dimer,
RNA
etc.
was further used for digestion with several
restriction enzymes for the genetic analysis by
PCR-RFLP. The restriction enzymes were chosen on
the basis of
COI
gene sequences in the laboratory.
2.2
Single digestion
Single digestion was performed using the purified
CO
I gene with enzymes
Dra
I
,
EcoR
I
,
Taq
I
,
Mbo
I
,
Bgl
II
and
MspI
(
Table 1). For all digestions the
fragments were visualized on 2.5% agarose gel. The
restriction enzymes
EcoR
I and
Bgl
II were not able to
digest the
COI
gene for any of the four species of
mosquitoes under study whereas
Dra
I produced two
fragments of equal sizes with
Cx. quinquefasciatus, Ae.
albopictus
,
An. stephensi,
at 280 bp & 300 bp.
Dra
I
restriction enzymes could not digest the
CO
I gene in
Ae. aegypti. Taq
I digested three mosquito species with
80
bp, 200 bp & 300 bp in
Cx. quinquefasciatus,
200
bp & 380 bp fragments in
Ae. albopictus
, 280
bp
& 300
bp in
An. stephensi.
However no digestion was
observed in
Ae. aegypti
.
Mbo
I was the only restriction
enzyme that could digest all the
CO
I gene of four
mosquito species with digested fragments of 90 bp,
190
bp & 290 bp in
Cx. quinquefasciatus,
120
bp, 180
and 280 bp in both
Ae. aegypti
and
Ae. albopictus
while in
An. stephensi
fragments were of 190 bp, 390 bp.
Msp
I
digested the gene into two fragments of 180 bp,
400
bp in
Ae. aegypti
and
260
bp & 320 bp in
Ae.
albopictus
.
But no digestion was observed in
Cx.
quinquefasciatus
and
An. stephensi.
The respective
fragment sizes with all the restriction enzymes as
single digestion have been summarized in Table 1.
2.3
Double digestion
When double digestion was performed using different
combinations of
Mbo
I
+ Dra
I
,
and
Mbo
I
+ Taq
I. The
double digestion with
Mbo
I
+Taq
I
revealed that
Ae.
aegypti, Ae. albopictus, An. stephensi
are similar in
their restriction pattern with visible fragments of 120,
180 & 280
bp sizes whereas a replacement of 90 bp
band in place of 120 bp in
Cx. quinquefasciatus
which differentiated it from all other mosquito species
under study. The combinations of
Mbo
I
+ Dra
I
used
were noteworthy as the digestion with these restriction
endonucleases was capable of differentiating all four
mosquito species under study from each other. The
double digested fragments of all the four species have
been summarized in Table 2.
2.4
Estimation of polymorphism and construction
of phylogenetic tree
Based on the double digestion with
Mbo
I
+ Dra
I
the