Journal of Mosquito Research, 2013, Vol.3, No.8, 58-64
ISSN 1927-646X
http://jmr.sophiapublisher.com
62
restriction enzymes and analyzing size of resulting
fragments by gel electrophoresis. PCR-RFLP has been
used in the study of genetic structure of malaria vector
Anopheles subpictus
in Iran using mitochondrial
cytochrome oxidase
CO
I and
CO
II
(
Karimian et al.,
2011).
Similar technique has been used to distinguish
between several
Diabrotica
species using PCR-RFLP
of the
COI
gene in which mitochondrial
COI
subunit
was amplified and digested (Alam et al., 2012). The
comparative linkage map for
Culex pipiens
(
Cx.
pipiens
)
and
Ae. aegypti
was also developed based on
RFLP using cDNA clones of
Ae. aegypti
as probes to
southern blot of
Cx. pipiens
genomic DNA.
Present study reveals that PCR-RFLP is a versatile
molecular tool to screen genetic polymorphism among
various mosquito species. The PCR-RFLP has been
able to demonstrate clear genetic diversity by using
the
COI
gene in the present study. The use of this gene
is arbitrary and it is highly probable that other
mitochondrial protein coding genes can be used to
differentiate these species. In preliminary studies
using other mitochondrial genes such as 16S rRNA,
CO
II and
ND
5
it was observed that many of the
mosquito species could be differentiated in a similar
manner (Alam et al. 2012; Ozdil et al., 2012 and Zitco
et al., 2011).
Double digestion performed with combination of
enzymes
Mbo
I
+ Dra
I
during the PCR-RFLP provided
significant genetic variation among the four mosquito
species under study reveals that
Aedes
species (
Ae.
aegypti, Ae. albopictus)
are closely related to the
Cx.
quinquefasciatus
as minimum genetic distance and
maximum genetic identity was observed among them.
The finding can be further substantiated by the facts
that these three belongs to the same family Culicidae.
Moreover, at the structural and behaviour level also
they share many characters such as their sitting
posture, larval movement in water, position of siphon
etc.
Amongst the two
Aedes
sps., the
Ae. albopictus
is
found more closer to
Cx. quinquefasciatus
compared
to
Ae. aegypti
,
having a genetic distance of 0.28. It
may be well thought-out that they might have evolved
from a similar origin during evolutionary process. The
study undertaken proposes that
Ae. aegypti
and
Ae.
albopictus
has evolved in the same line but
Ae.
albopictus
has diversed into another independent line
as the
Cx
.
quinquefasciatus
species. The most
genetically diverse species, the
An. stephensi
has
shown maximum genetic distance to other three
species. Thus it can be considered as the most
distantly related species among all species under study.
Whether the outcomes of our study are factual and can
be established with extensive assessment of literature
and studies carrying out by using some other
molecular markers. The insights of the genetic pattern
of a vector species are fundamental in understanding
the diseases epidemiology. Such an understanding
becomes crucial in devising vector control strategies.
4.
Materials and Methods
4.1.
Test insect
Laboratory reared
Cx. quinquefasciatus, Ae. aegypti,
Ae. albopictus, An. stephensi
were used as test insect.
The adult mosquitoes were collected with the help of
mechanical aspirator from the stock colony
maintained in the laboratory at (27± 2)
?
and relative
humidity of (70 ± 5)%.
4.2.
Extraction of mosquito DNA
The DNA extraction was done by using the method as
described in our previous publication (Sharma et al.,
2009).
In this each single adult mosquito was
homogenized in the micro centrifuge tube by adding
100
µL lysis buffer. The homogenate was immediately
kept on ice for 10 minutes and followed by heat
treatment at 65
?
for 30 minutes. Subsequently, 30 µL
5
M potassium acetate was added and immediately
transferred to ice for one hour followed by
centrifugation at 13000 rpm for 15 minutes at 10
?
.
To the supernatant obtained, a double volume of
absolute chilled ethanol was added for precipitation of
DNA and kept at -20
?
for overnight. After
centrifugation at 13 000 rpm for 15 minutes at 10
?
,
the precipitated DNA was washed in 70% ethanol
twice. The palleted DNA was allowed to air dry and
finally dissolved in 50 µL TE buffer.
4.3.
PCR amplification of
COI
gene
The PCR amplification reactions were performed in
25
µL reaction mixture per tube containing 2.5 µL of
10
×
assay buffer, 2.5 mM of MgCl
2
, 2
mM each
dNTP, 10 µL of forward and reverse primers for
COI