Journal of Mosquito Research 2015, Vol.5, No.15, 1-15
4
Ae. aegypti
from Mysore city and surrounding places.
Further, these enzymes were assayed using spectr-
ophotometer to understand the genetic resistance
mechanism involved. These investigations were aimed
to learn the genetic differentiation if any, between the
populations and also to correlate the insecticide
susceptibility difference with the biochemical data.
1 Material and Methods
1.1 Collection of larvae
Aedes agypti
larvae were collected from habitats in
Mysore urban areas viz., Vishveshwara nagar,
Chamundipuram, J.P. Nagar, Agrahara, Kuvempunagar
and surrounding rural areas viz., Mandya, Nanjangud,
Hunsur, Channarayapatna of Hassan district. They
were brought to laboratory and maintained.
1.2 Larval rearing
Aedes aegypti
larvae collected from the aforementioned
areas were transferred to a clean enamel/plastic bowls
and were maintained at 28±2
o
C and 80±5% humidity,
employing dechlorinated water. Hardness and pH of
the rearing water were measured periodically and
maintained at permissible range. The immature were
fed with yeast granules and transferred to fresh
enamel bowl on alternate days. Late third instar or
early fourth instar larvae were used for bioassay studies.
1.3 Larval Bioassay
Larval susceptibility tests on
Ae. aegypti
lavae were
carried out following the standard WHO procedure
(WHO, 1981). Synthetic pyrethroid such as Deltamethrin
was employed for the larval bioassay. Different test
concentrations of the said synthetic pyrethroid were
prepared using ethanol as solvent. One ml of the
known concentration of this was added to 249 ml of
the dechlorinated water in a 500ml glass beaker.
Twenty five healthy early fourth instar larvae were
released in to three replicates of each concentration.
Control tests were done with same volume of
dechlorinated water supplemented with one ml of
ethanol. Dead/moribund larvae were recorded after
24hr of exposure (larvae were considered to be
moribund if they failed to flex their head to siphon,
when stimulated). The percent mortality was calculated
for each concentration and corrected by using Abbott’s
(1925) formula; provided the control mortality was
less than 20%. All the tests were carried out at room
temperature of 28±2
o
C and 80±5% relative humidity.
1.4 Statistical Analysis
The LC
50
and LC
90
values for each population with
fiducial limits for the larval bioassay experiments
were calculated by probit regression analysis as per
Finney (1971); Difference in LC
50
values between the
populations for pyrethroid tested was also determined.
1.5 Enzyme Analysis - Qualitative Assay
Aedes aegypti
from different areas were employed for
the investigation so as to understand the isozyme
profiles. The mosquito immatures were reared in the
laboratory. Either larvae or adults were used for
enzyme assay as per the stage specific activity of the
enzymes. For the present study, five enzymes such as
Carboxylesterases Est-A and Est-B (EC 3.1.1.1),
G6PD (EC 1.1.1.49), AcPH (EC 3.1.3.2) and APH
(EC 3.1.3.1) were analyzed to establish the differential
isozyme profiles in the populations of
Aedes aegypti
.
1.5.1 Native polyacrylamide gel electrophoresis
Continuous nondenaturing polyacrylamide gel
electrophoresis (Native PAGE) was conducted to
detect the isozyme profiles of the urban and rural
populations of a dengue/chikungunya vectors according
to the procedures of Gopalan et al.
,
(1997) and
Bonning et al., (1991). A medium sized dual vertical
slab (10 x 12 cm) gel electrophoresis system was used
for the current study. Gels of 0.75 mm thickness were
cast employing Teflon spacers. Separating and stacking
gels were prepared with 8% and 4% acrylamide
respectively. The gel buffer with 8.8 and 6.8 pH was
prepared with tris (hydroxyl methyl aminomethane)
respectively for separating and stacking gels. A 12 slot
teflon comb was employed to make the wells for
sample loading. A constant power supply unit (Systronics,
India, model No. 610) was used as power source for
running the experiment.
1.5.2 Sample preparation and running the gels
Early instar or one day old adult mosquitoes were
individually homogenized under cold conditions with
25 µl of 40% sucrose solution in an eppendorf tube
using a Knot’s pestle. Later cell debris was flocculated
by spinning at 2400rpm for 5min at 4
o
C, supernatant
obtained were used for enzyme assays. For G6PD
enzyme profile, adult mosquitoes were employed. An
equal (15-20 µl) volume of supernatant from all the
different samples was carefully loaded to each well.
Sodium Borate buffer (0.3 M, pH 8.65) was used as
