Journal of Mosquito Research 2015, Vol.5, No.15, 1-15
6
(µM/mg protein) was calculated from the molar
extinction coefficient 6.22 x 10
6
. Specific activity was
expressed in µMoles of NADH reduced/min/mg of
protein.
1.6.4 AcPH and APH assays
Both the Phosphatases were quantified following the
method of Rupangi et al., (2013) using spectroph-
otometer with some modifications. For AcPH assay
0.2 ml of assay mixture (equal volume of 16 mM
p-nitrophenyl phosphate and 180 mM Sodium acetate
buffer of pH 5.0) was added to 25 µl of the sample
homogenate. This sample was incubated at 37
o
C for
20 min. The reaction was then blocked by adding 0.6
ml of 0.25 M NaOH. The samples were centrifuged at
15,000rpm for 2 min. Replicate control contained 25
µl homogenate and stopping reagent (0.25M NaOH)
before the addition of the 0.2 ml of assay mixture. The
absorbance values were measured at 410 nm. Total
enzyme activity was calculated using the published
extinction co-efficient value (920) for a known path
length (1 cm). For the assay of APH the assay mixture
is replaced by adding equal volume of 16mM
p-nitrophenyl phosphate and 250 mM sodium borate
NaOH buffer of 9.8 pH. To every 10ml of this assay
mixture 20 µl of 1.0 M MgCl
2
solution was added at
the time of reaction. Other condition was alike that of
AcPH assay and activity was expressed in µg/indivisual.
1.6.5 Protein Estimation
Protein assay of the early fourth instar larvae and one
day old adult mosquitoes were carried out as per the
standard procedures of Lowry et al., (1951).
2 Results
2.1 Insecticide Susceptibility Test
After successful rearing and treatment with synthetic
pyrethriods, the insecticide susceptibility test against
the dengue vector
Ae. aegypti
from urban and rural
area were carried out; where the LC
50
and LC
90
values
for insecticide Deltamethrin tested was determined in
order to check the frequency of vulnerability to
Deltamethrin wherein rural area population shows
high values lethal dose requirements especially in
Hunsur and Channarayapatna populations with the
IC
90
values of 13.9×10
-4
and 21.7×10
-4
ppm respectively
(Table 1 and Figure 1).
2.2 Qualitative Analysis of Enzymes
Native PAGE was employed for qualitative analysis
and to study the allelic frequency of enzymes such as
Est-A (Figure 2, Table 2), Est-B (Figure 3, Table 3),
G6PD, AcPH and APH (Figure 4, 5, 6, Table 4) which
degrades the organophosphorus and pyrethrenoids; a
common insecticides used to control the vectors in
both urban and rural areas. Qualitative assay revealed
the level of enzyme expression which was quantified
based on its band intensity and mobility.
2.3 Quantitative Analysis of Enzymes
The quantitative analysis of enzymes such as Est-A
(Figure 7), Est-B (Figure 8), G6PD (Figure 9), AcPH
(Figure 10) and APH (Figure 11) for
Ae.
aegypti
from
urban and rural area populations were carried out
(Table 5) in spite of qualitative assay in order to
understand the genetic resistance mechanism involved
and differentiation in resistance genetics and its product
expression that supports survival of insects among the
populations and also to correlate the insecticide
susceptibility difference with the biochemical data.
Table 1 Comparison of Susceptibility status (LC
50
and LC
90
) of
different populations of
Aedes aegypti
larvae to synthetic
pyrethroid deltamethrin
Populations
LC
†
50
(ppm
‡
)
LC
†
90
(ppm
‡
)
Urban Area
1. VISHVESHWARA NAGAR 1.2×10
-4
2.2×10
-4
2. CHAMUNDIPURAM
2.5×10
-4
8.4×10
-4
3. J.P NAGAR
2.6×10
-4
5.9×10
-4
4. AGRAHAR
1.9×10
-4
5.7×10
-4
5. KUVEMPUNAGAR
1.7×10
-4
7.1×10
-4
Rural Area
6. NANJUNGUD
3.6×10
-4
9×10
-4
7. HUNSUR
3.4×10
-4
13.9×10
-4
8. CHANNARAYAPATTANNA
13.1×10
-4
21.7×10
-4
9. MANDYA
3×10
-4
9.8×10
-4
Figure 1 LC50 of Deltamethrin against different poulations of
Aedes aegypti
Legend: VHN- Vishweshwara nagar, CPM- Chamundipuram,
JPN- J.P. Nagar, AGN- Agrahara, KPR- Kuvempunagar, NJD-
Nanjangud, HSR- Hunsur, CRP- Channarayapatna, MDA-
Mandya
