Journal of Mosquito Research 2015, Vol.5, No.15, 1-15
5
tray buffer for carboxyl esterases, Phosphatases and
Tris EDTA-Borate buffer (pH 9.0) for G-6-PD assay.
The gels were initially run at 40V for 25-35min and
then at 60V for nearly 5hr at 4
o
C employing a
refrigerator.
1.5.3 Staining Techniques
1.5.3.1 Qualitative assay of Est-A and Est-B
Substrate was prepared by amalgamation of A/B
naphthyl acetate (1.85 mg/ml) in 0.1M Sodium
phosphate buffer (pH 5.9) and Fastblue RR salt (2
mg/ml) in 0.1M phosphate buffer (pH 6.5) collectively
incubated with gel containing resolved native Esterase
at 37
0
C in dark for 20 min. in order to trace the
isozyme patterns (Patricia et al
.,
1988).
1.5.3.2 Qualitative assay of G6PD
G6PD zymogram was prepared according to the
method described by Umberto & Antoniettina (1973);
with slight modifications. In brief, Substrate containing 25
mg of each Glucose 6 phosphate and NADP, 75 mg of
each MgCl
2
and MnCl
2
, 25 mg NBT (Nitro Blue
Tetrazolium) and 5mg PMS (PhenazoniumMethosulphate)
were primed in 0.05M Tris HCl buffer (pH 8.5) and
were incubated with the zymogram having native
enzymes at 37
o
C for 2 hrs in obscurity.
1.5.3.3 Qualitative assay of APH and AcPH
125 mg of polyvinyl pyrrolidone, 25 mg of each
fastblue RR salt and sodium-1-Naphthyl phosphate,
15 mg of each MgCl
2
and MnCl
2
and 500mg NaCl
were made ready in 25 ml of 0.05 M tris buffer (pH
8.5) and the gels were stained for nearly 2 hour in
gloom at 37
o
C for APH assay. For AcPH, 125 mM
Sodium acetate buffer (pH 5.0) used while other
components are alike that of APH assay.
1.5.4 Fixatio
n
After the appearance of bands, the gels were washed
with distilled water and were fixed in 7% glacial
acetic acid solution. Zymograms were prepared
depending upon the mobility of the bands. The
commonest band appeared in both the population of
the species was designated as 1.00. Likewise bands
with higher mobility were designated with the number
greater than 1.00 in an increasing order (
i.e.,
1.02,
1.04, 1.05 likewise). Similarly the bands which had
lesser mobility, were designated with number in a
decreasing order (
i.e.,
0.98, 0.90, 0.89 likewise.). The
numbering has been made by considering the distance
traveled by individual bands.
1.6 Enzyme activity analysis - Quantitative assay
1.6.1 Sample preparation
Batches of thirty early fourth instar mosquito larvae or
one day old adult mosquitoes were individually
homogenized in 200 µl of distilled water under cold
conditions using a polypropelene pestle and spun at
5000rpm for 2min in a microcentrifuge at 4
o
C.
Replicate of the 20 µl supernatant from each sample
was used as enzyme source. For assays viz., Naphthyl
acetate esterase assays, assay of both the Phosphatases
and G6PD using microtiter plate.
1.6.2 Estimation of Naphthyl acetate esterase
Est-A activity was quantitatively assayed in microtiter
plate where, 200 µl of A-naphthyl acetate solution
(100 µl of 30 mM A-naphthyl acetate in 10 ml of 0.02
M phosphate buffer pH 7.2) was added to 20 µl of the
supernatant sample homogenates. The reaction was
carried for 30 min at room temperature before the
addition of 50 µl of Fastblue stain solution (22.5 mg
fastblue RR salt in 2.25 ml distilled water and 5.25 ml
5% sodium lauryl sulphate diluted in 0.1 M phosphate
buffer of pH 7.0) to each well to stop the reaction.
Replicate blanks were maintained where enzyme is
replaced by distilled water. The enzyme activity was
read at 570 nm as end point. Absorbance levels for
individual mosquitoes were compared with the help of
a standard curve of absorbance for known concentrations
of A-naphthol and the results are reported as µg of
A-naphthol produced per min per mg of larval protein
(Patricia et al., 1988). The methodology followed for
Est-B is similar except for the substrate used
i.e.,
instead of A-naphthyl acetate, B-naphthyl acetate was
employed. The results were reported as µg of B-naphthol
produced per min per mg of larval protein (Patricia et
al., 1988).
1.6.3 G6PD Assay
The total activity of G6PD was estimated following
the modified method of Kumar et al., (1991). In brief
100 µl of the larval homogenate was taken in 1 ml
capacity microcuvette and incubated with 400 µl of
1.5 M Tris HCl (pH 7.5) containing 3.8 x 10
-4
M
NADP, 10 µl of 0.3 M MgCl
2
, 500 µl of 0.03 M
D-Glucose-6-phosphate. It was read at 340 nm with a
UV spectrophotometer. The amount of NADP reduced
