Development, Characterization and Field Assessment of Multiple Insecticides ... 339
monocrotophos strain treated with monocroto-
phos for 30 and 120 min, respectively. A 2.92-
fold decreased specific activity found in the sus-
ceptible strain was treated with fenvalerate for
30 and 120 min. A. 1.25- and 1.4-fold decreased
specific activity found in fenvalerate tolerant
strain was treated with fenvalerate for 30 and
120 min, respectively (Table 6 ).
Michaelis constant (Km) values were calcu-
lated for the lab susceptible and tolerant strains
for the substrate 1-Napthyl acetate, the substrate
concentrations 0.025—1 mM were used. The
Km value for the susceptible, endosulfan toler-
ant, fenvalerate tolerant, monocrotophos tol-
erant, and MIHTTS was found to be 0.02, 0.1,
0.032, 0.0735, and 0.02 mM, respectively. In
terms of respective insecticides tolerance, endo-
sulfan, fenvalerate and monocrotophos tolerant
strains showed negative correlation toward es-
terase activity. Endosulfan tolerant strain showed
fivefold higher Km value compared to lab sus-
ceptible strain. Monocrotophos tolerant strain
showed 3.68-fold higher Km value compared to
lab susceptible strain. Fenvalerate tolerant strain
showed 1.6-fold higher Km value compared to
susceptible strain (Table 7 ).
Isoenzyme Analysis
The results of native PAGE of esterase electro-
morphs from susceptible, tolerant strains, sus-
ceptible strain treated with endosulfan, mono-
crotophos and fenvalerate separately and tolerant
strains treated with respective insecticides are
shown in the Fig. 3. Nondenaturing PAGE of
homogenates from each strain stained for 1-Nap-
thyl acetate showed difference in composition of
esterase isoenzyme. Esterase bands were desig-
nated E1, E2 and E3; E1 as the slowest migrat-
ing esterase and E3 as the fastest. E1, E2 and E3
bands were present in fenvalerate tolerant strain
treated with fenvalerate. E1 and E3 bands were
present in susceptible untreated and susceptible
strain treated with monocrotophos. E2 and E3
bands were present in endosulfan tolerant, fen-
valerate tolerant, monocrotophos tolerant strains
and also in endosulfan tolerant treated with endo-
sulfan and fenvalerate tolerant strain treated with
fenvalerate. However, monocrotophos tolerant
strain treated with monocrotophos showed only
one E3 band. The differences in electromorphs
may be related to individual isoenzyme activity
to the substrate used and the genetic variation
among strains. The results provide baseline infor-
mation for further research on the involvement
of esterases in the insecticides resistance mecha-
nism of T. chilonis.Microsomal Cytochrome C Reductase and
p-Nitroanisole O-demethylase
This activity was determined by measuring the
production of p-nitrophenol spectrophotometri-
cally. The activity of fenvalerate tolerant strain
was 5.65-fold more compared to susceptible
strain (Table 8 ).Microsomal Cytochrome p-Nitroanisole
O-demethylase
One unit will reduce 1.0 micro mole of oxidized
cytochrome c in the presence of 100 micro molar
NADPH per minute at pH 7.8 at 25°C. The activ-
ity of resistant strain to fenvalerate was 10.5-fold
more (Table 9 ).
Wu and Jiang ( 2003 ) reported insecticide re-
sistance was associated with insensitivity to ace-
tyl cholinesterase (AchE) in P. xylostella and D.
rapae based on the kinetic parameters. Again in
D. rapae insecticide resistance is due to AchE
insensitivity and detoxification enzymes. Shu-
sheng et al. ( 2003 ) found that fenvalerate resis-
tance in Cotesia plutellae is positively related to
mixed function oxidase (MFO), but not related to
carboxylesterase (CE) and esterase (Es) activity.
Perez-Mendoza ( 2000 ) studied the biochemical
mechanism of resistance in a malathion tolerant
strain of the Hebrobracon hebetor. It was found
that malathion resistance in H. hebetor is associ-
ated with both an increased activity of the ester-
ase E3 and null alleles of the esterases El and E2.