98 R. B. Veegala and S. Vemuri
titre of 114.3 μmols min−^1 mg−^1 protein and
5.66 μmols min−^1 mg−^1 protein while ICRISAT
population showed 155.2 μmols min−^1 mg−^1 pro-
tein and GST titre of 12.59 μmols min−^1 mg−^1
protein, respectively. The results revealed that
ICRISAT C. medinalis population had 1.35-fold
greater carboxyl esterase and 2.245-fold more
GST’s in its midgut homogenates over DRR pop-
ulation (Table 2 ). Similar findings were reported
by Mohan and Gujar (2003a), where 1.2–1.8-
fold increased CarE activity was observed in P.
xylostella for monocrotophos, cartap and fipro-
nil resistant populations. Yamamoto et al. ( 2008 )
reported that GST from RLF, C. medinalis, was
inhibited by fenitrothion, permethrin, and del-
tamethrin, suggesting GST may be involved in
metabolizing organophosphorus and pyrethroid
insecticides.
Esterase activity visualized in native PAGE
following incubation in substrate solution
(0.05 % α-naphthyl acetate in 0.1 % fast blue ®
revealed two isozyme bands. The major band
with diffused esterase activity was relatively of
more molecular mass than the other esterase ac-
tivity band (Fig. 1 ). Difference in esterase band-
ing pattern in ICRISAT C. medinalis population
was observed when using midgut and whole body
extracts. The midgut produced all three types of
esterase bands while whole body homogenates
produced only two bands (Fig. 2 ). Inhibitor stud-
ies with the esterase isozymes separated under
native PAGE when subjected to inhibition by
incubating with class-specific esterase inhibitors
in buffers containing dichlorvos (DDVP)10−^4 M,
eserinesulphate, 10−^6 and 10−^4 M concentrations
indicated that these two esterase isozymes are B
type esterases as eserinesulphate, a specific in-
hibitor of cholinesterase did not inhibit the ester-
ase activities at 10−^6 and 10−^4 M and esterases
were characterized to have carboxylesterase ac-
tivity (Fig. 2 ).
In insects, the esterase bands separated elec-
trophoretically under native condition are classi-
fied into three types by the substance which in-
hibits their activity (Aldridge 1953 ; Van Asperen
1962 ). The results of the present study are in
conformity with cypermethrin resistant Plutella
xylostella strain, wherein the carboxylesterase
levels from the first to the fourth instar, pupa and
adult showed 2.64-, 3.16-, 2.61-, 3.04-, 2.93-,
and 2.75-fold higher carboxylesterase activity
in comparison to P. xylostella susceptible strain
(Moharil et al. 2008 ) and also reported DDVPTable 2 Carboxylesterase and GST activity of third instar C. medinalis larvae
Location CarE (μmols/min/mg protein) CarE folds GST (μmols/min/mg protein) GST folds
DRR population 114.39 1.00 5.660 1.00
ICRISAT population 155.2 1.35 12.598 2.245
Fig. 1 Carboxylesterase, profiles from whole body and
midgut homogenates of ICRISAT, C. medinalis popula-
tion. Lanes 1–5 are whole body homogenates showing
two bands of esterase isozymes, Lanes 6–10 are midgut
homogenates showing three bands of esterase isozymes
Table 1 Toxicity of monocrotophos to third instar larvae C. medinalis 24 HAT
Location LC 50 (ppm) RR SLOPE ± SE ‘F’ limits χ^2 (degrees of freedom)
Lower Upper
DRR population 30 1.00 1.29 ± 0.21 0.0018 0.0057 4.0(4)
ICRISAT population 60 2.00 1.14 ± 0.21 0.002 0.011 2.31(4)
RR resistance ratio over one generation, F Limits fiducial limits, HAT hours after treatment