Development, Characterization and Field Assessment of Multiple Insecticides ... 329
(by T. chilonis) were kept in the glass vial for
emergence. After emergence, egg card was pro-
vided @ 50 eggs/female. The vials were kept in
above temperature to record percent mortality
and percent parasitism after 6 and 24 h of ex-
posure. The data on mortality and percent para-
sitism were analyzed by one-way ANOVA and
means were separated by LSD values.
Development of Population of T.
chilonis Having Multiple Insecticide and
High Temperature Tolerance
The experiment was conducted with exposing
field-collected population to three insecticides,
viz., endosulfan, monocrotophos, and fenvalerate
as well as to variable high temperature (32–38 °C)
simultaneously. The induction of tolerance in the
multiple insecticides tolerant strain of T. chilonis
to high temperature (32–38 °C) was carried out in
the laboratory in growth chamber maintained at
variable temperature for its ability to survive and
parasitize Corcyra cephalonica (Stainton) eggs.
Both strains were mixed together and allowed to
mate for 24 h before exposure to insecticides and
thereafter to higher temperature. The insecticides
used were endosulfan (2.0 ml/L), monocrotophos
(1.5 ml/L), and fenvalerate (0.4 ml/L). Parasit-
oids thus obtained were treated with same con-
centration till fixed parameters were achieved.
The parameters fixed were ≤ 30 % mortality after
6 h of constant exposure and ≥ 90 % parasitism
in sprayed condition. After 50 generations, con-
centrations were increased to double of field rec-
ommended that is, endosulfan (4.0 ml/L), mono-
crotophos (3.0 ml/L), and fenvalerate (0.4 ml/L).
The mortality and parasitism data were tabulated
for each generation.
LC 50 Values to Determine Increased
Tolerance to Insecticides and
Temperature
The experiment was carried out by serial dilution
method. The pesticide solution was prepared by
taking dosages higher than field recommended
dosages and serial dilution by reducing dilution
by 1/2. About 100 adults were released in each
vial in each concentration. The mortality was re-
corded after 6 and 24 h of constant exposure and
percent parasitism was recorded after 6 days of
exposure. The data obtained on mortality were
subjected to probit analysis by statistical program
SPSS version 8.0 and LC 50 , LC 90 , fiducial limits,
slope, and χ^2 values were calculated.Sequencing of Heat Shock Protein (HSP)
for Determination of High Temperature
Tolerance in Multiple Insecticides and
High Temperature Tolerant Strain
(MIHTTS) of T. chilonis
Heat tolerant adults were heat shocked at 40 °C
for 30 min and allowed to recover at room tem-
perature for an hour. The adults were freeze killed
in liquid nitrogen. Total mRNA was extracted
using Biogene RNA extraction kit as per manu-
facturers’ instruction. The first strand synthesis
was carried out by reverse transcriptase enzyme
on total RNA. The amplification reaction was
carried out in 200 μl PCR tubes using gradient
thermal cycler. The PCR amplified DNA samples
were electrophoresed on an Agarose gel along
with the marker DNA to check the presence of
heat shock protein (HSP).Biochemical Characterization
GST-Activity
The conjugative activity of GST was assayed
with CDNB as a substrate using standard proce-
dure.Carboxylesterase Activity
Five milligrams of T. chilonis adults were homog-
enized in a micro pestle and mortar with 0.2 ml
of 50 mM ice-cold buffer of pH 7.5, containing
0.5 % (w/vol) TritonX-100. The homogenates
were centrifuged at 4 ºC at 12,000 g for 10 min.
Supernatant was collected. This was repeated
for two more extractions and supernatants were