New Horizons in Insect Science Towards Sustainable Pest Management

338 T. Venkatesan and S. K. Jalali

that of a relatively susceptible natural population
(Rosenheim et al. 1989 ) (Table 4 ).

Sequencing Heat Shock Protein (HSP)

for Determination of High Temperature

Tolerance in Multiple Insecticides and

High Temperature Tolerant Strain

(MIHTTS) of T. chilonis

Sequencing of HSP PCR Products

A 300 bp HSP PCR product was gel eluted and
sequenced using HSP-specific primers. Sequenc-
ing was done using both forward and reverse
primers to check the accuracy of the sequencer.
The similarity of the obtained sequence was
found out using Blast program. Sequence showed
homology with other HSP-70 sequences submit-
ted in the database.

Biotin Labeling of HSP 300 bp Product

The 300 bp PCR was gel eluted and was la-
beled by random oligonucleotide DNA labeling
method. The tube was incubated in water bath for
5–10 min and was cooled on ice. Biotin labeling
mix 5 μl was added to klenow pol 1 μl and again
incubated at 37 °C in a water bath over night. The
biotin labeled probe was detected by spot hybrid-
ization using biotin chromogenic detection kit.
The biotin labeled probe was detected by spot
hybridization using biotin chromogenic detection
kit (Fig. 2 ).
Which resulted intense bright spot. The PCR
product was labeled with high efficiency.

Biochemical Characterization

GST-Activity

GST—Conjugative activity in the susceptible

was significantly lower than that of insecticide

tolerant strains (endosulfan, monocrotopho and

fenvalerate). Highest activity measured in MI-

HTTS followed by endosulfan, monocrotophos

and fenvalerate tolerant strains. MIHTS shows

2.13-fold increase in activity, endosulfan, mono-

crotophos and fenvalerate tolerant strains show

1.7-fold increases in activity compared with the

susceptible strain. However, there were no sig-

nificant differences in activity among individual

insecticides tolerant strains (Table 5 ).

Carboxylesterase (CE) Activity

Varied levels of CE activity found in the suscep-

tible and insecticides tolerant strains were treated

with endosulfan, monocrotophos and fenvalerate

for 30 and 120 min. Table 6 shows that 2.92-fold

decreased specific activity found in the suscep-

tible strain was treated with endosulfan for 30

and 120 min, respectively. However, 1.25- and

1.5-decreased specific activity found in endosul-

fan tolerant strain was treated with endosulfan

for 30 and 120 min, respectively. A 9.12- and

9.9-fold decreased specific activity found in sus-

ceptible strain treated with monocrotophos for 30

and 120 min, respectively. Similarly a 5.6-fold

and 10-fold decreased specific activity found in

Table 4 Dose mortality response (LC) of T. chilonis to three insecticides after 6 h of constant exposure of tolerant
population
Insecticide LC 50 95 % Fiducial limit LC 90 95 % Fiducial limit Slope ± SE χ^2
Lower Upper Lower Upper
After F 81 generations
Endosulfan 5.53 3.64 11.6 81.8 29.7 543.3 1.19 ± 1.17 2.60
Monocrotophos 4.39 – – 87.8 – – 0.99 ± 0.22 37.7
Fenvalerate 0.24 0.0 0.0 0.43 0.0 0.0 0.0 0.0

dark brown spot

Fig. 2 Biotin labeled probe was detected by spot hybrid-

ization using biotin chromogenic detection kit