Vol.3, No.4A, 1-4 (2013) Open Journal of Animal Sciences
http://dx.doi.org/10.4236/ojas.2013.34A1001
Susceptibility of Culex quinquefasciatus (Diptera:
Culicidae) to malathion in Sargodha district, Pakistan
Hafiz Muhammad Tahir1*, Kishwar Hussain1, Azhar Abbas Khan2, Sajida Naseem1,
Hamza Tanveer Malik3, Abid a Butt4, Rabia Yaqoob1
1Department of Biological Sciences, University of Sargodha, Sargodha, Pakistan; *Corresponding Author:
2Department of Agricultural Entomology, University College of Agriculture, University of Sargodha, Sargodha, Pakistan
3College of Medicine, American University of Antigua, New York, USA
4Department of Zoology, University of Punjab, Lahore, Pakistan
Received 18 August 2013; revised 21 September 2013; accepted 6 October 2013
Copyright © 2013 Hafiz Muhammad Tahir et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The present study was designed to evaluate the
susceptibility of Culex quinquefasciatuns against
malathion in Sargodha district of Pakistan. The
activities of detoxif ying enz ymes i.e., β-esterases,
GSTs and monooxy genases were also estimated.
Our results revealed 100 percent mortality in
insecticide treated groups after 24 hours of ex-
posure. There was no difference in the activities
of insecticide detoxifying enzymes between
control and treated groups. We concluded from
our work that malathion is still effective against
C. quinquefasciatuns in the area for control of
mosquito population.
Keywords: C. quinquefasciatuns; Susceptibility;
Esterases; Glutathione S-Transfereases; Mala thion
1. INTRODUCTION
Mosquitoes are vector agents that cause diseases by
transmitting the virus and parasite from one person to
another. Mosquito-borne diseases are responsible for
significant global morbidity and mortality, and are dis-
proportionately affecting children and adolescents [1].
Culex mosquitoes, especially C. quinquefasciatus, are the
chief vectors of Wuchereria bancrofti that cause a disease
known as bancroftian filariasis. This disease is common
in many regions of the world including the Middle East
and Eastern Mediterranean countries [2]. C. quinquefas-
ciatus may also cause protozoan, viral, parasitic and
helminthic diseases.
Insecticides are considered as the most important
components in the global mosquito control efforts [3],
but due to their injudicious use, Culex mosquitoes have
developed resistance against them [4]. More than 100
mosquito species are known to have developed resistance
to one or more insecticides. Resistance to pyrethroids
and organophosphates has been found in C. quinquefas-
ciatus [5,6].
The insecticide detoxifying enzymes have been de-
scribed to confer insecticide resistance in mosquitoes
which are esterases, cytochrome P-450-dependent mono-
oxygenases and glutathione S-transferases [7]. Esterases
are mostly involved in detoxification of organophos-
phates and carbamates [8]. Glutathione S-transferases are
important family of multifunctional enzymes [9]. The
monooxygenases P450 are phase-I metabolic enzymes
[10] and are considered as the only enzymes to oxidize
insecticides in insects.
For the successful implementation of mosquito control
in a specific area, it is necessary to assess their resistance
status in that area. The aim of present study was to con-
duct the susceptibility tests of C. quinquefasciatus
against malathion, so the resistance status of C. quinque-
fasciatus in the study area may be determined. Activity
of insecticide detoxifying enzymes was also determined
in insecticide treated and control group.
2. MATERIALS AND METHODS
Samples were collected from University of Sargodha,
Punjab, Pakistan by using aspirator. Only blood fed fe-
male mosquitoes were used for study. Bioassays were
performed to determine susceptibility of C. quinquefas-
ciatus against malathion. Three concentrations of mal-
athion insecticide [i.e., recommended field dose (1.5
µl/500 ml), 3/4th of recommended field dose (1.12
µl/500 ml) and 1/2 of recommended field dose (0.75
µl/500 ml) were used]. For residual bioassay specimens
were divided into control and treated groups (n = 30 in

H. M. Tahir et al. / Open Journal of Animal Sciences 3 (2013) 1-4
2
each group). Treated group was exposed to insecticide
impregnated filter paper, for one hour and then trans-
ferred to clean holding jar. Control group was exposed to
water impregnated filter paper. Mortality was observed at
discrete intervals for 24 hours. The bioassay tests were
repeated thrice.
In order to evaluate the role of esterases, glutathione-
S-ransferases and Monooxygenases in insecticide resis-
tance, mosquitoes were exposed to sub-lethal dose of
malathion for one hour and then shifted to clean jars.
After 24 hours of exposure, survivors were frozen at
−20˚C for 15 - 20 minutes in order to immobilize them,
and then their wings, legs and abdomen were removed.
Rest of the body was homogenized in 400 µl of chilled
phosphate buffer (0.1 M, pH 7.0) containing 0.01% (w/v)
of triton X-100. The crude homogenate was centrifuged
at 13,000 rpm for 5 minutes. The supernatant was col-
lected and used further as enzyme source for biochemical
estimation of non-specific esterase (β-esterases), glu-
tathione-S-transferases (GST) and monooxygenases.
To measure the activity of non-specific esterases
method described by Baker et al. [11] was followed. Β-
naphthyl acetate (Substrate B) was used as substrate. The
activity of Glutathione-S-transferases towards 1-chloro-2,
4-dinitrobenzene (CDNB) was estimated according to
methods elaborated by Habig et al. [12]. The activity of
monooxygenases was determined by the method de-
scribed by Vulule et al. [13]. To compare the activity of
enzymes between control and insecticide treated group
two sample t-tests was applied using Minitab 13.2.
3. RESULTS
C. quinquefasciatus population was found susceptible
to the all tested concentartions of malathion. After 24
hours of exposure 100 percent mortality of mosquitoes
(all mosquitoes in a group died treated with each dose)
was observed at each concentration. However, no mor-
tality was recorded in control group (Table 1).
When the activity of Esterases between control and
treated groups were compared, non-significance differ-
ence was observed (df = 11; T-value = −0.74; P-value =
0.474, Figure 1). Although the activities of Glutathione-
Table 1. Mortality in C. quinquefasciatus against different con-
centrations of malathion.
Number of musquitoes died at different time intervals
Total moqquitoes in each group were 30.
Concentraions
(µl/500 ml) of
malathion 4 hrs 8 hrs 12 hrs 16 hrs 24 hrs
0.00 (Control) 0 0 0 0 0
0.75 20 23 24 26 30
1.12 25 25 25 25 30
1.5 25 28 28 28 30
0
100
200
300
400
500
600
Control Treated
Activity of beta esterases (mM/ m i n/ m g of protein)
(a)
0
0.05
0. 1
0.15
0. 2
0.25
0. 3
0.35
0. 4
Control Treated
Activity of GST (nM/ng protein/m i n)
(b)
0
5
10
15
20
25
Control Treated
Activity of monooxygenases (u g/ m in/ m g of protein)
(c)
Figure 1. Activities of esterases (a) Glutathione-s-transferases
(b) and Monooxygenases (c) in control and treated group.
s-transferases and Monooxygenases in the treated group
were higher but statistically difference was non-signifi-
cant (df = 18; T-value = −0.40; P-value = 0.697 for Glu-
tathione-s-transferases and df = 11; T-value = −1.711;
P-value = 0.148 for Monooxygenases) as depicted in

H. M. Tahir et al. / Open Journal of Animal Sciences 3 (2013) 1-4 3
Figure 1.
4. DISCUSSION
Mosquitoes are the major public health pests and are
vectors for many diseases, such as malaria and West Nile
Virus [14]. Various methods are being used by research-
ers to control the mosquitoes. Insecticides are frequently
used to control the mosquitoes, but over time these can
build up a resistance to insecticides [8].
Results of present study showed malathion to be the
potent insecticide to produce a high level of mortality in
C. quinquefasciatus. Duran and Stevenson [15] also re-
ported malathion susceptibility in C. quinquefasciatus.
However, these results are contrary to Hamdan et al. [16]
who reported development of resistance in larvae of C.
quinquefasciatus against malathion in Malaysia. Kumar
et al. [17] also reported the malathion resistance in C.
quinquefasciatus from India. Difference among our
findings and those, who found resistance in C. quinque-
fasciatus against malathion, might be due to insecticide
usage profile.
Resistance development is a slow process which takes
several years and successive generations to set up. It also
depends upon the dosage and frequency of insecticide
applied [18]. According to the information collected,
University of Sargodha is not sprayed with insecticides
regularly. However, the use of insecticides in Laboratory
for research purpose is common but this usage is not
sufficient to develop resistance in insects. Along with
insecticide usage profile, there are many other co-factors
for development of insecticide resistance, such as tem-
perature, humidity [19] and rainfall [20].
Biochemical methods were used to detect the possible
resistance mechanism in insects. Our results from bio-
chemical estimation of enzymes showed that the activities
of non-specific esterases, GSTs and monooxygenases
among treated and control groups are not different statis-
tically. Our result of biochemical estimation is correlated
with bioassay. Malathion caused high mortality in mos-
quitoes in the study area and insect’s enzymatic detoxi-
fication pathways have also not been activated. So, we
concluded from the present study that malathion is still
effective in area for control of mosquito population.
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