Susceptibility of <i>Culex quinquefasciatus</i> (Diptera: Culicidae) to malathion in Sargodha district, Pakistan

doi:10.4236/ojas.2013.34A1001

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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: [email protected]

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

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

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

100

200

300

400

500

600

Control Treated

Activity of beta esterases (mM/ m i n/ m g of protein)

(a)

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)

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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