Monitoring the Effect of Insecticide Selection on Culex pipiens (Diptera:
Culicidae) Larval Susceptibility to Malathion and Lambda-Cyhalothrin
El-Sayed A. El-Sheikh,
Mohamed-Bassem A. Ashour,
Mohamed M. Aamir
Mohamed M. Gamal
The susceptibility of Culex pipiens collected from different localities
of the Sharkia Governorate, Egypt, to malathion and lambda-cyhalothrin was investigated
for 11 successive generations. Larval Cx. pipiens developed 57 and 305-fold
resistance to malathion and lambda-cyhalothrin, respectively after 11 successive
generations of selection pressure. The susceptibility of unselected generations,
due to non-exposure to both insecticides for 11 generations, was increased to
1.1-fold in F11 for malathion and to 1.5-fold in F11 for
lambda-cyhalothrin. Acetylcholinestrase (AChE) activity increased gradually
in both selected and unselected generations until the 11th generation. The activity
of AChE in generations (F1-F7) selected with malathion
was significantly lower than that of the lambda-cyhalothrin selected and unselected
generations. General esterase activity increased 3.7 and 3.0-fold (malathion)
and 4.2 and 3.6-fold (lambda-cyhalothrin) compared with the susceptible strain
(LS-CP), when either α-napthyl acetate (NA) or β-NA were used as general
substrates in F11 selected generations, respectively. A significant
increase in glutathione-S-transferase (GST) activity was noticed in the F11
generation recording 6400 and 8800 μmoL min-1 mg-1
protein for malathion-and lambda-cyhalothrin-selected generations, respectively.
The ratios recorded in the 11th generations were 3.6 and 4.9 fold as compared
with LS-CP for malathion and lambda-cyhalothrin selected generations, respectively.
Our results indicate that the Cx. pipiens mosquito strain from Egypt
can increase resistance to malathion and lambda-cyhalothrin if these insecticides
are continuously or rotationally used to control this species. Increased resistance
is likely to be associated with increased activity of target and metabolic enzyme
to cite this article:
El-Sayed A. El-Sheikh, Mohamed-Bassem A. Ashour, Mohamed M. Aamir and Mohamed M. Gamal, 2014. Monitoring the Effect of Insecticide Selection on Culex pipiens (Diptera:
Culicidae) Larval Susceptibility to Malathion and Lambda-Cyhalothrin. Journal of Entomology, 11: 14-24.
Received: September 04, 2013;
Accepted: October 19, 2013;
Published: March 08, 2014
Culex pipiens Linnaeus (Diptera: Culicidae) is an important vector of
several human pathogens such as West Nile virus, Rift Valley Fever virus and
Bancroftian filariasis (Claire and Callaghan, 1999).
Cx. pipiens is found in tropical areas (Bourguet
et al., 1998) and different species have been reported in parts of
Africa, Russia, Australia, North and South America (Azari-Hamidian,
2007). Cx. pipiens is both a nuisance and a disease vector (Dehghan
et al., 2011), affecting more than 700 million people annually (Taubes,
2000). In Egypt, Cx. pipiens is one of the most common mosquito species
in urban and rural areas and causes a human health risk (Zahran
and Abdelgaleil, 2011).
The control of mosquitoes depends primarily on continued applications of different
insecticide classes (Rozendaal, 1997), which rotational
use may result in continued satisfactory control against field populations of
house mosquitoes (Shin et al., 2012). Although
they are effective, their continuous and repeated use has resulted in the widespread
development of resistance (Perumalsamy et al., 2010).
As insects become resistant, more insecticides are used which can cause human
and environmental health problems. Widespread insecticide resistance to commonly
used and less expensive insecticides has been a major obstacle in implementing
cost-effective and safe integrated mosquito management.
Insecticide resistance in mosquitoes is essentially achieved through two mechanisms:
target insensitivity and increased detoxification (Nauen,
2007). The former is associated with target modifications that lower their
affinity for the considered insecticide. Increased detoxification results from
an increased activity of detoxifying enzymes such as esterases and GSTs and
the specific carboxylesterase which was reported for malathion resistance in
Drosophila melanogaster by Ashour et al.
The effect of resistance on the chemical control of mosquitoes is very difficult
to determine due to a large number of associated factors that may impact on
successful control in the field. Understanding the relationship between insecticide
resistance and metabolic resistance mechanisms is important in order to address
the knowledge gap between control strategies and developing resistance (Rajatileka
et al., 2011). The aim of this study was to test the susceptibility
of Cx. pipiens populations, collected from different localities at Sharkia
Governorate in Egypt, to the most commonly used insecticides for mosquito control
(malathion and lambda-cyhalothrin) and to assess the relative activities of
detoxification and target enzymes in association with resistance.
MATERIALS AND METHODS
Test mosquitoes: A mixed population of Cx. pipiens was established
from larvae collected from five field localities in Sharkia Governorate, Egypt,
during July and August of 2010. The collected Cx. pipiens larvae identified
in the Research Institute of Medical Entomology, Mosquito Department, Egyptian
Ministry of Health, which the source of a laboratory susceptible (SS) strains
(LS-CP strain) of the same species that maintained in the laboratory for 15
years without any insecticidal exposure. The collection localities were Al-Asher
of Ramadan (PR), Diarb Negm (PD), Faquos (PF), El-Salhia (PS) and El-Zagazig
(PZ) (Fig. 1). The collected larvae were transferred to an
insect rearing room and reared in plastic trays (30x20x10 cm) containing 3000
mL tap water and 1.0 g of sterilized rodent diet. The first generation (F1)
was obtained from rearing 2500 fourth instar larvae (500 larvae/locality) in
one tray. Pupae were subsequently transferred to adult cages for adult emergence.
Adult mosquitoes were maintained on a 10% sucrose solution and were allowed
to blood feed on a domestic pigeon for oviposition under an approved animal
use protocol. All stages were maintained at 27±2°C, 65-75% Relative
Humidity (RH) and 14:10 h light:dark cycle (WHO, 1975).
Insecticides and reagents: Malathion (Malathion, 57% EC) and lambda-cyhalothrin
(Lambda, 5% EC) were used in this study. Malathion and lambda-cyhalothrin formulations
were obtained from a local manufacturer, Kafr El-Zayat for Pesticides and Chemicals
Company, Kafr El-Zayat City, Gharbia Governorate, Egypt. α-Naphthyl acetate
(α-NA), β-naphthyl acetate (β-NA), α-naphthol and β-naphthol
were obtained from Alfa Aesar Co (Karlsruhe, Germany). 5,5-dithiobis nitrobenzoic
acid (DTNB), acetylcholine iodide substrate (ATChI), 1-chloro-2,4-dinitrobezene
(CDNB), reduced glutathione, fast blue B salt and Bovine Serum Albumin (BSA)
were from Sigma-Aldrich (St. Louis, MO, USA).
||Localities of the collected Culex pipiens populations,
(a) Whole Egypt map indicate Sharkia governorate in shadow and (b) Collecting
localities and their latitude and longitude in Sharkia governorate which
PR: Al-Asher of Ramadan, PD: Diarb Negm, PF: Faquos, PS: Salhia and PZ:
All commercial reagents and other chemicals used in this study were of analytical
quality with the highest purity available and purchased from commercial suppliers.
Selection pressure and bioassay: Fourth instar larvae of the first generation
(F1), which considered the parent generation, were divided into three
groups of approximately 3000 larvae each. Two of the three groups were subjected
separately to selection with either malathion or lambda-cyhalothrin for 11 successive
generations at concentrations that caused 50-60% mortality every generation
(selected strain). The third group was reared in parallel under the same conditions
for the same number of generations but without insecticidal contamination so
as to be used as an unselected strain. After 24 h of insecticide-exposure, the
surviving larvae of each group (selected strain) were transferred separately
into clean plastic trays containing fresh tap water until pupation. The resulted
pupae were transferred into adult cages until adult emergence and reared using
the same procedure described above.
Larval bioassays were performed on the selected and unselected generations
as well as LS-CP strain. Batches of 25 fourth instar larvae were exposed to
a range of insecticide concentrations prepared in 100 mL of tap water in 4 OZ
plastic cups (Bio-Serv, Frenchtown, NJ). For each tested insecticide, serial
concentrations were prepared to give mortalities between 10 and 90%. At least
five concentrations ranging 0.01-1.0 mg mL-1 (malathion) and 0.00001-0.01
mg mL-1 (lambda-cyhalothrin) were used for each assay with three
replicates for each concentration. After 24 h of treatment at 27±2°C,
the larval mortality was recorded. Three similar batches of 25 fourth instar
larvae each were introduced to clean plastic cups containing tap water only
to be used as a control. For each generation, the toxicity regression lines
of the three populations (malathion and lambda-cyhalothrin selected strains
and the unselected strain) were established and the LC50 and slope
values were determined. Cx. pipiens larval susceptibility to the tested
insecticides in each generation of the selected strain was expressed as a resistance
ratio (RR) obtained by dividing the LC50 values of the selected or
unselected generations by the LC50 values of LS-CP.
Preparation of mosquito extracts: For total protein, acetylcholinesterase,
general esterases and GST determinations, samples of larval homogenate ware
prepared by homogenizing 20 fourth instar larvae of each tested generation as
well as the LS-CP strain. Larvae were homogenized in 250 μL of 0.1 M ice-cold
sodium phosphate buffer, pH 7.4, containing 0.02% Triton X-100 using a plastic
mini pestles in 1.5 mL centrifuge tubes. Debris was pelleted at 10,000 rpm for
15 min at 4°C. The supernatant was then separated in clean 0.5 mL eppendorf
tubes and stored at -20°C until used within 15 days.
Acetylcholinesterase: AChE activity in whole larval homogenate was estimated
according to the procedure described by Ellman et al.
(1961). Twenty larvae/batch were homogenized as mentioned above and the
supernatant was decanted, kept on ice and used as the crude enzyme preparation.
In 10 mL glass test tubes, 10 μL of the crude enzyme was added to 1.5 mL
of phosphate buffer, pH 7.2, containing 0.39 μM of 5,5-dithiobis nitrobenzoic
acid (DTNB). The reaction was initiated with the addition of 50 μL of acetylcholine
iodide substrate (ATChI) (final conc. in 1560 μL = 7.8 μM). The 300
μL of the previous mixture was transferred into ELISA plate's wells in
triplicates. Absorbance was recorded initially after 5 min at 450 nm in 96-well
microplate using Microplate Autoreader, EL311S (Bio-TEK Instrument, Highland
Park, Winooski, VT) (Ashour et al., 1987b).
Reading was repeated after exactly 30, 60 and 90 sec. The mean absorbance change
per 30 sec (ΔA/30 sec) was determined. Blank contains the same components
except the substrate was used as control. Rates were converted to nmol min-1
mg-1 using the extinction coefficients of 9.25 mM-1 300
μL-1 for 2-nitro-5-mercaptobenzoate (Grant
et al., 1989).
Esterases: Colorimetric esterases (EST) activity assays were determined
using the general substrates of α and β-NA as described by Gomori
(1953) with modifications. Measurements were performed in 96-well microplates
using microplate autoreader. For each reaction mixtures, 480 μL phosphate
buffer (0.1 M, pH, 7.4) with 0.02% Triton X-100, 20 μL of protein solution,
500 μL of α or β-NA substrate solutions (final concentration
in 1500 μL total volume = 2.5 mM) and 500 μL of Fast Blue B salt solution
(consists of 2 parts of 1% Fast Blue B salt and 5 parts of 5% SDS) were mixed
in 10 mL glass tubes and incubated at 30°C for 5 min. A thousand five hundred
μL total volume of the mixture was measured in 5 replicates (300 μL
well-1) for each sample. The Optical Density (OD) was measured at
450 nm during the first 5 min of the reaction and rates were converted to nmol
min-1 mg-1 using the extinction coefficients of 9.25 mM-1
300 μL-1 for 1-naphthol (Grant et al.,
1989). Activities were corrected for non-enzymatic hydrolysis using reactions
without protein as controls.
Glutathione-S-transferase: GST activity assays were done by the modified
method of Grant and Matsumura (1988). Twenty five μL
of larval homogenate prepared as previously mentioned, 75 μL of chloro-2,
4-dinitrobenzen (CDNB) and 75 μL of reduced glutathione (fin. conc. 5 mM)
were mixed with 750 μL of phosphate buffer, pH 7.4. Reactions were allowed
to take place for 5 min at 37°C and then terminated by adding 75 μL
of trichloroacetic acid to make final assay volume of 1000 μL test-1
tube. Three replicates were used for each measurement and activities were corrected
for non-enzymatic hydrolysis using reactions without protein as controls. The
conjugation of glutathione to CDNB is accompanied by an increase in absorbance
at 340 nm. The rate of increase was directly proportional to the GST activity
in sample. These measurements were done using spectrophotometer (Spectronic
20, Bausch and Lomb, USA) by measuring absorbance at 340 nm at 30°C after
15 min from the addition of glutathione. Rates were converted to nmol min-1
mg-1 using the extinction coefficients of 8.5 O.D. mM-1
1000 μL-1 for CDNB (Grant et al.,
Total protein: Protein concentrations were determined according to the
method of Bradford (1976) by incubating 10 μL of
larval homogenate with 290 μL of Bio-Rad protein assay solution for 10
min. Absorption was then measured at 570 nm and bovine serum albumin was used
as the standard.
Statistical analysis: Mortality data were subjected to probit regression
analysis using a Probit polo pc plus software v 3.1 (LeOra Software Inc., Cary,
NC) which automatically corrected for control mortality according to the method
of Finney (1971) and the lethal concentrations which gave
50% (LC50) mortalities were calculated. Data of acetylcholinesterase,
esterases and GST in selected and unselected generations were subjected to SPSS
10.0 for Windows software package for statistical analyses. One-way analysis
of variance (ANOVA) was performed and variant among groups (selected, unselected
and susceptible strains) were determined by means of the Duncan test (Duncan,
Development of resistance: As a result of continuous exposure to malathion
or lambda-cyhalothrin for 11 successive generations, the resistance ratio to
malathion when compared with LS-CP strain (Table 1) was increased
from 24.7 in F1 to 57.2 fold in F11 based on LC50 values
(0.296 mg L-1 in F1 to 0.687 mg L-1 in F11).
The slope values of malathion-selected generations fluctuated around the value
of the F1 generation (2.22) suggesting that the field strain didnt
alter its homogeneity as it may be already homogenous from initial field collection
for response to malathion.
||Resistance development of Cx. pipiens either continuously
selected for resistance to malathion or unselected for 11 successive generationsa,
LC50 = Lethal concentration inducing 50% mortality, LS-CP refers
to an insecticide susceptible Cx. pipiens laboratory strain
|aData are shown from 3 replicates of bioassay,
bTotal number of larvae tested population-1, cCL
means confidence limit, dResistance ratio (RR) = LC50
of the selected or unselectedgenerations/LC50 of the susceptible
||Resistance development of Cx. pipiens either continuously
selected for resistance to lambda-cyhalothrin or unselected for 11 successive
generationsa, LC50 = Lethal concentration inducing
50% mortality, LS-CP refers to an insecticide susceptible Cx. pipiens
|aData are shown from 3 replicates of bioassay,
bTotal number of larvae tested population-1, cCL
means confidence limit, dResistance ratio (RR) = LC50
of the selected or unselected generations/LC50 of the susceptible
When unselected generations were tested against malathion (Table
1), the susceptibility increased as LC50 values decreased from
0.254 mg L-1 in F1 to 0.013 mg L-1 in F11.
There is a dramatic drop was noticed in malathion sensitivity of the unselected
generation from F1 to F3. Both slope and resistance ratio
of unselected generations decreased as the rearing in the laboratory was continued
(Table 1), suggesting an increase of susceptibility and heterogeneity.
Table 2 shows Cx. pipiens susceptibility to lambda-cyhalothrin
through 11 successive generations of selection as well as without any insecticidal
selection. The resistance ratio increased from 5 (F1) to 305 (F11)
fold, respectively in the selected generations. The slope of the log concentration-probit
line decreased in F11 compared with F1 indicating an increase
of heterogeneous individuals in the population. The unselected generations showed
a decrease in resistance to lambda-cyhalothrin, with the resistance ratio dropping
from 5 in F1 to 1.5 fold in F11 (Table 2).
In the same way of unselected generations to malathion, the slope of the unselected
to lambda-cyhalothrin was decreased indicating an increase of heterogeneity.
Biochemical analysis: AChE activity (Fig. 2) was markedly
decreased in the F1, F3, F5 and F7
malathion-selected generations compared with lambda-cyhalothrin selected or
unselected generations. The normal activity was recovered in the malathion-selected
F9 and F11 generations in which no significant differences
in activity were observed compared with unselected generations. AChE activity
in lambda-cyhalothrin-selected generations was always markedly high compared
with malathion-selected or unselected generations.
The EST activity was measured in the unselected, selected generations as well
as in the laboratory LS-CP (Table 3). The activity ratio was
calculated compared with that in LS-CP. General esterase activity was significantly
increased in generations selected with both insecticides. The highest activity
was scored in F11 following continuous exposure to malathion and
lambda-cyhalothrin. Continuous exposure to lambda-cyhalothrin showed markedly
high EST activity compared with the malathion selected F11 generation.
General esterase activity was not significantly altered through 11 successive
generations without exposure to insecticides.
||Acetylcholinesterase activity in malathion or lambda-cyhalothrin
selected and unselected Cx. pipiens larvae*, F1-F11 represent
unselected generations, F1M-F11M represent malathion-selected generations
and F1L-F11L represent lambda-cyhalothrin-selected generations, The same
letters on bars indicate that there are non-significant differences at p
= 0.05 among selected or unselected generations
||Esterase activity in Cx. pipiens malathion or lambda-cyhalothrin
selected and unselected larvae*, LS-CP refers to an insecticide
susceptible Cx. pipiens laboratory strain
|* Values are shown as Means±SD of three
determinations, Activity data are statistically different when numbers followed
by different letters within the same column at p<0.05, ** Ratio
is calculated as mean activity value in selected or unselected generations/mean
activity value in the LS-CP
||Glutathion-S-transferase activity in Cx. pipiens malathion
or lambda-cyhalothrin selected and unselected larvae*, LS-CP
refers to an insecticide susceptible Cx. pipiens laboratory strain
|*Values are shown as Means±SD of three determinations,
Activity data are statistically different when numbers followed by different
letters within the same column at p<0.05, **Ratio is calculated as mean
activity value in selected generations/mean activity value in the LS-CP
The activity ratios increased 3.7 and 3.0 fold in the malathion selected F11
generation compared with 2.0 and 1.7 fold in F1 generation when α-NA
or β-NA were used as general substrates, respectively. For lambda-cyhalothrin
selected generations, the activity ratio was increased from 2.0 and 1.8 fold
in F1 to 4.2 and 3.6 fold in F11 when α-NA or β-NA
were used as general substrates, respectively.
Based on mean values, GST activity (Table 4) was increased
in the F11 generation selected with either malathion or lambda-cyhalothrin.
The activity ratios were calculated by comparing them with the activity scored
for LS-CP, which increased from 2.0 (F1) to 3.6 fold (F11)
following malathion selection. GST activity increased from 2.2 to 4.9 fold through
11 generations of selection with lambda-cyhalothrin. The activity ratios in
the unselected generations, did not alter significantly through 11 generations.
Insecticide resistance in members of the Cx. pipiens mosquito complex
has been documented in many countries such as Tunisia (Daaboub
et al., 2008), Cote dIvoire and Burkina Faso (Chandre
et al., 1998), Saudi Arabia (Amin and Hemingway,
1998), Egypt (El-Sheikh, 2011), USA (McAbee
et al., 2004) and China (Li et al., 2002).
Our laboratory assays of Cx. pipiens, selected and unselected through
11 generations with either malathion or lambda-cyhalothrin, resulted in increased
resistance to the tested insecticides. Although the main purpose was not to
evaluate the resistance, Cx. pipiens developed high resistance levels
to lambda-cyhalothrin (305.0-fold) and malathion (57.2-fold) due to continuous
exposure to the tested insecticides through 11 generations. The case of developing
resistance to lambda-cyhalothrin was recorded in other mosquito species such
as Ae. Aegypti adults collected from Colombia and many other countries.
In the study of Ocampo et al. (2011), the susceptibility
of larvae and adults of Ae. Aegypti from Colombia to malathion, temephos
and fenitrothion showed adult resistance to all tested insecticides and larval
resistance to temephos and fenitrothion only which explained that temephos resistance
are important as it has been the primary chemical for controlling immature stages
of Ae. aegypti in Colombia. Additionally, temephos pressure on larvae
may generate cross-resistance to pyrethroids or in the adult stages to other
In contrast to the insecticide selected strains, the unselected strain of the
same population showed increasing susceptibility to malathion and lambda-cyhalothrin
through 11 generations. In both cases of developing or reverse resistance to
the tested insecticides, Cx. pipiens larvae were exhibited resistance
to malathion in a rate less than that of lambda-cyhalothrin and reverse to susceptibility
faster, suggesting that exposure to malathion in nature may be less than that
of lambada-cyhalothrin. Insecticide resistance has been reported in more than
100 mosquito species including Cx. pipiens and resistance to one insecticide
can induce cross-resistance to another insecticide from the same group or different
groups (Hemingway and Ranson, 2000). Regarding this
phenomenon, a colony of Cx. pipiens Marin in California rapidly developed
high levels of resistance following a few generations of selection with permethrin
and showed cross-resistance to lambda-cyhalothrin as well as to DDT (McAbee
et al., 2004). In Tunisia, Cx. pipiens populations from different
localities developed high resistance levels to pyrethroid (permethrin and deltamethrin)
and organophosphorus (chlorpyrifos) insecticides. The result of these high levels
of resistance was stopping use such insecticides in localities of Tunisia where
strong resistance was detected (Daaboub et al.,
AChE activity was affected by malathion-exposure. The activity was increased
by repeating Cx. pipiens larval exposure to malathion until no significant
differences were found in the 11th generation among selected and unselected
larvae, suggesting that an altered acetylcholinesterase as a target insensitivity
may result (Bonning and Hemingway, 1991). In our study,
the activities of esterases and GSTs in resistance selected Cx. pipiens
were determined because of their potential use as biochemical indicators of
insecticide resistance (Grant et al., 1989;
McAbee et al., 2004).
Field collected Cx. pipiens larvae showed 24.7 and 5.0 fold resistance
to malathion and lambda-cyhalothrin, respectively. Continuous exposure to these
insecticides through 11 generations resulted in 57.2 and 305.0 fold increases
in resistance to malathion and lambda-cyhalothrin, respectively. Malathion resistance
was associated with an altered acetylcholinesterase as well as increased activity
of detoxification enzymes (esterases and GST). These data suggest that Cx.
pipiens in Sharkia Governorate, Egypt, may also develop high levels of resistance
to these insecticides if exposed to them continuously for several generations.
On the other hand, reversions to susceptibility may occur if selection is relaxed
by discontinuing the use of these insecticides as part of a resistance management
This study was funded by a research Grant from Zagazig University (Project
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