Journal of Medical Entomology Advance Access published July 8, 2015

VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS

Selection and Preliminary Mechanism of Resistance to Profenofos

in a Field Strain of Musca domestica (Diptera: Muscidae)
from Pakistan
HAFIZ AZHAR ALI KHAN,1,2 WASEEM AKRAM,3 AND NAEEM IQBAL4

J. Med. Entomol. 1–5 (2015); DOI: 10.1093/jme/tjv095

ABSTRACT House flies are major insect pests at dairy farms in Pakistan and are mainly controlled with
insecticides of different classes, including organophosphates. To develop a better resistance management
strategy, a field strain of house flies was selected in the laboratory to study the potential for the develop-
ment of resistance, possible mechanisms of resistance and cross-resistance to other insecticides. The se-
lection of the field strain with profenofos for five consecutive generations resulted in the LC50 values to
increase from 50.49 to 176.03 mg/ml, and the resistance ratio increased from 29.70 to 103.55 as compared
with a laboratory-susceptible strain; however, the resistance was decreased significantly when
the selected strain was reared for the next five generations without exposure to any insecticide. The
profenofos-selected strain (Profen-SEL) showed cross-resistance to chlorpyrifos and deltamethrin
but no cross-resistance observed to spinosad. Synergism studies with piperonyl butoxide and S,S,S-
tributylphosphorotrithioate indicated that the resistance to profenofos was probably associated with es-
terase and, possibly, microsomal oxidase activity. Resistance to profenofos in the selected strain suggests
that the resistance, owing to instability, could be overcome by switching off profenofos use for few gener-
ations in the field or by rotation with different insecticides having different modes of action.

KEY WORDS house fly, insecticide resistance, dairy pest management, organophosphate

The house fly, Musca domestica L. (Diptera: Musci-
dae), a common pest in urban and rural environments,
is responsible for the transmission of a number of hu-
man and animal intestinal disease agents (Kaufman
et al. 2006, Khan et al. 2012) like those that cause chol-
era, diarrhea, dysentery, poliomyelitis, and viral hepati-
tis A and E (Khan and Akram 2014). Poorly maintained
dairy or livestock farms provide ample resources to the
house flies; for example, animal manure at the farms is
a major source for both feeding and breeding of flies,
which ultimately leads to their expansion in nearby
communities (Khan et al. 2012).
Globally, house flies have developed resistance to a
variety of insecticide classes (Liu and Yue 2000,
Acevedo et al. 2009, Kaufman et al. 2010a, Khan et al.
2013c). Several factors encourage their ability to de-
velop resistance, including adaptability to varying
environmental conditions, rapid development time,
cross-resistance potential, and public health and nui-
sance issues that encourage dairy producers to apply
insecticides excessively for fly control (Kaufman et al.
1
Institute of Agricultural Sciences, University of the Punjab, La-
hore, Pakistan.
2
Corresponding author, e-mail: azhar_naturalist@yahoo.com.
3
Department of Entomology, University of Agriculture, Faisalabad,
Pakistan.
4
Department of Entomology, Bahauddin Zakariya University, Mul-
tan, Pakistan.
C The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America.
V
All rights reserved. For Permissions, please email: journals.permissions@oup.com
2010b). Recently, the development of insecticide resis-
tance to organophosphate, carbamate, pyrethroid, and
new chemical classes in different strains of house flies
has been reported from dairies in Punjab, Pakistan
(Khan et al. 2013a,b).
Profenofos is a broad-spectrum and contact-effective
organophosphate insecticide and acaricide, widely used
to control crop pests and ecotoparasites of farm animals
(Abbas et al. 2014). In Pakistan, it is mainly used for the
management of cotton bollworms and sucking insect
pests of different crops (Abbas et al. 2014). However, it
is typical for regional crop growers to use leftover insec-
ticides for the management of their dairy animal pests,
including house flies (Khan et al. 2013c), which might
encourage house flies to develop resistance. House flies
from the dairies in Punjab have recently been found to
show low to moderate level of resistance to profenofos
(Khan et al. 2013a); however, the mechanism of profe-
nofos resistance and cross-resistance potential to other
insecticides is unclear. Therefore, the aims of the pre-
sent study were to investigate the potential of a field
strain of house flies to develop resistance to profenofos,
the possible mechanisms of resistance, and its cross-
resistance potential to other insecticides.
Materials and Methods
Insects. Two strains were used in the present study: a
laboratory reference strain (hereafter “Lab-susceptible”)
2 JOURNAL OF MEDICAL ENTOMOLOGY
reared in the laboratory without exposure to any
insecticide, and a field strain collected from a dairy
farm near Lahore (31 320 5900 N; 74 200 3700 E), Pun-
jab, Pakistan, and was consecutively selected in the
laboratory with profenofos to generate a profenofos-
selected strain (Profen-SEL). Both of the strains
were reared in the laboratory (25 6 2 C, 65 6 5% rel-
ative humidity, and a photoperiod of 12:12 [L: D] h),
as discussed previously (Khan 2014, Khan et al.
2014).
Chemicals. Four commercially available insecti-
cides were used both for bioassays and selection: chlor-
pyrifos 40 EC, profenofos 50 EC, deltamethrin 10.5
EC, and spinosad 24 SC. In addition, two synergists,
S,S,S-tributylphosphorotrithioate (DEF; Sigma Ltd,
UK) and piperonyl butoxide (PBO; Sigma Ltd, UK),
were also used in synergism studies.
Selection and Bioassays. The field strain of house
flies was selected with profenofos, as described previ-
ously (Khan et al. 2014, Khan et al. 2014). Briefly, the
selection and insecticidal bioassays were performed
using a no-choice feeding bioassay method. Unmated
house flies, <1 d old, were exposed to profenofos (70%
mortality level) via cotton wicks soaked in 20% sucrose
solution for five consecutive generations (G1–G5).
Mortality data were recorded after 48 h of exposure to
profenofos and the survivors were used as parents of
the next generation.
For bioassays, at least five concentrations of profeno-
fos (causing >0 and <100% mortality) were prepared
in 20% sucrose solution. The insecticide solution was
given by moistening two cotton dental wicks (per repli-
cate) to 3–5-d-old female house flies (n ¼ 20 flies per
concentration per replication) in 250-ml plastic jars
(Khan et al. 2014, Khan et al. 2014). All bioassays were
replicated three times.
To study the stability of profenofos resistance in the
field strain of house flies, the selection of the strain was
stopped at G5 and it was reared for the next five gener-
ations (G6–G10) without exposure to any insecticide.
Then the strain was bioassayed at G11 with profenofos
and the stability of resistance was assessed by the fol-
lowing formula (Sayyed et al. 2005):
R ¼ ½log ðfinal LC50 Þ  log ðinitial LC50 Þ=n
Where, n is the number of generations reared without
exposure to insecticide.
All mortality data were analyzed by probit analysis
using the software SPSS (version 10.0) to determine
median lethal concentrations (LC50) and 95% CIs. The
LC50 values of the respective bioassays were consid-
ered significantly different on the basis of nonoverlap-
ping of 95% CIs (Litchfield and Wilcoxon 1949).
To study the mechanism of profenofos resistance, as
described previously (Khan et al. 2014, Khan et al.
2014), female house flies of the Lab-susceptible and
Profen-SEL strains were exposed topically to the
enzyme inhibitors DEF and PBO at the sublethal dose
of 10 mg per fly, 1 h before the insecticide treatment
(Liu and Yue 2000). After an hour, the treated flies
were bioassayed to different concentrations of profeno-
fos as stated above. All bioassays were performed
at 25 6 2 C, and mortality data were recorded
after 48 h.
Results
Toxicity of Different Insecticides to the House
Fly Strains. Based on the LC50 values, the toxicity of
different insecticides was significantly different. Spino-
sad proved to be more toxic to the Lab-susceptible
strain compared with the rest of the insecticides (nono-
verlapping of 95% CIs; P < 0.01), followed by profeno-
fos, chlorpyrifos, and deltamethrin. Similarly, spinosad
also proved more toxic to the newly collected field
strain (G0) followed by chlorpyrifos, profenofos, and
deltamethrin (non-overlapping of 95% CIs; Table 1).
Selection Experiment. Selection of the field strain
with profenofos for five consecutive generations
resulted in the LC50 values that increased from 50.49
to 176.03 mg/ml, together with the resistance ratio
from 29.70 to 103.55, when compared with the Lab-
susceptible strain (Table 2). However, the resistance
was unstable when the selected strain (Profen-SEL)
was reared for the next five generations without expo-
sure to any insecticide. The LC50 value of the Profen-
SEL strain declined significantly from 176.03 to
105.29 mg/ml at G10 (Table 2).
Cross-resistance of Profen-SEL Strain to Other
Insecticides. Selection of the field strain with profe-
nofos significantly affected the toxicity to chlorpyrifos
and deltamethrin in the Profen-SEL strain (Table 3).
The LC50 value of chlorpyrifos and deltamethrin
increased from 10.73 to 19.88 mg/ml and 162.58 to
291.44 mg/ml at G5, respectively, when compared with
the toxicity at G0. However, the Profen-SEL strain did
not show any cross-resistance to spinosad (overlapping
of 95 % CIs; Table 3).
Effect of Enzyme Inhibitors on the Toxicity of
Profenofos Against House Flies Strains. Against
the Lab-susceptible strain, the enzyme inhibitors PBO
and DEF when used in combination with profenofos
had no effect on reducing the LC50 values when com-
pared with the profenofos used alone. However, the
toxicity of profenofos against the Profen-SEL strain
increased when used in combination with the enzyme
inhibitors (Table 4).
Discussion
Profenofos is a broad-spectrum organophosphorus
insecticide used to manage agricultural and household
insect pests around the globe, including Pakistan. The
field-collected strain of the house fly used in the
present study, before laboratory selection with profeno-
fos, proved to be more resistant to profenofos and del-
tamethrin than the rest of the tested insecticides. In
the previous report (Khan et al. 2013a), house flies
from different areas in Punjab, Pakistan, showed low to
moderate levels of resistance to profenofos and delta-
methrin. The results of the present study are in accord-
ance with the fact that the collection of the field strain
2015 KHAN ET AL.: RESISTANCE TO PROFENOFOS IN HOUSE FLIES 3

Table 1. Toxicity of different insecticides to Lab-susceptible and newly collected field strains of house flies

Strain Insecticide n* LC50** (95% CI***) (mg/ml) Fit of probit line RR****
2
Slope (SE) v df P

Lab-susceptible Profenofos 420 1.70 (1.46–1.99) 2.45 (0.20) 4.69 4 0.32

Lab-susceptible Chlorpyrifos 420 2.01 (1.72–2.35) 2.50 (0.22) 2.57 4 0.63
Lab-susceptible Deltamethrin 420 11.20 (9.45–13.27) 2.17 (0.18) 0.62 4 0.96
Lab-susceptible Spinosad 360 0.69 (0.58–0.81) 2.50 (0.25) 2.76 3 0.43
Field (G0) Profenofos 420 50.49 (41.34–63.67) 1.84 (0.18) 1.77 4 0.78 29.70
Field (G0) Chlorpyrifos 420 10.73 (9.02–12.98) 2.15 (0.19) 3.74 4 0.44 5.34
Field (G0) Deltamethrin 480 162.58 (117.69–229.05) 1.77 (0.14) 8.33 5 0.14 14.52
Field (G0) Spinosad 420 3.54 (2.89–4.49) 1.86 (0.19) 2.31 4 0.68 5.13
*n ¼ number of flies used in bioassays and controls.
**LC50, median lethal concentration (in microgram per millilitre) estimated to give 50 % mortality.
***CI, confidence intervals.
****RR, resistance ratio ¼ LC50 of field strain/LC50 of Lab- susceptible strain.
The same applies to the following tables.

Table 2. Selection history of the field strain of house flies with profenofos

Strain n LC50 (95% CI; mg/ml) Fit of probit line RR DR*

2
Slope (SE) v df P

Field (G0) 420 50.49 (41.34–63.67) 1.84 (0.18) 1.77 4 0.78 29.70
G1 480 54.55 (43.47–71.64) 1.60 (0.16) 1.63 5 0.90 32.09
G2 420 68.82 (54.18–93.29) 1.62 (0.18) 0.77 4 0.94 40.48
G3 420 79.40 (66.30–96.68) 2.02 (0.19) 3.96 4 0.41 46.71
G4 420 158.73 (127.95–208.73) 1.95 (0.22) 2.96 4 0.56 93.37
G5 (Profen-SEL) 420 176.03 (146.22–216.42) 1.97 (0.19) 1.47 4 0.83 103.55
G10 420 105.29 (87.16–130.75) 1.98 (0.20) 1.11 4 0.89 61.94 0.045
*Rate of decrease in resistance.
G0 ¼ Field strain before the start of selection experiment.
G5 ¼ Profenofos-selected strain after five generations of continuous selection with profenofos.
G10 ¼ Profen-SEL strain after five generations without exposure to insecticides.

Table 3. Cross-resistance analysis of the Profen-SEL strain of house flies to other insecticides

Insecticide n LC50 (95% CI; mg/ml) Fit of probit line RR

Slope (SE) v2 df P

Profenofos (G0) 420 50.49 (41.34–63.67) 1.84 (0.18) 1.77 4 0.78 29.70
Profenofos (Profen-SEL) 420 176.03 (146.22–216.42) 1.97 (0.19) 1.47 4 0.83 103.55
Chlorpyrifos (G0) 420 10.73 (9.02–12.98) 2.15 (0.19) 3.74 4 0.44 5.34
Chlorpyrifos (Profen-SEL) 360 19.88 (15.61–27.50) 1.69 (0.22) 1.70 3 0.64 9.89
Deltamethrin (G0) 480 162.58 (117.69–229.05) 1.77 (0.14) 8.33 5 0.14 14.52
Deltamethrin (Profen-SEL) 420 291.44 (242.57–355.10) 1.94 (0.17) 3.40 4 0.49 26.02
Spinosad (G0) 420 3.54 (2.89–4.49) 1.86 (0.19) 2.31 4 0.68 5.13
Spinosad (Profen-SEL) 420 4.08 (3.30–5.30) 1.83 (0.19) 3.04 4 0.55 5.91

Table 4. Effect of enzyme inhibitors on the toxicity of profenofos against house flies strains

Strain Compound n LC50 (95% CI; mg/ml) Fit of probit line SR*
2
Slope (SE) v df P

Laboratory Profenofos 420 1.70 (1.46–1.99) 2.45 (0.20) 4.69 4 0.32

þDEF 360 1.33 (1.08–1.66) 1.76 (0.20) 2.25 3 0.52 1.27
þPBO 360 2.22 (1.89–2.61) 2.52 (0.24) 2.84 3 0.42 0.77
Profen-SEL (G5) Profenofos 420 176.03 (146.22–216.42) 1.97 (0.19) 1.47 4 0.83
þDEF 420 62.68 (51.95–76.32) 1.87 (0.17) 3.70 4 0.45 2.81
þPBO 420 89.79 (75.73–108.14) 2.22 (0.21) 0.94 4 0.92 1.96
* SR ¼ LC50 of profenofos alone/LC50 of profenofos along with an enzyme inhibitor.
4 JOURNAL OF MEDICAL ENTOMOLOGY
was done from a dairy farm having a history of insecti-
cide usage from different classes, including profenofos
and deltamethrin for the management of different dairy
pests. Previously, development of the field evolved
resistance to profenofos in various insect pests has
been reported from Punjab, Pakistan (Ahmad et al.
2007, Khan et al. 2011, 2013a). In Punjab, pyrethroids,
particularly deltamethrin, have been considered as
a potential candidate for the management of
different dairy animals’ pests like tick, fleas, and flies
(Muhammad et al. 2008). Regional dairy farmers used
these chemicals by different methods like dipping,
pour-on, wall sprays, or direct sprays on animals. A sur-
vey of the dairy producers (Khan et al. 2013c) also
revealed that there was a lack of systematic manage-
ment/recommendation plan for the management of
flies at the dairy farms, which forced the farmers to use
insecticides on hit and trial basis. For example, farmers
used leftover insecticide from crop farming and the
type and application doses of insecticides were usually
determined based on the availability of insecticides and
insecticide volume. This resulted in an overuse of
insecticides, which might be the cause of resistance
development in the field strain of house flies.
The selection of the field strain with profenofos for
five continuous generations resulted in an increase of
resistance ratios from 29.70- to 103.55-fold, which sug-
gest that the selection had a marked effect on the
development of resistance to profenofos. However, the
resistance was unstable/declined when the Profen-SEL
strain was reared in the absence of selection pressure.
This decline in resistance suggests that the allele (s)
responsible for resistance to profenofos might be unsta-
ble. Initial gene frequencies and relative fitness differ-
ences in the selected strain of house flies are assumed
as important stimuli that determine the stability of
resistance under laboratory conditions (Roush and
Croft 1986).
Insecticide resistance mechanisms of different insec-
ticides can be estimated by studying cross-resistance
among different insecticides. In the present study,
cross-resistance of Profen-SEL strain was observed
with chlorpyrifos and deltamethrin, suggesting a com-
mon mechanism. However, no cross-resistance has
been observed between profenofos and spinosad,
which might be due to the unique mode of action of
spinosad (Shono and Scott 2003). This type of phenom-
enon (no cross-resistance) has also been reported in
different insect pests like Blattella germanica (L.) (Wei
et al. 2001), Leptinotarsa decemlineata (Say) (Mota-
Sanchez et al. 2006), and M. domestica (Kristensen and
Jespersen 2004, Khan et al. 2014). Deltamethrin resist-
ance could be because of enhanced activity of detoxify-
ing enzymes or changes in target site sensitivity (Morin
et al. 2002). Similarly, organophosphate resistance
could also be because of detoxifying enzymes (Gunning
et al. 1999) or modification of target site (i.e., acetyl-
cholinesterase) sensitivity (Hama 1983). Cross-
resistance between profenofos and chlorpyrifos has also
been reported previously in Plutella xylostella (L.)
(Sayyed et al. 2005) and Spodoptera litura (F.) (Saleem
et al. 2008, Abbas et al. 2014). Similarly, cross-
resistance between organophosphate and pyrethroid
insecticides has also been reported previously in M.
domestica (Liu and Yue 2000), P. xylostella (Sayyed
et al. 2005), S. litura (Saleem et al. 2008), and Aedes
albopictus (Skuse) (Khan et al. 2011). Resistance to
these compounds could be associated with monooxyge-
nase, esterase activity, or both (Sayyed et al. 2010). Syn-
ergism studies with the enzyme inhibitors PBO and
DEF showed the reduction in resistance to profenofos
two- and threefold, respectively. This suggests that pro-
fenofos resistance in the Profen-SEL strain could be
associated with the activities of monooxygenase and
esterase enzymes (Sayyed et al. 2010); however, there
is a further need to confirm these mechanisms.
The findings of the present study reveal that the field
strain of house flies rapidly develop resistance to profe-
nofos when selected under laboratory conditions, and
also have ability to develop cross-resistance to other
insecticides (such as chlorpyrifos and deltamethrin) to
which it was not exposed during the selection process.
In addition, the study also reveals the rapidity of resist-
ance development to profenofos under high selection
pressure, and the early appearance of that resistance in
the field situation. At this point, dairy farmers should
be informed that the development of profenofos resist-
ance will increase under high selection pressure.
Therefore, they should be cautious when choosing
insecticides for the management of flies at dairy farms.
Cross-resistance between organophosphate and pyreth-
roid insecticides suggests that alternative insecticides
with different modes of action should be used for the
management of flies. Lack of cross-resistance between
profenofos and spinosad in the present and previous
studies (Khan et al. 2014, Khan et al. 2014) suggest
that spinosad could be used in rotation to restore the
toxicity of profenofos in the field. Moreover, instability
of profenofos resistance suggests that the toxicity of
profenofos could be restored by releasing selection
pressure in the field.
Acknowledgments
Sincere thanks to Dr. Asad Shabbir, Assistant Professor,
Department of Botany, University of the Punjab, for helping
us in improving the English language and grammar of the
manuscript. Thanks are due to the University of the Punjab,
Lahore, Pakistan, for financial assistance in publication fee of
the manuscript.
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Received 12 November 2014; accepted 17 June 2015.