ORIGINAL PAPER

Resistance to new chemical insecticides in the house fly,Musca domestica L., from dairies in Punjab, Pakistan

Hafiz Azhar Ali Khan & Sarfraz Ali Shad & Waseem Akram

Received: 1 September 2012 /Accepted: 19 February 2013 /Published online: 3 March 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract The house fly, Musca domestica L., is one of themajor pests in dairy operations that has developed resistanceto a number of insecticides with different modes of action.Adult house fly populations from six dairies in Punjab,Pakistan were evaluated for resistance to insecticides withnovel modes of action (abamectin, emamectin benzoate,fipronil, imidacloprid, indoxacarb, and spinosad). Signifi-cant levels of resistance to most of the insecticides testedwere observed in the present study. For avermectins at LC50

level, the resistance ratios were in the range of 38.40 to94.44-fold for abamectin and 13.16 to 36.30-fold foremamectin benzoate. Fipronil LC50 resistance ratiosexceeded 10-fold in three house fly populations, while allthe populations had >10-fold resistance ratios forimidacloprid. Indoxacarb and spinosad had the lowest resis-tance ratios that ranged from 3.02 to 7.12-fold forindoxacarb and 2.91 to 9.0-fold for spinosad. As the resis-tance to fipronil, indoxacarb, and spinosad are emerging,therefore these chemicals should be used cautiously in man-agement programs to retain the efficacy for longer times.

Introduction

The house fly, Musca domestica L. (Diptera: Muscidae), isone of the major pests of dairies with the potential to transmitpathogens, cause irritation in animals and humans, and has theability to disperse off-farm areas (Khan et al. 2012). A varietyof insecticides from pyrethroid, organophosphate, carbamate,

and new chemical classes have been used for the managementof house flies worldwide (Shi et al. 2011). Unfortunately,house flies have developed resistance to most of the insecti-cides; therefore, this phenomenon has become one of themajor global concerns of present day research (Akiner andCaglar 2006; Butler et al. 2007).

Insecticide resistance is one of the major constraints inthe management of insect pests of agricultural and publichealth importance (Khan et al. 2011). Resistance often re-sults in increased application rates and frequency of insec-ticide use, and ultimately loss of efficacy. Therefore,resistance management programs are of utmost importanceso as to decrease pesticide usage and limit its harmfulimpacts on humans and the environment. In Pakistan, thereis no control program or systematic study on monitoringresistance to insecticides in house flies (Ahmed andIrfanullah 2007). Owing to lack of systematic managementplans, different insecticide usage has been observed in dairyfarmers’ survey in Punjab, during 2011–2012 (Khan,unpublished data). For example, farmers had no concernsabout the type or specificity of insecticides against a partic-ular dairy pest. The majority of the farmers were also en-gaged in the crop or vegetable farming and used differenttypes of pesticides (organochlorine, organophosphates, car-bamates, pyrethroids, and new chemicals) for the manage-ment of insect pests. Farmers reported the use of leftoverpesticides from crop farming against a wide variety of pestslike ticks, fleas, buffalo lice, and flies. The survey suggestedthat the dose and time of application largely depended onthe volume and availability of leftover pesticides. The pes-ticides used in the dairies were from organochlorine, organ-ophosphate, carbamate, pyrethroid, and new chemicalclasses. Moreover, farmers used their own experience orthat of fellow farmers in these practices rather than consult-ing entomologists or veterinarians. These practices couldexert resistance selection pressure in different insect pestsexposed to pesticides in or around dairies.

H. A. A. Khan (*) : S. A. ShadDepartment of Entomology, Bahauddin Zakariya University,Multan, Pakistane-mail: azhar_naturalist@yahoo.com

W. AkramDepartment of Agri-Entomology, University of Agriculture,Faiasalabd, Pakistan

Parasitol Res (2013) 112:2049–2054DOI 10.1007/s00436-013-3365-8

Insecticide resistance in house flies from different partsof the world has been reported quite frequently (Deacutis etal. 2006; Kaufman et al. 2006; Acevedo et al. 2009; Memmi2010); however, to the best of the authors’ knowledge, thereis no report on the status of new chemical insecticide resis-tance in Punjab, Pakistan. We conducted a survey on resis-tance to insecticides from different classes in six localities ofPunjab, Pakistan. The aims were to develop proactive in-secticide resistance management/prevention program and todefine baseline data for future monitoring efforts.

Materials and methods

Insects

Adult house flies were collected by sweep netting from thedairies located in six cities of Punjab province, Pakistan:Multan (30° 11′ 44 N; 71° 28′ 31 E), Shorkot (30° 46′ 60 N;72° 15′ 0 E), Okara (30° 49′ 34″N; 73° 32′ 28″ E), Faisalabad(31° 26′ 0″N; 73° 6′ 0 E), Lahore (31° 32′ 59 N; 74° 20′ 37 E),and Sargodha (32° 1′ N; 72° 40′ E). The dairies were morethan 50 km apart; therefore, it could be assumed that thecollected flies were distinct populations. The selected dairieshad heavy usage of pesticides (see above) for the managementof various dairy pests including flies. The adults were broughtto the laboratory and colonies of each strain were establishedto ensure flies were healthy and pathogen free. The adultswere held in mesh cages provided with sugar, milk powder,and water. The larvae were reared on a medium of wheat bran,grass meal, yeast, powdered milk, and sugar with a ratio of4:2:1:0.3:0.3 by weight, respectively (Bell et al. 2010). Houseflies were maintained at 25±2 °C, 60±5 % RH, and 12:12light/dark photoperiod. The F1 generation of field-collectedflies was used for bioassays. The laboratory susceptible strainwas collected from very low chemical use zone andmaintained in the laboratory without exposure to insecticides.This strain was used for resistance monitoring.

Chemicals

The commercial formulations of six new chemical insecticidesused for bioassays comprised fipronil (Regent® 36EC, BayerCrop Sciences), spinosad (Tracer® 24SC, Dow Agro-Sciences), emamectin benzoate (Proclaim® 019EC,Syngenta), indoxacarb (Steward® 15SC, DuPont), abamectin(Agrimec™, Syngenta), and imidacloprid (Confidor® 20SL,Bayer Crop Sciences).

Bioassays

The toxicity of different insecticides was assessed by feed-ing bioassay method (Kaufman et al. 2006; Khan et al.

2013). Briefly, 20 female flies (3–5 days old) were intro-duced in 250 ml plastic container. Two pieces of cottondental wick (2 cm length) moistened with 20 % sucrosesolution having different concentrations of insecticides wereplaced in each plastic container. At least five to sevenconcentrations were prepared for each insecticide and eachconcentration was replicated three times. The control wasconsisted of a cotton wick soaked in 20 % sucrose solution,without the addition of any insecticide. To avoid dryingeffects, cotton wicks were hydrated at 24 and 48 h. Bio-assays were conducted at ±2 °C, 60±5 % RH, and 12:12light/dark photoperiod. Final mortality was assessed after72 h of exposure to insecticides. The flies were assumeddead if these were ataxic.

Data analysis

The data of three replicates were pooled and, if necessary,were corrected by Abbott’s formula (1925) and analyzed byprobit analysis (Finney 1971) using SPSS software (Version10.0 for Windows, SPSS Inc., Chicago, USA). The levels ofresistance were classified based on the following scale: resis-tance ratio (RR) = 1, no resistance; RR = 2–10, very lowresistance; RR = 11–20, low resistance; RR = 21–50, moder-ate resistance; RR = 51–100, high resistance; and RR = >100,very high resistance (Ahmed and Arif 2009; Khan et al. 2013).

Results

The results of bioassays revealed that the highest level ofresistance was found with abamectin (Table 1). The level ofresistance was moderate to high with resistance ratios rang-ing from 38.40 to 94.44-fold. All the field-collectedpopulations of flies were more resistant to abamectin thanthe lab susceptible strain with the highest resistance levelobserved in the Okara population (94.44-fold RR).

Low to moderate levels of resistance to emamectin ben-zoate were found in all the field-collected populations ofhouse flies with the resistance ratios ranging from 13.16 to36.30-fold. The highest level of resistance was found in theSargodha strain (36.30-fold RR) followed by the Shorkotstrain (29.29-fold RR) (Table 1).

Among the six populations tested for fipronil, three hadvery low level of resistance (6.3–9.64-fold RR), while therest of the populations had low to moderate levels of resis-tance (10.01–22.61-fold RR). The Sargodha and Multanpopulations had relatively higher resistance ratios than thoseof the remaining populations (Table 1).

All the tested populations of house flies had significantlylow to moderate levels of resistance to imidacloprid withvalues ranging from 10.02 to 23.81-fold RR. The Lahoreand Sargodha populations had relatively high levels of

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Table 1 Toxicity of new chemical insecticides to adults of M. domestica from the dairies in Punjab, Pakistan

Population n LC50 (95 % CI) (μg/ml) Fit of probit line RR

Slope (SE) χ2 df Pa

Abamectin Lab susceptible 360 1.03 (0.71–1.52) 2.56 (0.24) 5.67 3 0.13 –

Multan 420 45.56 (37.84–56.12) 1.98 (0.19) 1.94 4 0.75 44.23b

Shorkot 480 77.65 (61.38–101.98) 1.41 (0.13) 5.65 5 0.34 75.39b

Okara 360 97.28 (62.40–218.19) 1.36 (0.23) 2.25 3 0.52 94.44b

Faisalabad 480 43.37 (36.13–53.17) 2.00 (0.18) 7.22 5 0.21 42.11b

Lahore 360 70.68 (56.01–95.55) 1.70 (0.21) 3.47 3 0.32 68.62b

Sargodha 480 39.55 (30.50–51.99) 1.16 (0.12) 3.20 5 0.67 38.40b

Emamectin Lab susceptible 420 2.92 (2.47–3.45) 2.21 (0.19) 4.71 4 0.32 –

Multan 480 63.44 (52.50–77.87) 1.80 (0.16) 1.53 5 0.91 21.73b

Shorkot 420 85.53 (66.65–115.) 1.35 (0.15) 3.14 4 0.54 29.29b

Okara 420 38.43 (31.20–48.52) 1.65 (0.17) 1.92 4 0.75 13.16b

Faisalabad 360 64.21 (52.50–82.04) 1.95 (0.23) 0.75 3 0.86 21.99b

Lahore 480 56.70 (45.18–72.85) 1.39 (0.13) 2.39 5 0.79 19.42b

Sargodha 480 106.38 (84.85–139.37) 1.60 (0.15) 6.66 5 0.25 36.30b

Fipronil Lab susceptible 360 2.01 (1.37–3.01) 2.43 (0.24) 5.62 3 0.13 –

Multan 360 42.57 (30.00–76.99) 1.72 (0.28) 1.25 3 0.72 21.18b

Shorkot 420 13.98 (11.07–18.60) 1.56 (0.17) 4.42 4 0.35 6.96b

Okara 480 19.37 (12.65–38.43) 1.69 (0.18) 10.2 5 0.07 9.64b

Faisalabad 360 12.66 (10.42–15.83) 1.91 (0.21) 0.86 3 0.83 6.30b

Lahore 420 20.12 (16.50–25.12) 1.76 (0.17) 4.23 4 0.38 10.01b

Sargodha 420 45.44 (27.76–59.40) 1.71 (0.20) 8.80 4 0.07 22.61b

Imidacloprid Lab susceptible 420 4.91 (4.16–5.79) 2.28 (0.20) 2.74 4 0.60 –

Multan 420 59.93 (41.74–103.44) 1.28 (0.17) 2.23 4 0.69 12.21b

Shorkot 420 71.76 (48.82–120.85) 1.31 (0.18) 3.90 4 0.42 14.62b

Okara 420 49.18 (38.03–67.42) 1.33 (0.15) 0.61 4 0.96 10.02b

Faisalabad 420 81.96 (64.75–107.88) 1.43 (0.16) 5.99 4 0.20 16.69b

Lahore 420 116.92 (91.80–157.98) 1.52 (0.17) 4.53 4 0.34 23.81b

Sargodha 420 99.15 (70.41–163.47) 1.24 (0.17) 2.78 4 0.59 20.19b

Indoxacarb Lab susceptible 420 1.62 (1.34–1.94) 2.07 (0.19) 1.21 4 0.88 –

Multan 420 7.74 (6.37–9.52) 1.78 (0.17) 2.94 4 0.57 4.78b

Shorkot 480 9.85 (6.78–15.89) 1.54 (0.14) 8.73 5 0.12 6.08b

Okara 480 4.89 (4.05–5.89) 1.90 (0.17) 3.08 4 0.55 3.02b

Faisalabad 480 11.53 (8.03–18.47) 1.70 (0.16) 9.05 5 0.11 7.12b

Lahore 420 8.23 (6.55–10.58) 1.43 (0.15) 0.77 4 0.94 5.08b

Sargodha 360 8.49 (6.77–11.34) 1.73 (0.21) 3.68 3 0.30 5.24b

Spinosad Lab susceptible 360 0.75 (0.63–0.88) 2.38 (0.24) 0.57 3 0.90 –

Multan 420 2.18 (1.74–2.79) 1.50 (0.16) 0.81 4 0.94 2.91b

Shorkot 420 6.75 (5.33–9.00) 1.53 (0.17) 3.87 4 0.42 9b

Okara 480 3.87 (3.15–4.86) 1.61 (0.14) 6.13 5 0.29 5.16b

Faisalabad 480 4.04 (3.36–4.93) 1.88 (0.16) 7.79 5 0.17 5.39b

Lahore 360 2.68 (2.15–3.44) 1.61 (0.20) 1.40 3 0.71 3.57b

Sargodha 420 4.09 (3.43–4.94) 2.02 (0.18) 4.92 4 0.30 5.45b

RR resistance ratio = LC50 of field population/LC50 of lab susceptible populationaP values are based on Chi-square goodness of fit test. P values >0.05 suggest goodness of fit of the modelb Statistically different from lab susceptible, based on non-overlapping of 95 % confidence interval (CI)

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resistance while the remaining populations had considerablylow level of resistance (Table 1).

Concerning indoxacarb and spinosad, all the testedpopulations had very low levels of resistance ranging from3.02 to 7.12-fold for indoxacarb and 2.91–9.0-fold forspinosad. Resistance to indoxacarb was relatively high inthe Faisalabad strain compared to the other populationstested. Resistance to spinosad was relatively high in theShorkot population compared to the other populations tested(Table 1).

In all the cases, field-collected house fly populations hada significantly shallower regression line slopes compared tothe laboratory susceptible population (P<0.05).

The resistance to indoxacarb had significant (P<0.05) pos-itive correlation with emamectin bemzoate and imidaclopridinsecticides (Table 2).

Discussion

The present study was conducted to assess the toxicity of sixinsecticides having novel modes of action against house fliesfrom the dairies in Punjab, Pakistan. The results indicate thathouse fly populations have moderate to high levels of resis-tance to abamectin, emamectin benzoate, and fipronil. Resis-tance to imidacloprid was low to moderate while theresistance to spinosad and indoxacarb was very low. It hasbeen recommended that any insect population should not beassumed resistant until and unless the resistance ratio of 10 isobserved (Valles et al. 1997). In the present study, all the field-collected populations of house flies had resistance ratios <10to both indoxacarb and spinosad, while three populations hada resistance ratio <10 to fipronil. Therefore, we would con-sider these populations as tolerant rather than resistant. Thehouse fly resistance to insecticides having novel modes ofaction has previously been reported from different parts of theworld (Kaufman et al. 2006, 2010a, b; Acevedo et al. 2009;Memmi 2010). The present study suggests that house flieshave evolved resistance to various new chemical insecticidesdue to possible cross-resistance mechanisms. Moreover, asignificant correlation between indoxacarb and emamectin orindoxacarb and imidacloprid also points to the occur-rence of a common insecticide resistance mechanismfor the chemicals despite different modes of action.

The insecticides tested in the present experiment haddifferent action sites. For instance, avermectins andfipronil groups act on the GABA-gated chloride channel(Kristensen et al. 2004; Ahmed et al. 2008). However,the level of resistance in house flies to emamectin andfipronil was nearly identical.

In the present study, moderate to high level of resistance toavermectins could be attributed to the intensive use of thesechemicals for the management of cattle and buffalo ticks andlice. Avermectins have been strongly recommended for thesepests in the dairies of Punjab (Muhammad et al. 2008). Adairy farmers’ survey in Punjab during 2011–2012 (Khan,unpublished data) revealed that the majority of the farmersused leftover insecticides from crop farming without anysystematic plan, for the management dairy insect pests. Theyhad no concern about the nature or class of insecticides used inpesticide applications. Moreover, the dose of pesticides andmodes of application largely depended on the availability,amount of chemicals, and application equipments. The inap-propriate and injudicious use together with improper applica-tion techniques could also be responsible for the developmentof resistance to various insecticides that might be more severein the future. Low to moderate resistance levels to imidaclopridwere detected in the present study. The resistance toimidacloprid has previously been reported from different partsof the world (Kaufman et al. 2006, 2010a, b; Memmi 2010).

Spinosad is an effective chemical, derived from the soilbacterium Saccharopolyspora spinosa. It has very lowmammalian toxicity, relatively low affinity towards non-target or beneficial insects and has been used extensivelyfor the management of different insect pests including houseflies worldwide (Shi et al. 2011). However, various insectpests have developed resistance to spinosad in differentparts of the world, e.g., Plutella xylostella (Mau andGusukuma-Minuto 2001), Helicoverpa armigera (Wang etal. 2009), Spodoptera exigua (Wang et al. 2006), Drosoph-ila melanogaster (Perry et al. 2007), and M. domestica (Shiet al. 2001). Fipronil and indoxacarb belongs tophenylpyrazole and oxadiazine groups, respectively, andhave strong potential against different insect pests includinghouse flies (Kristensen et al. 2004). In the present study,very low level of resistance to spinosad, indoxacarb, andfipronil (from three sampling sites) is intriguing. This phe-nomenon may reflect two points: (1) low usage of these

Table 2 Pairwise comparison ofcorrelation coefficients of toxic-ity of different insecticides tofield populations of M.domestica

*P>0.05; **P<0.05

Abamectin Emamectin Fipronil Imidacloprid Indoxacarb

Emamectin 0.30*

Fipronil 0.10* 0.67*

Imidacloprid 0.42* 0.70* 0.46*

Indoxacarb 0.33* 0.78** 0.30* 0.77**

Spinosad 0.62* 0.69* 0.06* 0.42* 0.66*

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chemicals and/or (2) the presence of an independent resistancemechanism (Ahmed et al. 2008). Further studies are needed toexplore this phenomenon by selecting the population in thelaboratory. The experiments on the selection of house flies withspinosad are currently under evaluation in our laboratory tostudy mechanisms of resistance in the regional population.

The work presented here reveals that owing lack ofsystematic management programs, dairy farmers practicechemical approaches for the management of dairy insectpest on opportunistic and/or hit and trial basis. Thus, theoccurrence of insecticide resistance in dairy pests includinghouse flies is inevitable. A systematic plan for the manage-ment of dairy pests including flies is needed so that thedevelopment of resistance could be delayed at maximum.Some examples of resistance management strategies inhouse flies may include the application of insecticide mix-tures with different modes of action, restriction of insecti-cide use to which resistance has been developed, and/orrotation of different insecticides (Kaufman et al. 2001;Memmi 2010; Khan et al. 2013). The use of insect growthregulators in combination with biocontrol agents may alsoprove effective in resistance management (Srinivasan andAmalraj 2003). Since very low levels of resistance havebeen observed to spinosad, indoxacarb, and fipronil, thesechemicals should therefore be used cautiously in manage-ment plans to retain their effectiveness for longer times.Moreover, continuous resistance monitoring should beplanned that will help us to identify the efficacy of insecti-cides for the management of house flies.

Fipronil, indoxacarb, and spinosad resistance are emerg-ing, therefore these chemicals should be used cautiously inmanagement plans to retain their efficacy for longer times.Besides insecticide resistance, developing countries includ-ing Pakistan have the problems of insecticide usage for themanagement of different insect pests of agricultural impor-tance (Sayyed et al. 2005). Some important problems in-clude: the use of inappropriate insecticides at incorrectdoses, the use of adulterated or substandard pesticide for-mulations, and the use of ineffective application methods.With this, dairy farmers should be trained regarding theproper use of appropriate chemicals to minimize the occur-rence of insecticide resistance.

Acknowledgments The authors thank the Higher Education Com-mission, Pakistan, for providing funds to carry out this study.

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