Introduction

Conventional crop farming systems still rely heavily on the use of agrochemicals to manage pests and diseases and to ensure a high yield of crops to feed a continuously increasing human population1. The use of insecticides, one of the agrochemicals, to manage target insect pests is among the major agricultural management practices that may affect negatively non-target species (e.g., insects, nematodes, earthworms, molluscs) and other connected environmental resources2,3. Therefore, it is the need of the hour to protect the environment and non-target insect species while managing target insect pests1.

Non-target species in or around insecticides-treated areas receive insecticides via residual, topical or through dietary exposures that result in various toxicity symptoms4. Acute and chronic toxicity of insecticides in non-target species usually depends on several factors such as the type of insecticides, the frequency of application, the level of uptake and metabolization, non-target species in question, its age, life stage, tolerance and adaptation to insecticides5,6,7. In addition, the time of exposure to a particular insecticide is also important in risk assessment studies that results in the accumulation of insecticide residues in the exposed species and produce a delayed toxic effect with the passage of time8,9. As a result of acute and chronic exposure to insecticides, exposed species exhibited arrested growth and lengthened-development period10 or may evolve resistance to insecticides due to continued selection pressure11.

Insecticide resistance is a genetic change in response to continued selection pressure by insecticides that ultimately results in impaired chemical control in the field12. The development of resistance to insecticide in non-target species could be more alarming in situations where the species is a major pest in another situation13. For instance, the presence of human- or animal-diseases vectors such as mosquitoes and flies would be non-target species in cropping areas, and if they developed resistance to insecticides, they become difficult to control when expended to nearby human populated areas14,15,16.

Pakistan is among the major cotton (Gossypium hirsutum L.) producing countries of the world, with around 2.79 million hectares cultivated area annually17. Cotton is mainly cultivated in the two major provinces of Pakistan i.e., Punjab and Sindh. Management of insect pests of cotton is important to ensure a high yield of the crop. Therefore, farmers heavily rely on the use of insecticides as a major insect pest management tool in both of the provinces18,19. Methomyl is among the most widely and commonly used insecticides for the management of a number of insect pests of cotton such as bollworms, armyworm, aphids, mealy bug, dusky bug, jassids and whiteflies20,21. It is a broad-spectrum insecticide and belongs to the carbamate class of insecticides. In Pakistan, it is being used usually in the form of spray applications for the last four decades, due to which resistance have been reported in different target insect species21,22,23,24,25,26. It is believed that the use of insecticides on crops also results in lethal and sublethal exposure to non-target insect species around farming areas as well1,27. Previously, resistance to mathomyl in the non-target mosquito Aedes albopictus (Skuse) was reported from the cotton fields of Punjab, Pakistan28. The house fly, Musca domestica Linnaeus, is a public health pest and one of the most common non-target insect species in cotton cultivated areas29. Musca domestica has been reported as one of pollinator and nectar feeder species of cotton crop29,30,31,32,33. In this way, it is expected that M. domestica get residues of methomyl after its application during flight, pollination and/or nectar feeding activities into the cotton fields, and develop resistance to methomyl after prolonged and repeated exposures.

Therefore, the present study was planned to check the hypothesis that the use of methomyl in cotton cultivation has caused resistance development in the non-target M. domestica collected from the cotton fields of major cotton producing areas of the Punjab and Sindh provinces of Pakistan.

Materials and methods

Musca domestica strains

Twenty field strains of M. domestica were collected from major cotton producing localities of the Punjab and Sindh provinces of Pakistan (Fig. 1). Localities from the Punjab province included: Bhakkar (31.6082° N, 71.0854° E), Kasur (31.1179° N, 74.4408° E), Jhang (31.2781° N, 72.3317° E), Rajanpur (29.1044° N, 70.3301° E), Lodhran (29.5467° N, 71.6276° E), Bahawalpur (29.3544° N, 71.6911° E), Bahawalnagar (30.0025° N, 73.2412° E), Dera Ghazi (D.G.) Khan (30.0489° N, 70.6455° E), Rahim Yar (R.Y.) Khan (28.4212° N, 70.2989° E), Mianwali (32.5839° N, 71.5370° E), Toba Tek Singh (30.9709° N, 72.4826° E), Muzaffargarh (30.0736° N, 71.1805° E), Layyah (30.9693° N, 70.9428° E) and Vehari (30.0442° N, 72.3441° E)34. Localities from the Sindh province included: Sukkur (27.7244° N, 68.8228° E), Khairpur (27.5256° N, 68.7551° E), Jamshoro (25.4304° N, 68.2809° E), Ghotki (28.0271° N, 69.3235° E), Sanghar (26.0436° N, 68.9480° E) and Dadu (26.7341° N, 67.7795° E)34. Cotton fields for M. domestica collection in the above localities were chosen based on history of methomyl use for the management of insect pests of cotton (personal communication with regional farmers and agriculture extension workers). An insecticide susceptible reference strain (Lab-susceptible) of M. domestica35,36 was used in bioassays for the estimation of resistance to methomyl in field strains. Field strains were collected at the adult stage and brought to the laboratory of entomology, University of the Punjab, Lahore (31.5204° N, 74.3587° E)34. All strains were reared under the laboratory conditions (12:12 h dark/light photoperiod, 26 ± 2 °C, and 65 ± 5% relative humidity) following a well-established methodology using a sugar–milk-based diet37,38. Flies were reared in mesh cages, and pupae of specific date/time duration of a preceding generation were separated and kept in a new/empty cage for starting the subsequent generation. Adults from pupae usually emerged in 4–5 days; in this way flies of required age could easily be collected for bioassays. The first generation (F1) of field-collected strains was used for bioassays.

Figure 1
figure 1

Collection sites of Musca domestica from the cotton fields of Punjab and Sindh provinces of Pakistan (Source Wikimedia commons).

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Bioassays and data analyses

Technical-grade methomyl (> 95% purity; Chem Service Inc, West Chester PA) was used for resistance detection bioassays in field strains of M. domestica. Topical bioassay method was used to apply methomyl doses on M. domestica as stated earlier in author's previous reports 15,39:

“Briefly, 0.5 μL of insecticide in acetone solution was applied by using a micropipette (0.1–2 µL, Acura ® manual 825, Socorex, Switzerland) on thoracic notum of 3–5-day-old female M. domestica. M. domestica were exposed to a range of methomyl doses that caused > 0 and < 100% mortality, and each bioassay was consisted of 20 M. domestica per dose. In the control treatment, flies were treated with acetone alone. Treated flies were kept in plastic jars (250 mL) provided with a cotton dental wick soaked with 20% sugar solution. All the bioassays were conducted at 26 ± 2 °C, 60 ± 5% RH, 12:12 (L/D) photoperiod, and replicated three times on different days. Mortality counts were made 48-h post-treatment and the data were analyzed by Probit analysis (Finney 1971) to determine median lethal doses (LD50s) of insecticides tested. Resistance ratios (RRs) were calculated by dividing LD50 values of different field strains to those obtained with the Lab-susceptible reference strain, and categorized as high resistance (RR = 51–100 fold), moderate resistance (RR = 21–50 fold), low resistance (RR = 11–20 fold), very low resistance (RR = 2–10 fold) and no resistance (RR = 1)”15,39.

For synergism bioassays, M. domestica were exposed topically to enzyme inhibitors: piperonyl butoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) (Chem Service Inc, West Chester PA), with the maximum sublethal dose of 10 µg fly−1, one hour before the insecticide treatment40,41. Treated M. domestica were then exposed to methomyl doses as stated above. Synergism ratio (SR) was calculated by dividing the LD50 value of a particular strain to methomyl alone by the LD50 value of methomyl of the corresponding strain along with PBO or DEF. The SR value was considered significantly different if its 95% fiducial limit (FL) did not include “1” on the basis of the ratio test42.

Results

Field strains of M. domestica collected from different localities of the Punjab and Sindh provinces exhibited variable toxicity and resistance levels to methomyl compared with the Lab-susceptible strain (Table 1). The Lab-susceptible strain was the most susceptible one among all strains of M. domestica with the LD50 value 0.77 µg fly−1. Toxicity values of methomyl for Punjabi strains ranged from 28.07 to 136.16 µg fly−1. Amomg Punjabi strains of M. domestica, the Mianwali strain exhibited the lowest LD50 value (28.07 µg fly−1) for methomyl followed by R.Y. Khan (42.65 µg fly−1), Muzaffargarh (47.10 µg fly−1), Layyah (49.14 µg fly−1), Vehari (49.71 µg fly−1), Kasur (62.88 µg fly−1), Bhakkar (72.22 µg fly−1), Jhang (80.16 µg fly−1), Rajanpur (87.13 µg fly−1), Bahawalnagar (116.01 µg fly−1), Toba Tek Singh (117.78 µg fly−1), Bahawalpur (116.01 µg fly−1), Lodhran (124.38 µg fly−1) and D.G. Khan (136.16 µg fly−1) strains. In the case of Sindhi strains of M. domestica, the highest toxicity of methomyl was recorded in the Dadu strain (29.32 µg fly−1) followed by Ghotki (44.92 µg fly−1), Jamshoro (50.68 µg fly−1), Khairpur (74.64 µg fly−1), Sanghar (80.41 µg fly−1) and Sukkur (136.87 µg fly−1) strains (Table 1).

Table 1 Toxicity and resistance levels of Musca domestica strains to methomyl.
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Resistance ratio (RR) values ranged from 36.45 to 176.83 fold for Punjabi strains, and 38.08 to 177.75 fold for Sindhi strains, compared with the Lab-susceptible strain of M. domestica (Table 1). Among Punjabi strains, D.G. Khan, Lodhran, Bahawalpur, Toba Tek Singh, Bahawalnagar, Rajanpur and Jhang strains exhibited very high levels of resistance (RR > 100) to methomyl; Bhakkar, Kasur, Vehari, Layyah, Muzaffargarh and R.Y. Khan showed high resistance (RR = 51–100 fold), while the Mianwali strain showed a moderate level of resistance to methomyl (RR = 36.45 fold). In case of Sindhi strains, very high levels of resistance (> 100 fold) were reported for Sukkar and Sanghar strains, high levels of resistance (RR 51–100 fold) for Khairpur, Jamshoro and Ghotki, and moderate resistance to methomyl (38.08 fold) in the Dadu strain (Table 1).

Except the Lab-susceptible strain, toxicity of methomyl in all field strains increased significantly, based on non-overlapping 95% FL of LD50 values and significant synergism ratios (SR), when bioassayed along with either PBO or DEF (Table 2). For instance, the LD50 value of the Bhakkar strain reduced (toxicity increased) from 72.22 to 24.33 and 26.74 µg fly−1 when bioassayed in the presence of PBO and DEF, respectively. The LD50 value of the Kasur strain reduced from 62.88 to 33.05 and 32.21 µg fly−1 when bioassayed in the presence of PBO and DEF, respectively. The LD50 value reduced from 80.16 to 33.15 and 24.47 µg fly−1 for the Jhang strain; from 87.13 to 20.40 and 29.17 µg fly−1 for the Rajanpur strain; from 124.38 to 77.00 and 67.61 µg fly−1, when bioassayed in the presence of PBO and DEF, respectively. A similar trend of increased toxicity was observed with the rest of the Punjabi and Sindhi strains of M. domestica. Synergism ratios were significant in all the cases of field strains based on 95% FL of SR values did not include 01 (Table 2).

Table 2 Synergism of methomyl toxicity by the enzyme inhibitors in different strains of Musca domestica.
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Discussion

It is a matter of great concern that the use of pesticides in agriculture often poses negative impact on non-target organisms, including insect species43,44. Based on the data of last few years, it has been observed that the use of insecticides in cropping systems resulted in the occurrence of resistance in the target and non-target insect species in Pakistan, which shows that this phenomenon is extremely widespread1,15. The present work could be considered as a continuation of our efforts to explore side-effects of insecticidal usage in agriculture on non-target insect species, providing additional data of the impact of methomyl on M. domestica from localities not already explored. The data clearly indicates the occurrence of field-evolved resistance to methomyl in field strains of M. domestica in comparison to the Lab-susceptible strain. According to Valles et al.45, an insect strain should be assumed resistant to a particular insecticide if it shows more than tenfold RR value in comparison to the susceptible or reference strain. The data of the present study revealed that all of the field strains were resistant to methomyl and exhibited more than tenfold RR values in comparison to the Lab-susceptible strain.

The susceptibility of reference strains of M. domestica to methomyl varies in different reports, depending upon strain origin, rearing conditions, and/or bioassay methods. While this does not undermine such studies, it is valuable to refer literature estimates as a rough means of comparison46. In the present study, the LD50 value of methomyl for the Lab-susceptible strain (0.77 µg fly−1), is greater than UCR (0.58 µg fly−1)46, WHO (0.10 µg fly−1)47 and Cooper (0.07 µg fly−1)48 strains. Resistance ratio values for both Punjabi and Sindhi strains ranged from moderate to high levels, compared with the Lab-susceptible strain of M. domestica. Among Punjabi strains, D.G. Khan, Lodhran, Bahawalpur, Toba Tek Singh, Bahawalnagar, Rajanpur and Jhang strains exhibited very high levels of resistance (RR > 100) to methomyl; Bhakkar, Kasur, Vehari, Layyah, Muzaffargarh and R.Y. Khan showed high resistance (RR = 51–100 fold), while the Mianwali strain showed a moderate level of resistance to methomyl (RR = 36.45 fold). In case of Sindhi strains, very high levels of resistance (> 100 fold) were reported for Sukkar and Sanghar strains, high levels of resistance (RR 51–100 fold) for Khairpur, Jamshoro and Ghotki, and moderate resistance to methomyl (38.08 fold) in the Dadu strain. Resistance to methomyl could be due to the fact that field strains were collected from the cotton fields where methomyl was being used as one of the major insecticides to manage different insect pests such as bollworms, armyworm, aphids, mealy bug, dusky bug, jassids and whiteflies20,49. It is assumed that variation in resistance levels or toxicity in different strains might be due to the differences in origin of strains, climatic factors of collection sites and/or history of insecticide exposure. Variations in toxicity to insecticides due to these reasons have also been documented for different insect pests50,51,52,53,54,55,56.

Variable levels of resistance to methomyl in M. domestica have been reported from different countries in the past46,57,58,59. Previously, we also have reported low levels of resistance to methomyl in M. domestica strains collected from dairy farms in different localities, other than the ones in the present work, of Punjab, Pakistan37. Methomyl was used to target/manage M. domestica in dairy farms. However, in the present study, M. domestica strains were collected from the cotton fields where these are non-target species. Insecticidal usage in crops, besides controlling target pests, usually results in the lethal and sublethal exposures to non-target species that ultimately make these species resistant to insecticides with the passage of time10,11. Recently, resistance development has been reported in M. domestica and Aedes albopictus due to non-targeted exposure to insecticides used in rice farming1,15. Methomyl formulation has been registered in the form of emulsifiable concentrate (EC) and applied as sprays to manage different insect pests of cotton in Pakistan. Sprays of insecticides contaminate plant parts, soil, water and the surrounding air for a certain period of time11,15. It is believed that M. domestica get direct and/or indirect exposure to methomyl sprays during their routine life activities and developed resistance to methomyl as evidenced by the data of the present study.

Resistance to methomyl could be due to the activation of metabolic enzymes such as microsomal oxidases, esterases, etc., which can be initially checked by the use of enzyme inhibitors along with insecticides in bioassays51,60. Synergism of methomyl by PBO and DEF in Helicoverpa armigera (Hübner)60, M. domestica61 and Oxycarenus hyalinipennis Costa26 inferred that resistance may be attributable to microsomal oxidase and esterase detoxification. In the present work, synergism of methomyl with PBO and DEF in all the field strains was observed suggesting the possibility of metabolic mechanism of resistance. More in vitro studies are needed to further confirm the role of metabolic resistance mechanism in field strains of M. domestica.

Conclusion

The finding that non-target M. domestica has evolved resistance to methomyl used for the management of insect pests of cotton is troubling evidence of the side-effects of insecticidal usage in crop farming. The development of insecticide resistance in non-target species as a result of insecticide application against the targeted species usually lead to the outbreak of former species11. M. domestica is one of the major medical and veterinary pests and the development of resistance to insecticides may promote its outbreak coupled with an increased incidence of fly-borne diseases. Therefore, it is important to perform risk assessment studies in order to determine side-effects of a particular insecticide on non-target species before and after its approval for use in cropping systems.