Development of Resistance in Field Housefly (Musca Domestica): Comparison of Effects of Classic Spray Regimes versus Integrated Control Methods

Koãi‰ová A. , P. Novák, J . Toporãák, M. Petrovsk ̆: Development of Resistance in Field Housefly (Musca domestica): Comparison of Effects of Classic Spray Regimes versus Integrated Control Methods. Acta Vet. Brno 2002, 71: 401-405. The development of resistance in the housefly (Musca domestica) to azamethiphos, pirimiphosmethyl, bendiocarb, permethrin, cypermethrin and deltamethrin was investigated on pig farms over a 4-year period. The results obtained in laboratory tests were compared with those obtained under practical conditions in pig houses. An intensive use of insecticides induced resistance in the course of 2 to 3 seasons. The classic spray regimes of insecticides led to the development of high resistance after one or two seasons. Integrated control, based on rotational application – organophosphate, pyrethroid, carbamate, pyrethroid, organophosphate – retained the resistance at low to moderate levels. Because of the variability of resistance factor (RF) in the field populations observed, the monofactorial and rotational selective pressure of insecticides was investigated. The rotational application of azamethiphos and permethrin or cypermethrin having sufficient insecticidal effect retained the resistance at low to moderate levels over a 4-year period. Although the alternation of insecticides cannot prevent the development of resistance, it can extend several times the period of their successful application on farms. This knowledge can help to formulate the strategies for fly control programmes. Muscidae, nuisance flies, resistance, organophosphates, carbamates, pyrethroids, control Because of increasing awareness of risks of insects, great attention of veterinarians is paid not only to well-known blowfly species causing myiases of birds and mammals, but also to other non-biting and blood-sucking flies that affect animal welfare and health in a variety of ways, including nuisance, fly worry and dissemination of diseases (F ischer 1999 and 2000). The house fly, Musca domestica (L.) can be managed to some extent by sanitation measures that reduce accumulation of waste materials that serve as breeding sites. For the most part, however, fly control is most commonly achieved with insecticides, but unfortunately, house flies have shown a remarkable ability to develop resistance to these. On the other hand, knowledge about the immunosuppressive effects of insecticides and their possible interference with the genetic material of live organisms indicates that it is necessary to restrict gradually the extensive use of a broad spectrum of pesticides through accentuated application of scientifically justified agrotechnical procedures (Kaãmár et al. 1999). The control of flies in animal houses where they find both attractive substrates for feeding and breeding and an all-year-round suitable environmental temperature important for their rapid reproduction is not a simple task and is becoming a worldwide problem. The main reason is that they have high reproductive potential and acquire resistance to insecticides rather rapidly. European countries, including Slovakia, are no exception to this rule. After many years of the use of pyrethroids, organophosphates and carbamates we are confronted ACTA VET. BRNO 2002, 71: 401–405 Address for correspondence: MVDr. A. Koãi‰ová PhD Department of Environmental Protection University of Veterinary Medicine Komenského 73, 041 81 Ko‰ice, Slovak Republic Phone: +421 55 333 8175 Fax: + 421 55 638 3666 E-mail: kocisova@uvm.sk http://www.vfu.cz/acta-vet/actavet.htm with the serious problem of resistance to these compounds (Koãi‰ová 1998). Flies are so flexible that it is completely absurd to consider their total eradication. We recognize several important factors that have undoubtedly an unfavorable effect on the successful control of flies. One of the most important is the limited range of really potent insecticides due to the present state of resistance. Therefore, it is necessary to apply as extensively as possible the rules of integrated control of flies which means the use of combinations of the greatest possible variety of products, application methods and the ways of fly control. In our study we present results of the comparison of effects of monofactorial and rotational and combinational insecticide selection pressure on the development of resistance. Investigations of resistance stability in field populations can help to formulate strategies for fly control programmes in practice. Materials and Methods Populat ions of f l ies tes ted Wild populations of flies were collected on 9 farms for rearing of pigs. Flies were kept and prepared for individual tests in an insectary at 24–27 oC under standard conditions and methods (Rupe‰ and Rett ich 1998) complying with the Slovak legislation dealing with scientific experiments on live organisms (Bugarsk ̆ et al. 1999). The tests for determination of resistance level were carried out on female flies, 4to7-day-old, of F1-F3 laboratory generations. The results were compared with those obtained for adult flies of the susceptible strain SRS/WHO Standard Reference Strain/World Health Organization, kept under identical laboratory conditions as the wild populations. Factor of res is tance and evaluat ion of the resis tance The resistance factor (RF) was calculated from the ratio of mean values of LC50 (lethal concentration for 50% of tested flies) of the wild population tested and the respective values for the sensitive strain SRS/WHO. The resistance was evaluated on the basis of RF for LC50 in four categories: low RF < 10; moderate RF = 11 40; high RF = 41 – 160 and very high RF > 160. A method of tarsal contact was used to determine the values of LC50 (Rupe‰ et al. 1975). The impregnation was carried out with insecticides the doses of which were expressed as concentration of the active ingredient in mg per 63.5 cm2 area of filtration paper (Table 1). A minimum of 6 different concentrations of preparations were tested each time. Fifteen females of the tested population of flies were exposed to the impregnated paper for 24 h. During this time good access of flies to water was ensured, but the impregnated filter paper remained dry. The mortality of flies was determined after 24 h. Controls were carried out simultaneously by exposing flies to a filter paper impregnated with drinking water dry at the time of test. In control groups with mortality ranging from 5 to 20%, the correction of experimental values was carried out according to the equation by Abbott (1925). The final values of LC50 were calculated using the probit method (Roth et al. 1962). The obtained results were processed with the statistic program Prism 3.0. Pract ical appl icat ion of insect ic ides observat ion of select ion pressure Practical application of insecticides was carried out in three selected animal houses for mating and pregnant sows, housed in group-pens with bedding. The treated surfaces were approximately 1200 m2. The emulsions or suspensions for spraying were prepared by diluting the preparations with water to concentrations recommended by the manufacturer (Actellic 25 EC 4%; Alfacron 50 WP 2.5 %; Ficam 80 W 0.3 %; Coopex 25 WP 0.25 %; Kordon 10 WP – 1 %; K-Othrine 25 Flow – 0.5 %). A pressure sprayer Maruyama MS 055 S with a storage tank of 23 l in volume, maximum pump output of 2.5 MPa, and a nozzle with two control settings producing 250 and 400 μm size particles was employed. 402 Preparation Active ingredient (a. i.) The concentration used (mg·63.5·cm-2) ACTELLIC 25 EC pirimiphos-methyl (250g.l-1) 6.0-0.0117 ALFACRON 50 WP azamethiphos (500g·kg-1) 7.5-0.0073 FICAM 80 W bendiocarb (800 g·kg-1) 1.44-0.0014 COOPEX 25 WP permethrin (250 g·kg-1) 0.38-0.0004 KORDON 10 WP cypermethrin (100 g.kg-1) 0.6-0.0006 K-OTHRINE 25 FLOW deltamethrin (25 g·l-1) 0.03-0.0001 Table 1 List of preparations used in the experiment Results and Discussion The study presents results obtained by classic and rotational regimes under practical conditions. The products Alfacron 50 WP, Actellic 25 EC, Ficam 80 W, Coopex 25 WP, Kordon 10 WP and K-Othrine 25 Flow were applied separately on individual farms in the course of 4 years. Intensive repeated application of a product containing the same active ingredient suggests the danger of rapid development of resistance in practice already during one or two seasons (Table 2). The monofactorial selection pressure of pirimiphos-methyl led to the development of high resistance within 10 weeks in 1999. The application of azamethiphos resulted in a 7-fold increase in the resistance of houseflies when expressed by the resistance factor (RF) value from low (RF = 7.3) to high (RF = 49). Similar results were obtained after intensive applications of carbamate bendiocarb and pyrethroids cypermethrin and deltamethrin, a high resistance developing after two seasons. LC50 – Lethal concentration On the other hand, the interruption of monofactorial selective pressure by rotational regimes based on different active ingredients (organophosphate – pyrethroid – carbamate –pyrethroid – organophosphate) results in the deceleration of the development of resistance (Table 3). The rotational application of azamethiphos, permethrin and cypermethrin having sufficient insecticidal effect retained resistance at low to moderate levels over a 4-year period. Although alternation of insecticides cannot prevent the development of resistance, it can extend several times the period of their successful application on farms. 403 Table 2 Development of resistance in housefly populations after classic spray regimes (4-6 times during the season) (Mean ± SEM) Table 3 Development of resistance in housefly populations after integrated control – rotational regimes (2-3 times during the season) Resistance factor (RF) for LC50 during 4 years Before 1998 1999 200

The development of resistance in the housefly (Musca domestica) to azamethiphos, pirimiphosmethyl, bendiocarb, permethrin, cypermethrin and deltamethrin was investigated on pig farms over a 4-year period.The results obtained in laboratory tests were compared with those obtained under practical conditions in pig houses.An intensive use of insecticides induced resistance in the course of 2 to 3 seasons.The classic spray regimes of insecticides led to the development of high resistance after one or two seasons.Integrated control, based on rotational application -organophosphate, pyrethroid, carbamate, pyrethroid, organophosphate -retained the resistance at low to moderate levels.Because of the variability of resistance factor (RF) in the field populations observed, the monofactorial and rotational selective pressure of insecticides was investigated.The rotational application of azamethiphos and permethrin or cypermethrin having sufficient insecticidal effect retained the resistance at low to moderate levels over a 4-year period.Although the alternation of insecticides cannot prevent the development of resistance, it can extend several times the period of their successful application on farms.This knowledge can help to formulate the strategies for fly control programmes.

Muscidae, nuisance flies, resistance, organophosphates, carbamates, pyrethroids, control
Because of increasing awareness of risks of insects, great attention of veterinarians is paid not only to well-known blowfly species causing myiases of birds and mammals, but also to other non-biting and blood-sucking flies that affect animal welfare and health in a variety of ways, including nuisance, fly worry and dissemination of diseases (Fischer 1999 and2000).The house fly, Musca domestica (L.) can be managed to some extent by sanitation measures that reduce accumulation of waste materials that serve as breeding sites.For the most part, however, fly control is most commonly achieved with insecticides, but unfortunately, house flies have shown a remarkable ability to develop resistance to these.On the other hand, knowledge about the immunosuppressive effects of insecticides and their possible interference with the genetic material of live organisms indicates that it is necessary to restrict gradually the extensive use of a broad spectrum of pesticides through accentuated application of scientifically justified agrotechnical procedures (Kaãmár et al. 1999).
The control of flies in animal houses where they find both attractive substrates for feeding and breeding and an all-year-round suitable environmental temperature important for their rapid reproduction is not a simple task and is becoming a worldwide problem.The main reason is that they have high reproductive potential and acquire resistance to insecticides rather rapidly.European countries, including Slovakia, are no exception to this rule.After many years of the use of pyrethroids, organophosphates and carbamates we are confronted with the serious problem of resistance to these compounds (Koãi‰ová 1998).Flies are so flexible that it is completely absurd to consider their total eradication.We recognize several important factors that have undoubtedly an unfavorable effect on the successful control of flies.One of the most important is the limited range of really potent insecticides due to the present state of resistance.Therefore, it is necessary to apply as extensively as possible the rules of integrated control of flies which means the use of combinations of the greatest possible variety of products, application methods and the ways of fly control.
In our study we present results of the comparison of effects of monofactorial and rotational and combinational insecticide selection pressure on the development of resistance.Investigations of resistance stability in field populations can help to formulate strategies for fly control programmes in practice.

Populations of flies tested
Wild populations of flies were collected on 9 farms for rearing of pigs.Flies were kept and prepared for individual tests in an insectary at 24-27 o C under standard conditions and methods (Rupe‰ and Rettich 1998) complying with the Slovak legislation dealing with scientific experiments on live organisms (Bugarsk˘et al. 1999).The tests for determination of resistance level were carried out on female flies, 4-to-7-day-old, of F 1 -F 3 laboratory generations.The results were compared with those obtained for adult flies of the susceptible strain SRS/WHO -Standard Reference Strain/World Health Organization, kept under identical laboratory conditions as the wild populations.

Factor of resistance and evaluation of the resistance
The resistance factor (RF) was calculated from the ratio of mean values of LC 50 (lethal concentration for 50% of tested flies) of the wild population tested and the respective values for the sensitive strain SRS/WHO.The resistance was evaluated on the basis of RF for LC 50 in four categories: low RF < 10; moderate RF = 11 -40; high RF = 41 -160 and very high RF > 160.A method of tarsal contact was used to determine the values of LC 50 (Rupe‰ et al. 1975).The impregnation was carried out with insecticides the doses of which were expressed as concentration of the active ingredient in mg per 63.5 cm 2 area of filtration paper (Table 1).A minimum of 6 different concentrations of preparations were tested each time.Fifteen females of the tested population of flies were exposed to the impregnated paper for 24 h.During this time good access of flies to water was ensured, but the impregnated filter paper remained dry.The mortality of flies was determined after 24 h.Controls were carried out simultaneously by exposing flies to a filter paper impregnated with drinking water dry at the time of test.In control groups with mortality ranging from 5 to 20%, the correction of experimental values was carried out according to the equation by Abbott (1925).The final values of LC 50 were calculated using the probit method (Roth et al. 1962).The obtained results were processed with the statistic program Prism 3.0.

Practical application of insecticides -observation of selection pressure
Practical application of insecticides was carried out in three selected animal houses for mating and pregnant sows, housed in group-pens with bedding.The treated surfaces were approximately 1200 m 2 .The emulsions or suspensions for spraying were prepared by diluting the preparations with water to concentrations recommended by the manufacturer (Actellic 25 EC -4%; Alfacron 50 WP -2.5 %; Ficam 80 W -0.3 %; Coopex 25 WP -0.25 %; Kordon 10 WP -1 %; K-Othrine 25 Flow -0.5 %).A pressure sprayer Maruyama MS 055 S with a storage tank of 23 l in volume, maximum pump output of 2.5 MPa, and a nozzle with two control settings producing 250 and 400 µm size particles was employed.

Results and Discussion
The study presents results obtained by classic and rotational regimes under practical conditions.The products Alfacron 50 WP, Actellic 25 EC, Ficam 80 W, Coopex 25 WP, Kordon 10 WP and K-Othrine 25 Flow were applied separately on individual farms in the course of 4 years.Intensive repeated application of a product containing the same active ingredient suggests the danger of rapid development of resistance in practice already during one or two seasons (Table 2).The monofactorial selection pressure of pirimiphos-methyl led to the development of high resistance within 10 weeks in 1999.The application of azamethiphos resulted in a 7-fold increase in the resistance of houseflies when expressed by the resistance factor (RF) value from low (RF = 7.3) to high (RF = 49).Similar results were obtained after intensive applications of carbamate bendiocarb and pyrethroids cypermethrin and deltamethrin, a high resistance developing after two seasons.
LC 50 -Lethal concentration On the other hand, the interruption of monofactorial selective pressure by rotational regimes based on different active ingredients (organophosphate -pyrethroid -carbamate -pyrethroid -organophosphate) results in the deceleration of the development of resistance (Table 3).The rotational application of azamethiphos, permethrin and cypermethrin having sufficient insecticidal effect retained resistance at low to moderate levels over a 4-year period.Although alternation of insecticides cannot prevent the development of resistance, it can extend several times the period of their successful application on farms.Studies of insecticide resistance should eventually result in preparation strategies that can prevent or slow down its development.Insecticides still remain the primary means of the control of flies in animal production.It is paradoxical that the development and spreading of resistance accelerates while the development of new preparations decelerates and it becomes more and more difficult to discover new, more effective insecticides with different modes of action.The development of new insecticides must be based on essential criteria related to the preservation of high effectiveness on noxious insects, low acute and chronic toxicity (Legáth 2000;Pistl et al. 2001), harmlessness to non-target organisms (Dudriková et al. 2000), low persistence in the environment and prevention of the development of additional resistance.The use of insecticides with long residual action against flies in closed facilities is counter-productive because such insecticides strongly affect selection for resistance.One cannot rely on migration of sensitive individuals from the environment, i.e. on the so-called "natural dilution" of resistant populations in animal houses.The efforts aimed at deceleration of the development of resistance in closed animal houses are counteracted by the fact that the environment in them is suitable for selection.Flies can multiply inside them rapidly in great numbers (Koãi‰ová 2001) and their killing should be more intensive than anywhere else.Frequent and thorough application of insecticides guarantees very strong selection pressure.The result of such interventions is, as presented in our study, that insect control measures may fail as soon as after one or two seasons.This is evidenced by the one-sided action of azamethiphos, bendiocarb and deltamethrin.The frequency of resistance in housefly populations results mostly, as it has been already mentioned, from the selection pressure by an insecticide.The starting point of verification of the effectiveness of the developed strategy are the results of resistance before and after realization of individual management principles.Integrated fly control regimes have to be viewed as a challenge requiring a continued high level of organization and cooperation between the agrochemical industry, entomologists and advisers, a sound biological foundation based on resistance monitoring, crossresistance studies, and versatility of response based upon feedback on the current state of the programme.

Table 2
Development of resistance in housefly populations after classic spray regimes (4-6 times during the season) (Mean ± SEM)