Alpha-Cypermethrin Resistance in Musca domestica: Resistance Instability, Realized Heritability, Risk Assessment, and Insecticide Cross-Resistance

Simple Summary The common house fly, Musca domestica L., is a major carrier of serious diseases in humans and livestock. The common house fly has developed resistance to many insecticides used against it. In the present study, resistance to alpha-cypermethrin increased from 46.4-fold to 474.2-fold in alpha-cypermethrin-selected (Alpha-Sel) females and 41.0-fold to 253.2-fold in Alpha-Sel males, when compared with an alpha-cypermethrin-unselected strain (Alpha-Unsel). However, alpha-cypermethrin resistance was unstable when a field population was reared without exposure for 24 generations. The realized heritability (h2) of alpha-cypermethrin resistance was 0.17 and 0.18 for males and females, respectively, in G1–G24. The Alpha-Sel strain revealed low cross-resistance (CR) to two pyrethroids and five organophosphates and moderate CR to bifenthrin (15.5-fold), deltamethrin (28.4-fold), or cyfluthrin (16.8-fold). The results of instability of resistance trait, low h2, and lack of CR associated with alpha-cypermethrin resistance will provide an opportunity to stakeholders and entomologists to plan better and more effective insect pest and vector management programs in Saudi Arabia. Abstract Musca domestica L., the common house fly, is a cosmopolitan carrier of human and livestock disease pathogens. The species exhibits resistance to many insecticides; therefore, effective M. domestica insecticide resistance management programs are required worldwide. In the present study, the development of alpha-cypermethrin resistance, realized heritability (h2), instability of resistance trait (DR), and cross-resistance (CR) was investigated in an alpha-cypermethrin-selected M. domestica strain (Alpha-Sel) across 24 generations (Gs). Compared with an alpha-cypermethrin-unselected strain (Alpha-Unsel), resistance to alpha-cypermethrin increased from 46.4-fold (G5) to 474.2-fold (G24) in Alpha-Sel females and 41.0-fold (G5) to 253.2-fold (G24) in Alpha-Sel males. Alpha-cypermethrin resistance declined by between –0.10 (G5) and –0.05 (G24) in both M. domestica sexes without insecticide exposure for 24 generations. The h2 of alpha-cypermethrin resistance was 0.17 and 0.18 for males and females, respectively, in G1–G24. With selection intensities of 10–90%, the G values required for a tenfold increase in the LC50 of alpha-cypermethrin were 6.3–53.7, 4.1–33.8, and 3.0–24.7, given h2 values of 0.17, 0.27, and 0.37, respectively, and a constant slope of 2.1 for males and h2 values of 0.18, 0.28, and 0.38, respectively, and a constant slope of 2.0 for females. Compared with Alpha-Unsel, Alpha-Sel M. domestica exhibited moderate CR to bifenthrin (15.5-fold), deltamethrin (28.4-fold), and cyfluthrin (16.8-fold), low CR to two pyrethroids and five organophosphates, and no CR to insect growth regulators. The instability of resistance trait, low h2, and absent or low CR associated with alpha-cypermethrin resistance in M. domestica indicate resistance could be managed with rotational use of the insecticide.


Introduction
Synthetic pyrethroid insecticides are commonly used to manage vector pests worldwide owing to their efficacy against the pests' adult and larval stages, lack of persistence in the environment, and low mammalian toxicity [1,2]. However, the indiscriminate use of and over-reliance on these insecticides can increase environmental pollution, negatively affect human health through exposure, and lead to insecticide resistance in the target insect vectors [3][4][5][6][7]. Following the development of insecticide resistance, the public may increase the dosages of insecticides used to suppress resistant insect vectors, thereby compounding the negative effects on the environment and fauna [8,9]. Therefore, characterization of insecticide resistance development is necessary to manage resistant insect vectors and minimize insecticide-related effects on the environment and nontarget organisms [10].
Musca domestica L., the common house fly, is a vector pest of humans and livestock worldwide; it can carry approximately 100 pathogens and is responsible for several deadly diseases [11][12][13]. M. domestica carries pathogens acquired during feeding under unsanitary conditions, transferring these pathogens when it moves to sanitary areas [14,15]. Additionally, M. domestica adults cause annoyance to livestock, and M. domestica larvae feed voraciously, sometimes resulting in serious injury to affected animals [9]. Cultural practices, chemicals, and biological agents are employed to control and manage M. domestica [16]. For example, the adult stage is targeted with synthetic pyrethroid insecticides in dairy facilities and urban environments [17]. Among these pyrethroids, alpha-cypermethrin is commonly used to control dipteran pests, including M. domestica [18,19]. However, the over-reliance on alpha-cypermethrin has led to resistance development and increased control costs [20,21]. Indeed, alpha-cypermethrin resistance has been reported in various insect pests worldwide [5,[20][21][22][23][24], forcing insecticide users to increase dosages, leading to the aforementioned negative effects.
The risk of insecticide resistance development can be determined through laboratory selection and realized heritability (h 2 ) estimation, i.e., the fraction of genetic variance to phenotypic variance [25], providing data that help improve resistance management plans and restore insecticide efficacy [10,26]. Resistance to pyrethroids, i.e., lambda-cyhalothrin and permethrin, has been studied in M. domestica through laboratory selection and h 2 estimation [6,8]. Additionally, the continuous use of pyrethroids is known to reduce their efficacy in controlling M. domestica owing to resistance development and the possibility of cross-resistance (CR) to unexposed insecticides [27]. CR is the phenomenon in which the selection pressure of one insecticide on insect pests favors the development of resistance to other insecticides not used in the field, thereby reducing the effectiveness of several insecticides [2,11,28]. Thus, CR analyses are conducted in pyrethroid-resistant strains of insect pests to inform the rotational use of insecticides [2,11]. Indeed, CR to unexposed pesticides with different or similar modes of action has been studied extensively in various insecticide-resistant M. domestica strains [2,8,10,11,[29][30][31].
In recent years, low resistance levels to alpha-cypermethrin were observed in Riyadh, Saudi Arabia [5]. However, data on the (1) risk of alpha-cypermethrin resistance, (2) pace at which alpha-cypermethrin resistance changes, and (3) presence or absence of CR are lacking. Such data are crucial for controlling M. domestica and insecticide pollution [10,30]. Therefore, the objectives of the present study were to (1) assess the risk of alpha-cypermethrin resistance through laboratory selection of M. domestica and h 2 estimations, (2) measure the stability of alpha-cypermethrin resistance, and (3) explore the CR phenomenon in alpha-cypermethrin-selected M. domestica to inform the rotational use of insecticides.

Chemicals
Fifteen insecticides belonging to pyrethroid, organophosphate, and insect growth regulator classes were used in the bioassays (Table 1).

Collection and Rearing of M. domestica
More than 200 adults of M. domestica (both sexes) were trapped in plastic jars (19 × 33 cm) at a dairy farm located in Dirab, Riyadh, Saudi Arabia (24.49 • N, 46.60 • E). The trapped flies were moved to an aerated cage (40 × 40 cm) in the laboratory and maintained according to the protocol described by Abbas and Hafez [16]. The adult flies were fed from Petri dishes (9 cm in diameter) containing (1) a mixture of powdered milk (1 mg) and sugar (1 mg) and (2) a cotton wick (~3 cm) soaked with deionized water, which were placed in the rearing cages and changed every two days. Plastic cups containing a mixture (500 mL total volume) of wheat bran (20.0 g, Second Milling Company, Riyadh, Saudi Arabia), yeast (5.0 g, S.I. Lesaffre, Marcq-en-Baroeul, France), sugar (1.5 g, Al-Osra Company, Jeddah, Saudi Arabia), dry milk powder (1.5 g, Almarai Company, Riyadh, Saudi Arabia), and deionized water (70 mL, Gesellschaft für Labortechnik mbH, Burgwedel, Germany) were also placed in the rearing cages to encourage egg laying and provide larval food. Cups containing eggs were covered with cloth secured by a rubber band to prevent the escape of larvae. Once the larvae had consumed the food provided, fresh food was provided in a glass beaker, and the larvae were allowed to pupate in these beakers. The emerged flies were moved into rearing cages to obtain the next progeny. All M. domestica stages were reared under controlled laboratory conditions (27 ± 2 • C, 65 ± 5% relative humidity, and a 12:12 h light:dark photoperiod).

Selection of M. domestica with Alpha-Cypermethrin
The M. domestica population collected from the dairy farm, named Field-Pop at generation one (G 1 ), was separated into two lines: the alpha-cypermethrin-unselected strain (Alpha-Unsel) was maintained for 24 generations (G 24 ) with no chemical treatment in the laboratory; the alpha-cypermethrin-selected strain (Alpha-Sel) was screened continuously with different concentrations of alpha-cypermethrin for 24 generations ( Table 2). The first selection was started with the LC 50  each generation were screened with alpha-cypermethrin through a feeding bioassay [10]. The surviving flies were moved to rearing cages and maintained under the aforementioned laboratory conditions. Survival data were not recorded at G 24 but were selected with 1000 ppm.

Bioassay of Adults
The toxicities of pyrethroid and organophosphate classes against adults were evaluated via a feeding bioassay as described previously by Hafez [5]. Five concentrations (with >0% to <100% mortality) of an insecticide were prepared in 20% sugar solution via serial dilution. Each concentration for each bioassay was replicated three times. For each insecticide, 10, 30, and 150 adults (either males or females) per replicate, concentration, and bioassay were used, respectively. For the control, 10 adults per replicate were used (30 adults in total). The adults were placed in perforated plastic jars (11 cm diameter × 15 cm height), and the mouth of the jar was covered with cloth tightened by rubber bands to prevent the escape of adults. For 2 h before each bioassay, the adults were starved. For each insecticide concentration solution, cotton wicks (~3 cm) were saturated and placed in Petri dishes (9 cm in diameter), which were placed in the plastic jars to feed the starved adults. In the control, cotton wicks saturated with 20% sugar solution only were used. Bioassays were conducted under the aforementioned laboratory conditions. The mortality of adults was assessed after 48 h of exposure owing to the fast action of the tested insecticides [4].

Bioassay of Larvae
The toxicity of insect growth regulators to M. domestica larvae was evaluated through a diet incorporation bioassay following Abbas and Hafez [16]. Five concentrations of insect growth regulator (with >0% to <100% mortality) were prepared via serial dilution. For each concentration, 140 mL of insect growth regulator solution was mixed with the larval food consisting of wheat bran (40.0 g), yeast (10.0 g), sugar (3.0 g), and dry milk powder (3.0 g). Each concentration for each bioassay was replicated three times. Second instar larvae were used in the bioassays with 10, 30, and 150 larvae per replicate, concentration, and bioassay, respectively. For the control, the larval diet was mixed with deionized water only, and 3 replicates were used (10 larvae per replicate). Bioassays were performed under the aforementioned laboratory conditions. Mortality was recorded based on the emergence of adults, with unemerged pupae counted as dead [16].

Alpha-Cypermethrin Resistance Stability in M. domestica
The Field-Pop (G 1 ) was raised without alpha-cypermethrin selection pressure in the laboratory for 24 generations (G 1 -G 24 ) to determine the stability of alpha-cypermethrin resistance. The decline in alpha-cypermethrin resistance (DR) was calculated using the equation of Tabashnik et al. [32] where N is the number of generations with no exposure to any chemical. DR ranges from −1 to +1: DR value of −1 illustrates decline in resistance and DR value of +1 illustrates no decline in resistance.

h 2 Values for Alpha-Cypermethrin Resistance
The h 2 values for alpha-cypermethrin resistance were assessed using the equations of Tabashnik [33] and Abbas et al. [10] where R is the alpha-cypermethrin selection response and S is the alpha-cypermethrin selection differential. h 2 ranging from 0 to 1: 0 means that most of the differences are not genetic and 1 means that the most of differences are genetic. R was measured using following equation: where n is the total number of generations (G 1 -G 24 ) screened with alpha-cypermethrin. S was measured as follows: where i is the selection intensity (mortality), determined following the method of Tabashnik and McGaughey [34] where "p" is the survival percentage of Alpha-Sel (G 1 -G 24 ) screened with alpha-cypermethrin. σp was measured as follows: The number of generations (G) required to produce a tenfold increase in the median lethal concentration (LC 50 ) of alpha-cypermethrin was determined following Abbas et al. [10].
Each of h 2 , R, and S were measured in the first phase (G 1 -G 12 ) and second phase (G 13 -G 24 ) separately (12 generations in each phase) as well as G 1 -G 24 to determine their changes. Each phase was defined on the basis of half of the total selected generations. The influence of the calculated and assumed slope and h 2 values on alpha-cypermethrin resistance was assessed through G and selection intensity.

Bioassay Data Analyses
To determine the LC 50 , fiducial limits (FLs), chi-square value (χ 2 ), and slope (±standard error), the toxicity data of each insecticide were subjected to probit analyses [35] via POLO PLUS Software [36]. The formula of Abbott [37] was considered to correct the mortalities of each bioassay using the mortality of its control treatment. Resistance ratios (RRs) and performance ratios (PRs) were determined using the following equation: The criteria used to classify the RR and PR levels in M. domestica were those described by Torres-Vila et al. [38] and Ullah et al. [39], i.e., >100, very high resistance; 31-100, high resistance; 11-30, moderate resistance; 2-10, low resistance; and <2, no resistance.

Alpha-Cypermethrin Resistance Selection in Alpha-Sel
On average, the survival rate of male and female M. domestica was 59.8% and 60.2%, respectively, in G 1 -G 24 at different alpha-cypermethrin concentrations (  Figure 1).

h 2 of Alpha-Cypermethrin Resistance in M. domestica
In M. domestica females and males, the overall h 2 values of alpha-cypermethrin resistance in G 1 -G 24 were 0.18 and 0.17, respectively. In females, in the first (G 1 -G 12 ) and second (G 13 -G 24 ) phases of selection, the estimated h 2 was 0.15 and 0.24, respectively (Table 5). In males, in G 1 -G 12 and G 13 -G 24 , the estimated h 2 was 0.14 and 0.20, respectively (Table 5).  3 Mean surviving males and females in selection. 4 Intensity of selection. 5 Phenotypic variation. 6 Selection differential. h 2 = Realized heritability of alpha-cypermethrin resistance.

Projected Rate of Alpha-Cypermethrin Resistance Development
With each selection causing 10-90% mortality for female M. domestica, the generations required for a tenfold increase in the LC 50 Figure 3B). These results indicate that fluctuations in h 2 and slope cause variation in the alpha-cypermethrin resistance development rate.

Discussion
Hafez previously found low resistance (2-to 4-fold) to alpha-cypermethrin in M. domestica females and almost no resistance to low resistance (0.5-to 7.0-fold) in M. domestica males [5]. However, in the present study, the reselection of M. domestica adults with alphacypermethrin for 24 generations increased resistance by 253.2-and 474.2-fold in males and females, respectively. Therefore, M. domestica adults can quickly gain very high resistance to alpha-cypermethrin after continuous exposure in the laboratory. Similarly, high resistance to other pyrethroids, including lambda-cyhalothrin, deltamethrin, and permethrin, has been found in M. domestica [6,8,40]. Nevertheless, M. domestica populations collected from Saudi dairies exhibited little or no field-evolved resistance [5], although inappropriate pesticide use in these dairies could lead to the development of alpha-cypermethrin resistance in M. domestica. Indeed, the present selection experiment revealed that alpha-cypermethrin selection pressure markedly affected the field population, leading to the rapid development of resistance after 24 generations. In addition to M. domestica, alpha-cypermethrin resistance had been found worldwide in pests such as Anopheles stephensi Liston [41], Rhipicephalus microplus Canestrini [42], Bactrocera oleae Rossi. [20,22], Blattella germanica L. [43], and Stomoxys calcitrans L. [24].
Estimating h 2 using a quantitative genetic model can support predictions of variation in a specific trait (e.g., insecticide resistance) when the variation is genetically linked to the trait. The expression of such traits depends on the nature of resistance genes and environmental factors [10,33], and the rate of developing resistance is directly proportional to the h 2 value of any insecticide [34]. A high h 2 value indicates a higher risk of genetic resistance development because more resistance genes are inherited by the next generation [44]. In contrast, a low h 2 value indicates higher phenotypic variation that may arise from gene mutation, population migration, selection pressure, insecticide rotation, and environmental influences under laboratory and field conditions [45]. In the present study, the low h 2 values of 0.18 and 0.17 for female and male M. domestica, respectively, indicate low genetic variation and high phenotypic variation, i.e., M. domestica exhibited a low probability of developing genetic resistance to alpha-cypermethrin. Previous studies have also found low h 2 values in insecticide-selected M. domestica strains, e.g., 0.07 for lambda-cyhalothrin [6], 0.23 for permethrin [8], 0.05 for fipronil [9], 0.17 for methoxyfenozide [46], 0.03 for pyriproxyfen [47], 0.02 for flonicamid [10], and 0.08 for diflubenzuron [30]. In the current study, R and S declined as the alpha-cypermethrin selection pressure was increased, producing a lower h 2 in the first half of alpha-cypermethrin selection than in the second half. Therefore, the alleles responsible for developing alpha-cypermethrin resistance existed at low levels in the first half of selection, whereas these levels increased after further alpha-cypermethrin exposure in the second half of selection. Random drift might also explain these observations. The present results contrast with those of Abbas and Shad [6] and Khan [8], who found additive genetic changes in the first half of selection that decreased in the second half of selection after further exposure of M. domestica strains, although our results are similar to those of Shah et al. [47] and Abbas et al. [9]. The h 2 of insecticide resistance might fluctuate because of changes in allele frequency and the environment over time [33]; consequently, forecasts based on h 2 estimation in laboratory-selected strains must be interpreted prudently in relation to M. domestica management. However, the conditions in the field are not counterparts to the controlled conditions in a laboratory, although the estimated h 2 of alpha-cypermethrin resistance mediated with laboratory selection has implications for resistance management programs [10,34]. The lower h 2 value in this study reveals that many generations may be needed before M. domestica reaches a significant resistance level, although, alpha-cypermethrin should be used rotationally for controlling this pest specie to prolong its usefulness.
Estimating the rate of resistance development through the number of generations (G = h 2 S −1 ) is a valuable step toward establishing rational resistance management strategies for insect vectors [6]. Such estimated rates have been determined previously in M. domestica strains selected with lambda-cyhalothrin [6], permethrin [8], flonicamid [10], clothianidin [31], and diflubenzuron [30]. According to the results in Figures 1 and 2, the risk of M. domestica males and females developing alpha-cypermethrin resistance increases when the h 2 value is increased. This reveals that as the h 2 value increases, the number of generations needed for a ten-fold increase in alpha-cypermethrin resistance decreases. Therefore, the populations with a high h 2 may become resistant after few generations when exposed to intense selection pressure of insecticide under field conditions. Therefore, alpha-cypermethrin should be applied prudently for the control of M. domestica.
The instability of resistance to any insecticide is essential for its prolonged potency, and determining this instability is useful for developing effective resistance management strategies. For instance, when insecticide resistance is unstable, the potency of a specific insecticide may persist if it is rotated with another insecticide. However, when insecticide resistance is stable, the insecticide should not be included in insecticide resistance management plans to avoid resistance complications [48][49][50]. The present results indicated that alpha-cypermethrin resistance was unstable in an M. domestica population collected from a Saudi dairy farm and reared for 24 generations without insecticide exposure. Indeed, the LC 50 values decreased greatly from G 1 to G 24 (from 64.1 to 4.4 ppm in males and from 90.1 to 4.5 ppm in females). This indicates that the mechanism of alpha-cypermethrin resistance is unstable and the unstable resistant alleles require higher fitness costs for their development and survival [51], so the population reverts towards susceptibility after 24 generations. Genetic drift and gene mutation due to stop of selection pressure might be the other reasons for instability of alpha-cypermethrin resistance [32]. Similarly, unstable insecticide resistance to lambda-cyhalothrin was found previously in M. domestica [52]. In contrast, stable resistance to permethrin was found previously in M. domestica [8].
Information on CR is required to choose alternative insecticides for rational management programs [10,30]. The present CR bioassay results revealed moderate CR between alpha-cypermethrin and bifenthrin, deltamethrin, or cyfluthrin, low CR between alpha-cypermethrin and cypermethrin, fenitrothion, chlorpyrifos, malathion, diazinon, pirimiphos-methyl, triflumuron, or pyriproxyfen, and no CR between alpha-cypermethrin and diflubenzuron, cyromazine, or methoxyfenozide in Alpha-Sel M. domestica. CR between alpha-cypermethrin and bifenthrin, deltamethrin, cyfluthrin, or cypermethrin was expected in Alpha-Sel M. domestica as these insecticides have a similar mode of action, a sodium channel modulator. However, the low CR of cypermethrin is interesting in the Alpha-Sel, despite the same molecular target and mode of action. CR between alpha-cypermethrin and the organophosphates (fenitrothion, chlorpyrifos, malathion, diazinon, and pirimiphos-methyl) and insect growth regulators (triflumuron, diflubenzuron, pyriproxyfen, cyromazine, and methoxyfenozide) was not expected as they are in different chemical classes and possess different modes of action [53]. The present results suggest that the no or low CR of organophosphates and insect growth regulators with alpha-cypermethrin may be due to differences in their mode of actions and lower metabolic detoxification. In a previous study, lambda-cyhalothrin-selected M. domestica exhibited very low CR with indoxacarb and abamectin and no CR with bifenthrin and methomyl [2]. In addition, permethrin-selected M. domestica exhibited low CR with β-cyfluthrin and deltamethrin and no CR with imidacloprid and spinosad [8]. Similarly, a thiamethoxam-resistant strain of Aphis gossypii Glover exhibited low CR with alpha-cypermethrin [54]. In contrast, permethrin-resistant Aedes aegypti L. exhibited high CR with deltamethrin, lambda-cyhalothrin, cypermethrin, alpha-cypermethrin, and zetacypermethrin but no CR with bifenthrin [55]. Conversely, diflubenzuron-resistant M. domestica exhibited no CR with alpha-cypermethrin, cypermethrin, bifenthrin, deltamethrin, cyfluthrin, malathion, pyriproxyfen, and methoxyfenozide [30]. Given the lack of or low CR between alpha-cypermethrin and cypermethrin, fenitrothion, chlorpyrifos, malathion, diazinon, pirimiphos-methyl, triflumuron, pyriproxyfen, diflubenzuron, cyromazine, or methoxyfenozide, these insecticides likely represent good alternatives to alpha-cypermethrin for controlling M. domestica.

Conclusions
In summary, Alpha-Sel M. domestica exhibited rapid development of alpha-cypermethrin resistance under continuous selection pressure in the laboratory, which may reflect the possibility of alpha-cypermethrin resistance development in this pest species if the insecticide is applied continuously for a long period in Saudi dairies or urban settings. However, the low h 2 values observed in the present study are encouraging in terms of establishing resistance management programs for alpha-cypermethrin and prolonging its potency against M. domestica. In addition, the absence of or low CR between alpha-cypermethrin and eleven other insecticides (cypermethrin, fenitrothion, chlorpyrifos, malathion, diazinon, pirimiphos-methyl, triflumuron, pyriproxyfen, diflubenzuron, cyromazine, or methoxyfenozide) indicates that several options are available for insecticide rotation in M. domestica control strategies.