Biological Fitness Cost, Demographic Growth Characteristics, and Resistance Mechanism in Alpha-Cypermethrin-Resistant Musca domestica (Diptera: Muscidae)

Simple Summary The house fly is a pest of animals and humans that has developed resistance to alpha-cypermethrin, a commonly used pyrethroid insecticide used to control medically important pests in Saudi Arabia. This study demonstrated that the alpha-cypermethrin selected strain of house fly had a 405.93-fold increase in resistance to alpha-cypermethrin compared with the alpha-cypermethrin–susceptible strain; the increased resistance was attributed to the presence of glutathione S-transferases, specific esterases, and cytochrome P450 monooxygenases. The alpha-cypermethrin resistant strain exhibited lower relative fitness (0.50), longevity, survival rate, life expectancy, reproductive values, intrinsic rate of increase, net reproductive rate, fecundity, maternity, and finite rate of increase, along with shorter larval, female preadult, and adult durations than the susceptible strain. These results demonstrated that alpha-cypermethrin resistance may pose fitness costs in house flies. These findings will aid in the development of rational house fly control methods. Abstract Musca domestica L., a pest of animals and humans, has developed resistance to alpha-cypermethrin, a pyrethroid insecticide commonly used to control medically important pests in many countries, including Saudi Arabia. We investigated the mechanism underlying the development of alpha-cypermethrin resistance and life history characteristics of alpha-cypermethrin–susceptible (Alpha-SS) and alpha-cypermethrin-resistant (Alpha-RS) M. domestica using the age-stage, two-sex life table theory, which is crucial for developing a future rational management strategy and minimizing the negative effects of alpha-cypermethrin on the environment. Our results showed that Alpha-RS M. domestica had a 405.93-fold increase in resistance to alpha-cypermethrin relative to Alpha-SS M. domestica. This increase in the resistance toward insecticide was attributed to metabolic enzymes, such as glutathione S-transferases, specific esterases, and cytochrome P450 monooxygenases. Furthermore, Alpha-RS M. domestica exhibited lower relative fitness (0.50), longevity, survival rate, life expectancy, reproductive values, intrinsic rate of increase, net reproductive rate, fecundity, maternity, and finite rate of increase, along with shorter larval, female preadult, and adult durations than Alpha-SS M. domestica, indicating fitness costs associated with most parameters. However, no significant differences were found between the strains in the following parameters: egg, pupa, and male preadult durations; adult preoviposition, total preoviposition, and oviposition periods; female ratio; and total generation time. Additionally, Alpha-RS M. domestica had a markedly lower intrinsic rate of increase, net reproductive rate, and finite rate of increase than Alpha-SS M. domestica. The results of this study suggest that alpha-cypermethrin resistance may lead to dominant fitness costs in M. domestica. Overall, these findings will aid in the development of rational control strategies for M. domestica as well as help to reduce pesticide pollution.


Introduction
Musca domestica L. (Diptera: Muscidae), the common house fly, is a globally distributed insect pest that transmits diseases to humans and animals [1,2]. The adult stage of M. domestica not only causes annoyance but also serves as a carrier for approximately 200 pathogens responsible for several life-threatening diseases, such as avian influenza, diarrhea, bovine respiratory diseases, typhoid fever, aspergillosis, tuberculosis, cholera, poliomyelitis, and hepatitis [2][3][4][5]. These pathogens are attached to the body of M. domestica adults either through direct contact or while feeding on garbage and animal waste in filthy habitats [5,6]. Subsequently, the pathogens are transmitted to humans and animals through physical contact when the flies enter sanitary settings [7].
Various measures, including the maintenance of proper waste hygiene around dairies and homes, chemical control, and biological agents such as predators and parasitoids, are taken to manage M. domestica [1]. Among these measures, chemical control is a common practice due to its knockdown effects. Alpha-cypermethrin, a synthetic pyrethroid insecticide, is used worldwide to control various insect pests, including M. domestica [8][9][10]. Unfortunately, overreliance on alpha-cypermethrin can lead to resistance development, increased control costs, and environmental pollution [11,12]. In fact, alpha-cypermethrin resistance has previously been observed in M. domestica, Bactrocera oleae Rossi., Rhipicephalus microplus Canestrini, and Stomoxys calcitrans L. [11,[13][14][15]. As a result, increasing the dose of insecticide has become inevitable, which exerts adverse effects on the environment.
Fitness is an individual's ability to reproduce in a particular environment and pass on their reproductive traits to subsequent generations [26,27]. Fitness costs are exhibited in insecticide-resistant individuals of pest strains that are less fit than insecticide-susceptible individuals in the absence of insecticides. These costs tremendously contribute to delaying the evolution of insecticide resistance and reducing its extent in the absence of insecticides [28]. The discontinuation of insecticide selective pressure can prevent the occurrence and spread of insecticide resistance when insecticide-resistant pests exhibit dominant fitness costs [29]. Insecticide resistance affects the fitness of insects, resulting in lower survival rates, fecundity (number of eggs per female), finite or intrinsic rates of increase, and net reproductive rates, along with longer developmental durations and generation times in insecticide-resistant pests than in insecticide-susceptible pests [29,30]. The fitness costs of resistance to various insecticide classes have been described in M. domestica [29,31], Dysdercus koenigii Fabricius [32], Aedes aegypti L. [33], Bemisia tabaci (Gennadius) [34], and Oxycarenus hyalinipennis Costa [35]. These insecticide-resistant strains have shown unfavorable biological and demographic growth characteristics compared with their susceptible counterpart strains. However, the fitness cost of alpha-cypermethrin resistance in M. domestica is still unknown.
Understanding fitness costs and mechanisms of insecticide resistance is crucial for the development of effective strategies to manage insecticide resistance. In our previous study, we assessed the resistance risk, quantitative genetics, and cross-resistance patterns in alphacypermethrin-resistant M. domestica [14]. However, the fitness costs and the mechanisms of alpha-cypermethrin resistance in M. domestica remain unknown. Therefore, in this study, the fitness costs of alpha-cypermethrin-resistant (Alpha-RS) and alpha-cypermethrinsusceptible (Alpha-SS) M. domestica were investigated based on the age-stage, two-sex life table theory. Furthermore, the mechanisms of alpha-cypermethrin resistance were explored using three synergists.

Strains of M. domestica
The rearing protocol for M. domestica strains is outlined in our previous studies [10,14,36]. Briefly, we collected a population of M. domestica from a dairy facility in Dirab, Riyadh, Saudi Arabia (24.49 • N, 46.60 • E) and divided it into two groups: Alpha-SS, which was maintained for 41 generations with no insecticide exposure; and Alpha-RS, which was continuously exposed to various concentrations of alpha-cypermethrin (90-1000 ppm) for 41 generations. Selection pressure was maintained by exposing subsequent generations to increased concentrations of alpha-cypermethrin while ensuring sufficient survival of adults for offspring production. The median lethal concentration (LC 50 ) of alpha-cypermethrin in the field strain (90 ppm) was used for the first eight generations. In each generation, approximately 1000 mixed-sex adults (2-3 days old) were screened with alpha-cypermethrin using a feeding method (IRAC method# 026) [37], and mortality data were recorded after exposure to insecticide for 48 h. Surviving adults were transferred to rearing cages (40 × 40 cm 2 ) for obtaining the next generation. Both strains were reared in the laboratory under controlled conditions: 27 • C ± 2 • C, 65% ± 5% relative humidity, and a 12:12 h light:dark photoperiod.

Concentration-Response Bioassay
Concentration-response bioassays were conducted to determine the resistance levels in the tested M. domestica following the selection pressure by alpha-cypermethrin over 41 generations. We used a concentration-response bioassay that has been described in our previous studies [10,14,36], with slight modifications. Briefly, the toxicity of alphacypermethrin against M. domestica adults was assessed using a feeding bioassay. Seven alpha-cypermethrin concentrations, whose mortality rates ranged from >0% to <100%, were prepared in 20% sugar solution through serial dilution and replicated three times. Each desired concentration was prepared by diluting a specific amount from the stock solution of alpha-cypermethrin into the relevant volume of the 20% sugar solution. We used alpha-cypermethrin concentrations ranging from 0.977 to 62.5 µg mL −1 and from 125 to 8000 µg mL −1 for Alpha-SS and Alpha-RS, respectively. For each replicate, concentration, and bioassay, 10, 30, and 210 mixed-sex adults were used, respectively. Thirty adults were used as controls, with ten adults included in each replicate. These adults were starved for 2 h in perforated plastic jars (11 cm diameter; 15 cm height) prior to the bioassay. Cotton wicks (3 cm in length) soaked in each insecticide concentration were placed in Petri dishes (9 cm in diameter); these Petri dishes were kept in plastic jars for feeding the insects. For the control group, cotton wicks soaked in 20% sugar solution were provided for feeding. Concentration-response bioassays were performed under the laboratory conditions mentioned in Section 2.1. Mortality was assessed after 48 h of exposure, considering the fast action of alpha-cypermethrin.

Synergism Experiment
Synergism bioassays were conducted to identify the possible mechanism of alphacypermethrin resistance in the tested M. domestica following the selection pressure by alphacypermethrin over 41 generations. In these synergism bioassays, analytical-grade acetone (Fisher Scientific, Loughborough, UK) was used to dissolve PBO, DEF, and DEM. The result-ing solutions were mixed with serially diluted concentrations of alpha-cypermethrin. The concentrations of alpha-cypermethrin that were mixed with PBO, DEF or DEM ranged from 0.977 to 62.5 µg mL −1 and from 62.5 to 4000 µg mL −1 for Alpha-SS and Alpha-RS, respectively. Based on the results of preliminary tests, a nonlethal concentration of 2 mg/L for PBO and DEF was used against both Alpha-RS and Alpha-SS M. domestica, and a nonlethal concentration of 0.5 mg/L was used for DEM against both strains. The control consisted of acetone mixed with deionized water only. Synergism bioassays were performed under the laboratory conditions mentioned in Section 2.1. Mortality was recorded after 48 h of exposure, and the synergism ratios (SRs) were calculated as the LC 50 of alpha-cypermethrin alone divided by the LC 50 of alpha-cypermethrin plus PBO, DEF, or DEM. The 95% confidence limits (CLs) were determined using the method described by Robertson et al. [38].

Assessment of Life Tables of Alpha-RS and Alpha-SS M. domestica
The life table parameters of Alpha-SS and Alpha-RS M. domestica were studied to assess the fitness cost of alpha-cypermethrin resistance. Beginning with the 41st generation, three replications of 150 randomly selected freshly hatched first instar larvae (50 larvae per replicate) from Alpha-SS and Alpha-RS M. domestica were used as experimental populations. The larvae were placed in 1000 mL glass beakers containing artificial diet (described in detail by Abbas and Hafez [14] and protected with a fine mesh cloth to prevent larvae from escaping. Larvae were allowed to pupate in glass jars, and the durations of larval and pupal stages as well as mortality rates were recorded for both strains. Within 24 h, the emerged males and females were paired in plastic jars (15 cm height; 11 cm diameter). Adult diet (1 mg of dry powdered milk and 1 mg of sugar) and water-soaked cotton wicks (~3 cm) were placed in separate Petri dishes and provided to adult flies for feeding. To examine fecundity, 50 ♂× 50 ♀ (1:1 ratio) and 30 ♂× 30 ♀ (1:1 ratio) pairs of Alpha-SS and Alpha-RS, respectively, were made based on the availability of adults. One pair of M. domestica was used as one replication for both strains. After 2 days of adult pairing, Petri dishes containing larval diet were provided for egg laying. Petri dishes were removed from jars daily, and eggs were counted using a fine hair brush. All life history parameters were recorded for both strains under the aforementioned laboratory conditions.
The demographic life table parameters were calculated using methods outlined in our previous studies [1,32,39].
"The age-stage survival rate" (s xj ), i.e., the probability that a newly laid egg will survive to age x and stage j, was calculated using the method described by Chi and Liu [40]. The age-specific survival rate (l x ), i.e., the pooled probability that a newly laid egg will survive to age x, was determined as follows: Stage differentiation is not possible because l x represents the pooled survival rates of all stages at age x. The age-specific fecundity (m x ), i.e., the mean fecundity of all individuals at age x, was determined as follows: where f xj is the mean fecundity of individuals at age x and stage j, and m is the number of stages. The net reproductive rate (R 0 ), i.e., the total number of offspring that an individual can produce during its lifetime, was determined as follows: The intrinsic rate of increase (r), i.e., the population growth rate when the time approaches infinity and the population reaches the stable age-stage distribution, was calculated using the Euler-Lotka equation [41,42] with an age index of zero [43] as follows: The finite rate of increase (λ) was determined using the following equation: The mean generation time (T), i.e., the length of time that a population requires to increase R 0 -fold in its size at the stable age-stage distribution, was calculated as follows: The life expectancy (e xj ), i.e., the expected duration of time that an individual of age x and stage j will survive after age x, was determined using the following equation: where s iy is the probability that an individual of age x and stage j will survive until age i and stage y [44]. The reproductive value (v xj ), i.e., the value of an individual at age x and stage j to future offspring [45], was calculated as follows: The curves for l x , s xj , m x , age-stage specific maternity (l x m x ), e xj , and v xj were plotted using Sigma Plot 11.0.
Relative fitness (Rf ) was calculated as follows:

Data Analysis
Mortality and life table data analyses were conducted using methods outlined in our previous studies [1,14,32,39]. Probit analysis was performed using Polo Plus Software to analyze mortality data [46] and obtain LC 50 values, fiducial limits (FLs), slopes (±standard errors (SEs)), and chi-square values (χ 2 ). Abbott's formula [47] was used to correct for control treatment mortality. Resistance ratios (RRs) were calculated by dividing the LC 50 of alpha-cypermethrin in Alpha-RS M. domestica by that in Alpha-SS M. domestica. SRs were calculated by dividing the LC 50s of alpha-cypermethrin alone in Alpha-SS and Alpha-RS M. domestica by the LC 50s of alpha-cypermethrin +PBO, DEF, or DEM in Alpha-SS and Alpha-RS M. domestica. The 95% CLs of RRs and SRs were assessed using the method described by Robertson et al. [38], and RRs were considered significantly different if the CLs were not 1. The life table data were analyzed using the TWO-SEX-MS Chart program using 100,000 replicates and a paired bootstrap test [48] to calculate the means and SEs. The differences in life table traits between Alpha-RS and Alpha-SS M. domestica were compared using confidence intervals (CIs) calculated via the paired bootstrap test [44,49,50]. A significant difference among life history traits of strains was indicated by CIs that did not include the value 0 at p ≤ 0.05.

Toxicity and Resistance of Alpha-RS M. domestica to Alpha-Cypermethrin
After 41 generations of selection in the laboratory, Alpha-RS had 405.93-fold increase in resistance to alpha-cypermethrin relative to Alpha-SS (Table 1).

Effect of Synergists on Alpha-Cypermethrin Toxicity in Alpha-RS and Alpha-SS M. domestica
The synergists PBO, DEF, and DEM did not exhibit any synergistic effect on alphacypermethrin toxicity in Alpha-SS M. domestica (Table 2). However, PBO, DEF, and DEM significantly reduced alpha-cypermethrin LC 50 values in Alpha-RS M. domestica from 1530.36 ppm to 412.18 ppm, 254.23 ppm, and 439.71 ppm, respectively, with SRs of 3.71, 6.02, and 3.48, respectively. These findings suggested that mechanisms of alpha-cypermethrin resistance in Alpha-RS M. domestica may be associated with specific esterases, glutathione S-transferases, and cytochrome P450 monooxygenases (Table 2).

Biological and Demographic Parameters of Alpha-RS and Alpha-SS M. domestica
The egg duration, pupal duration, ♂preadult duration, adult preoviposition period, total preoviposition period, oviposition period, generation time, and female ratio were not significantly different between Alpha-RS and Alpha-SS M. domestica. However, the larval duration, ♀preadult duration, overall adult duration, ♂total longevity, ♀total longevity, and overall total longevity were significantly shorter in Alpha-RS M. domestica than in Alpha-SS M. domestica. Additionally, reproductive female ratio, fecundity, r, and λ were considerably lower in Alpha-RS M. domestica than in Alpha-SS M. domestica. Ro of Alpha-RS M. domestica was negatively affected when compared with that of Alpha-SS M. domestica. Compared with Alpha-SS M. domestica, the relative fitness value of Alpha-RS M. domestica was 0.50 (Table 3).

Discussion
Alpha-cypermethrin is a relatively modern pyrethroid insecticide used in Saudi Arabia for controlling medically important pest species, including M. domestica [10,14]. In this study, Alpha-RS M. domestica had 405.93-fold resistance to alpha-cypermethrin after 41 generations of selection than Alpha-SS M. domestica. This rapid increase in the resistance of M. domestica to alpha-cypermethrin suggests that continuous exposure to this insecticide enhanced their insecticide resistance. Enzymatic detoxification is the most likely reason for the increase in alpha-cypermethrin resistance, as found in this study. The involvement of metabolically mediated resistance to insecticides can be preliminarily assessed using enzyme inhibitors such as DEF, PBO, and DEM [17,51]. The current findings revealed that the LC50 of alpha-cypermethrin in Alpha-RS M. domestica was significantly decreased in the presence of PBO, DEF, or DEM compared to when alpha-cypermethrin was used alone, indicating a possible involvement of the specific esterases, glutathione S-transferases, and cytochrome P450 monooxygenases in the development of alpha-cypermethrin resistance in Alpha-RS M. domestica. This result suggests that specific esterases, glutathione S-transferases, and cytochrome P450 monooxygenases can be used to mitigate the toxicity of alpha-cypermethrin. Previous

Discussion
Alpha-cypermethrin is a relatively modern pyrethroid insecticide used in Saudi Arabia for controlling medically important pest species, including M. domestica [10,14]. In this study, Alpha-RS M. domestica had 405.93-fold resistance to alpha-cypermethrin after 41 generations of selection than Alpha-SS M. domestica. This rapid increase in the resistance of M. domestica to alpha-cypermethrin suggests that continuous exposure to this insecticide enhanced their insecticide resistance. Enzymatic detoxification is the most likely reason for the increase in alpha-cypermethrin resistance, as found in this study. The involvement of metabolically mediated resistance to insecticides can be preliminarily assessed using enzyme inhibitors such as DEF, PBO, and DEM [17,51]. The current findings revealed that the LC 50 of alpha-cypermethrin in Alpha-RS M. domestica was significantly decreased in the presence of PBO, DEF, or DEM compared to when alpha-cypermethrin was used alone, indicating a possible involvement of the specific esterases, glutathione S-transferases, and cytochrome P450 monooxygenases in the development of alpha-cypermethrin resistance in Alpha-RS M. domestica. This result suggests that specific esterases, glutathione S-transferases, and cytochrome P450 monooxygenases can be used to mitigate the toxicity of alpha-cypermethrin. Previous studies have indicated that M. domestica has developed pyrethroid resistance through cytochrome P450-mediated detoxification [18,21,52,53]. Our results are in agreement with those of Abbas et al. [17] and Khan et al. [54], who found that cytochrome P450 monooxygenases and specific esterases were involved in mitigating the toxicity of lambda-cyhalothrin and deltamethrin in M. domestica. However, Zhang et al. [55] reported no involvement of cytochrome P450 monooxygenases and glutathione S-transferases in mitigating the toxicity of beta-cypermethrin in M. domestica.
It is crucial to understand the fitness costs that accompany insecticide resistance in pest strains for developing effective strategies to manage insecticide resistance [26,28]. Insecticideresistant individuals often exhibit unfavorable biological traits [39]. In this study, Alpha-RS M. domestica had a relative fitness of 0.50 when compared with Alpha-SS M. domestica; it also had shorter larval and adult durations, female preadult period, lower male and female total longevity, smaller reproductive female ratio, and lower fecundity than Alpha-SS, which shows the fitness costs associated with alpha-cypermethrin resistance. Previous studies also suggest that if the selection pressure of alpha-cypermethrin is discontinued for a period, the population of Alpha-RS M. domestica does not increase as rapidly as that of Alpha-SS M. domestica. Previous studies have also detected fitness costs in different insecticide-resistant strains, such as M. domestica [29,31,[56][57][58], Phlebotomus papatasi (Scopoli) [59], Ae. aegypti [60,61], O. hyalinipennis [62,63], Plutella xylostella L. [64], Chrysodeixis includens Walker [65], Rhopalosiphum padi L. [66], and Ceratitis capitata (Wiedemann) [67].
Demographic growth features, such as r, λ, and Ro, are indicators of potential population growth in certain environments. Changes in these features can disrupt the growth rate of insects [39]. In this study, r, λ, and Ro were significantly lower among Alpha-RS M. domestica than among Alpha-SS M. domestica, indicating the effect of alpha-cypermethrin selection on the reproduction and growth of M. domestica. The decrease in the number of eggs produced by a female in Alpha-RS M. domestica may be responsible for the reduced r, λ, and Ro values and subsequent decrease in relative fitness. Previous studies have also reported significantly reduced r and Ro in pyrethroid-resistant M. domestica, C. includens, C. capitata, Anopheles funestus Giles, Ae. aegypti, P. papatasi, and O. hyalinipennis compared with their pyrethroid-susceptible counterparts [29,59,61,62,65,67,68]. Additionally, s xj , m x , l x m x , e xj , and v xj were markedly reduced in Alpha-RS M. domestica compared with Alpha-SS M. domestica, indicating fitness costs associated with alpha-cypermethrin resistance. Consistent with our results, previous studies have reported significant reductions in these age-stage, two-sex life table traits in different insecticide-resistant insect pests [32,69]. Due to time constraints, we were not able to perform the required crosses of Alpha-SS and Alpha-RS to ascertain whether the fitness costs were due to a heterosis effect relative to deleterious resistant alleles or genetic drift in the heterozygotes leading to recovery of biological parameters [30,70]. In general, our results support the common notion that insecticide-resistant strains exhibit fitness costs compared with their insecticide-susceptible counterparts. Furthermore, our study provides a comprehensive understanding of fitness traits in both Alpha-RS and Alpha-SS M. domestica.
The presence of deleterious life table parameters in Alpha-RS M. domestica highlights the negative impact of resistant alleles on the fitness and mechanisms of insect resistance, which may result from a trade-off between resources and energy. The selection pressure of insecticides, including alpha-cypermethrin, on pest strains requires significant consumption of resources and energy [30] contributing to fitness costs and adverse biological and reproduction parameters in Alpha-RS M. domestica. To address this issue, temporary withdrawal of alpha-cypermethrin, and the use of insect growth regulators, which are effective against M. domestica [1], can help in restoring the susceptibility of these insects to pyrethroids and aid successful M. domestica management. Rational insect resistance management plans, including limited use of insecticides, biological control agents, and cultural practices, should also be considered while planning M. domestica management.

Conclusions
After 41 generations, Alpha-RS M. domestica developed a 405.93-fold higher resistance to alpha-cypermethrin than Alpha-SS M. domestica, primarily via specific esterases, cytochrome P450 monooxygenases, and glutathione S-transferases. However, fitness costs were observed in this strain, as evinced by its unfavorable life history parameters compared with those of Alpha-SS M. domestica, indicating that the development of alpha-cypermethrin resistance could be delayed by withdrawing the use of insecticide for a given period of time. Hence, implementing resistance management strategies, such as alternating the use of alpha-cypermethrin with new insecticides exhibiting different chemistry, could delay alpha-cypermethrin resistance development. Moreover, integrated pest management (such as chemical, cultural, and biological control) should be implemented for the management of M. domestica. These findings provide valuable insights for developing effective pyrethroid resistance management against M. domestica, ultimately preserving the environmental fauna by minimizing the use of higher insecticide doses.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available from the corresponding author on a reasonable request.