Single and Combined Mutations of Acetylcholinesterase Gene Giving Resistance to Pirimiphos-Methyl in Musca domestica Slaughterhouse Populations

Simple Summary The house fly is a worldwide public health pest associated with humans and livestock. The use of insecticides is still the main method of house fly management. The extensive use of insecticides is the main reason for the development of resistance in many insect species. The development of resistance in house flies was documented for numerous insecticides. Pirimiphos-methyl is used for house fly control in Saudi Arabia. This study was conducted to evaluate the resistance of M. domestica field populations against pirimiphos-methyl. The field-collected populations of M. domestica displayed different levels of resistance. The samples collected from Riyadh city exhibited the highest resistance, followed by populations from Jeddah and Taif. We investigated the genetic mutations of the acetylcholinesterase (Ace) gene in field-collected flies and the survivors after exposure to pirimiphos-methyl. The outcomes of the present study provide valuable information that may help in house fly management in Saudi Arabia. Abstract The house fly Musca domestica L. (Diptera: Muscidae) is a worldwide medical and veterinary pest, causing great economic losses. Organophosphate insecticides have been widely used to control house fly populations. The main objectives of the present study were to evaluate the resistance levels of M. domestica slaughterhouse populations, collected from Riyadh, Jeddah, and Taif, against the organophosphate insecticide pirimiphos-methyl and investigate the genetic mutations of the Ace gene associated with pirimiphos-methyl resistance. The obtained data showed that there were significant differences among pirimiphos-methyl LC50 values of the studied populations, where the highest LC50 was recorded for the Riyadh population (8.44 mM), followed by Jeddah and Taif populations (2.45 mM and 1.63 mM, respectively). Seven nonsynonymous SNPs were detected in the studied house flies. The Ile239Val and Glu243Lys mutations are reported for the first time, whereas Val260Leu, Ala316Ser, Gly342Ala, Gly342Val, and Phe407Tyr were previously reported in M. domestica field populations from other countries. Considering three mutations associated with insecticide resistance, at amino acid positions 260, 342, and 407 of acetylcholinesterase polypeptide, 17 combinations were recovered in this study. Three out of these seventeen combinations were frequently found both worldwide and in the three Saudi house fly field populations, as well as their pirimiphos-methyl-surviving flies. Overall, the single and combined Ace mutations are apparently associated with pirimiphos-methyl resistance, and the obtained data can be useful in managing house fly field populations in Saudi Arabia.


Introduction
The house fly Musca domestica (Diptera: Muscidae) is a worldwide medical and veterinary pest. It is often found in areas of human activities, e.g., houses, workplaces, food markets, restaurants, slaughterhouses, animal and poultry farms. These places are suitable for M. domestica reproduction due to the availability of organic materials [1]. In addition to being a nuisance pest, house flies are responsible for the transmission of more than 200 pathogens, including bacteria, viruses, fungi, and parasites, which cause serious diseases in humans and animals [2,3].
House fly populations can be controlled using a variety of strategies, including environmental sanitation, chemical, physical, and mechanical control. Insecticide chemicals, which come in a variety of formulations and treatment techniques, are the greatest options for quick, inexpensive, and practical house fly management. In several countries, organophosphate (OP) insecticides have been used extensively to control house flies [4][5][6]. In Saudi Arabia, the extensive use of insecticides for a long time has led to the development of resistance in house fly populations [7][8][9][10].
Resistance to OP insecticides has been linked to metabolic resistance via the increased activity of detoxifying enzymes and/or acetylcholinesterase (AChE) insensitivity [6,11]. Carbamate and OP insecticides are structural analogs of acetylcholine, and they can inactivate the insect AChE enzyme, leading to an excess of acetylcholine at cholinergic synapses, eventually causing paralysis and death of insects [12,13]. To overcome the sensitivity of AChE to OP and carbamate insecticides, house flies develop resistance by creating mutations in the acetylcholinesterase (Ace) gene that produces AChE insensitivity to insecticides [6,[14][15][16]. Currently, seven nonsynonymous mutations (A/G-495 > Ile162Met, G/C-787 > Val260Leu, C/T-929 > Thr310Met, G/T-946 > Ala316Ser, G/C/T-1025 > Gly342Ala/Val, T/A-1220 > Phe407Tyr, and G/C-1334 > Gly445Ala), existing singly or in combination and associated with OPs resistance, have been identified in the house fly Ace gene [5,15,17,18]. The emergence of resistance may lead to an increase in the quantity and frequency of insecticide applications in both domestic and commercial livestock and poultry farms, raising the expense of control and having detrimental environmental effects [19,20]. The main objectives of the present study are to (1) evaluate the resistance levels of M. domestica slaughterhouse populations, collected from Riyadh, Jeddah, and Taif, against OP insecticide pirimiphos-methyl (PM); and (2) investigate the genetic mutations of the Ace gene in collected field flies and their counterpart survivors after exposure to PM. The outcomes of the present study provide valuable information that may help in controlling house fly populations in Saudi Arabia. The live house flies were designated as the parental generation and were allowed to randomly mate. The F1 progeny (3-5-days-old) were used for toxicity bioassays. The susceptible laboratory strain (LAB, bred since 2006) was brought from the Public Health Pests Laboratory (PHLP-Jeddah, Jeddah, Saudi Arabia). The LAB strain and field house flies were kept at 25 ± 2 • C, 30-40% relative humidity, and in a 12:12 h light-dark cycle. Adult house flies were fed on a mixture of 2% milk powder and 10% sucrose. Eggs were collected from the cages and cultured in a larval medium containing wheat bran, yeast, milk powder, and water at the proportions of 20:1:2:20, respectively [21].

Bioassays
The topical application method was used for assessing the resistance levels in the house flies towards PM, according to Scott et al. [22]. A 32.75 mM stock solution of 90.5% PM was first prepared in acetone and then diluted to at least 5 serially diluted concentrations. Based on the preliminary dose-response, the 5 PM concentrations for the LAB strain, Riyadh, Jeddah, and Taif were 0.0033, 0.0066, 0.013, 0.026, and 0.052 mM; 3.28, 6.55, 9.83, 13.1, and 16.4 mM; 1.64, 2.46, 3.28, 4.9, and 6.55 mM; and 0.33, 0.66, 1.31, 2.62, and 5.24 mM, respectively. An amount of 1 µL of each diluted insecticide concentration was applied on the thoracic notum of 3-5-day-old flies. For control treatments, 1 µL of acetone was applied for each fly. Four replicates were used for each concentration and each replicate had twenty flies. Flies were anesthetized by CO 2 for 20 s before their treatments. The treated flies were maintained in glass cups (250 mL) with cotton inside that had been wet with a 20% sugar solution at 25 ± 2 • C, under a 12:12 h light-dark cycle. Mortality was assessed 24 h after treatment. Flies that were not moving when touched with a soft brush were scored as dead.

Ace Genotyping
Genomic DNA was extracted from individual house flies preserved in 95% ethanol using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocols. The quality and quantity of DNA solutions were determined with a Nanodrop spectrophotometer (Thermo Scientific™, Waltham, MA, USA) and agarose gels, according to Sambrook and Russell [23]. For PCR reactions, the concentrations of extracted DNA solutions were adjusted to 20 ng/µL and then stored at −20 • C for further molecular work [23].

Statistical Analysis
The median lethal concentration (LC 50 ) for PM was determined by probit analysis [24]. Significant differences between LC 50 values were based on no overlapping, 95% confidence intervals [25]. Resistance ratios (RRs) were calculated by dividing LC 50 of a field population by LC 50 of the laboratory strain [26]. To assess the relationship between the frequency of single or combined mutations of the Ace gene and PM-RRs, Pearson correlation coefficient (r) was calculated. The analyses were conducted using SPSS 26 software (IBM, Armonk, NY, USA). In order to reveal possible genetic relationships among the three house fly field populations, the frequencies of Ace genotypes recovered from them were tested for conformity to Hardy-Weinberg equilibrium, using the Exact HWE test embedded in Genepop 4.7.5 [27]. In addition, pairwise F ST estimates [28] and number of migrants (Nm) [29] were calculated with Genepop.

Susceptibility of House Flies to PM
The LC 50 values of adult M. domestica slaughterhouse populations, collected from Riyadh, Jeddah, and Taif regions in Saudi Arabia, were significantly higher than that of the LAB strain (Table 1). Moreover, there were significant differences among PM-LC 50 values of the three slaughterhouse populations ( Table 1). The highest LC 50 was recorded for the Riyadh population (8.44 mM), followed by Jeddah and Taif populations, with LC 50 values of 2.45 and 1.63 mM, respectively. The recorded RRs were 602.9, 175, and 116.4-fold for Riyadh, Jeddah, and Taif populations, respectively (Table 1).

Genotyping of Ace Resistance Alleles
An 842 bp fragment of the Ace gene was amplified by PCR from 172 individual house flies. The Ace fragment contained one 85 bp intron. A total of 16 SNPs were detected: 7/16 from the exon and 9/16 from the intron. The seven SNPs in exon region were nonsynonymous, causing amino acid changes in AChE protein. These mutations were Ile239Val, Val260Leu, Glu243Lys, Ala316Ser, Gly342Ala, Gly342Val, and Phe407Tyr (Table 2). Single, double, and triple peaks per SNP were detected in the Ace fragment (data not shown). There were six individuals that showed triple peaks at position G/C/T-1025: two from Taif and four from Jeddah slaughterhouse populations. * Number of individual flies. ** Survived F1 flies from Riyadh (Riyadh/S), Jeddah (Jeddah/S), and Taif (Taif/S), exposed to different doses of pirimiphos-methyl. *** The mutation positions are underlined and indicated by arrows.

Ace Genotypes Recovered from Field House Fly Populations and Their PM-Surviving Counterparts
When the whole Ace fragment was considered for analysis, 31 Ace genotypes were found among the 48 individual house flies of the Riyadh field population. Among the 31 Ace genotypes, there were 26 singletons and 5 multi-individual genotypes with 8, 3, 6, 2, and 3 individuals each (Table 5). An amount of 10 Ace genotypes were recovered from 12 individuals who survived F1 offspring flies from the Riyadh population exposed to PM. The 10 heterozygous genotypes included 8 singletons and 2 multi-individual genotypes, with 2 individuals each. Interestingly, there were 5 genotypes not recovered from the field population. The Jeddah population showed 33 Ace genotypes, that included 26 singletons and 7 multi-individual genotypes, with the following frequencies: 6, 4, 3, 2, 2, 2, and 2 each (Table 5). A total of 1 out of the 26 singleton genotypes had the susceptible Ace allele. Ten Ace genotypes (nine singletons and one genotype with two individuals) were recovered from the eleven survivors of Jeddah F1 flies exposed to PM. Among the 10 surviving Ace genotypes of Jeddah, 5 were not recovered from the field population. From the 44 house flies of the Taif population, 29 genotypes were found, with 23 singletons and 6 multi-individual genotypes with 2, 2, 2, 3, 3, and 8 individuals each (Table 5). One susceptible genotype was represented among the 23 singletons. A total of 8 Ace genotypes (5 were not recovered from the field population) were found among the 10 survivors of Taif F1, with 7 singletons; the eighth one had 3 individual flies (Table 5). Based on the F is estimates, with probabilities very close to zero, the three field populations showed deviation from Hardy-Weinberg equilibrium ( Table 5). The values of the pairwise F ST estimates for the Ace genotypes of three housefly field populations were very low, along with high gene flow (Nm ≥ 8), indicating that there is little genetic differentiation among these populations (Table 6). Table 5. Ace genotypes recovered from Saudi field house fly populations and their F is estimates of HWE test.

Recovered Ace Genotype
Riyadh Jeddah Taif

Discussion
The use of insecticides is still the primary means of controlling house flies in places such as animal production and slaughterhouses [5]. The extensive use of insecticides is the main reason for the development of resistance to these chemicals in various insect species [30]. The development of resistance in house flies was documented for numerous insecticides [5,16,31,32]. Pirimiphos-methyl is one of the common OP insecticides used for house fly control in Saudi Arabia [33]. This study was conducted to evaluate the resistance of M. domestica field populations, collected from slaughterhouses in three cities (Riyadh, Jeddah, and Taif), against PM. The field-collected populations of M. domestica displayed different levels of resistance to PM. The samples collected from Riyadh city exhibited the highest resistance, followed by populations from Jeddah and Taif, with RR values of 602.9, 175, and 116.4-fold, respectively. Several studies have documented house fly resistance to OP insecticides worldwide [5,34,35]; however, few studies documented M. domestica resistance to PM. Kočišová et al. [36] reported 40-fold increase in the house fly resistance to PM after exposing the flies to the insecticide for 10 weeks. The resistance to PM has also been found in other important insects, such as Rhyzopertha dominica, Bemisia tabaci, Aedes aegypti, and Anopheles gambiae [37][38][39][40][41]. In Saudi Arabia, OP insecticides have been used to control public health pests, and the development of house fly resistance has been reported [9,10,42,43]. House fly populations collected from Riyadh city showed an increase in the resistance rate over time to OPs [9,10]. For example, Abobakr et al. [10] reported higher RRs (62.47-309.78 folds) to diazinon compared with those reported earlier, by Alzahrani et al. [9], (6.8-72 folds) in field populations collected from the same slaughterhouses in Riyadh. Indeed, the use of insecticides with a long residual effect against flies, in closed facilities, can lead to a rapid development of resistance due to selection pressure, rapid replication rate, and absence of sensitive fly dilution effect [36].
Understanding the genetic basis of insecticide resistance in house fly field populations is critical to develop effective and successful control strategies. Many mutations detected in the Ace gene have been found to be associated with OP resistance in house flies (Table 7) [15][16][17][18]34,44]. However, three point mutations at Ace nt positions, G/C-787, G/C/T-1025, and T/A-1220, are considered the most important in offering OP resistance in house flies [15][16][17]. These point mutations lead to changes in the amino acid composition of the AChE enzyme as follows: G/C-787 > Val260Leu, G/C/T-1025 > Gly342Ala/Val, and T/A-1220 > Phe407Tyr [15][16][17]. In this study, an 842 bp fragment of the Ace gene was amplified from individual house flies representing three field populations, and their survivors were exposed to PM-insecticide. Seven nonsynonymous SNPs (A/G-715 > Ile239Val, G/A-727 > Glu243Lys, G/C-787 > Val260Leu, G/T946 > Ala316Ser, G/C-1025 > Gly342Ala, G/T-1025 > Gly342Val, and T/A-1220 > Phe407Tyr) were detected in the Ace fragment of studied flies. The detected SNPs were mostly represented by single peaks in the chromatograms. However, double and triple peaks in single positions were also detected. The double peaks indicate the presence of two alleles at a single nucleotide position (a heterozygous SNP). The triple peaks, detected at the G/C/T-1025 of Ace sequences, indicate more than two alleles at that position, suggesting the existence of more than one copy of Ace in the M. domestica genome. The triple peaks in house fly Ace sequences were previously detected in different populations, e.g., in the USA [5]. Many studies reported the existence of Ace gene duplication in mosquitoes, e.g., Anopheles gambiae and Culex pipiens [45][46][47]. Further studies are needed to confirm the existence of Ace gene duplication and to investigate their fitness cost, associated with OP resistance, in field populations of house flies.  [34,44] * The mutation positions are underlined and indicated by arrows. ** The mutation not detected (-), not available (ND) or detected (+).
The individual contribution of single amino acid changes, resulting from single Ace point mutations, can differ in their influence on the house fly insensitivity to OP insecticides [15,17]. Kozaki et al. [17] and Walsh et al. [15] reported that Val260Leu mutation in house fly AChE may confer relatively limited levels of insecticide insensitivity. In this study, there was a positive correlation between RRs and Val260Leu mutation; however, the correlation was not significant. For the Gly342Ala/Val mutation, Walsh et al. [15] showed that it resulted in much stronger resistance in house fly populations. The change of Gly to either Ala or Val, at the amino acid position 342, results in increasing the size of its side chains and decreasing the area of the active site of AChE [48]. The strong positive correlation between PM-RRs and Gly342Ala mutation (r = 0.99, p = 0.03) supports the previous findings. The replacement of Phe with Tyr, at 407 position of AChE polypeptide, showed a minor effect on its insensitivity [15]. In this study, the three amino acid replacements at positions 260, 342, and 407 were detected in both field populations and their PM-surviving counterparts. Moreover, wild type amino acids (Val260, Gly342, and Phe407) at the three positions were also detected in both field populations and their counterpart survivors, except the Jeddah survivors ( Table 2). In addition, the homozygous genotypes of these wild type amino acids were detected in field populations, except for the Riyadh population, where Val/Val and Gly/Gly, at positions 260 and 342, respectively, were not detected ( Table 3). As in other worldwide house fly populations, Val/Val homozygotes at the 342 position were not recovered [4,6,16,44]. Based on previous studies and this study, the three mutations at positions 260, 342, and 407 confer different levels of insecticide insensitivity in house flies [4,15,17,18,48,49].
When the three individual mutations (at amino acid positions 260, 342, and 407) are combined, they show a strong effect on AChE activity [15,17,18,49]. For example, 342Val mutation always co-occurs with the 407Tyr mutation in insecticide-resistant house flies [15,32,44]. In this study, seventeen combinations, four homologous and thirteen heterozygous, of Val260Leu, Gly342Ala/Val, and Phe407Tyr mutations were recovered. The sensitive wild type (Val + Gly + Phe) was one of four homologous combinations and was represented by only one individual fly in Jeddah and Taif field populations. The homozygous Leu + Ala + Tyr was the most frequently detected combination, with frequencies of 46%, 32%, and 19% in Riyadh, Jeddah, and Taif field populations, respectively. In addition, the same combination was recovered from PM-surviving flies. This most frequent combination was strongly, positively correlated with the corresponding PM-RR 602.9, 175, and 116.4 values for Riyadh, Jeddah, and Taif, respectively. Walsh et al. [15] reported that house fly strain 77M, possessing Leu + Ala + Tyr combination, showed 48-fold DDVP resistance compared with the wild type. This combination was also frequently detected in house fly field populations in the USA, Turkey, Japan, Kazakhstan, and China [4,6,16,17,35,48]. Among the 13 heterogeneous combinations, Val/Leu + Ala/Val + Tyr and Leu + Ala + Phe/Tyr were well represented in the surveyed regions. Although these two combinations were recovered from the PM-survivors of Riyadh and Jeddah flies, they were not recovered from the surviving flies of Taif. Both Val/Leu + Ala/Val + Tyr and Leu + Ala + Phe/Tyr were prevalent combinations in field populations in Turkey, Japan, and China [4,6,17,48].
Selection pressure is one of the driving forces that stimulates insects to overcome insecticides. The selection pressure affects genes whose products are targets for insecticides [50,51]. In most cases, the pesticide-resistant insect populations depart from Hardy-Weinberg equilibrium [52][53][54]. In this study, the three field house fly populations departed from Hardy-Weinberg equilibrium. Moreover, the three field populations showed little genetic differentiation and high gene flow, suggesting that Ace genotypes may move among these populations.

Conclusions
In conclusion, the house fly field-collected populations from slaughterhouses in Riyadh, Jeddah, and Taif in Saudi Arabia displayed high levels of resistance to PM. Single and combined Ace mutations are apparently associated with this resistance. Based on the obtained data, the OP insecticides used in the house fly control programs in Saudi Arabia should be replaced/rotated with insecticides from other insecticide groups, targeting sites other than AChE. The outcomes of the present work can be useful in managing house fly field populations in Saudi Arabia.