Polymorphism and Distribution of Ace Gene Involved in the Resistance of Musca Domestica to Organophosphates in Guizhou Province of China

Acetylcholinesterase is the primary target of organophosphates (OPs) and carbamates in insects. As gene mutation has been veri�ed as an important mechanism of insecticide resistance in insects, in this study, we investigated the status of OPs resistance and the polymorphism of ace gene (that encodes acetylcholinesterase) in house�y (Musca domestica L) �eld populations in Guizhou Province, China. Bioassays showed that the house�ies had 142.16– 303.54-fold resistance to dichlorvos (DDVP) and 122.13–363.98-fold resistance to temephos. Molecular analysis revealed resistance-causing mutations of ace gene at loci of 260, 342 and 407 in the house�y populations, with a total frequency of 55%, 100% and 94%, respectively. In addition, 11 combinations of ace mutation were observed across the detected populations. The most frequently detected combination was L/V+A/V+Y, followed by L+A+Y and L/V+A+Y. No signi�cant relationship was found between single mutation/combination mutations and DDVP resistance. These results indicate that the OPs resistance is prevalent among the house�y populations in Guizhou Province, with a similar pattern of allele mutation of ace across China. The target resistance can not fully account for the resistance of house�ies to OPs in Guizhou.


Introduction
House ies, Musca domestica, are a transmission vector of more than 100 human and animal pathogens, including bacteria, parasites, viruses, and rickettsia [1]. Until now, chemicals with strong insect lethality have been widely used to control house ies. Currently, the use of harmful insecticides has amounted to 1.8 billion kilograms in China and the amount of hazardous insecticide use in the USA has reached 31.75 million kilograms in urban settings [2,3]. Although the chemical insecticides can effectively control house ies and the diseases they carry, the long-term, extensive use of insecticides can lead to the development of insecticide resistance in house ies [4,5]. A study in 48 Chinese cities has suggested that the house ies have developed a strong resistance to several common insecticides, such as dichlorvos (DDVP), temephos and deltamethrin [6]. This indicates that the resistance to organophosphate (OP) insecticides including DDVP and temephos has become a serious concern in the control of local house ies in China. It has been revealed that the resistance of house ies to OPs is mainly attributed to the insensitive target-site acetylcholinesterase (AChE) [3,7,8]. AChE (EC 3.1.1.7), encoded by the ace gene, is the key enzyme of the cholinergic system in insects [9]. OPs and carbamates (CBs) can irreversibly bind with AChE and cause phosphorylation or carbamylation of the enzyme at the active serine site, leading to the accumulation of acetylcholine at synapses [10,11]. This in turn leaves the acetylcholine receptor permanently open, resulting in the death of the insect [10]. However, modi cation or mutation of the ace gene can change the structure of AChE, thereby reducing or eliminating the binding a nity of insecticides at the target-site and inducing the resistance to OPs and CBs[8, 12,13]. For house ies, there is only a single ace gene in the whole genome [14]. Currently, six mutations (V260L, A316S,G342A/V, F407Y and G445A) have been veri ed to be present separately or in combination within a particular active site of AChE that can change the current of the catalytic triad and restrict the bind ability to insecticides, thus being responsible for the development of resistance [8, 15,16].
In Guizhou Province located in the southwest part of China, the insecticide resistance of house ies has been reported only by one study (1999) [17]. With the campaign of establishing the national sanitary cities across China, the resistance of house ies to propoxur and DDVP has been reported in some major cities of Guizhou, such as Anshun, Guiyang and Xingyi, in recently years [18][19][20]. However, little is known about the underlying genetic variability of AChE that confers the resistance to OPs and CBs. In this study, we set out to explore the resistance status of house ies to OPs and investigate the genetic mutations of ace in the house y (Musca domestica L) eld populations across Guizhou Province.

Materials And Methods
Collection and rearing of house ies The house y observation, adult house y collection, and eld studies in Guizhou Province were conducted from April 2018 to December 2019. Adult M. domestica house ies were collected in urban or suburban areas distributed in 7 different regions in Guizhou Province (Fig. 1). In the current study, about 100 house ies were collected by the sweep net mainly in waste transfer stations and refused dump of the farmer markets or old residential buildings, and then mixed to represent a local population. All of the eld-collected populations were routinely reared with a mixture of milk powder and granulated sugar at a ratio of 1:1 in an appropriate amount of water, at a constant indoor temperature of 25±1 ℃, with humidity of 70±10% under a 12 h light/12 h dark cycle. House y eggs were laid in wheat bran (100 g), with milk powder (5 g), granulated sugar (5 g) and water (130 mL), as the random mating of house ies occurred in the breeding cage. The eggs were hatched to pupate on the dry surface of the feed within 7 days. The adult susceptibility bioassay and molecular tests were conducted using the rst-generation, eld-collected house ies of 3-5 days old with a similar body weight of 18-22 mg.

Bioassays
Bioassays were performed in accordance with the Bioassay Methods for Musca domestica (GB/T 26350 (2010)) released by the Standardization Administration of the People's Republic of China [21].
The 97.6% DDVP and 87.4% temephos solutions provided by the Chinese Center for Disease Control and Prevention (CDC) were rst dissolved in acetone and then half-diluted to a series of concentrations. The customized 0.35 μL pippets purchased from Nanjing Agricultural University (Nanjing, China) were used to conduct bioassays for each house y population. The assays at each concentration of DDVP and temephos were performed in three replicates, with acetone being used as a negative control. The regression equations were obtained using the mortality 24 h after drug exposure in each test recorded after. The LD 50 (lethal dose, 50%) value was calculated based on the corresponding insecticide concentrations. The mortality of the control group was below 5%. The resistance ratio (RR) was obtained from dividing the LD 50 of different populations by the LD 50 of the susceptible house y.

Extraction of genomic DNA
The genomic DNA of house ies was extracted according to the description by Yang et al. [20]. A whole adult house y was homogenized in 300 μL extraction buffer in a 1.5 mL Eppendorf tube, and proteinase K (50 μg) was added. The homogenates were incubated at 56℃ overnight. Then a solution (300 μL) of chloroform and isoamyl alcohol (24:1) was added. After shaking violently for several times, the samples were centrifuged at 10000 rpm/min at 4℃ for 10 min. The supernatant was transferred to a new tube, and a 0.1-fold volume of 3 M sodium acetate (4℃) and a 2-fold volume of pure ethanol were added to precipitate DNA for 2 h. Afterwards, the supernatant was discarded after centrifugation at 12000 rpm/min for 5 min at 4 ℃, and the DNA was washed twice with 70% ethanol (1 mL). Finally, the DNA was resolved with ddH 2 O and stored at -20℃ until use.

Statistical analysis
The LD 50 of insecticides in the tested populations was calculated with a probit analysis based on the recorded concentration-mortality data. Association between ace genotype and phenotypical resistance was analyzed with the Spearman's correlation coe cient. All analyses were conducted using SPSS 22.0 software (IBM, Armonk, NY, USA).

Resistance of house y populations to insecticides
The resistance of house ies to DDVP and temephos was analyzed in 5 and 4 populations, respectively. The LD 50 values of DDVP and temephos ranged 0.56865-1.21415 μg/house y and 13.8005-41.12605 μg/house y, respectively. The two OPs showed a high resistance ratio, 122.13~363.95-fold for temephos and 142.16~303.54-fold for DDVP ( Table 1).
The distribution and frequency of ace mutations A total of 237 PCR products of ace gene were obtained and sequenced. Nonsynonymous mutations were detected only at the loci of V260, G342, and F407 among the eld-collected populations (Fig. 2). The frequencies of these mutant alleles were 55%, 100%, and 94% respectively. In addition, 23.63% and 4.24% of the house ies were homozygous for L260 and V260 respectively, whereas 72.13% were heterozygous. Only 2 mutation genotypes (A/V or A/A) were detected at the locus 342, and the frequencies were 63.94% and 36.06%, respectively. At the locus 407, three genotypes (F/F F/Y and Y/Y) were found and 91.7% of the house ies were homozygotes for Y/Y ( Table 2). The KL and GY were two top populations for the simultaneous mutation frequency of the three loci, while the CH and ZY populations had the lowest simultaneous mutation frequency of the three loci.
Eleven combinations of ace mutation were detected among the populations (Fig. 3). The combination of L/V+A/V+Y had the highest frequency (59.9%), followed by the L+A+Y (22.8%) and L/V+A+Y (8%). The ZY population had seven combinations, the AS population had ve combinations, the LPS, HS and GY populations each had four combinations, and the CS population had the least 2 combinations. No distinct differences among the populations had been found.

Relationship between ace mutation and insecticide resistance
According to the bioassay results of DDVP in 5 eld populations, we conducted the correlation analysis to explore the relationship between single or combined mutations of ace gene and DDVP resistance phenotype (Fig.4). However, no signi cant correlation was detected between them.

Discussion
In China, OPs and CBs have been widely used in the public health eld since 1950s, because of the low costs and high e ciency [23]. The development of insecticide resistance during house y control has been widely reported in China. Previous studies on house ies collected from Xingyi City and Anshun City in Guizhou have also shown a high resistance to OPs and CBs [18][19][20]. The current bioassay results further revealed the presence of a high resistance to OPs in house ies in Guizhou Province.
The ace gene of house y has been demonstrated as an indicator for the response to OPs and CBs stress [22]. In this study, 6 alleles at loci 260, 342, and 407 were detected in all 7 house y populations in Guizhou Province. It is noteworthy that the frequency of allele mutation was 100% at locus 342 and 94% at locus 407. This is consistent with previous report on ace gene in house ies in Guangdong and Shanghai, China [24]. Previous studies have shown that both G342A/V and F407Y mutations, which are located close to the active-site triad of AChE at the base of the gorge, can cause resistance to OPs and CBs, with G342A/V mutation likely to affect the orientation of the catalytic serine and F407Y decreasing the available space within the acyl-binding pocket[8].
compared to the wild-type enzyme [8,25]. In the present study, the frequency of the 342A/V genotype (61.9%) was higher than that of the 342A/A genotype (36.1%), which might suggest that the 342A/V genotype confers higher resistance to OPs. Interestingly, 342V homozygotes have not been detected in the present or previous studies, either in China or in other countries outside of China[16,22,24].It is further suggested that house ies with the 342V mutation carry higher tness cost [24,26]. For mutations at the locus 407, 407Y conferred 1.8-fold resistance to DDVP[8], and the genotype 407Y/Y (91.7%) ranked top in frequency among three genotypes in all detected populations. Similar results have been found in other places in China [24].
It has been found that combined mutations of ace often confer higher resistance than single mutations [8,25]. Here, 11 combinations of three mutations (V260L, G342A/V, and F407Y) were observed in the eld populations. House ies with any of these combinations showed resistance to OPs and CBs. V/L+A/V+Y and L+A+Y were the top 2 combinations in frequency, which caused 48-fold resistance to DDVP compared to the wild-type[8]. This mutation pattern of ace has also been found in other regions of China [24], and in Japan, Turkey [22] and Europe [25], but not in the USA [16], which indicates that the resistance alleles vary globally in different regions. The combination V/L + A + Y has been detected in Guangdong Province in south of China, but not in Shanghai, Shandong, Beijing, or Jilin, which are located in east and north of China [24]. In the present study, the combination V/L + A + Y was found in the house y populations, and also ranked third in frequency. This suggests that this pattern of mutation is common in south China.
There was no distinct distribution division of ace resistance alleles either for single mutation or combined mutations, among the different eld populations in Guizhou. This might be the result of two evolutionary forces (mutation and migration). The allelic diversity in eld populations might enable them survive against the different insecticides [27]. Correspondingly, application of a multiplicity of treatments on eld populations might result in the population heterogeneity, composed of a mixture of different alleles.
Although the current study showed a high resistance phenotype, no signi cant relationship was found between either single mutation or combined mutations and DDVP resistance. This indicates that target resistance is not the only mechanism involved in the house y resistance to OPs in Guizhou. Further studies are needed to clarify the resistance mechanism.

Conclusion
In conclusion, the resistance to OPs exists widely in house ies in Guizhou Province. A similar pattern of allele mutation of ace has been found across China. The ndings imply a need for monitoring on insecticide use to avoid the abuse of insecticides. It also demonstrates that target resistance is not enough to account for house y resistance to OPs in Guizhou.