Exploration of New Mutations for Resistance of Houseflies to Organophosphate Insecticides in Housefly Populations in Guizhou Province, China


 Background： The campaign to establish the national sanitary city has been launched across major places in Guizhou Province, which leads to the extensive use of insecticide to eradicate the disease-carrier Musca domestica found everywhere while keeping the cleanliness of environment.Methods: In order to perceive the resistance of houseflies to the commonly used organophosphate insecticides in 7 housefly populations belonging to Guizhou province (China), the susceptibility bioassays, detection of resistance-associated mutations, and the carboxylesterase activity assay were conducted.Results: The bioassays exhibited 142.16~303.54-fold to dichlorvos (DDVP) and 122.13~363.98-fold to temephos. The molecular analysis unveiled mutant ACE gene at loci of 260, 342 and 407 in all populations, which led to high frequencies at 27.4~73.8% of 260L, 59.1~76.7% of 342A, 23.7~40.9% of 342V, and 83.4~100.0% of 407Y, with inclusion of 8 genotypes and 10 mutant combinations. Further analysis of mutations showed a linkage disequilibrium of L/V+A+Y at locus 260 & 342, indicating a significant association with the DDVP resistance. The Hardy-Weinberg equilibrium test demonstrated that the observed ACE heterozygosity mostly exceeded 0.5 and deviated from the equilibrium. In the fixation index, an insignificant differentiation was noted among the 7 housefly populations. Conclusions: However, further research should concentrate on the use of insecticides to avoid the abuse of insecticides, and to regularly monitor the resistance of houseflies using novel methods.

makes the selection pressure works and forces house y to activate various mechanisms for adaptation and resistance to insecticide. A study concentrated on 48 Chinese cities demonstrated that the house ies showed a strong resistance to several commonly used insecticides, such as dichlorvos (DDVP), temephos, etc. [5], Moreover, the resistance of house ies to deltamethrin and DDVP was reported [5]. This indicated that the resistance to the organophosphate (OP) insecticides, including DDVP and temephos, may cause a serious concern to control local house ies.
It has been previously revealed that the resistance of house ies to OP is attributable to insensitive acetylcholinesterase (AChE) and enhances carboxylesterase activity. The AChE enzyme is a serine hydrolase vital for regulating the neurotransmitter acetylcholine in mammals, birds, and insects [6,7]. However, the modi cation or mutation of ACE gene encoding AChE may change the structure of enzyme, thereby reducing or eliminating the binding a nity of insecticide with the target-site [8]. It was suggested that V260L, G342A/V, and F407Y mutations of ACE gene in a particular active site are responsible for the resistance to changing the current of the catalytic triad, as well as restricting binding to insecticides [9,10]. Carboxylesterase is widely found in insects, and changes in carboxylesterase activity were proven to be associated with resistance to OP insecticide in some species of house ies, mosquito, aphids, blow ies, and western ower thrips where the resistant strains of ies showed a tendency to suppress the level of the carboxylesterase activity. The main reason is that structural mutation and overexpression of carboxylesterase inhibit hydrolyzation of methyl butyrate and naphthyl acetate, triggering increase of OP [11,12]. An evidence suggested that multiple carboxylesterase genes were co-upregulated in resistant house ies [12,13].
Regrettably, since 1999, only a research conducted by Lin & Zhu (1999) concentrated on house y resistance in Guizhou province (China). Subsequently, in the campaign of establishing the national sanitary city that was spread gradually across mainland China [14], Guizhou province played an active role using a series of insecticides for reducing the number of house ies, which could be an indicator to determine whether that city was quali ed to be a national sanitary city or not. Thereafter, the resistance of house ies to propoxur and DDVP in Anshun, Guiyang and Xingy, major cities of Guizhou, was reported to be terrible [15][16][17]. In the present study, an attempt was made to understand the current status of the resistance of house ies to OP in other places across Guizhou province, and to predict the future trend of resistance.

Collection and rearing of house ies
Adult Musca domestica house ies were collected from urban or suburban areas distributed in 7 different places across Guizhou province, as shown in Fig. 1. In the current study, there were around 100 house ies with the sweep net mainly in waste transfer stations and refuse dump of the farmer markets or old residential buildings, and those were mixed to represent a local population. All of the collected house ies were routinely reared with the mixture of milk powder: granulated sugar (1:1) plus 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 [11]. House ies' eggs were laid in wheat bran (100 g) containing milk powder (5 g), granulated sugar (5 g), and water (130 mL) and hatched to pupate in some dry surface of the feed during within 7 days [18]. The World Health Organization (WHO) susceptibility bioassay, as well as chemical and molecular tests were conducted using the rst-generation of collected house ies that aged 3 ~ 5 days old with a similar body weight of 18-22 mg. The insecticide-susceptible house y population was transferred to the laboratory of the Chinese Center for Disease Control and Prevention (CDC), without exposure to any insecticide for decades and treated as the control group [19].

Bioassays
The resistance of house ies to DDVP in 5 populations and to temephos in 4 populations were examined. The LD 50 (µg/house y) of house ies to DDVP and temephos ranged from 0.56865-1.21415 and 13.8005-41.12605, respectively. Two OPs were characterized by an extremely high resistance, which reached 122.13 ~ 363.95-fold in temephos and 142.16 ~ 303.54-fold in DDVP, respectively (Table 1).

Extraction Of Genomic Dna
A whole adult house y was fully homogenized in a 300 µL extraction buffer (100 mM Tris-HCl (pH, 8.8), 50 mM NaCl, 10 mM EDTA, with 1% SDS) in a 1.5 mL Eppendorf tube, and then, proteinase K (50 µg) was added to each sample. The homogenates were incubated in a water bath at 56 ℃ overnight. On the next-day, solution of chloroform (300 µL) and isoamyl alcohol (24:1) was added to the tube for separation of the protein from DNA. After that, it was turned upside down for several times and centrifuged at 10000 rpm/min for 10 min at 4 ℃. The supernatant with aqueous phase containing DNA was sucked up to a new tube, added with sodium acetate (3 M) of 0.1-fold volume and absolute ethanol of 2-fold volume supernatant, and frozen to precipitate for longer than 2 h. Afterwards, the supernatant was discarded after centrifugation at 12000 rpm/min for 5 min at 4 ℃. Moreover, 70% ethanol (1 mL) was used to wash the DNA for once or twice after centrifugation at 12000 rpm/min, 4℃, for 10 min. Finally, the supernatant in the tube was discarded and the DNA remained on the tube bottom or the wall, in form of mixed with ddH 2 O (15 µL). DNA (10-15 µg) was extracted from a house y and more than 30 DNA samples were isolated from each eld-collected population as described previously [20,21].

Ampli cation And Sequencing Of Ace Gene
The ACE gene fragment was ampli ed in a 25 µL reaction containing Premix Taq™ (12.5µL), with Taq

Assay Of Carboxylesterase Activity
Preparation and quanti cation of crude enzyme: A decapitated house y (age, 4 days old; body weight, 18-22 mg) was homogenized with 500 µL cold phosphate-buffered saline (PBS; 0.1 M, pH 7.5, containing 0.1% Triton-x100) in a pre-cold Eppendorf tube (1.5 mL). Subsequently, the homogenate was centrifuged at 10000 g for 15 min, and the supernatant was stored at 4 ℃. A total of 150 house ies were chosen from the eld-collected population of AS, GY, and HS. The enzyme was quanti ed using the Thermo Scienti c Pierce BCA Protein Assay Kit [23].
Standard curve of α-naphthol: 600 µL of α-naphthol solution (1, 0.8, 0.5, 0.25, 0.125, and 0.0625 mM) and 100 µL of Fast Blue BB salt solution (3 mM containing 5% SDS, Shanghai Yuanye Biotech Co., Ltd., Shanghai, China) were added into each well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate. The mixtures exhibited a different level of yellow-brown color and were measured with the Bio-tech Epoch spectrophotometer at wavelength of 570 nm after 5 min. The average absorbance of the diluting solutions minus that of the PBS control was calculated to plot the curve. An equation of y = ax + b was formulated (x = 1, 0.8, 0.5, 0.25, 0.125, and 0.0625 mM). Then, the following assay was conducted to calculate the unknown concentration of the produced α-naphthol using the standard curve [24,25].
Measurement of carboxylesterase activity: Carboxylesterase catalyzes α-naphthylacetate and produces α-naphthol, and can be detected by the reaction product of α-naphthol. The mixture with 20 µL of crude enzyme and 200 µL of α-naphthylacetate (0.3 mM Shanghai Yuanye Biotech Co., Ltd., Shanghai, China) was incubated for 15 min at room temperature. Subsequently, the Fast Blue BB salt solution (50 µL) was added for 5 min, and the absorption was recorded at 570 nm with the assistance of the Bio-tech Epoch spectrophotometer. Three duplicates were required for each house y and PBS control. According to the quanti ed enzyme (µg/µL) and the standard curve of α-naphthol (mM), the carboxylesterase activity was converted to α-naphthyl acetate (µmol α-naphthol/min/mg protein) [24,25].

Statistical Analysis
The LD 50 of tested house ies was calculated by conducting probit analysis of concentration-mortality data using SPSS 17.0 software (IBM, Armonk, NY, USA). The resistance ratio (RR) was obtained from the LD 50 of different insecticides dividing the respective LD 50 of the susceptible house y. The Pearson's correlation coe cient between the mutation frequency of each house y population and the respective insecticide resistance was analyzed by GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). The mutation frequency was examined by Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium with Arlequin 3.5, while the xation index (F ST ) was applied to analyze the population differentiation by the Analysis of Molecular Variance (AMOVA) [34]. The analysis of carboxylesterase activity was carried out using GraphPad Prism 6.0 software.

Bioassays
The resistance of house ies to DDVP in 5 populations and to temephos in 4 populations were examined. The LD 50 (μg/house y) of house ies to DDVP and temephos ranged from 0.56865-1.21415 and 13.8005-41.12605, respectively. Two OPs were characterized by an extremely high resistance, which reached 122.13~363.95-fold in temephos and 142.16~303.54-fold in DDVP, respectively (Table 1).

Ace Mutation And Aa Substitution
Eight genotypes were detected at loci of 260, 342, and 407 in all eld-collected populations, as shown in Fig. 2. New mutations of the ACE gene were at loci of 260, 342, and 407 in all populations. The frequency of heterozygous substitution L/V at locus 260 was 53.3%, whole house ies were completely found at locus 342 substitution of A/V or A/A, and Y/Y substitution occurred at locus 407 in house ies that occupied more than 80% (Table 2). KL had the highest substitution in 260L, and GY had in 342A. With respect to 407Y, CS and LPS even reached 100% substitution, as shown in Table 2. Besides, 10 combinations of ACE were observed among all detected samples, where ZY involved 7 combinations and CS involved only 2 (Fig. 3). The combination of L/V + A/V + Y topped each eld-collected population, followed by L + A + Y and L/V + A + Y. According to the Pearson's correlation coe cient, combination of 342A/V substitution (r = 0.844/-0.835, P < 0.05) and L/V + A + Y substitution (r = 0.970, P = 0.003) exhibited a strong correlation with LD 50 of DDVP (Table 3).   The observed and the projected heterozygosity of the 10 combinations among 7 populations were validated by analysis of variance (ANOVA) using Arelequin 3.5 software. CS and LPS were detected without any heterozygosity, as presented in Table 4.  (Table 5). AMOVA results unveiled a F ST of 0.02819 (P < 0.05), suggesting that the differences in ACE genotypes in each population were statistically signi cant ( Table 6). The F ST between ZY and CS reached the maximum (0.08246, P < 0.05), followed by ZY and KL (0.07023, P < 0.05), while the F ST between other populations mainly ranged from 0 to 0.05 (P < 0.05), as shown in Table 7.

Carboxylesterase Activity
The increased metabolic detoxci cation is another signi cant resistant mechanism, in which carboxylesterase has indicated a close association with OP resistance. In a previous research, a variety of carboxylesterases were detected to be associated with OP metabolism and resistance [32], which is attributed to changes or overexpression of the carboxylesterase. For instance, a recent duplication event was noted in L. cuprina that was resulted in the duplication of the chromosomal region containing αE7, with two copies of this gene and others from the α-esterase cluster being carried on one chromosome [33]. In the current study, we attempted to assess carboxylesterase activity of house ies in three places in Guizhou province. AS house y population was considered to have a high carboxylesterase activity. However, measurement of enzymatic activity demonstrated that the HS house y catalyzed more substrate (Fig. 4). Thus, it can be concluded that structural changes in carboxylesterase contributed to a reduced a nity to the substrate. Therefore, further research needs to be conducted to validate this hypothesis. Recently, Feng and Liu (2018) suggested that up-regulation of carboxylesterase genes is a major component of insecticide resistant mechanisms in insects, and concluded that multiple carboxylesterase genes are coupregulated in resistant house ies, providing further evidence for their involvement in the detoxi cation of insecticides and development of insecticide resistance.

Discussion
China is faced with a widespread resistance of house ies to the commonly used insecticides, with even an extremely high resistance to certain chemical products (e.g., propoxur). In recent years, several researches have assessed the resistance of house ies to insecticides in some cities across mainland China (e.g., Anshun, Guiyang, and Xingyi), which is consistent with a higher resistance to OP than that of pyrethroid [15]. Compared with previously obtained results, DDVP resistance was found more serious than pyrethroid in the present research. To our knowledge, pyrethroid is a less harmful insecticide to human and environment, while DDVP is being slathered in several places and resistance of house ies to DDVP has gradually increased. A house y obtained from the eld-collection of Guizhou exhibited an extremely high resistance to DDVP with a LD 50 of 0.56865 ~ 1.21415 µg/house y, in which the RR of GY reached 303.54, and a relatively high resistance to temephos was noted with a LD 50 of 13.8005 ~ 41.12605 µg/house y; besides, the RR of KL reached 363.95, and that of AS stood in the second place (212.51). The results of bioassays unveiled that the house y resistance in different populations varied, and prompted a speci c control program for house ies according to local conditions. Although house ies in Hangzhou, Wuhan, and Zhangjiagang cities (China) showed a resistance to DDVP and pyrethroid to some extent between 2014 and 2016, the average resistance to DDVP in Guizhou province was signi cantly higher than that in other provinces [26][27][28]. This resistant was found to be associated with duration of pest control in China [29]. For temephos, it is more frequently used as mosquito larvicide, and few reports are available, highlighting the resistance of house ies to temephos [30]. In the present research, the resistance of house ies to temephos seemed hardly optimistic in 4 detected places of Guizhou, especially KL for the RR that reached 363.95-fold. Therefore, continuous application of DDVP and temephos might be harmful to control house ies in Guizhou province, while the application of insecticide could be well coordinated and a regular supervision was enforced.

The Relationship Between Ace Mutation And Resistance To Op
The ACE mutation was associated with resistance to OP, including V260L, G342A/V, F407Y, etc. [6,7,11]. ACE gene fragment of 7 house y populations was ampli ed and sequenced, and 8 genotypes were identi ed ( HWE test revealed a disequilibrium that the observed heterozygosity was more than expectation in AS, CS, GY, HS, and LPS at locus 260, in AS, CS, LPS and ZY at locus 342, and in ZY at 407, while an observed disequilibrium indicated that the heterozygosity was less than expectation in AS at 407. Due to excessive heterozygosity, the disequilibrium occurred, suggesting that all the populations survived at the highest heterozygosity. When it could not reach the genetic equilibrium, the relative gene from the population was mutated for a survival bene t [31]. In order to avoid the possible xed mutant population, we may adjust the use of various insecticides before the resistance of house ies to OP become steady. For balance-keeping resistant population, it may exhibit a stable inheritance of resistance, demonstrating loss of the best opportunity to take containment measures. In this survey, KL showed the equilibrium at the three loci and the resistance ratio reached 363.95, while it needs to be reduced in the future control program of house ies in KL. The LD test disclosed a linkage of 260 and 342 in all detected populations, a linkage of 260 and 407 in AS, HS and ZY, and a linkage of 342 and 407 in GY and HS. It was previously uncovered that 342V and multiple mutations could lead to increased resistance [9]. Thus, the correlation among the three loci increased the existence of L/V + A + Y, leading to a threatening resistance to OP.

Carboxylesterase Activity
The increased metabolic detoxci cation is another signi cant resistant mechanism, in which carboxylesterase has indicated a close association with OP resistance. In a previous research, a variety of carboxylesterases were detected to be associated with OP metabolism and resistance [32], which is attributed to changes or overexpression of the carboxylesterase. For instance, a recent duplication event was noted in L. cuprina that was resulted in the duplication of the chromosomal region containing αE7, with two copies of this gene and others from the α-esterase cluster being carried on one chromosome [33]. In the current study, we attempted to assess carboxylesterase activity of house ies in three places in Guizhou province. AS house y population was considered to have a high carboxylesterase activity. However, measurement of enzymatic activity demonstrated that the HS house y catalyzed more substrate (Fig. 4). Thus, it can be concluded that structural changes in carboxylesterase contributed to a reduced a nity to the substrate.
Therefore, further research needs to be conducted to validate this hypothesis. Recently, Feng and Liu (2018) suggested that up-regulation of carboxylesterase genes is a major component of insecticide resistant mechanisms in insects, and concluded that multiple carboxylesterase genes are coupregulated in resistant house ies, providing further evidence for their involvement in the detoxi cation of insecticides and development of insecticide resistance.

Conclusion
In summary, the different house y populations in Guizhou province caused a terrible resistance to the OP insecticide, and bioassays exhibited the large mutant ACE with stable inheritance of partial loci to the offspring was widespread and kept the Hardy-Weinburg equilibrium, and the carboxylesterase activity assays presented a possible strong OP hydrolysis. However, further research should concentrate on the use of insecticides to avoid the abuse of insecticides, and to regularly monitor the resistance of house ies using various methods.