Polymorphism and Geographical Distribution of vgsc and ace-1 Gene in Anopheles Sinensis Field Populations in Guizhou Province, China

Background: Vector control has been a key strategy in malaria prevention and elimination for decades. However, insecticide resistance is becoming a serious threat to vector control. Anopheles sinensis is one of the important transmission vectors for malaria in Guizhou Province, China. However, little is known on insecticide resistance status and related mechanism. In this study, the diversity and frequency of the major insecticide resistance associated genes such as voltage-gated sodium channel (vgsc) and acetylcholinesterase-1 ( ace-1 ) genes that encoded the target proteins of Pyrethroids and OPs were investigated in eld populations. Methods: Adult mosquitoes were collected from 12 sampling sites across Guizhou by lamp trapping. Female An. sinensis were identied by morphological and molecular identication. Genomic DNA was extracted to amplify vgsc and ace-1 gene fragments. PCR products were sequenced bi-directly. Mutations of vgsc gene at locus 1014 and that of ace-1 at locus 119 were analyzed using MEGA 7.1 software, and the frequencies of mutations were calculated respectively. Results: 5 kdr mutation alleles at the locus 1014 of vgsc gene as a result of three amino acid replacements ( namely 1014F/C/S) in 548 samples of 12 An. sinensis populations. The total frequency of kdr mutation alleles was 27.4%, of which the TTT/C (F) allele had a highest mutation frequency of 22.5%. The top three mutation genotypes were from XiShui, TongZhi and DeJiang populations collected in north Guizhou. There were three alleles at locus 119 in ace-1 gene with 49.47% of GGC/G, 0.17% of GGT/G and 50.36% of AGC/S. The 100% frequency of mutation genotypes (GS, SS) was found in CeHeng, LuoDian and SanDu populations gathered in southwest Guizhou. Conclusion: A diverse genetic mutations of vgsc and ace-1 genes are found in An. sinensis in Guizhou. There are a signicant geographical heterogeneities of allele frequency among different populations in A high frequency of mutation (>44 %) in north Guizhou. The 119S mutation of ace-1 gene is present at a high frequency in most An. sinensis populations in Guizhou, especially in the previously highly endemic malaria regions. These ndings suggest continued monitoring of the genotypic diversity of insecticides resistance genes may assist to formulate a region-customized resistance management strategies. In this study, we investigated the mutation and distribution of two target-site resistance genes in 12 eld populations of Anopheles sinensis across Guizhou Province. We discovered 5 different kdr mutation alleles at the locus 1014 and 4 different ace-1 alleles at the locus 119 in these samples, of which the TGG/C allele of kdr gene and TTG/G allele of ace-1 were found for the rst time. The results showed a diverse genetic mutations in An. sinensis in Guizhou. A high frequency of kdr mutation (> 44 %) in DeJiang, TongZhi, and Xishui indicates a risk of resistance to pyrethroids and DDT in An. sinensis in these areas. The G119S mutation is widespread in An. sinensis in Guizhou, especially in the previously identied high-risk malaria endemic areas which implicate that cautions should be paid when using OPs and CBs as control agents for vectors. Continued monitoring of insecticide resistance and the genotypic diversity of resistance genes may assist to formulate effective region-customized resistance management strategies before implementing malaria control strategies.


Introduction
Malaria remains an important public health problem in tropical and sub-tropical countries. According to the report from World Health Organization (WHO), there were 229 million clinical cases of and approximately 409,000 deaths from malaria infection in 2019, and nearly 92% of them occurred in Africa [1]. It also ranked the rst among top ve important parasitosis in China in the past. In mainland China, no indigenous malaria cases have been reported since 2017 because 'an action plan for malaria elimination' with a goal of eliminating malaria throughout the country by the end of 2020 was launched in 2010 by the National Ministry of Health [2,3]. However, imported malaria cases are posing a serious challenge to malaria control for China.
Due to the lack of effective vaccine for malaria, vector control is still one of the effective measures to control and eliminate malaria transmission in the world. Insecticides remain the most important vector control method. Currently, four groups of insecticides such as DDT, pyrethroids, organophosphates (OPs) and carbamates (CBs) are recommended to control mosquito by WHO [4]. Massive sprayings of insecticides greatly limited mosquito-borne diseases and even eradicated malaria in a few areas in the past [5]. However, the widespread development of resistance in mosquitoes to insecticides is now causing serious problems in many areas [6].
As we well known, target-site insensitivity has been veri ed to be one of two major mechanisms that are involved in insecticide resistance. The insect voltage-gated sodium channels (VGSCs) are the target of pyrethroids and organochlorine insecticides. Many studies have demonstrated that mutations at 1014 locus are able to confer knockdown resistance (kdr) in many arthropod species including Anophelines [7]. To date, in Anopheles, a total of 4 types of kdr mutations (L1014F, L1014C, L1014S and L1014W) that are related to insecticide resistance were reported [8-10], of which L1014F was evidenced to be the most common mutation in Africa, Asia and America [11]. Further, organophosphates (OPs) and carbamates (CBs) can target acetylcholinesterases (AChEs) of insects which are encoded by two acetylcholinesterase genes ace-1 and ace-2 in mosquito, but only ace-1 has been found to be signi cantly associated with resistance to OPs and CBs [12]. The G119S mutation of ace-1 gene causes a spatial shift of AChE structure, which alters the interaction between the enzyme and insecticides, hereby resulting in insecticide resistance [13]. Multiple lines of evidence have revealed that the G119S mutation occurs at high frequency in many eld populations of An. sinensis in Asia [8,13].
There are mainly 4 species of Anopheles that can transmit malaria in China, of which Anopheles sinensis was found to be the most concerned vector because of its wide distribution and species predominance in the country [14].Currently, the insecticide resistance level and resistance mechanism on An. sinensis have been widely reported in many places in China, including Sichuan Province, Guangxi Province and Chongqing that neighbor Guizhou [15][16][17][18][19][20]. However, little information on the insecticide resistance status of An. sinensis has been reported in Guizhou Province, southwest of China. In this study, the distribution and frequency of target-site mutation in vgsc and ace-1 genes of An. sinensis across Guizhou were detected to reveal the molecular resistance status.

Mosquito samples
Adult mosquitoes were collected in 12 sites (Fig. 1) across Guizhou province by light trap (wave length 365 nm) in August 2017 and July-August 2018. In each site, three or four houses (with a distance > 50 m) were each equipped with a light trap. The light trap was placed in the pig pen or cattle pen, 1.5-2.0 m above the ground. The mosquitoes were collected from 1 hour before sunset to 1 hour after sunrise, for consecutive three days. The captured mosquitoes individually underwent morphological identi cation and those con rmed to be An. sinensis adults were put into Eppendorf tubes containing 100% ethanol and kept at -4℃ until use.

Extraction of genomic DNA and species identi cation
The genomic DNA of mosquitoes was extracted with TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver. 5.0, according to the manufacturer's instructions. Genomic DNA was kept at -20℃ until use. The extracted DNA was used immediately for PCR assay or stored at -20°C for later use. Molecular identi cation of An.sinensis species was performed with primers for species-speci c mitochondrial DNA cytochrome oxidase subunit I (mtDNA-COI) gene (COI-F: 5' -GGTCAACAAATCATAAAGATATTGG − 3'; COI-R: 5'-TAAACTTCAGGGTGACCAAAAAATCA − 3') [21]. The mtDNA-COI gene fragment was ampli ed in a 15 µL reaction system containing Premix TaqTM (7.5µL) (TAKARA Bio Inc., Shiga, Japan), ddH2O (4.9 µL), DNA template (1 µL), and 10 µM primers (1µL each). PCR reactions were performed in a SimpliAmpTM Thermal Cycler (Thermo Fisher Scienti c Inc., Waltham, MA, USA) under the following conditions: 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and terminated with a nal extension at 72°C for 10 min. PCR products (5 µL) were identi ed and bi-directionally sequenced by Sangon Biotech (Shanghai, China).

Data analysis
The sequencing data were manually checked and cleaned. All con rmed DNA sequences were aligned with the sequence at locus 1014 in vgsc gene or locus 119 in ace-1 gene in the MEGA 7.1 software [22].
The nucleotide and amino-acid sequences of vgsc (Genbank ID:KP763810.1) and ace-1 (Genbank ID: KM875636.1) of An. sinensis were used as the reference for alignment. Data were imported into Excel 2019 to construct the database for analysis.
The polymorphism of kdr mutation genotypes varied signi cantly among the 12 populations. The TZ and HX populations had highest diversity of genotypes for vgsc gene (Fig. 1). The top 3 mutation alleles frequency were from the populations of XS(55%), TZ(56%) and DJ(44%), north Guizhou.
The distribution and frequency of mutations at locus 119 of ace-1 gene in An. sinensis Three alleles (GGC/G, GGT/G and AGC/S) were identi ed at the locus 119 of ace-1 gene in 551 samples of An. sinensis (Fig. 2). The GGT/G allele was found for the rst time and in only one sample of the ZJ population. The total frequency of the resistance allele (AGC/S) was 50.36%. Speci cally, the frequency of the resistance allele AGC/S ranged 6% (LP)-77% (CH) among the 12 populations (Table 3). The frequency of mutation genotypes (119GG, 119GS) was over 66% in all 12 eld populations, except for the LP population in which the frequency was 12% (Fig. 1). The populations of CH, LD, and SD ranked top in the frequency of mutation genotypes (100%), which were from the once highly epidemic areas of malaria.

Discussion
Guizhou Province is located in the Yunnan-Guizhou Plateau with complex landforms, humid climate, diverse natural environment and abundant mosquito species. As a once prominent malaria-endemic province, no indigenous cases were reported since 2016 when a long-term strategy on malaria elimination in Guizhou was implemented [23]. In spite of this, the imported malaria infection cases reported every year become the new concern [24]. In this study, 12 eld populations were selected across Guizhou Province, according to the previous distribution feature of malaria epidemics. After morphological identi cation and molecular identi cation, the distribution and frequencies of kdr and ace-1 mutations were detected for the rst extensive survey.
We discovered 5 kdr mutation alleles at the locus 1014 of vgsc gene as a result of three amino acid replacements ( namely 1014F/C/S) in 548 samples of 12 An. sinensis populations. Of the three replacements at the amino acids level, the highest allele frequency was found for 1014F (22.5%), implying that 1014F is a predominant mutation allele in Guizhou Province, similarly to the previous studies [9,10,16,19,25,26].1014C is also a common variant in southeast Asia and China. In addition to the reported variant, TGC/C allele was found for the rst time in this study which indicated it had the most variants in Guizhou. It might be related to the environment variety in Guizhou. Though 1014C was demonstrated to be the predominant in northeast of Guangxi [15], a low frequency was detected in most of places in China, including Guizhou. Guizhou is located in north of Guangxi, and the low frequency of L1014C suggests the geographical barriers limiting gene ow imposed by the mountainous landscapes of Guizhou. 1014S has been widely reported in Anopheles in the Greater Mekong Subregion and Africa [25,26]. However, to date it is only detected in An. sinensis in some provinces of South China, such as Guangdong[26], Sichuan[16], Guangxi [15] and Guizhou.
The frequency of kdr mutation has been evidenced to be signi cantly positively correlated with the resistance phenotype to Pyrethroids and DDT in Anopheles [29][30][31]. In this study, distinct distribution patterns for kdr mutation genotypes were observed in the 12 eld populations across Guizhou. The frequency for kdr mutation genotypes including homozygotes and heterozygotes in north part of Guizhou was higher than that in other locates, which warn it should be strengthened to surveil the pyrethroids resistance in north Guizhou. A high frequency of kdr mutation were also observed in An. sinensis from Sichuan Province [16] and Chongqing [19], the neighbors next to north part of Guizhou, which indicates it is possible that migration of An. sinensis population occurs from Sichuan to north Guizhou.
Previous genotyping results on ace-1 gene have revealed a modest-to-high frequency of 119S resistance allele in An. sinensis populations in Guangxi province (80%) [32], at the China-Vietnam border in Guangxi province (73%)[8], Hainan Island (45 ~ 75%) [33], Sichuan province (56%) [16], and Yunnan (38.5%) and Anhui (58.9%) provinces [20]. Here we found a modest frequency of the 119S resistance allele (53.6%) in An. sinensis across Guizhou (Table 1). These results suggest that the insecticide resistance to OPs and CBs may be widely distributed in An. sinensis populations in China, including Guizhou province. It is well known that OPs have been used for pest control in China since 1950s and the high frequency of mutation genotypes may be a consequence of the long-term use of OPs in agriculture industry [19]. It is worth to note that 100% frequency mutation genotypes (119GS, 119SS) occurred in 3 regions in Guizhou: Ceheng, Luodian and Sandu, which were previously considered as the highest endemic malaria regions in Guizhou. It further indicates a strong risk of resistance to OPs and CBs in these regions.
In general, mutation and migration are the main driving forces for the occurrence of a novel resistance allele in a given population [34]. It is noted that the LP population had lowest mutation frequency for kdr and ace-1 gene among all 12 populations, suggesting that this population faces low insecticide selection pressure and has no migration force.

Conclusion
In this study, we investigated the mutation and distribution of two target-site resistance genes in 12 eld populations of Anopheles sinensis across Guizhou Province. We discovered 5 different kdr mutation alleles at the locus 1014 and 4 different ace-1 alleles at the locus 119 in these samples, of which the TGG/C allele of kdr gene and TTG/G allele of ace-1 were found for the rst time. The results showed a diverse genetic mutations in An. sinensis in Guizhou. A high frequency of kdr mutation (> 44 %) in DeJiang, TongZhi, and Xishui indicates a risk of resistance to pyrethroids and DDT in An. sinensis in these areas. The G119S mutation is widespread in An. sinensis in Guizhou, especially in the previously identi ed high-risk malaria endemic areas which implicate that cautions should be paid when using OPs and CBs as control agents for vectors. Continued monitoring of insecticide resistance and the genotypic diversity of resistance genes may assist to formulate effective region-customized resistance management strategies before implementing malaria control strategies.

Declarations Ethics approval and participant consent
All the eld studies on mosquito were approved by the Institutional Animal Care and Use Committee of Guizhou Medical University (China).

Consent for publication
No applicable.

Availability of data and material
No.

Competing interests
The authors declare that they have no competing interests. Authors` Contribution WJH designed the whole study, genetic analysis and wrote the manuscript. CJZ and LQG performed mosquito collection, genotyping and data process. YX and WYM performed house y collection and data processing. TWL proofread the manuscript. All authors read and approved the nal manuscript.

Figure 1
Distribution and frequency of genotypes in Anopheles sinensis in Guizhou