Distribution and frequency of G119S mutation in ace-1 gene within Anopheles sinensis populations from Guangxi, China
- Xiangyang Feng†1,
- Chan Yang†2, 3,
- Yichao Yang1,
- Jun Li1,
- Kangming Lin1,
- Mei Li2 and
- Xinghui Qiu2, 3Email author
© Feng et al. 2015
Received: 7 September 2015
Accepted: 19 November 2015
Published: 25 November 2015
Malaria is one of the most serious vector-borne diseases in the world. Vector control is an important measure for malaria prevention and elimination. However, this strategy is under threat as disease vectors are developing resistance to insecticides. Therefore, it is important to monitor mechanisms responsible for insecticide resistance. In this study, the presence of G119S mutation in the acetyl cholinesterase-encoding gene (ace-1) was investigated in nine Anopheles sinensis populations sampled across Guangxi Zhuang Autonomous Region China.
PCR–RFLP (polymerase chain reaction-restriction fragment length polymorphism) method was used to genotype each individual adult of An. sinensis. Direct sequencing of PCR products was performed to verify the accuracy of PCR–RFLP genotyping result. Population genetics analysis was conducted using Genepop programme.
The frequencies of susceptible homozygotes, heterozygotes and resistant homozygotes in the nine populations ranged between 0–0.296, 0.143–0.500 and 0.333–0.857, respectively. Overall, a high frequency (0.519–0.929) of mutant 119S allele was observed and the genotype frequency of the ace-1 gene of An. sinensis was at Hardy–Weinberg equilibrium in each of the nine examined populations.
The G119S mutation has become fixed and is widespread in An. sinensis field populations in Guangxi, China. These findings are useful in helping design strategies for An. sinensis control.
Malaria is one of the most serious vector-borne diseases, representing a major threat to global public health . Vector control has been proven to be an important component in malaria prevention and elimination programmes. The use of different classes of insecticides has played an essential role in controlling mosquitoes, but also has resulted in the development of insecticide resistance [1, 2]. Insecticide resistance is well regarded as a major obstacle in vector control, thus resistance monitoring is critical for establishing smart vector management strategies .
Organophosphates (OP) and carbamates (CM) have been used for agriculturally important pest and disease-vector control. The primary molecular target of OP and CM is the acetylcholinesterase (AchE, EC 18.104.22.168). Inhibition of insect AchE leads to the accumulation of acetylcholine, thus terminates nerve impulses in cholinergic synapses and eventually causes death . Previous studies have demonstrated that point mutations in AchE are associated with insecticide resistance against OP and CM . For example, a point mutation leading to a single amino acid substitution of glycine to serine at position 119 (G119S, Torpedo californica numbering) in the AchE1 , is associated with OP and CM resistance in several important mosquito species [4–16]. This resistance-associated mutation provides a molecular marker for detecting or monitoring CM and OP resistance in these mosquitoes.
Guangxi Zhuang Autonomous Region was once a malaria-endemic region. Since the launch of the National Malaria Control Programme in 1955 in China, the malaria morbidity rate in Guangxi reduced from 296.67 per 10,000 people in 1954 to below 1 per 100,000 people during 2000 to 2011 . Although indigenous malaria has been basically eliminated in Guangxi, many imported cases of malaria have been identified in returning workers from Africa and southern Asia . In addition, increased population migration (especially cross-border migration) and the possible change of vectorial capacity may enhance the risk of malaria re-emergence . This situation underlines the necessity of malaria prevention through effective vector control.
Anopheles sinensis collection
Anopheles sinensis adults used in the study were caught by light (wave length 365 nm) trap from July to September in 2014, around farmers’ houses in Nanning, Yulin, Hezhou, Baise, Wuzhou, Liuzhou, Guilin, Hechi, and Guigang of Guangxi (Fig. 1). Individual mosquitoes were morphologically identified and the confirmed An. sinensis adults with a high level of confidence were put into 100-μl Eppendorf tubes containing 100 % ethanol solution, and kept at 4 °C until use. Ten randomly selected adults from each population were further identified using the rDNA-ITS2 method . The identities of the molecularly identified specimens matched perfectly to their morphological identifications.
Genomic DNA extraction
Genomic DNA of individual mosquitoes was prepared according to the method of Rinkevich . Genomic DNA samples were stored at −20 °C.
PCR–RFLP was conducted for genotyping the ace-1 gene at codon 119. Primers As-ace-F and As-ace-R  (commercially synthesized by Invitrogen, China Service) were used to amplify a fragment encompassing codon 119 of the ace-1 gene of An. sinensis. Reaction system contained 10 × Buffer 3 μl, dNTP 3 μl, rTaq DNA polymerase (Takara) 0.3 μl, DNA template 5 μl, As-ace-F 0.6 μl, As-ace-R 0.6 μl, ddH2O 17.5 μl. PCR parameters were set as 95 °C for 5 min, 36 cycles of 95 °C for 30 s, 52 °C for 30 s and 72 °C for 40 s, followed by 72 °C for 10 min and 4 °C forever. PCR products were detected on a 1.2 % agarose gel.
Restriction endonuclease AluI (New England Biolabs) was used for genotyping. This enzyme can cut the 119S-type (mutant) PCR product into two bands, but cannot cut the G119-type (wild) PCR product due to its specific recognition site (AGCT). The digestion reaction consisted of PCR product 10 μl, Cutsmart buffer 2 μl, AluI (10 unit/L) 0.4 μl, ddH2O 8 μl in a total volume of 20 μl. After reaction for 4 h at 37C°, the digestion products were detected on a 1.2 % agarose gel. Direct sequencing was performed for confirming the reliability of PCR–RFLP in genotyping by GBI tech (Beijing Service, China).
Bioinformatics analysis was conducted using online programmes. Population genetic parameters were calculated using Genepop 3.4, and Chi-test was performed by SAS 9.2.
Allele frequency of ace-1 in Anopheles sinensis populations sampled across Guangxi, China
Genotype χ2-test (p value)
Heterozygote excess (p-value)
Heterozygote deficiency (p-value)
No heterozygote excess or heterozygote deficiency was observed in all the nine populations (Table 1). Chi-test indicated that there was no significant difference between observed number and expected number of individuals of each genotype in each population (Table 1). These parameters suggested that all the nine populations of An. sinensis in Guangxi were at Hardy–Weinberg equilibrium (HWE).
Genotyping results reveal that there was a high frequency of 119S resistance allele in each of the nine field populations of An. sinensis collected across Guangxi (Table 1). Notably, the average frequency of resistance allele was close to 0.8, and an even higher mutation frequency (higher than 0.9) was observed in Wuzhou and Hechi populations. These observations clearly demonstrate that the G119S mutation is prevalent throughout Guangxi. Also in China, modest to high (0.45–0.75) frequency of resistance allele was observed in An. sinensis populations from Hainan Island , and Yunnan (0.385) and Anhui (0.589) provinces . Similar to results obtained in this study, the frequency of the 119S (mutant) allele was determined to range from 0.744 to 0.972 in ten local field populations of An. sinensis in Korea . These results indicate that the G119S mutation is widely distributed in Asia.
In contrast to the study in West Africa observing a significant departure from Hardy–Weinberg equilibrium (HWE) in some field samples of An. gambiae , population genetics analysis shows that the genotype frequency at codon 119 of the ace-1 gene was at HWE in all the nine examined populations of An. sinensis from Guangxi, with no heterozygote excess or heterozygote deficiency being observed (Table 1). These parameters indicate that mosquitoes carrying G119S mutation may suffer no fitness cost under current natural conditions. If this is the case, actions to eliminate resistant individuals and limit the spread of the resistant population should be taken. Whether G119S genotype expresses fitness cost in An. sinensis remains to be characterized.
This study demonstrates that G119S mutation has become fixed, and is widespread in An. sinensis field populations across Guangxi, China. The high frequency of G119S mutation and high ratio of mutant homozygotes may allow these mosquito populations to be resistant to OP and CM. These findings emphasize the need to monitor insecticide resistance and to establish efficient resistance management tactics before implementing malaria control programmes in Guangxi region.
XHQ and XYF conceived the study. CY performed the molecular experiments. XYF, YCY, JL, KML identified the species. CY, XYF and XHQ analysed the data. CY, XYF and XHQ wrote the paper. XYF, YCY, JL, ML, and KML contributed to sample collection. All authors read and approved the final manuscript.
The authors thank the State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, China for the gift of susceptible adults of Anopheles sinensis and the reviewers for their suggestions. This work was supported by grants from National Basic Research Programme of China (973 Program, No. 2012CB114103) and National Health and Family Planning Commission of the People’s Republic of China. The funders had no role in the study design, data collection, analysis, decision to publish or preparation of the manuscript.
The authors declare that they have no competing interests.
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