State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Malar J. 2017 Nov 28;16(1):482. doi: 10.1186/s12936-017-2133-0.
Anopheles sinensis is a major vector of malaria in China. The gamma-aminobutyric acid (GABA)-gated chloride channel, encoded by the RDL (Resistant to dieldrin) gene, is the important target for insecticides of widely varied structures. The use of various insecticides in agriculture and vector control has inevitably led to the development of insecticide resistance, which may reduce the control effectiveness. Therefore, it is important to investigate the presence and distribution frequency of the resistance related mutation(s) in An. sinensis RDL to predict resistance to both the withdrawn cyclodienes (e.g. dieldrin) and currently used insecticides, such as fipronil.
Two hundred and forty adults of An. sinensis collected from nine locations across Guangxi Zhuang Autonomous Region were used. Two fragments of An. sinensis RDL (AsRDL) gene, covering the putative insecticide resistance related sites, were sequenced respectively. The haplotypes of each individual were reconstructed by the PHASE2.1 software, and confirmed by clone sequencing. The phylogenetic tree was built using maximum-likelihood and Bayesian inference methods. Genealogical relations among different haplotypes were also analysed using Network 5.0.
The coding region of AsRDL gene was 1674 bp long, encoding a protein of 557 amino acids. AsRDL had 98.0% amino acid identity to that from Anopheles funestus, and shared common structural features of Cys-loop ligand-gated ion channels. Three resistance-related amino acid substitutions (A296S, V327I and T345S) were detected in all the nine populations of An. sinensis in Guangxi, with the 296S mutation being the most abundant (77-100%), followed by 345S (22-47%) and 327I (8-60%). 38 AsRDL haplotypes were identified from 240 individuals at frequencies ranging from 0.2 to 34.8%. Genealogical analysis suggested multiple origins of the 345S mutation in AsRDL.
The near fixation of the 296S mutation and the occurrence of the 327I and 345S mutations in addition to 296S, in all the nine tested An. sinensis populations in Guangxi, strongly indicate a risk of multiple insecticide resistance. The haplotype diversity plus genetic heterogeneities in the geographical distribution, and multiple origins of AsRDL alleles call for a location-customized strategy for monitoring and management of insecticide resistance.
中华按蚊是中国疟疾的主要传播媒介。γ-氨基丁酸（GABA）门控氯离子通道，由 RDL（对狄氏剂的抗性）基因编码，是结构广泛多样的杀虫剂的重要靶标。农业和病媒控制中各种杀虫剂的使用不可避免地导致了抗药性的发展，这可能会降低控制效果。因此，重要的是要调查中华按蚊 RDL 中抗性相关突变的存在和分布频率，以预测对已停用的环戊二烯（如狄氏剂）和目前使用的杀虫剂（如氟虫腈）的抗性。
使用从广西壮族自治区 9 个地点收集的 240 只中华按蚊成虫。分别对中华按蚊 RDL（AsRDL）基因的两个片段（涵盖假定的杀虫剂抗性相关位点）进行测序。使用 PHASE2.1 软件重建每个个体的单倍型，并通过克隆测序进行确认。使用最大似然法和贝叶斯推断法构建系统发育树。还使用网络 5.0 分析不同单倍型之间的系统发生关系。
AsRDL 基因的编码区长 1674bp，编码 557 个氨基酸的蛋白质。AsRDL 与致倦库蚊的氨基酸同一性为 98.0%，并具有 Cys 环配体门控离子通道的共同结构特征。在广西的 9 个中华按蚊种群中均检测到 3 个与抗药性相关的氨基酸取代（A296S、V327I 和 T345S），其中 296S 突变最为丰富（77-100%），其次是 345S（22-47%）和 327I（8-60%）。从 240 个个体中鉴定出 38 种 AsRDL 单倍型，其频率范围为 0.2-34.8%。系统发生分析表明，AsRDL 中的 345S 突变有多个起源。
在广西测试的 9 个中华按蚊种群中，296S 突变的近固定以及 327I 和 345S 突变的发生除 296S 外，强烈表明存在多种杀虫剂抗性的风险。地理分布中的单倍型多样性加上遗传异质性，以及 AsRDL 等位基因的多个起源，需要根据地点制定监测和管理杀虫剂抗性的策略。
