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Background    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 sinensis  RDL to predict resistance to both the withdrawn cyclodienes (e.g. dieldrin) and currently used insecticides, such as fipronil.    Methods  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.    Results  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 .    Conclusions  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.

RDL mutations predict multiple insecticide resistance in Anopheles sinensis in Guangxi, China