Resistance to pyrethroid and organophosphate insecticides, and the geographical distribution and polymorphisms of target-site mutations in voltage-gated sodium channel and acetylcholinesterase 1 genes in Anopheles sinensis populations in Shanghai, China

Abstract

Background: In the final phase of China's national programme to eliminate malaria by 2020, it is vitally important to monitor the resistance of malaria vectors for developing effective vector control strategies. In 2017 Shanghai declared that it had eliminated malaria; however, the insecticide resistance status of the primary malaria vector Anopheles sinensis remains unknown.

Methods: We examined the pyrethroid and organophosphate resistance of An. sinensis via a bioassay of two populations from the Chongming District of Shanghai. The voltage-gated sodium channel (VGSC) and acetylcholinesterase 1 (ace-1) genes were partially sequenced to examine the association between resistance phenotype and target site genotype. In addition, the geographical distribution, polymorphism and genotype frequencies of insecticide resistance genes were examined using samples collected during routine mosquito surveillance in 2016 and 2017 from Chongming, Songjiang, Jiading and Qingpu Districts.

Results: In Chongming District, the An. sinensis population near Dongtan National Nature Reserve showed resistance to pyrethroids, sensitivity to organophosphate, no knockdown resistance (kdr) mutations in the VGSC gene, and a low frequency (1.71%) of the ace-1 119S allele. An An. sinensis population near the Chongming central area (CM-Xinhe population) showed high resistance to pyrethroids and organophosphates and high frequencies of kdr 1014F and 1014C (80.73%) and ace-1 119S (85.98%) alleles. A significant association was detected between the homozygous kdr mutation 1014F/1014F and pyrethroid resistance in the CM-Xinhe population, indicating that the kdr mutation is probably recessive. Eight kdr genotypes with 1014F and 1014C substitutions were detected in the four surveyed districts of Shanghai. TTT and GGC/AGC were the dominant kdr allele and ace-1 genotype, respectively, and were prevalent in most Shanghai An. sinensis populations.

Conclusions: On the basis of our assessment of insecticide resistance gene mutations in Shanghai, we identified a kdr mutation-free population in Chongming Dongtan. However, high frequencies of target-site mutations of insecticide resistance genes were observed in most areas of Shanghai. Bioassays of An. sinensis populations in the central Chongming District indicated the high insecticide resistance status of An. sinensis populations in Shanghai. We accordingly recommend a restriction on insecticide usage and development of effective integrated pest/vector management interventions to support disease control efforts.

Keywords: Acetylcholinesterase 1; Insecticide bioassay; Knockdown resistance; Malaria; Organophosphate; Pyrethroid.

Fig. 1

Map of the five sampling sites for kdr and ace-1 genotype detection in Shanghai. Pie charts show the kdr genotypes and their frequencies. Histograms show the ace-1 genotypes and their frequencies. Red and green stars represent the sampling sites for the CM-Dongtan and CM-Xinhe populations, respectively. Abbreviations: HP, Huangpu District; XH, Xuhui District; CN, Changning District; JA, Jing’an District; PT, Putuo District; HK, Hongkou District; YP, Yangpu District

Fig. 2

Chromatograms and alignments of the kdr and ace-1 genotypes detected in Anopheles sinensis from Shanghai. a Eight kdr genotypes; codon 1014 of the para-type voltage-gated sodium channel gene is indicated by a red box. b Three ace-1 genotypes; codon 119 of the ace-1 gene is indicated by a red box. Different peak colours distinguish the four bases T (red), C (blue), A (green) and G (black). K = G/T; Y = T/C; R = A/G