Knockdown resistance (kdr) mutations within seventeen field populations of Aedes albopictus from Beijing China: first report of a novel V1016G mutation and evolutionary origins of kdr haplotypes

Abstract

Background: Aedes albopictus (Skuse) is an important vector of chikungunya, dengue, yellow fever and Zika viruses. In the absence of anti-viral medication and with limited availability of a commercial vaccine for public health use, vector control remains an effective means for reducing Aedes-borne disease morbidity. Knowledge about genetic mutations associated with insecticide resistance (IR) is a prerequisite for developing rapid resistance diagnosis, and the distribution and frequency of IR conferring mutations is important information for making smart vector control decisions.

Methods: Partial DNA sequences of domain II and domain III of Ae. albopictus voltage gated sodium channel (VGSC) gene were amplified from a total of 426 individuals, collected from 17 sites in the Beijing municipality. These DNA fragments were sequenced to discover the possible genetic mutations mediating knockdown resistance (kdr) to pyrethroids. The frequency and distribution of kdr mutations were assessed in the 17 Ae. albopictus populations. The origin of kdr mutations was investigated by haplotype clarification and phylogenetic analysis.

Results: Sequence alignments revealed the existence of multiple mutations (V1016G, I1532T, F1534S and F1534L) in VGSC. The highest frequency of the mutant 1016G allele (0.647) was found in Haidian, while 1016G was not detected in Huai Rou, Yan Qing, Ping Gu and Shun Yi. The frequency of 1532T was highest (0.537) in the population from the Olympic Forest Park (OFP, Chao Yang District), but not detectable in Huai Rou and Mi Yun. Two mutations were observed at codon 1534 with different distribution patterns: 1534L was only found in Tong Zhou (TZ) with a frequency of 0.017, while 1534S was distributed in TZ, OFP, Fang Shan, Da Xing and Shi Jing Shan with frequencies ranging from 0.019 (OFP) to 0.276 (TZ). One 1016G, one 1532T, one 1534L and two 1534S haplotypes were identified.

Conclusions: Multiple mutations (V1016G, I1532T, F1534L/S) in VGSC were found in Ae. albopictus in Beijing. This represents the first report of V1016G in Ae. albopictus. Sequence alignment and phylogenetic analysis revealed multiple origins of 1534S. The spatial heterogeneity in distribution and frequency of kdr mutations calls for a site-specific strategy for the monitoring of insecticide resistance. The relatively high frequencies of V1016G warn of a risk of pyrethroid resistance in mosquitoes in the urban zones.

Keywords: Aedes albopictus; Beijing; Haplotype; Insecticide resistance; Voltage-gated sodium channel; kdr.

Fig. 1

Schematic representation of the two regions of Ae. albopictus VGSC gene analyzed in this study. The intron-exon structure between the predicted initiation codon and stop codon is identified based on the genomic DNA sequence (MNAF02001058.1) and cDNA sequence (XM_019696540.1)

Fig. 2

Example chromatograms showing the non-synonymous mutations identified in this study

Fig. 3

Distribution and frequency of kdr mutations in Ae. albopictus populations in Beijing. a Geographical position of the sampling sites. The shape file was downloaded from the GADM database of Global Administrative Areas (http://gadm.org/) on December 24, 2018. b 1016G mutation. c 1532T mutation. d 1534L/S mutations

Fig. 4

Alignment of nucleotide sequences of two 1534S haplotypes. The intron is highlighted in yellow. The codon encoding the 1534 residue is underlined. Asterisks indicate identical nucleotides

Fig. 5

Alignment of nucleotide sequences of mutant VGSC haplotypes. The 5' and 3' regions that are identical are not presented in the figure. The intron is highlighted in yellow. The codons encoding 1532 and 1534 residues are underlined. Asterisks indicate identical nucleotides

Fig. 6

Molecular phylogenetic analysis by the maximum likelihood method based on D2 sequences. The GenBank accessions for the sequences identified in this study (named as D2Hx) are provided in Table 4. The tree with the highest log likelihood (−917.40) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with the superior log-likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 27 nucleotide sequences. There were a total of 317 positions in the final dataset

Fig. 7

Molecular phylogenetic analysis by the Maximum Likelihood method based on D3 sequences. The GenBank accessions for the sequences identified in this study (named as D3Hx) are provided in Table 5. The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model [1]. The tree with the highest log-likelihood (−744.91) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with the superior log-likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 16 nucleotide sequences. There were a total of 306 positions in the final dataset

Fig. 8

Alignment of nucleotide sequences of kdr haplotypes. The sequences (c.300 bp in length) in each alignment are highly similar, differing by 1 or 2 nucleotides. Only the region with variations is showed in the figure. The intron is highlighted in yellow. The codons encoding the insecticide resistance related amino acid residues are underlined. Asterisks indicate identical nucleotides