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Dengue is one of the most serious mosquito-borne infectious diseases in the world. Aedes albopictus is the most invasive mosquito and one of the primary vectors of dengue. Vector control using insecticides is the only viable strategy to prevent dengue virus transmission. In Guangzhou, after the 2014 pandemic, massive insecticides have been implemented. Massive insecticide use may lead to the development of resistance, but few reports are available on the status of insecticide resistance in Guangzhou after 2014. In this study, Ae. albopictus were collected from four districts with varied dengue virus transmission intensity in Guangzhou from 2015 to 2017. Adult Ae. albopictus insecticide susceptibility to deltamethrin (0.03%), permethrin(0.25%), DDT(4%), malathion (0.8%) and bendiocarb (0.1%) was determined by the standard WHO tube test, and larval resistance bioassays were conducted using temephos, Bacillus thuringiensis israelensis (Bti), pyriproxyfen (PPF) and hexaflumuron. Mutations at the voltage-gated sodium channel (VGSC) gene and acetylcholinesterase (AChE) gene were analyzed. The effect of cytochrome P450s on the resistance of Ae. albopictus to deltamethrin was tested using the synergistic agent piperonyl butoxide (PBO). The results showed that Ae. albopictus populations have rapidly developed very high resistances to multiple commonly used insecticides at all study areas except malathion, Bti and hexaflumuron. We found 1534 codon mutations in the VGSC gene that were significantly correlated with the resistance to pyrethroids and DDT, and 11 synonymous mutations were also found in the gene. The resistance to deltamethrin can be significantly reduced by PBO but may generated cross-resistance to PPF. Fast emerging resistance in Ae. albopictus may affect mosquito management and threaten the prevention and control of dengue, similar to the resistance in Anopheles mosquitoes has prevented the elimination of malaria and call for timely and guided insecticide management.

Wide and improper application of pyrethroid insecticides for mosquito control has resulted in widespread resistance in Aedes albopictus mosquitoes, an important dengue vector. Therefore, understanding the molecular regulation of insecticide resistance is urgently needed to provide a basis for developing novel resistance diagnostic methods and vector control approaches. We investigated the transcriptional profiles of deltamethrin-resistant and -susceptible Ae. albopictus by performing paired-end sequencing for RNA expression analysis. The analysis used 24 independent libraries constructed from 12 wild-caught resistant and 12 susceptible Ae. albopictus female adults. A total of 674,503,592 and 612,512,034 reads were obtained, mapped to the Ae. albopictus genome and assembled into 20,091 Ae. albopictus transcripts. A total of 1,130 significantly differentially expressed genes included 874 up-regulated genes and 256 down-regulated genes in the deltamethrin-resistant individuals. These differentially expressed genes code for cytochrome P450s, cuticle proteins, glutathione S-transferase, serine proteases, heat shock proteins, esterase, and others. We selected three highly differentially expressed candidate genes, CYP6A8 and two genes of unknown function (CCG013931 and CCG000656), to test the association between these 3 genes and deltamethrin resistance using RNAi through microinjection in adult mosquitoes and oral feeding in larval mosquitoes. We found that expression knockdown of these three genes caused significant changes in resistance. Further, we detected 1,162 single nucleotide polymorphisms (SNPs) with a frequency difference of more than 50%. Among them, 5 SNPs in 4 cytochrome P450 gene families were found to be significantly associated with resistance in a genotype-phenotype association study using independent field-collected mosquitoes of known resistance phenotypes. Altogether, a combination of novel individually based transcriptome profiling, RNAi, and genetic association study identified both differentially expressed genes and SNPs associated with pyrethroid resistance in Ae. albopictus mosquitoes, and laid a useful foundation for further studies on insecticide resistance mechanisms.

BackgroundAedes albopictus is an important dengue vector because of its aggressive biting behavior and rapid spread out of its native home range in Southeast Asia. Pyrethroids are widely used for adult mosquito control, and resistance to pyrethroids should be carefully monitored because vector control is the only effective method currently available to prevent dengue transmission. The voltage-gated sodium channel gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). Previous studies reported various mutations in the voltage-gated sodium channel (VGSC) gene, but the spatial distribution of kdr mutations in Ae. albopictus has not been systematically examined, and the association between kdr mutation and phenotypic resistance has not been established.MethodsA total of 597 Ae. albopictus individuals from 12 populations across Asia, Africa, America and Europe were examined for mutations in the voltage-gated sodium channel gene. Three domains for a total of 1,107 bp were sequenced for every individual. Two populations from southern China were examined for pyrethroid resistance using the World Health Organization standard tube bioassay, and the association between kdr mutations and phenotypic resistance was tested.ResultsA total of 29 synonymous mutations were found across domain II, III and IV of the VGSC gene. Non-synonymous mutations in two codons of the VGSC gene were detected in 5 populations from 4 countries. A novel mutation at 1532 codon (I1532T) was found in Rome, Italy with a frequency of 19.7%. The second novel mutation at codon 1534 (F1534S) was detected in southern China and Florida, USA with a frequency ranging from 9.5-22.6%. The WHO insecticide susceptibility bioassay found 90.1% and 96.1% mortality in the two populations from southern China, suggesting resistance and probable resistance. Positive association between kdr mutations with deltamethrin resistance was established in these two populations.ConclusionsTwo novel kdr mutations, I1532T and F1534S were found in Ae. albopictus. This is the first report of I1532T mutations in Italy and F1534S mutation in China and US. Significant association between kdr mutation and protection from deltamethrin raised the possibility that kdr mutation may be a viable biomarker for pyrethroid resistance surveillance in Ae. albopictus. The patchy distribution of kdr mutations in Ae. albopictus mosquitoes calls for developing global surveillance plan for pyrethroid resistance and developing countermeasures to mitigate the spread of resistance.