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Insecticide resistance in Aedes mosquitoes presents a major challenge to the control of arboviral diseases. However, resistance mechanisms for many of the insecticides remain unknown. A commonly used insecticide, deltamethrin, was used to select a resistance strain of the vector mosquito, Aedes albopictus, and we identified an F1534S substitution in the voltage-gated sodium channel (VGSC) gene product as the first event in generating resistance. Engineering an F1534S substitution using Cas9/gRNA technologies conferred deltamethrin resistance on a previously susceptible strain. Crosses that removed this mutation restored the susceptible phenotype. Predicted protein structural changes and differences in transcript accumulation levels were correlated with the resistance phenotype. Furthermore, F1534S mutations were detected in all resistant Ae. albopictus populations collected in the field. We conclude that the VGSC F1534S mutation is essential for resistance to deltamethrin in Ae. albopictus, and is a suitable molecular index for pyrethroid resistance detection and monitoring in this species.

BackgroundAedes albopictus is one of the most invasive species in the world as well as the important vector for mosquito-borne diseases such as dengue fever, chikungunya fever and zika virus disease. Chemical control of mosquitoes is an effective method to control mosquito-borne diseases, however, the wide and improper application of insecticides for vector control has led to serious resistance problems. At present, there have been many reports on the resistance to pyrethroid insecticides in vector mosquitoes including deltamethrin to Aedes albopictus. However, the fitness cost and vector competence of deltamethrin resistant Aedes albopictus remain unknown. To understand the impact of insecticide resistant mosquito is of great significance for the prevention and control mosquitoes and mosquito-borne diseases.Methodology/principal findingsA laboratory resistant strain (Lab-R) of Aedes albopictus was established by deltamethrin insecticide selecting from the laboratory susceptible strain (Lab-S). The life table between the two strains were comparatively analyzed. The average development time of Lab-R and Lab-S in larvae was 9.7 days and 8.2 days (P < 0.005), and in pupae was 2.0 days and 1.8 days respectively (P > 0.05), indicating that deltamethrin resistance prolongs the larval development time of resistant mosquitoes. The average survival time of resistant adults was significantly shorter than that of susceptible adults, while the body weight of resistant female adults was significantly higher than that of the susceptible females. We also compared the vector competence for dengue virus type-2 (DENV-2) between the two strains via RT-qPCR. Considering the results of infection rate (IR) and virus load, there was no difference between the two strains during the early period of infection (4, 7, 10 day post infection (dpi)). However, in the later period of infection (14 dpi), IR and virus load in heads, salivary glands and ovaries of the resistant mosquitoes were significantly lower than those of the susceptible strain (IR of heads, salivary glands and ovaries: P < 0.05; virus load in heads and salivary glands: P < 0.05; virus load in ovaries: P < 0.001). And then, fourteen days after the DENV-2-infectious blood meal, females of the susceptible and resistant strains were allow to bite 5-day-old suckling mice. Both stains of mosquito can transmit DENV-2 to mice, but the onset of viremia was later in the mice biting by resistant group as well as lower virus copies in serum and brains, suggesting that the horizontal transmission of the resistant strain is lower than the susceptible strain. Meanwhile, we also detected IR of egg pools of the two strains on 14 dpi and found that the resistant strain were less capable of vertical transmission than susceptible mosquitoes. In addition, the average survival time of the resistant females infected with DENV-2 was 16 days, which was the shortest among the four groups of female mosquitoes, suggesting that deltamethrin resistance would shorten the life span of female Aedes albopictus infected with DENV-2.Conclusions/significanceAs Aedes albopictus developing high resistance to deltamethrin, the resistance prolonged the growth and development of larvae, shorten the life span of adults, as well as reduced the vector competence of resistant Aedes albopictus for DENV-2. It can be concluded that the resistance to deltamethrin in Aedes albopictus is a double-edged sword, which not only endow the mosquito survive under the pressure of insecticide, but also increase the fitness cost and decrease its vector competence. However, Aedes albopictus resistant to deltamethrin can still complete the external incubation period and transmit dengue virus, which remains a potential vector for dengue virus transmission and becomes a threat to public health. Therefore, we should pay high attention for the problem of insecticide resistance so that to better prevent and control mosquito-borne diseases.

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.

BACKGROUND:Aedes albopictus (Skuse) is an invasive mosquito that has become an important vector of chikungunya, dengue and Zika viruses. In the absence of specific antiviral therapy or a vaccine, vector management is the sole method available for reducing Aedes-induced disease morbidity. Determining the resistance status of Ae. albopictus to insecticides and exploring the resistance mechanisms is essential for future vector control planning. METHODS:Aedes albopictus larvae and pupae were sampled from six sites (two sites each from urban, suburban and rural) in Guangzhou. The resistance bioassays were conducted against Bacillus thuringiensis israelensis (Bti): deltamethrin, propoxur and malathion for larvae; and deltamethrin, DDT, propoxur and malathion for adults. P450 monooxygenase (P450s), glutathione S-transferase (GSTs) and carboxylesterase (COEs) activities of adult mosquitoes were measured. Mutations at the knockdown resistance (kdr) gene were analyzed, and the association between kdr mutations and phenotypic resistance was tested. RESULTS:Adult bioassays revealed varied susceptibility against DDT, deltamethrin and propoxur in the six Ae. albopictus populations. Significantly lower mortality rates were found in urban populations than suburban and rural populations. Urban mosquito populations showed resistance against DDT, deltamethrin and propoxur, while one rural population was resistant to DDT. All populations tested were susceptible to malathion. Larval bioassays results indicated that all populations of Ae. albopictus were sensitive to the larvicide Bti and malathion. Resistance to deltamethrin and propoxur was common in larval populations. The F1534S and F1534 L mutations were found to be significantly associated with deltamethrin resistance. Biochemical assays indicated elevated detoxification enzyme activities in the field mosquito populations. CONCLUSIONS:Aedes albopictus populations in Guangzhou, especially in urban areas, have developed resistance to the commonly used insecticides, primarily DDT and deltamethrin. This finding calls for resistance management and developing counter measures to mitigate the spread of resistance.

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.

Interactions between bacterial microbiota and mosquitoes play an important role in mosquitoes' capacity to transmit pathogens. However, microbiota assemblages within mosquitoes and the impact of microbiota in environments on mosquito development and survival remain unclear. This study examined microbiota assemblages and the effects of aquatic environment microbiota on the larval development of the Aedes albopictus mosquito, an important dengue virus vector. Life table studies have found that reducing bacterial load in natural aquatic habitats through water filtering and treatment with antibiotics significantly reduced the larva-to-adult emergence rate. This finding was consistent in two types of larval habitats examined-discarded tires and flowerpots, suggesting that bacteria play a crucial role in larval development. Pyrosequencing of the bacterial 16S rRNA gene was used to determine the diversity of bacterial communities in larval habitats and the resulting numbers of mosquitoes under both laboratory and field conditions. The microbiota profiling identified common shared bacteria among samples from different years; further studies are needed to determine whether these bacteria represent a core microbiota. The highest microbiota diversity was found in aquatic habitats, followed by mosquito larvae, and the lowest in adult mosquitoes. Mosquito larvae ingested their bacterial microbiota and nutrients from aquatic habitats of high microbiota diversity. Taken together, the results support the observation that Ae. albopictus larvae are able to utilize diverse bacteria from aquatic habitats and that live bacteria from aquatic habitats play an important role in larval mosquito development and survival. These findings provide new insights into bacteria's role in mosquito larval ecology.