Metabolic resistance of the major malaria vector Anopheles gambiae (s.l.) to insecticides is operationally significant, particularly in combination with target site resistance. However, detection of metabolic resistance is not trivial and relies on laborious bioassays, unspecific biochemical methods, or sophisticated and expensive molecular approaches using transcriptomics. Rapid one-step multiplex TaqMan-probe based RT-qPCR assays were developed and optimised to measure the expression levels of genes associated with metabolic insecticide resistance in An. gambiae (s.l.). Primers and probes were designed to target the mRNA of cytochrome P450-dependent monooxygenases CYP6P3, CYP6M2, CYP9K1, CYP6P4 and CYP6Z1, and the glutathione-S-transferase GSTE2. The novel assays were validated versus gold standard methods with a range of phenotyped mosquito specimens. The assays were also tested directly on lysates of RNAlater®-preserved mosquitoes without an RNA extraction step. The novel assays are efficient (reaction efficiencies = 95–109%), sensitive (covering a > 10.0 Ct range with R2 values > 0.99), specific (TaqMan chemistry), reproducible (%CV = 4.46–12.07%), as well as readily expandable to capture additional loci as they evolve or to cover additional species. The assays were successfully validated in terms of expression levels against standard two-step singleplex qPCR assays (overall % difference = -17.6%, 95% CI = -38.7–3.43%) and microarrays, using laboratory strains and field-caught samples. The assays can also be applied directly on lysates of mosquito specimens, without RNA extraction or DNase treatment. The novel multiplex assays for monitoring the levels of major detoxification genes and metabolic resistance in An. gambiae (s.l.) are simple to perform, robust and rapid. They may complement current diagnostic assays to provide evidence-based and operationally relevant information for insecticide resistance management.

Previous studies have indicated widespread insecticide resistance in malaria vector populations from Cameroon. However, the intensity of this resistance and underlying mechanisms are poorly known. Therefore, we conducted three cross-sectional resistance surveys between April 2018 and October 2019, using the revised World Health Organization protocol, which includes resistance incidences and intensity assessments. Field-collected Anopheles gambiae s.l. populations from Nkolondom, Nkolbisson and Ekié vegetable farms in the city of Yaoundé were tested with deltamethrin, permethrin, alpha-cypermethrin and etofenprox, using 1× insecticide diagnostic concentrations for resistance incidence, then 5× and 10× concentrations for resistance intensity. Subsamples were analyzed for species identification and the detection of resistance-associated molecular markers using TaqMan® qPCR assays. In Nkolbisson, both An. coluzzii (96%) and An. gambiae s.s. (4%) were found together, whereas only An. gambiae s.s. was present in Nkolondom, and only An. coluzzii was present in Ekié. All three populations were resistant to the four insecticides (<75% mortality rates―MR1×), with intensity generally fluctuating over the time between mod-erate (<98%―MR5×; ≥98%―MR10×) and high (76–97%―MR10×). The kdr L995F, L995S, and N1570Y, and the Ace-1 G280S-resistant alleles were found in An. gambiae from Nkolondom, at 73%, 1%, 16% and 13% frequencies, respectively, whereas only the kdr L995F was found in An. gambiae s.s. from Nkolbisson at a 50% frequency. In An. coluzzii from Nkolbisson and Ekié, we detected only the kdr L995F allele at 65% and 60% frequencies, respectively. Furthermore, expression levels of Cyp6m2, Cyp9k1, and Gste2 metabolic genes were highly upregulated (over fivefold) in Nkolondom and Nkolbisson. Pyrethroid and etofenprox-based vector control interventions may be jeopardized in the prospected areas, due to high resistance intensity, with multiple mechanisms in An. gambiae s.s. and An. coluzzii.

Bioassays and molecular diagnostics are routinely used for the monitoring of malaria vector populations to support insecticide resistance management (IRM), guiding operational decisions on which insecticides ought to be used for effective vector control. Previously developed TaqMan assays were optimised to distinguish the wild-type L1014 from the knockdown resistance (kdr) point mutations 1014F and 1014S (triplex reaction), and the N1575 wild-type from the point mutation 1575Y (duplex reaction). Subsequently, artificial pools of Anopheles gambiae (An. gambiae) specimens with known genotypes of L1014F, L1014S, and N1575Y were created, nucleic acids were extracted, and kdr mutations were detected. These data were then used to define a linear regression model that predicts the allelic frequency within a pool of mosquitoes as a function of the measured ΔCt values (Ct mutant − Ct wild type probe). Polynomial regression models showed r² values of >0.99 (p < 0.05). The method was validated with populations of variable allelic frequencies, and found to be precise (1.66–2.99%), accurate (3.3–5.9%), and able to detect a single heterozygous mosquito mixed with 9 wild type individuals in a pool of 10. Its pilot application in field-caught samples showed minimal differences from individual genotyping (0.36–4.0%). It allowed the first detection of the super-kdr mutation N1575Y in An. gambiae from Mali. Using pools instead of individuals allows for more efficient resistance allele screening, facilitating IRM.

Insecticide resistance inAnophelesmosquitoes is seriously threatening the success of insecticide-based malaria vector control. Surveillance of insecticide resistance in mosquito populations and identifying the underlying mechanisms enables optimisation of vector control strategies. Here, we investigated the molecular mechanisms of insecticide resistance in threeAnopheles coluzziifield populations from southern Côte d’Ivoire, including Agboville, Dabou and Tiassalé. All three populations were resistant to bendiocarb, deltamethrin and DDT, but not or only very weakly resistant to malathion. The absence of malathion resistance is an unexpected result because we found the acetylcholinesterase mutationAce1-G280S at high frequencies, which would typically confer cross-resistance to carbamates and organophosphates, including malathion. Notably, Tiassalé was the most susceptible population to malathion while being the most resistant one to the pyrethroid deltamethrin. The resistance ratio to deltamethrin between Tiassalé and the laboratory reference colony was 1,800 fold. By sequencing the transcriptome of individual mosquitoes, we found numerous cytochrome P450-dependent monooxygenases – including CYP6M2, CYP6P2, CYP6P3, CYP6P4 and CYP6P5 – overexpressed in all three field populations. This could be an indication for negative cross-resistance caused by overexpression of pyrethroid-detoxifying cytochrome P450s that may activate pro-insecticides, thereby increasing malathion susceptibility. In addition to the P450s, we found several overexpressed carboxylesterases, glutathioneS-transferases and other candidates putatively involved in insecticide resistance.

Background: The threat of mosquito-borne diseases is increasing in continental Europe as demonstrated by several autochthonous chikungunya, dengue and West Nile virus outbreaks. In Switzerland, despite the presence of competent vectors, routine surveillance of arboviruses in mosquitoes is not being carried out, mainly due to the high costs associated with the need of a constant cold chain and laborious processing of thousands of mosquitoes. An alternative approach is using honey-baited nucleic acid preserving cards (FTA cards) to collect mosquito saliva that may be analysed for arboviruses. Here, we evaluate whether FTA cards could be used to detect potentially emerging viruses in an area of low virus prevalence in combination with an effective mosquito trap.Methods: In a field trial in southern Switzerland we measured side-by-side the efficacy of the BG-Sentinel 2, the BG-GAT and the Box gravid trap to catchAedesandCulexmosquitoes in combination with honey-baited FTA cards during 80 trapping sessions of 48 hours. We then screened both the mosquitoes and the FTA cards for the presence of arboviruses using reverse-transcription PCR. The efficacy of the compared trap types was evaluated using generalized linear mixed models.Results: The Box gravid trap collected over 11 times more mosquitoes than the BG-GAT and BG-Sentinel 2 trap. On average 75.9% of the specimens fed on the honey-bait with no significant difference in feeding rates between the three trap types. From the total of 1401 collected mosquitoes, we screened 507Aedesand 500Culexfemales for the presence of arboviruses. A pool of sixCx. pipiens/Cx. torrentiummosquitoes and also the FTA card from the same Box gravid trap were positive for Usutu virus. Remarkably, only two of the sixCulexmosquitoes fed on the honey-bait, emphasising the high sensitivity of the method. In addition, twoAe. albopictuscollections but no FTA cards were positive for mosquito-only flaviviruses.Conclusions: Based on our results we conclude that honey-baited FTA cards, in combination with the Box gravid trap, are an effective method for arbovirus surveillance in areas of low prevalence, particularly where resources are limited for preservation and screening of individual mosquitoes.

Background: Insecticide-based malaria vector control is increasingly undermined due to the development of insecticide resistance in mosquitoes. Insecticide resistance may partially be related to the use of pesticides in agriculture, while the level and mechanisms of resistance might differ between agricultural practices. The current study aimed to assess whether phenotypic insecticide resistance and associated molecular resistance mechanisms in Anopheles gambiae sensu lato differ between agricultural practices. Methods: We collected An. gambiae s.l. larvae in six sites with three different agricultural practices, including rice, vegetable and cocoa cultivation. We then exposed the emerging adult females to discriminating concentrations of bendiocarb (0.1%), deltamethrin (0.05%), DDT (4%) and malathion (5%) using the standard World Health Organization insecticide susceptibility test. To investigate underlying molecular mechanisms of resistance, we used multiplex TaqMan qPCR assays. We determined the frequency of target-site mutations, including Vgsc-L995F/S and Vgsc-N1570Y, and Ace1-G280S. In addition, we measured the expression levels of genes previously associated with insecticide resistance in An. gambiae s.l., including the cytochrome P450-dependent monooxygenases CYP4G16, CYP6M2, CYP6P1, CYP6P3, CYP6P4, CYP6Z1 and CYP9K1, and the glutathione S-transferase GSTe2. Results: The An. gambiae s.l. populations from all six agricultural sites were resistant to bendiocarb, deltamethrin and DDT, while the populations from the two vegetable cultivation sites were additionally resistant to malathion. Most tested mosquitoes carried at least one mutant Vgsc-L995F allele that is associated with pyrethroid and DDT resistance. In the cocoa cultivation sites, we observed the highest 995F frequencies (80–87%), including a majority of homozygous mutants and several in co-occurrence with the Vgsc-N1570Y mutation. We detected the Ace1 mutation most frequently in vegetable-growing sites (51–60%), at a moderate frequency in rice (20–22%) and rarely in cocoa-growing sites (3–4%). In contrast, CYP6M2, CYP6P3, CYP6P4, CYP6Z1 and CYP9K1, previously associated with metabolic insecticide resistance, showed the highest expression levels in the populations from rice-growing sites compared to the susceptible Kisumu reference strain. Conclusion: In our study, we observed intriguing associations between the type of agricultural practices and certain insecticide resistance profiles in the malaria vector An. gambiae s.l. which might arise from the use of pesticides deployed for protecting crops. Graphical