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Resistance to insecticides in Anopheles mosquitoes threatens the effectiveness of malaria control, but the genetics of resistance are only partially understood. We performed a large scale multi-country genome-wide association study of resistance to two widely used insecticides: deltamethrin and pirimiphos-methyl, using sequencing data from An. gambiae and An. coluzzii from ten locations in West Africa. Resistance was highly multi-genic, multi-allelic and variable between populations. While the strongest and most consistent association with deltamethrin resistance came from Cyp6aa1, this was based on several independent copy number variants (CNVs) in An. coluzzii, and on a non-CNV haplotype in An. gambiae. For pirimiphos-methyl, signals included Ace1, cytochrome P450s, glutathione S-transferases and the nAChR target site of neonicotinoid insecticides. The regions around Cyp9k1 and the Tep family of immune genes showed evidence of cross-resistance to both insecticides. These locally-varying, multi-allelic patterns highlight the challenges involved in genomic monitoring of resistance, and may form the basis for improved surveillance methods., (© 2023. Springer Nature Limited.)

The spread of resistance to insecticides in disease-carrying mosquitoes poses a threat to the effectiveness of control programmes, which rely largely on insecticide-based interventions. Monitoring mosquito populations is essential, but obtaining phenotypic measurements of resistance is laborious and error-prone. High-throughput genotyping offers the prospect of quick and repeatable estimates of resistance, while also allowing resistance markers to be tracked and studied. To demonstrate the potential of highly-mulitplexed genotypic screening for measuring resistance-association of mutations and tracking their spread, we developed a panel of 28 known or putative resistance markers in the major malaria vector Anopheles gambiae, which we used to screen mosquitoes from a wide swathe of Sub-Saharan Africa (Burkina Faso, Ghana, Democratic Republic of Congo (DRC) and Kenya). We found resistance association in four markers, including a novel mutation in the detoxification gene Gste2 (Gste2-119V). We also identified a duplication in Gste2 combining a resistance-associated mutation with its wild-type counterpart, potentially alleviating the costs of resistance. Finally, we describe the distribution of the multiple origins of kdr resistance, finding unprecedented diversity in the DRC. This panel represents the first step towards a quantitative genotypic model of insecticide resistance that can be used to predict resistance status in An. gambiae.

Background: Strategies for combatting residual malaria by targeting vectors outdoors are gaining importance as the limitations of primary indoor interventions are reached. Strategies to target ovipositing females or her offspring are broadly applicable because all mosquitoes require aquatic habitats for immature development irrespective of their biting or resting preferences. Oviposition site selection by gravid females is frequently studied by counting early instar larvae in habitats; an approach which is valid only if the number of larvae correlates with the number of females laying eggs. This hypothesis was tested against the alternative, that a higher abundance of larvae results from improved survival of a similar or fewer number of families., Methods: In a controlled experiment, 20 outdoor artificial ponds were left uncovered for 4 days to allow oviposition by wild mosquitoes, then covered with netting and first and second instar larvae sampled daily. Natural Anopheles habitats of two different types were also identified, and all visible larvae sampled. All larvae were identified to species, and most samples of the predominant species, Anopheles arabiensis, were genotyped using microsatellites for sibling group reconstructions using two contrasting softwares, BAPS and COLONY., Results: In the ponds, the number of families reconstructed by each software significantly predicted larval abundance (BAPS R 2  = 0.318, p = 0.01; COLONY R 2  = 0.476, p = 0.001), and suggested that around 50% of females spread larvae across multiple ponds (skip oviposition). From natural habitats, the mean family size again predicted larval abundance using BAPS (R 2  = 0.829, p = 0.017) though not using COLONY (R 2  = 0.218, p = 0.68), but both softwares once more suggested high rates of skip oviposition (in excess of 50%)., Conclusion: This study shows that, whether in closely-located artificial habitats or natural breeding sites, higher early instar larval densities result from more females laying eggs in these sites. These results provide empirical support for use of early instar larval abundance as an index for oviposition site preference. Furthermore, the sharing of habitats by multiple females and the high skip-oviposition rate in An. arabiensis suggest that larviciding by auto-dissemination of insecticide may be successful.

Culex quinquefasciatus plays an important role in transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF), as well as many arboviral diseases. Currently, efforts to tackle C. quinquefasciatus vectored diseases are based on either mass drug administration (MDA) for LF, or insecticide-based interventions. Widespread and intensive insecticide usage has resulted in increased resistance in mosquito vectors, including C. quinquefasciatus. Herein, the transcriptome profile of Ugandan bendiocarb-resistant C. quinquefasciatus was explored to identify candidate genes associated with insecticide resistance. High levels of insecticide resistance were observed for five out of six insecticides tested, with the lowest mortality (0.97%) reported to permethrin, while for DDT, lambdacyhalothrin, bendiocarb and deltamethrin the mortality rate ranged from 1.63-3.29%. Resistance to bendiocarb in exposed mosquitoes was marked, with 2.04% mortality following 1 h exposure and 58.02% after 4 h. Genotyping of the G119S Ace-1 target site mutation detected a highly significant association (p < 0.0001; OR = 25) between resistance and Ace1-119S. However, synergist assays using the P450 inhibitor PBO, or the esterase inhibitor TPP resulted in markedly increased mortality (to ≈80%), suggesting a role of metabolic resistance in the resistance phenotype. Using a novel, custom 60 K whole-transcriptome microarray 16 genes significantly overexpressed in resistant mosquitoes were detected, with the P450 Cyp6z18 showing the highest differential gene expression (>8-fold increase vs unexposed controls). These results provide evidence that bendiocarb resistance in Ugandan C. quinquefasciatus is mediated by both target-site mechanisms and over-expression of detoxification enzymes.

Unfortunately, the original article [1] contained an error mistakenly carried forward by the Production department handling this article whereby some figures and their captions were interchanged. The correct figures (Figs. 1, 2, 3, 4, 5) and captions are presented in this erratum. The original article has also been updated to reflect this correction.

Background: The spread of insecticide resistance (IR) is a major threat to vector control programmes for mosquito-borne diseases. Early detection of IR using diagnostic markers could help inform these programmes, especially in remote locations where gathering reliable bioassay data is challenging. Most current molecular tests for genetic IR markers are only suitable for use in well-equipped laboratory settings. There is an unmet need for field-applicable diagnostics., Methods: A single-cartridge test was designed to detect key IR mutations in the major African vector of malaria, Anopheles gambiae. Developed on the portable, rapid, point-of-care compatible PCR platform - Genedrive® (genedrive® plc), the test comprises two assays which target single nucleotide polymorphisms (SNPs) in the voltage gated sodium channel (VGSC) gene that exert interactive effects on knockdown resistance (kdr): L1014F, L1014S and N1575Y., Results: Distinct melt peaks were observed for each allele at each locus. Preliminary validation of these assays using a test panel of 70 An. gambiae samples showed complete agreement of our assays with the widely-used TaqMan assays, achieving a sensitivity and specificity of 100%., Conclusion: Here we show the development of an insecticide resistance detection assay for use on the Genedrive® platform that has the potential to be the first field-applicable diagnostic for kdr.

Background: Insecticide resistance threatens malaria control in sub-Saharan Africa. Knockdown resistance to pyrethroids and organochlorines in Anopheles gambiae sensu lato (s.l.) is commonly caused by mutations in the gene encoding a voltage-gated sodium channel which is the target site for the insecticide. The study aimed to examine risk factors for knockdown resistance in An. gambiae s.l. and its relationship with malaria infection in children in rural Gambia. Point mutations at the Vgsc-1014 locus, were measured in An. gambiae s.l. during a 2-year trial. Cross-sectional surveys were conducted at the end of the transmission season to measure malaria infection in children aged 6 months-14 years., Results: Whilst few Anopheles arabiensis and Anopheles coluzzii had Vgsc-1014 mutations, the proportion of An. gambiae sensu stricto (s.s.) mosquitoes homozygous for the Vgsc-1014F mutation increased from 64.8 to 90.9% during the study. The Vgsc-1014S or 1014F mutation was 80% higher in 2011 compared to 2010, and 27% higher in the villages with indoor residual spraying compared to those without. An increase in the proportion of An. gambiae s.l. mosquitoes with homozygous Vgsc-1014F mutations and an increase in the proportion of An. gambiae s.s. in a cluster were each associated with increased childhood malaria infection. Homozygous Vgsc-1014F mutations were, however, most common in An. gambiae s.s. and almost reached saturation during the study meaning that the two variables were colinear., Conclusions: As a result of colinearity between homozygous Vgsc-1014F mutations and An. gambiae s.s., it was not possible to determine whether insecticide resistance or species composition increased the risk of childhood malaria infection.

Metabolic resistance to pyrethroid insecticides is widespread in Anopheles mosquitoes and is a major threat to malaria control. DNA markers would aid predictive monitoring of resistance, but few mutations have been discovered outside of insecticide-targeted genes. Isofemale family pools from a wild Ugandan Anopheles gambiae population, from an area where operational pyrethroid failure is suspected, were genotyped using a candidate-gene enriched SNP array. Resistance-associated SNPs were detected in three genes from detoxification superfamilies, in addition to the insecticide target site (the Voltage Gated Sodium Channel gene, Vgsc). The putative associations were confirmed for two of the marker SNPs, in the P450 Cyp4j5 and the esterase Coeae1d by reproducible association with pyrethroid resistance in multiple field collections from Uganda and Kenya, and together with the Vgsc-1014S (kdr) mutation these SNPs explained around 20% of variation in resistance. Moreover, the >20 Mb 2La inversion also showed evidence of association with resistance as did environmental humidity. Sequencing of Cyp4j5 and Coeae1d detected no resistance-linked loss of diversity, suggesting selection from standing variation. Our study provides novel, regionally-validated DNA assays for resistance to the most important insecticide class, and establishes both 2La karyotype variation and humidity as common factors impacting the resistance phenotype.

Background: The success of malaria vector control is threatened by widespread pyrethroid insecticide resistance. However, the extent to which insecticide resistance impacts transmission is unclear. The objective of this study was to examine the association between the DDT/pyrethroid knockdown resistance mutation Vgsc-1014S, commonly termed kdr, and infection with Plasmodium falciparum sporozoites in Anopheles gambiae., Methods: WHO standard methods were used to characterize susceptibility of wild female mosquitoes to 0.05 % deltamethrin. PCR-based molecular diagnostics were used to identify mosquitoes to species and to genotype at the Vgsc-L1014S locus. ELISAs were used to detect the presence of P. falciparum sporozoites and for blood meal identification., Results: Anopheles mosquitoes were resistant to deltamethrin with mortality rates of 77.7 % [95 % CI 74.9-80.3 %]. Of 545 mosquitoes genotyped 96.5 % were A. gambiae s.s. and 3.5 % were Anopheles arabiensis. The Vgsc-1014S mutation was detected in both species. Both species were predominantly anthropophagic. In A. gambiae s.s., Vgsc-L1014S genotype was significantly associated with deltamethrin resistance (χ2 = 11.2; p < 0.001). The P. falciparum sporozoite infection rate was 4.2 %. There was a significant association between the presence of sporozoites and Vgsc-L1014S genotype in A. gambiae s.s. (χ2 = 4.94; p = 0.026)., Conclusions: One marker, Vgsc-1014S, was associated with insecticide resistance and P. falciparum infection in wild-caught mixed aged populations of A. gambiae s.s. thereby showing how resistance may directly impact transmission.

Functionally constrained genes are ideal insecticide targets because disruption is often fatal, and resistance mutations are typically costly. Synaptic acetylcholinesterase (AChE) is an essential neurotransmission enzyme targeted by insecticides used increasingly in malaria control. In Anopheles and Culex mosquitoes, a glycine-serine substitution at codon 119 of the Ace-1 gene confers both resistance and fitness costs, especially for 119S/S homozygotes. G119S in Anopheles gambiae from Accra (Ghana) is strongly associated with resistance, and, despite expectations of cost, resistant 119S alleles are increasing significantly in frequency. Sequencing of Accra females detected only a single Ace-1 119S haplotype, whereas 119G diversity was high overall but very low at non-synonymous sites, evidence of strong purifying selection driven by functional constraint. Flanking microsatellites showed reduced diversity, elevated linkage disequilibrium and high differentiation of 119S, relative to 119G homozygotes across up to two megabases of the genome. Yet these signals of selection were inconsistent and sometimes weak tens of kilobases from Ace-1. This unexpected finding is attributable to apparently ubiquitous amplification of 119S alleles as part of a large copy number variant (CNV) far exceeding the size of the Ace-1 gene, whereas 119G alleles were unduplicated. Ace-1 CNV was detectable in archived samples collected when the 119S allele was rare in Ghana. Multicopy amplification of resistant alleles has not been observed previously and is likely to underpin the recent increase in 119S frequency. The large CNV compromised localization of the strong selective sweep around Ace-1, emphasizing the need to integrate CNV analysis into genome scans for selection., (© 2015 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Insecticide resistance is a major impediment to the control of vectors and pests of public health importance and is a strongly selected trait capable of rapid spread, sometimes even between closely related species. Elucidating the mechanisms generating insecticide resistance in mosquito vectors of disease, and understanding the spread of resistance within and between populations and species are vital for the development of robust resistance management strategies. Here, we studied the mechanisms of resistance in two sympatric members of the Anopheles gambiae species complex-the major vector of malaria in sub-Saharan Africa-to understand how resistance has developed and spread in eastern Uganda, a region with some of the highest levels of malaria. In eastern Uganda, where the mosquitoes Anopheles arabiensis and An. gambiae can be found sympatrically, low levels of hybrids (0.4 %) occur, offering a route for introgression of adaptively important variants between species. In independent microarray studies of insecticide resistance, Gste4, an insect-specific glutathione S-transferase, was among the most significantly up-regulated genes in both species. To test the hypothesis of interspecific introgression, we sequenced 2.3 kbp encompassing Gste4. Whilst this detailed sequencing ruled out introgression, we detected strong positive selection acting on Gste4. However, these sequences, followed by haplotype-specific qPCR, showed that the apparent up-regulation in An. arabiensis is a result of allelic variation across the microarray probe binding sites which artefactually elevates the gene expression signal. Thus, face-value acceptance of microarray data can be misleading and it is advisable to conduct a more detailed investigation of the causes and nature of such signal. The identification of positive selection acting on this locus led us to functionally express and characterise allelic variants of GSTE4. Although the in vitro data do not support a direct role for GSTE4 in metabolism, they do support a role for this enzyme in insecticide sequestration. Thus, the demonstration of a role for an up-regulated gene in metabolic resistance to insecticides should not be limited to simply whether it can metabolise insecticide; such a strict criterion would argue against the involvement of GSTE4 despite the weight of evidence to the contrary.

Background: In areas where the morphologically indistinguishable malaria mosquitoes Anopheles gambiae Giles and An. arabiensis Patton are sympatric, hybrids are detected occasionally via species-diagnostic molecular assays. An. gambiae and An. arabiensis exhibit both pre- and post-reproductive mating barriers, with swarms largely species-specific and male F1 (first-generation) hybrids sterile. Consequently advanced-stage hybrids (back-crosses to parental species), which would represent a route for potentially-adaptive introgression, are expected to be very rare in natural populations. Yet the use of one or two physically linked single-locus diagnostic assays renders them indistinguishable from F1 hybrids and levels of interspecific gene flow are unknown., Methods: We used data from over 350 polymorphic autosomal SNPs to investigate post F1 gene flow via patterns of genomic admixture between An. gambiae and An. arabiensis from eastern Uganda. Simulations were used to investigate the statistical power to detect hybrids with different levels of crossing and to identify the hybrid category significantly admixed genotypes could represent., Results: A range of admixture proportions were detected for 11 field-collected hybrids identified via single-locus species-diagnostic PCRs. Comparison of admixture data with simulations indicated that at least seven of these hybrids were advanced generation crosses, with backcrosses to each species identified. In addition, of 36 individuals typing as An. gambiae or An. arabiensis that exhibited outlying admixture proportions, ten were identified as significantly mixed backcrosses, and at least four of these were second or third generation crosses., Conclusions: Our results show that hybrids detected using standard diagnostics will often be hybrid generations beyond F1, and that in our study area around 5% (95% confidence intervals 3%-9%) of apparently 'pure' species samples may also be backcrosses. This is likely an underestimate because of rapidly-declining detection power beyond the first two backcross generations. Post-F1 gene flow occurs at a far from inconsequential rate between An. gambiae and An. arabiensis, and, especially for traits under strong selection, could readily lead to adaptive introgression of genetic variants relevant for vector control.

Insecticide resistance in the malaria vector Anopheles gambiae s.l. (Diptera: Culicidae) threatens insecticide-based control efforts, necessitating regular monitoring. We assessed resistance in field-collected An. gambiae s.l. from Jinja, Uganda using World Health Organization (WHO) bioassays. Only An. gambiae s.s. and An. arabiensis (≈70%) were present. Female An. gambiae exhibited extremely high pyrethroid resistance (permethrin LT50 > 2 h; deltamethrin LT50 > 5 h). Female An. arabiensis were resistant to permethrin and exhibited reduced susceptibility to deltamethrin. However, while An. gambiae were DDT resistant, An. arabiensis were fully susceptible. Both species were fully susceptible to bendiocarb and fenitrothion. Kdr 1014S has increased rapidly in the Jinja population of An. gambiae s.s. and now approaches fixation (≈95%), consistent with insecticide-mediated selection, but is currently at a low frequency in An. arabiensis (0.07%). Kdr 1014F was also at a low frequency in An. gambiae. These frequencies preclude adequately-powered tests for an association with phenotypic resistance. PBO synergist bioassays resulted in near complete recovery of pyrethroid susceptibility suggesting involvement of CYP450s in resistance. A small number (0.22%) of An. gambiae s.s. ×An. arabiensis hybrids were found, suggesting the possibility of introgression of resistance alleles between species. The high levels of pyrethroid resistance encountered in Jinja threaten to reduce the efficacy of vector control programmes which rely on pyrethroid-impregnated bednets or indoor spraying of pyrethroids., (© 2012 The Royal Entomological Society.)