Contemporary gene flow between wild An. gambiae s.s. and An. arabiensis

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

Hybridization in parasites: consequences for adaptive evolution, pathogenesis and public health in a changing world

[No abstract available

Candidate-gene based GWAS identifies reproducible DNA markers for metabolic pyrethroid resistance from standing genetic variation in East African Anopheles gambiae.

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

Genetic diversity of the African malaria vector Anopheles gambiae

The sustainability of malaria control in Africa is threatened by the rise of insecticide resistance in Anopheles mosquitoes, which transmit the disease1. To gain a deeper understanding of how mosquito populations are evolving, here we sequenced the genomes of 765 specimens of Anopheles gambiae and Anopheles coluzzii sampled from 15 locations across Africa, and identified over 50 million single nucleotide polymorphisms within the accessible genome. These data revealed complex population structure and patterns of gene flow, with evidence of ancient expansions, recent bottlenecks, and local variation in effective population size. Strong signals of recent selection were observed in insecticide-resistance genes, with several sweeps spreading over large geographical distances and between species. The design of new tools for mosquito control using gene-drive systems will need to take account of high levels of genetic diversity in natural mosquito populations

Parallel evolution or purifying selection, not introgression, explains similarity in the pyrethroid detoxification linked GSTE4 of Anopheles gambiae and An. arabiensis

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 – in order 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 2.3kbp 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, facevalue 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

Anopheles species composition and insecticide resistance patterns in Uganda

Background: Long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are essential to malaria control, but are threatened by insecticide resistance. This thesis aimed to: 1) investigate malaria vector species collected indoor and outdoor using different methods; 2) describe species composition of Anopheles vectors collected from different areas; 3) determine susceptibility of Anopheles to pyrethroid insecticides, and 4) evaluate the association between genotypic markers of pyrethroid resistance and mosquito survival. Methods: Between October 2011 and November 2021, mosquitoes were collected from 13 sites across Uganda – indoors: human landing catches (HLC), CDC light traps (CDC LT) and prokopack aspirators; outdoors – HLCs and pit traps. Implementation of vector control, including LLINs only and LLINs + IRS, differed across time and space. Anopheles species composition was assessed using PCR. Standard WHO tube assays were done for permethrin and deltamethrin, with and without piperonyl butoxide (PBO). Mosquitoes were genotyped for Vgsc-L995S/L1014S and Vgsc-L995F/L1014F, Cyp6aa1, Cyp6p4, ZZB-TE, Cyp4j5, and Coeae1d using locked nucleic acid (LNA) and TaqMan assays. Results: Overall, 165,739 female Anopheles mosquitoes were collected from 13 sites using different collection methods. Of these, 160,657 were collected using CDC light traps (objectives 1 and 3), 349 using prokopack aspirators, 746 using human landing catches (indoor and outdoor), 1,234 using pit traps and 2,753 using larval collections. In the assessment of the impact of different mosquito collection methods, the vector density (mosquitoes per unit collection) using CDC light traps was 4.24 compared to 2.96 using indoor HLCs (4.24 vs 2.96, density ratio [DR] 0.70, 95% CIs 0.63–0.77, p < 0.001) and 4.24 compared to 1.82 using prokopacks (4.24 vs 1.82, DR 0.43, 95% CIs 0.37–0.49, p < 0.001). Sporozoite rates were similar between indoor methods, although precision was limited. Considering outdoor collections, the vector density was 3.53 using HLCs compared to 6.43 using pit traps (3.53 vs 6.43, DR 1.82, 95% CIs 1.61–2.05, p < 0.001). However, the sporozoite rate using pit trap collections was significantly lower at 0.004 compared to 0.018 using outdoor HLCs (0.018 vs 0.004, rate ratio [RR] 0.23, 95% CIs 0.07–0.75, p = 0.008). Prokopacks collected a higher proportion of Anopheles funestus (75.0%) than indoor HLCs (25.8%), while pit traps collected a higher proportion of Anopheles arabiensis (84.3%) than outdoor HLCs (36.9%) At least 158,095 female Anopheles mosquitoes were collected from 3 sites with varying malaria transmission intensities to assess the impact of control interventions. In the low transmission site, LLIN distribution was associated with a decline in Anopheles funestus vector density (0.07 vs 0.02 mosquitoes per house per night, density ratio [DR] 0.34, 95% CI: 0.18–0.65, p = 0.001), but not in Anopheles gambiae s.s. (hereafter An. gambiae) or Anopheles arabiensis. In the moderate transmission site, over 98% of mosquitoes were An. gambiae and LLIN distribution was associated with a decline in An. gambiae vector density (4.00 vs 2.46, DR 0.68, 95% CI: 0.49–0.94, p = 0.02). In the high transmission site, the combination of LLINs and multiple rounds of IRS was associated with significantly lower density of An. gambiae (28.0 vs 0.17, DR 0.004, 95% CI: 0.002–0.009, p < 0.001), and An. funestus sensu lato (s.l.) (3.90 vs 0.006, DR 0.001, 95% CI: 0.0005–0.004, p < 0.001), with a less pronounced decline in An. arabiensis (9.18 vs 2.00, DR 0.15 95% CI: 0.07–0.33, p < 0.001). In total, 2,753 An. gambiae s.l. were subjected to phenotype bioassays. Overall, mortality rates in An. gambiae and An. arabiensis following exposure to pyrethroids were 18.8% (148/788) and 74.6% (912/1,222) respectively. Pre-exposure to PBO resulted in higher mortality for both An. gambiae (permethrin: 12.9% to 56.5%; deltamethrin: 25.2% to 68.7%), and An. arabiensis (permethrin: 65.5% to 93.3%; deltamethrin: 82.4% to 89.8%). Most An. gambiae had the Vgsc-995S/F mutation (95% frequency) and the Cyp6p4 resistance allele (87%), while the frequency of Cyp4j5 and Coeae1d were lower (52% and 55%, respectively). Conclusions: The density and species of mosquitoes collected with alternative methods varied, reflecting the feeding and resting characteristics of the common vectors and the different collection approaches. LLIN distribution was associated with reductions in An. funestus s.l. in the lowest transmission site and An. gambiae in the moderate transmission site. In the high transmission site, a combination of LLINs and IRS and multiple rounds of IRS was associated with the significant reduction of An. gambiae and An. funestus s.l. Following IRS, An. arabiensis, a behaviorally resilient vector, was the predominant species. Resistance to pyrethroids was widespread in the study area, and the mortality rate was higher in An. arabiensis compared to An. gambiae. Further surveillance of insecticide resistance and assessment of correlations between genotypic markers and phenotypic outcomes are needed to better understand mechanisms of pyrethroid resistance in conferring resistance to guide vector control

Contemporary evolution of resistance at the major insecticide target site gene Ace-1 by mutation and copy number variation in the malaria mosquito Anopheles gambiae.

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 A. 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. Multi-copy 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. This article is protected by copyright. All rights reserved