Studies of insecticide resistance provide insights into the capacity of populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs). Resistance in the major malaria vectors has increased in concert with the expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance is crucial for the development and deployment of resistancebreaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae

Accurately monitoring insecticide resistance in target mosquito populations is important for combating malaria and other vector-borne diseases, and robust methods are key. The “WHO susceptibility bioassay” has been available from the World Health Organization for 60+ years: mosquitoes of known physiological status are exposed to a discriminating concentration of insecticide. Several changes to the test procedures have been made historically, which may seem minor but could impact bioassay results. The published test procedures and literature for this method were reviewed for methodological details. Areas where there was room for interpretation in the test procedures or where the test procedures were not being followed were assessed experimentally for their impact on bioassay results: covering or uncovering of the tube end during exposure; the number of mosquitoes per test unit; and mosquito age. Many publications do not cite the most recent test procedures; methodological details are reported which contradict the test procedures referenced, or methodological details are not fully reported. As a result, the precise methodology is unclear. Experimental testing showed that using fewer than the recommended 15–30 mosquitoes per test unit significantly reduced mortality, covering the exposure tube had no significant effect, and using mosquitoes older than 2–5 days old increased mortality, particularly in the resistant strain. Recommendations are made for improved reporting of experimental parameters

Insecticide resistance provides both an increasingly pressing threat to the control of vector-borne diseases and insights into the remarkable capacity of natural populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on deployment of pyrethroid insecticides, primarily in long lasting insecticide treated nets (LLINs), but resistance in the major malaria vectors has increased over the last 15 years in concert with dramatic expansion of LLIN distributions. Identifying genetic mechanisms causing high-level resistance in mosquitoes, which may almost entirely overcome pyrethroid efficacy, is crucial for the development and deployment of potentially resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000G) data we identified a very recent selective sweep in Ugandan mosquitoes which localized to a cluster of cytochrome P450 genes, including some frequently implicated in resistance. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border region around lake Victoria, with stepwise replacement of the double-mutant (Zanzibar TE and Cyp6p4-236M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolise pyrethroids; is strongly predictive of resistance to pyrethroids most notably deltamethrin, a commonly-used LLIN insecticide, but importantly, appears less effective against LLINs co-treated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable even within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid co-treated LLINs across African countries. Duplications of Cyp6aa1 gene are common in An. gambiae across Africa and are likely to be a useful diagnostic for high levels of pyrethroid resistance.

Irrigation schemes provide an ideal habitat for Anopheles mosquitoes particularly during the dry season. Reliable estimates of outdoor host-seeking behaviour are needed to assess the impact of vector control options and this is particularly the case for Anopheles arabiensis which displays a wide range of behaviours that circumvent traditional indoor-insecticide based control. In this study we compared the sampling efficiency of the host decoy trap (HDT) with the human landing catch (HLC) and Suna trap in a repeated Latin square design in two villages (Lengwe and Mwanza) on an irrigated sugar estate in southern Malawi. Over the course of 18 trapping nights, we caught 379 female Anopheles, the majority of which were identified as An. arabiensis. Across both villages, there was no detectable difference in Anopheles catch between the HDT compared with the HLC (RR = 0.85, P = 0.508). The overall sensitivity of the HLC was greater than the Suna trap regardless of mosquito density (Lengwe, α = 2.75, 95% credible interval: 2.03–3.73; Mwanza, α = 3.38, 95% credible interval: 1.50–9.30) whereas the sensitivity of the HDT was only greater than the Suna trap when mosquito numbers were high (Lengwe, α = 2.63, 95% credible interval: 2.00–3.85).We conclude that the HDT is an effective sampling device for outdoor host seeking An. arabiensis in southern Malawi. The presence of An. arabiensis in irrigated lands during the dry season poses a challenge for ongoing indoor vector control efforts.