Insecticide Resistance in Areas Under Investigation by the International Centers of Excellence for Malaria Research: A Challenge for Malaria Control and Elimination

Scale-up of the main vector control interventions, residual insecticides sprayed on walls or structures and/or impregnated in bed nets, together with prompt diagnosis and effective treatment, have led to a global reduction in malaria transmission. However, resistance in vectors to almost all classes of insecticides, particularly to the synthetic pyrethroids, is posing a challenge to the recent trend of declining malaria. Ten International Centers of Excellence for Malaria Research (ICEMR) located in the most malaria-endemic regions of the world are currently addressing insecticide resistance in the main vector populations, which not only threaten hope for elimination in malaria-endemic countries but also may lead to reversal where notable reductions in malaria have been documented. This communication illustrates the current status of insecticide resistance with a focus on the countries where activities are ongoing for 9 out of the 10 ICEMRs. Most of the primary malaria vectors in the ICEMR countries exhibit insecticide resistance, albeit of varying magnitude, and spanning all mechanisms of resistance. New alternatives to the insecticides currently available are still to be fully developed for deployment. Integrated vector management principles need to be better understood and encouraged, and viable insecticide resistance management strategies need to be developed and implemented

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

[No abstract available

A Push-Pull System to Reduce House Entry of Malaria Mosquitoes.

Mosquitoes are the dominant vectors of pathogens that cause infectious diseases such as malaria, dengue, yellow fever and filariasis. Current vector control strategies often rely on the use of pyrethroids against which mosquitoes are increasingly developing resistance. Here, a push-pull system is presented, that operates by the simultaneous use of repellent and attractive volatile odorants. Experiments were carried out in a semi-field set-up: a traditional house which was constructed inside a screenhouse. The release of different repellent compounds, para-menthane-3,8-diol (PMD), catnip oil e.o. and delta-undecalactone, from the four corners of the house resulted in significant reductions of 45% to 81.5% in house entry of host-seeking malaria mosquitoes. The highest reductions in house entry (up to 95.5%), were achieved by simultaneously repelling mosquitoes from the house (push) and removing them from the experimental set-up using attractant-baited traps (pull). The outcome of this study suggests that a push-pull system based on attractive and repellent volatiles may successfully be employed to target mosquito vectors of human disease. Reductions in house entry of malaria vectors, of the magnitude that was achieved in these experiments, would likely affect malaria transmission. The repellents used are non-toxic and can be used safely in a human environment. Delta-undecalactone is a novel repellent that showed higher effectiveness than the established repellent PMD. These results encourage further development of the system for practical implementation in the field

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

Genome variation and population structure among 1142 mosquitoes of the African malaria vector species Anopheles gambiae and Anopheles coluzzii

Mosquito control remains a central pillar of efforts to reduce malaria burden in sub-Saharan Africa. However, insecticide resistance is entrenched in malaria vector populations, and countries with a high malaria burden face a daunting challenge to sustain malaria control with a limited set of surveillance and intervention tools. Here we report on the second phase of a project to build an open resource of high-quality data on genome variation among natural populations of the major African malaria vector species Anopheles gambiae and Anopheles coluzzii. We analyzed whole genomes of 1142 individual mosquitoes sampled from the wild in 13 African countries, as well as a further 234 individuals comprising parents and progeny of 11 laboratory crosses. The data resource includes high-confidence single-nucleotide polymorphism (SNP) calls at 57 million variable sites, genome-wide copy number variation (CNV) calls, and haplotypes phased at biallelic SNPs. We use these data to analyze genetic population structure and characterize genetic diversity within and between populations. We illustrate the utility of these data by investigating species differences in isolation by distance, genetic variation within proposed gene drive target sequences, and patterns of resistance to pyrethroid insecticides. This data resource provides a foundation for developing new operational systems for molecular surveillance and for accelerating research and development of new vector control tools. It also provides a unique resource for the study of population genomics and evolutionary biology in eukaryotic species with high levels of genetic diversity under strong anthropogenic evolutionary pressures

Local selection in the presence of high levels of gene flow: Evidence of heterogeneous insecticide selection pressure across Ugandan Culex quinquefasciatus populations

Background: Culex quinquefasciatus collected in Uganda, where no vector control interventions directly targeting this species have been conducted, was used as a model to determine if it is possible to detect heterogeneities in selection pressure driven by insecticide application targeting other insect species. Methodology/Principal findings: Population genetic structure was assessed through microsatellite analysis, and the impact of insecticide pressure by genotyping two target-site mutations, Vgsc-1014F of the voltage-gated sodium channel target of pyrethroid and DDT insecticides, and Ace1-119S of the acetylcholinesterase gene, target of carbamate and organophosphate insecticides. No significant differences in genetic diversity were observed among populations by microsatellite markers with HE ranging from 0.597 to 0.612 and low, but significant, genetic differentiation among populations (FST = 0.019, P = 0.001). By contrast, the insecticide-resistance markers display heterogeneous allelic distributions with significant differences detected between Central Ugandan (urban) populations relative to Eastern and Southwestern (rural) populations. In the central region, a frequency of 62% for Vgsc-1014F, and 32% for the Ace1-119S resistant allele were observed. Conversely, in both Eastern and Southwestern regions the Vgsc-1014F alleles were close to fixation, whilst Ace1-119S allele frequency was 12% (although frequencies may be underestimated due to copy number variation at both loci). Conclusions/Significance: Taken together, the microsatellite and both insecticide resistance target-site markers provide evidence that in the face of intense gene flow among populations, disjunction in resistance frequencies arise due to intense local selection pressures despite an absence of insecticidal control interventions targeting Culex

Resistance to pirimiphos-methyl in West African Anopheles is spreading via duplication and introgression of the Ace1 locus

Vector population control using insecticides is a key element of current strategies to prevent malaria transmission in Africa. The introduction of effective insecticides, such as the organophosphate pirimiphos-methyl, is essential to overcome the recurrent emergence of resistance driven by the highly diverse Anopheles genomes. Here, we use a population genomic approach to investigate the basis of pirimiphos-methyl resistance in the major malaria vectors Anopheles gambiae and A. coluzzii. A combination of copy number variation and a single non-synonymous substitution in the acetylcholinesterase gene, Ace1, provides the key resistance diagnostic in an A. coluzzii population from Coˆte d’Ivoire that we used for sequence-based association mapping, with replication in other West African populations. The Ace1 substitution and duplications occur on a unique resistance haplotype that evolved in A. gambiae and introgressed into A. coluzzii, and is now common in West Africa primarily due to selection imposed by other organophosphate or carbamate insecticides. Our findings highlight the predictive value of this complex resistance haplotype for phenotypic resistance and clarify its evolutionary history, providing tools to for molecular surveillance of the current and future effectiveness of pirimiphos-methyl based interventions

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