Identifying permethrin resistance loci in malaria vectors by genetic mapping

Identification of the major loci responsible for insecticide resistance in malaria vectors would aid the development andimplementation of effective resistance management strategies, which are urgently needed to tackle the growing threat posedby resistance to the limited insecticides available for malaria control. Genome-wide association studies in the major malariavector, Anopheles gambiae, have been hindered by the high degree of within-population structuring and very low levels oflinkage disequilibrium hence we revisited the use of quantitative trait loci (QTL) mapping to study resistance phenotypesin this vector species. Earlier work, identified two major QTL associated with pyrethroid resistance in A. gambiae s.s. fromEast Africa using genetic crossing of laboratory-colonized resistant and susceptible strains. In this study, we report theresults from genetic mapping of pyrethroid resistance in three isofemale pedigrees established from wild-caught femaleA. gambiae s.s. mosquitoes from Benin. We identified two QTL on chromosomes 2L and 3R in these field populations, insimilar genomic locations to theQTLidentified in laboratory strains. The relative merits of two alternative study designs arediscussed and suggestions made for future genetic mapping studies of insecticide resistance in mosquitoes

Gene amplification and microsatellite polymorphism underlie a recent insect host shift

Host plant shifts of herbivorous insects may be a first step toward sympatric speciation and can create new pests of agriculturally important crops; however, the molecular mechanisms that mediate this process are poorly understood. Certain races of the polyphagous aphid Myzus persicae have recently adapted to feed on tobacco (Myzus persicae nicotianae) and show a reduced sensitivity to the plant alkaloid nicotine and cross-resistance to neonicotinoids a class of synthetic insecticides widely used for control. Here we show constitutive overexpression of a cytochrome P450 (CYP6CY3) allows tobacco-adapted races of M. persicae to efficiently detoxify nicotine and has preadapted them to resist neonicotinoid insecticides. CYP6CY3, is highly overexpressed in M. persicae nicotianae clones from three continents compared with M. persicae s.s. and expression level is significantly correlated with tolerance to nicotine. CYP6CY3 is highly efficient (compared with the primary human nicotine-metabolizing P450) at metabolizing nicotine and neonicotinoids to less toxic metabolites in vitro and generation of transgenic Drosophila expressing CYP6CY3 demonstrate that it confers resistance to both compounds in vivo. Overexpression of CYP6CY3 results from the expansion of a dinucleotide microsatellite in the promoter region and a recent gene amplification, with some aphid clones carrying up to 100 copies. We conclude that the mutations leading to overexpression of CYP6CY3 were a prerequisite for the host shift of M. persicae to tobacco and that gene amplification and microsatellite polymorphism are evolutionary drivers in insect host adaptation

Positional cloning of rp2 QTL associates the P450 genes CYP6Z1, CYP6Z3 and CYP6M7 with pyrethroid resistance in the malaria vector Anopheles funestus

Pyrethroid resistance in Anopheles funestus is threatening malaria control in Africa. Elucidation of underlying resistance mechanisms is crucial to improve the success of future control programs. A positional cloning approach was used to identify genes conferring resistance in the uncharacterised rp2 quantitative trait locus (QTL) previously detected in this vector using F6 advanced intercross lines (AIL). A 113 kb BAC clone spanning rp2 was identified and sequenced revealing a cluster of 15 P450 genes and one salivary protein gene (SG7-2). Contrary to A. gambiae, AfCYP6M1 is triplicated in A. funestus, while AgCYP6Z2 orthologue is absent. Five hundred and sixty-five new single nucleotide polymorphisms (SNPs)were identified for genetic mapping from rp2 P450s and other genes revealing high genetic polymorphisms with one SNP every 36 bp. A significant genotype/phenotype association was detected for rp2 P450s but not for a cluster of cuticular protein genes previously associated with resistance in A. gambiae. QTL mapping using F6 AIL confirms the rp2 QTL with an increase logarithm of odds score of 5. Multiplex gene expression profiling of 15 P450s and other genes around rp2 followed by individual validation using qRT–PCR indicated a significant overexpression in the resistant FUMOZ-R strain of the P450s AfCYP6Z1, AfCYP6Z3, AfCYP6M7 and the glutathione-s-transferase GSTe2 with respective fold change of 11.2,6.3, 5.5 and 2.8. Polymorphisms analysis of AfCYP6Z1 and AfCYP6Z3 identified amino acid changes potentially associated with resistance further indicating that these genes are controlling the pyrethroid resistance explained by the rp2 QTL. The characterisation of this rp2 QTL significantly improves our understanding of resistance mechanisms in A. funestus

Genomic Footprints of Selective Sweeps from Metabolic Resistance to Pyrethroids in African Malaria Vectors Are Driven by Scale up of Insecticide-Based Vector Control

Insecticide resistance in mosquito populations threatens recent successes in malaria prevention. Elucidating patterns of genetic structure in malaria vectors to predict the speed and direction of the spread of resistance is essential to get ahead of the `resistance curve' and to avert a public health catastrophe. Here, applying a combination of microsatellite analysis, whole genome sequencing and targeted sequencing of a resistance locus, we elucidated the continent-wide population structure of a major African malaria vector, Anopheles funestus. We identified a major selective sweep in a genomic region controlling cytochrome P450-based metabolic resistance conferring high resistance to pyrethroids. This selective sweep occurred since 2002, likely as a direct consequence of scaled up vector control as revealed by whole genome and fine-scale sequencing of pre- and post-intervention populations. Fine-scaled analysis of the pyrethroid resistance locus revealed that a resistanceassociated allele of the cytochrome P450 monooxygenase CYP6P9a has swept through southern Africa to near fixation, in contrast to high polymorphism levels before interventions, conferring high levels of pyrethroid resistance linked to control failure. Population structure analysis revealed a barrier to gene flow between southern Africa and other areas, which may prevent or slow the spread of the southern mechanism of pyrethroid resistance to other regions. By identifying a genetic signature of pyrethroid-based interventions, we have demonstrated the intense selective pressure that control interventions exert on mosquito populations. If this level of selection and spread of resistance continues unabated, our ability to control malaria with current interventions will be compromised

Susceptibility profiles of helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) to deltamethrin reveal a contrast between the northern and the southern Benin

Open Access Journal; Published online: 28 May 2019Helicoverpa armigera is an indigenous species in Africa and has been reported in the destruction of several crops in Benin. Management of H. armigera pest is mainly focused on the use of synthetic pyrethroids, which may contribute to resistance selection. This study aimed to screen the susceptibility pattern of field populations of H. armigera to deltamethrin in Benin. Relevant information on the type of pesticides used by farmers were gathered through surveys. Collected samples of Helicoverpa (F0) were reared to F1. F0 were subjected to morphological speciation followed by a confirmation using restriction fragment length polymorphism coupled with a polymerase chain reaction (RFLP-PCR). F1 (larvae) were used for insecticide susceptibility with deltamethrin alone and in the presence of the P450 inhibitor Piperonyl Butoxide (PBO). Deltamethrin and lambda-cyhalothrin were the most used pyrethroids in tomato and cotton farms respectively. All field-sampled Helicoverpa were found to be H. armigera. Susceptibility assays of H. armigera to deltamethrin revealed a high resistance pattern in cowpea (resistance factor (RF) = 2340), cotton (RF varying from 12 to 516) and tomato (RF=85) farms which is a concern for the control of this major polyphagous agricultural pest. There was a significant increase of mortality when deltamethrin insecticide was combined with piperonyl butoxide (PBO), suggesting the possible involvement of detoxification enzymes such as oxidase. This study highlights the presence of P450 induced metabolic resistance in H. armigera populations from diverse cropping systems in Benin. The recorded high levels of deltamethrin resistance in H. armigera is a concern for the control of this major agricultural pest in Benin as the country is currently embarking into economical expansion of cotton, vegetables and grain-legumes cropping systems

Target-site resistance mutations (kdr and RDL), but not metabolic resistance, negatively impact male mating competiveness in the malaria vector Anopheles gambiae

The implementation of successful insecticide resistance management strategies for malaria control is currently hampered by poor understanding of the fitness cost of resistance on mosquito populations, including their mating competiveness. To fill this knowledge gap, coupled and uncoupled Anopheles gambiae s.l. males (all M form (Anopheles coluzzii)) were collected from mating swarms in Burkina Faso. This multiple insecticide resistant population exhibited high 1014F kdrR allele frequencies (460%) and RDLR (480%) in contrast to the Ace-1R allele (o6%). Kdr heterozygote males were more likely to mate than homozygote resistant (OR=2.36; Po0.001), suggesting a negative impact of kdr on An. coluzzii mating ability. Interestingly, heterozygote males were also more competitive than homozygote susceptible (OR=3.26; P=0.006), suggesting a heterozygote advantage effect. Similarly, heterozygote RDLR/RDLS were also more likely to mate than homozygote-resistant males (OR=2.58; P=0.007). Furthermore, an additive mating disadvantage was detected in male homozygotes for both kdr/RDL-resistant alleles. In contrast, no fitness difference was observed for the Ace-1 mutation. Comparative microarray-based genome-wide transcription analysis revealed that metabolic resistance did not significantly alter the mating competitiveness of male An. coluzzii mosquitoes. Indeed, no significant difference of expression levels was observed for the main metabolic resistance genes, suggesting that metabolic resistance has a limited impact on male mating competiveness. In addition, specific gene classes/GO terms associated with mating process were detected including sensory perception and peroxidase activity. The detrimental impact of insecticide resistance on mating competiveness observed here suggests that resistance management strategies such as insecticide rotation could help reverse the resistance, if implemented early

Midgut microbiota of the malaria mosquito vector Anopheles gambiae and Interactions with plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.Institut de Recherche pour le Developpement (IRD); French Agence Nationale pour la Recherche [ANR-11-BSV7-009-01]; European Community [242095, 223601]info:eu-repo/semantics/publishedVersio

Underpinning Sustainable Vector Control through Informed Insecticide Resistance Management

Background: There has been rapid scale-up of malaria vector control in the last ten years. Both of the primary control strategies, long-lasting pyrethroid treated nets and indoor residual spraying, rely on the use of a limited number of insecticides. Insecticide resistance, as measured by bioassay, has rapidly increased in prevalence and has come to the forefront as an issue that needs to be addressed to maintain the sustainability of malaria control and the drive to elimination. Zambia’s programme reported high levels of resistance to the insecticides it used in 2010, and, as a result, increased its investment in resistance monitoring to support informed resistance management decisions. Methodology/Principal Findings: A country-wide survey on insecticide resistance in Zambian malaria vectors was performed using WHO bioassays to detect resistant phenotypes. Molecular techniques were used to detect target-site mutations and microarray to detect metabolic resistance mechanisms. Anopheles gambiae s.s. was resistant to pyrethroids,DDT and carbamates, with potential organophosphate resistance in one population. The resistant phenotypes were conferred by both target-site and metabolic mechanisms. Anopheles funestus s.s. was largely resistant to pyrethroids and carbamates, with potential resistance to DDT in two locations. The resistant phenotypes were conferred by elevated levels of cytochrome p450s. Conclusions/Significance: Currently, the Zambia National Malaria Control Centre is using these results to inform their vector control strategy. The methods employed here can serve as a template to all malaria-endemic countries striving to create a sustainable insecticide resistance management pla

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

[No abstract available

A marker of glutathione S-transferase-mediated resistance to insecticides is associated with higher Plasmodium infection in the African malaria vector Anopheles funestus

Metabolic resistance to insecticides is threatening malaria control in Africa. However, the extent to which it impacts malaria transmission remains unclear. Here, we investigated the association between a marker of glutathione S-transferase mediated metabolic resistance and Plasmodium infection in field population of Anopheles funestus s.s. in comparison to the A296S-RDL target site mutation. The 119F-GSTe2 resistant allele was present in southern (Obout) (56%) and central (Mibellon) (25%) regions of Cameroon whereas the 296S-RDL resistant allele was detected at 98.5% and 15% respectively. The whole mosquito Plasmodium and sporozoite infection rates were 57% and 14.8% respectively in Obout (n = 508) and 19.7% and 5% in Mibellon (n = 360). No association was found between L119F-GSTe2 genotypes and whole mosquito infection status. However, when analyzing oocyst and sporozoite infection rates separately, the resistant homozygote 119F/F genotype was significantly more associated with Plasmodium infection in Obout than both heterozygote (OR = 2.5; P = 0.012) and homozygote susceptible (L/L119) genotypes (OR = 2.10; P = 0.013). In contrast, homozygote RDL susceptible mosquitoes (A/A296) were associated more frequently with Plasmodium infection than other genotypes (OR = 4; P = 0.03). No additive interaction was found between L119F and A296S. Sequencing of the GSTe2 gene showed no association between the polymorphism of this gene and Plasmodium infection. Glutathione S-transferase metabolic resistance is potentially increasing the vectorial capacity of resistant An. funestus mosquitoes. This could result in a possible exacerbation of malaria transmission in areas of high GSTe2-based metabolic resistance to insecticides