Detection and quantitation of copy number variation in the voltage-gated sodium channel gene of the mosquito Culex quinquefasciatus

Insecticide resistance is typically associated with alterations to the insecticidal target-site or with gene expression variation at loci involved in insecticide detoxification. In some species copy number variation (CNV) of target site loci (e.g. the Ace-1 target site of carbamate insecticides) or detoxification genes has been implicated in the resistance phenotype. We show that field-collected Ugandan Culex quinquefasciatus display CNV for the voltage-gated sodium channel gene (Vgsc), target-site of pyrethroid and organochlorine insecticides. In order to develop field-applicable diagnostics for Vgsc CN, and as a prelude to investigating the possible association of CN with insecticide resistance, three assays were compared for their accuracy in CN estimation in this species. The gold standard method is droplet digital PCR (ddPCR), however, the hardware is prohibitively expensive for widespread utility. Here, ddPCR was compared to quantitative PCR (qPCR) and pyrosequencing. Across all platforms, CNV was detected in ≈10% of mosquitoes, corresponding to three or four copies (per diploid genome). ddPCR and qPCR-Std-curve yielded similar predictions for Vgsc CN, indicating that the qPCR protocol developed here can be applied as a diagnostic assay, facilitating monitoring of Vgsc CN in wild populations and the elucidation of association between the Vgsc CN and insecticide resistance

Fine scale spatial investigation of multiple insecticide resistance and underlying target-site and metabolic mechanisms in Anopheles gambiae in central Côte d’Ivoire

Routine monitoring of occurrence, levels and mechanisms of insecticide resistance informs effective management strategies, and should be used to assess the effect of new tools on resistance. As part of a cluster randomised controlled trial evaluating a novel insecticide-based intervention in central Côte d’Ivoire, we assessed resistance and its underlying mechanisms in Anopheles gambiae populations from a subset of trial villages. Resistance to multiple insecticides in An. gambiae s.s. and An. coluzzii was detected across villages, with dose–response assays demonstrating extremely high resistance intensity to the pyrethroid deltamethrin (> 1,500-fold), and mortality following exposure to pyrethroid-treated bednets was low (< 30% mortality in cone bioassays). The 1014F kdr mutation was almost fixed (≥ 90%) in all villages but the 1575Y kdr-amplifying mutation was relatively rare (< 15%). The carbamate and organophosphate resistance-associated Ace-1 G119S mutation was also detected at moderate frequencies (22–43%). Transcriptome analysis identified overexpression of P450 genes known to confer pyrethroid resistance (Cyp9K1, Cyp6P3, and Cyp6M2), and also a carboxylesterase (COEAE1F) as major candidates. Cyp6P3 expression was high but variable (up to 33-fold) and correlated positively with deltamethrin resistance intensity across villages (r2 = 0.78, P = 0.02). Tools and strategies to mitigate the extreme and multiple resistance provided by these mechanisms are required in this area to avoid future control failures

Costs of insensitive acetylcholinesterase insecticide resistance for the malaria vector Anopheles gambiae homozygous for the G119S mutation

<p>Abstract</p> <p>Background</p> <p>The G119S mutation responsible for insensitive acetylcholinesterase resistance to organophosphate and carbamate insecticides has recently been reported from natural populations of <it>Anopheles gambiae </it>in West Africa. These reports suggest there are costs of resistance associated with this mutation for <it>An. gambiae</it>, especially for homozygous individuals, and these costs could be influential in determining the frequency of carbamate resistance in these populations.</p> <p>Methods</p> <p>Life-history traits of the AcerKis and Kisumu strains of <it>An. gambiae </it>were compared following the manipulation of larval food availability in three separate experiments conducted in an insecticide-free laboratory environment. These two strains share the same genetic background, but differ in being homozygous for the presence or absence of the G119S mutation at the <it>ace-1 </it>locus, respectively.</p> <p>Results</p> <p>Pupae of the resistant strain were significantly more likely to die during pupation than those of the susceptible strain. Ages at pupation were significantly earlier for the resistant strain and their dry starved weights were significantly lighter; this difference in weight remained when the two strains were matched for ages at pupation.</p> <p>Conclusions</p> <p>The main cost of resistance found for <it>An. gambiae </it>mosquitoes homozygous for the G119S mutation was that they were significantly more likely to die during pupation than their susceptible counterparts, and they did so across a range of larval food conditions. Comparing the frequency of G119S in fourth instar larvae and adults emerging from the same populations would provide a way to test whether this cost of resistance is being expressed in natural populations of <it>An. gambiae </it>and influencing the dynamics of this resistance mutation.</p

Evidence of Introgression of the ace-1R Mutation and of the ace-1 Duplication in West African Anopheles gambiae s. s

Background: The role of inter-specific hybridisation is of particular importance in mosquito disease vectors for predicting the evolution of insecticide resistance. Two molecular forms of Anopheles gambiae s.s., currently recognized as S and M taxa, are considered to be incipient sibling species. Hybrid scarcity in the field was suggested that differentiation of M and S taxa is maintained by limited or absent gene flow. However, recent studies have revealed shared polymorphisms within the M and S forms, and a better understanding of the occurrence of gene flow is needed. One such shared polymorphism is the G119S mutation in the ace-1 gene (which is responsible for insecticide resistance); this mutation has been described in both the M and S forms of A. gambiae s.s. Methods and Results: To establish whether the G119S mutation has arisen independently in each form or by genetic introgression, we analysed coding and non-coding sequences of ace-1 alleles in M and S mosquitoes from representative field populations. Our data revealed many polymorphic sites shared by S and M forms, but no diversity was associated with the G119S mutation. These results indicate that the G119S mutation was a unique event and that genetic introgression explains the observed distribution of the G119S mutation within the two forms. However, it was impossible to determine from our data whether the mutation occurred first in the S form or in the M form. Unexpectedly, sequence analysis of some resistant individuals revealed a duplication of the ace-1 gene that was observed in both A. gambiae s.s. M and S forms. Again, the distribution of this duplication in the two forms most likely occurred through introgression. Conclusions: These results highlight the need for more research to understand the forces driving the evolution of insecticide resistance in malaria vectors and to regularly monitor resistance in mosquito populations of Africa

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

Dynamics of multiple insecticide resistance in the malaria vector Anopheles gambiae in a rice growing area in South-Western Burkina Faso

<p>Abstract</p> <p>Background</p> <p>Insecticide resistance of the main malaria vector, <it>Anopheles gambiae</it>, has been reported in south-western Burkina Faso, West Africa. Cross-resistance to DDT and pyrethroids was conferred by alterations at site of action in the sodium channel, the Leu-Phe <it>kdr </it>mutation; resistance to organophosphates and carbamates resulted from a single point mutation in the oxyanion hole of the acetylcholinesterase enzyme designed as <it>ace-1</it>^<it>R</it>.</p> <p>Methods</p> <p>An entomological survey was carried out during the rainy season of 2005 at Vallée du Kou, a rice growing area in south-western Burkina Faso. At the Vallée du Kou, both insecticide resistance mechanisms have been previously described in the M and S molecular forms of <it>An. gambiae</it>. This survey aimed i) to update the temporal dynamics and the circumsporozoite infection rate of the two molecular forms M and S of <it>An. gambiae </it>ii) to update the frequency of the Leu-Phe <it>kdr </it>mutation within these forms and finally iii) to investigate the occurrence of the <it>ace-1</it>^{<it>R </it>}mutation.</p> <p>Mosquitoes collected by indoor residual collection and by human landing catches were counted and morphologically identified. Species and molecular forms of <it>An. gambiae</it>, <it>ace-1</it>^{<it>R </it>}and Leu-Phe <it>kdr </it>mutations were determined using PCR techniques. The presence of the circumsporozoite protein of <it>Plasmodium falciparum </it>was determined using ELISA.</p> <p>Results</p> <p><it>Anopheles gambiae </it>populations were dominated by the M form. However the S form occurred in relative important proportion towards the end of the rainy season with a maximum peak in October at 51%. Sporozoite rates were similar in both forms. The frequency of the Leu-Phe <it>kdr </it>mutation in the S form reached a fixation level while it is still spreading in the M form. Furthermore, the <it>ace</it>-<it>1</it>^{<it>R </it>}mutation prevailed predominately in the S form and has just started spreading in the M form. The two mutations occurred concomitantly both in M and S populations.</p> <p>Conclusion</p> <p>These results showed that the Vallée du Kou, a rice growing area formerly occupied mainly by M susceptible populations, is progressively colonized by S resistant populations living in sympatry with the former. As a result, the distribution pattern of insecticide resistance mutations shows the occurrence of both resistance mechanisms concomitantly in the same populations. The impact of multiple resistance mechanisms in M and S populations of <it>An. gambiae </it>on vector control measures against malaria transmission, such as insecticide-treated nets (ITNs) and indoor residual spraying (IRS), in this area is discussed.</p

Cotton pest management practices and the selection of pyrethroid resistance in Anopheles gambiae population in Northern Benin

<p>Abstract</p> <p>Background</p> <p>Pyrethroid insecticides, carbamate and organophosphate are the classes of insecticides commonly used in agriculture for crop protection in Benin. Pyrethroids remain the only class of insecticides recommended by the WHO for impregnation of bed nets. Unfortunately, the high level of pyrethroid resistance in <it>Anopheles gambiae </it>s.l., threatens to undermine the success of pyrethroid treated nets. This study focuses on the investigation of agricultural practices in cotton growing areas, and their direct impact on larval populations of <it>An. gambiae </it>in surrounding breeding sites.</p> <p>Methods</p> <p>The protocol was based on the collection of agro-sociological data where farmers were subjected to semi-structured questionnaires based on the strategies used for crop protection. This was complemented by bioassay tests to assess the susceptibility of malaria vectors to various insecticides. Molecular analysis was performed to characterize the resistance genes and the molecular forms of <it>An. gambiae</it>. Insecticide residues in soil samples from breeding sites were investigated to determine major factors that can inhibit the normal growth of mosquito larvae by exposing susceptible and resistant laboratory strains.</p> <p>Results</p> <p>There is a common use by local farmers of mineral fertilizer NPK at 200 kg/ha and urea at 50 kg/hectare following insecticide treatments in both the Calendar Control Program (CCP) and the Targeted Intermittent Control Program (TICP). By contrast, no chemicals are involved in Biological Program (BP) where farmers use organic and natural fertilizers which include animal excreta.</p> <p>Susceptibility test results confirmed a high resistance to DDT. Mean mortality of <it>An. gambiae </it>collected from the farms practicing CCP, TICP and BP methods were 33%, 42% and 65% respectively. <it>An. gambiae </it>populations from areas using the CCP and TICP programs showed resistance to permethrin with mortality of 50% and 58% respectively. By contrast, bioassay test results of <it>An. gambiae </it>from BP areas gave a high level of susceptibility to permethrin with an average mortality of 94%.</p> <p>Molecular analysis identified <it>An. gambiae </it>s.s, and <it>An. arabiensis </it>with a high predominance of <it>An. gambiae s.s </it>(90%). The two molecular forms, M and S, were also determined with a high frequency of the S form (96%).</p> <p>The <it>Kdr </it>gene seemed the main target- site resistance mechanism detected in CCP, TICP, and BP areas at the rates ranging from 32 to 78%. The frequency of <it>ace-1R </it>gene was very low (< 0.1).</p> <p>The presence of inhibiting factors in soil samples under insecticide treatments were found and affected negatively in delaying the development of <it>An. gambiae </it>larval populations.</p> <p>Conclusions</p> <p>This research shows that <it>Kdr </it>has spread widely in <it>An. gambiae</it>, mainly in CCP and TICP areas where pyrethroids are extensively used. To reduce the negative impact of pesticides use in cotton crop protection, the application of BP-like programs, which do not appear to select for vector resistance would be useful. These results could serve as scientific evidence of the spread of resistance due to a massive agricultural use of insecticides and contribute to the management of pesticides usage on cotton crops hence reducing the selection pressure of insecticides on <it>An. gambiae </it>populations.</p

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

Insecticide resistance status in Anopheles gambiae in southern Benin

BACKGROUND: The emergence of pyrethroid resistance in Anopheles gambiae has become a serious concern to the future success of malaria control. In Benin, the National Malaria Control Programme has recently planned to scaling up long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) for malaria prevention. It is, therefore, crucial to monitor the level and type of insecticide resistance in An. gambiae, particularly in southern Benin where reduced efficacy of insecticide-treated nets (ITNs) and IRS has previously been reported. METHODS: The protocol was based on mosquito collection during both dry and rainy seasons across forty districts selected in southern Benin. Bioassay were performed on adults collected from the field to assess the susceptibility of malaria vectors to insecticide-impregnated papers (permethrin 0.75%, delthamethrin 0.05%, DDT 4%, and bendiocarb 0.1%) following WHOPES guidelines. The species within An. gambiae complex, molecular form and presence of kdr and ace-1 mutations were determined by PCR. RESULTS: Strong resistance to permethrin and DDT was found in An. gambiae populations from southern Benin, except in Aglangandan where mosquitoes were fully susceptible (mortality 100%) to all insecticides tested. PCR showed the presence of two sub-species of An. gambiae, namely An. gambiae s.s, and Anopheles melas, with a predominance for An. gambiae s.s (98%). The molecular M form of An. gambiae was predominant in southern Benin (97%). The kdr mutation was detected in all districts at various frequency (1% to 95%) whereas the Ace-1 mutation was found at a very low frequency (<or= 5%). CONCLUSION: This study showed a widespread resistance to permethrin in An. gambiae populations from southern Benin, with a significant increase of kdr frequency compared to what was observed previously in Benin. The low frequency of Ace-1 recorded in all populations is encouraging for the use of bendiocarb as an alternative insecticide to pyrethroids for IRS in Benin