Helen Irving

Two duplicated P450 genes are associated with pyrethroid resistance in Anopheles funestus, a major malaria vector.

Pyrethroid resistance in Anopheles funestus is a potential obstacle to malaria control in Africa. Tools are needed to detect resistance in field populations. We have been using a positional cloning approach to identify the major genes conferring pyrethroid resistance in this vector. A quantitative trait locus (QTL) named rp1 explains 87% of the genetic variance in pyrethroid susceptibility in two families from reciprocal crosses between susceptible and resistant strains. Two additional QTLs of minor effect, rp2 and rp3, were also detected. We sequenced a 120-kb BAC clone spanning the rp1 QTL and identified 14 protein-coding genes and one putative pseudogene. Ten of the 14 genes encoded cytochrome P450s, and expression analysis indicated that four of these P450s were differentially expressed between susceptible and resistant strains. Furthermore, two of these genes, CYP6P9 and CYP6P4, which are 25 and 51 times overexpressed in resistant females, are tandemly duplicated in the BAC clone as well as in laboratory and field samples, suggesting that P450 gene duplication could contribute to pyrethroid resistance in An. funestus. Single nucleotide polymorphisms (SNPs) were identified within CYP6P9 and CYP6P4, and genotyping of the progeny of the genetic crosses revealed a maximum penetrance value f(2) = 1, confirming that these SNPs are valid resistance markers in the laboratory strains. This serves as proof of principle that a DNA-based diagnostic test could be designed to trace metabolic resistance in field populations. This will be a major advance for insecticide resistance management in malaria vectors, which requires the early detection of resistance alleles.

Pyrethroid Resistance in an Anopheles funestus Population from Uganda

Background The susceptibility status of Anopheles funestus to insecticides remains largely unknown in most parts of Africa because of the difficulty in rearing field-caught mosquitoes of this malaria vector. Here we report the susceptibility status of the An. funestus population from Tororo district in Uganda and a preliminary characterisation of the putative resistance mechanisms involved. Methodology/Principal Findings A new forced egg laying technique used in this study significantly increased the numbers of field-caught females laying eggs and generated more than 4000 F1 adults. WHO bioassays indicated that An. funestus in Tororo is resistant to pyrethroids (62% mortality after 1 h exposure to 0.75% permethrin and 28% mortality to 0.05% deltamethrin). Suspected DDT resistance was also observed with 82% mortality. However this population is fully susceptible to bendiocarb (carbamate), malathion (organophosphate) and dieldrin with 100% mortality observed after exposure to each of these insecticides. Sequencing of a fragment of the sodium channel gene containing the 1014 codon conferring pyrethroid/DDT resistance in An. gambiae did not detect the L1014F kdr mutation but a correlation between haplotypes and resistance phenotype was observed indicating that mutations in other exons may be conferring the knockdown resistance in this species. Biochemical assays suggest that resistance in this population is mediated by metabolic resistance with elevated level of GSTs, P450s and pNPA compared to a susceptible strain of Anopheles gambiae. RT-PCR further confirmed the involvement of P450s with a 12-fold over-expression of CYP6P9b in the Tororo population compared to the fully susceptible laboratory colony FANG. Conclusion This study represents the first report of pyrethroid/DDT resistance in An. funestus from East Africa. With resistance already reported in southern and West Africa, this indicates that resistance in An. funestus may be more widespread than previously assumed and therefore this should be taken into account for the implementation and management of vector control programs in Africa.

Directionally selected cytochrome P450 alleles are driving the spread of pyrethroid resistance in the major malaria vector Anopheles funestus

Pyrethroid insecticides are critical for malaria control in Africa. However, resistance to this insecticide class in the malaria vector Anopheles funestus is spreading rapidly across Africa, threatening the success of ongoing and future malaria control programs. The underlying resistance mechanisms driving the spread of this resistance in wild populations remain largely unknown. Here, we show that increased expression of two tandemly duplicated P450 genes, CYP6P9a and CYP6P9b, is the main mechanism driving pyrethroid resistance in Malawi and Mozambique, two southern African countries where this insecticide class forms the mainstay of malaria control. Genome-wide transcription analysis using microarray and quantitative RT-PCR consistently revealed that CYP6P9a and CYP6P9b are the two genes most highly overexpressed (>50-fold; q < 0.01) in permethrin-resistant mosquitoes. Transgenic expression of CYP6P9a and CYP6P9b in Drosophila melanogaster demonstrated that elevated expression of either of these genes confers resistance to both type I (permethrin) and type II (deltamethrin) pyrethroids. Functional characterization of recombinant CYP6P9b confirmed that this protein metabolized both type I (permethrin and bifenthrin) and type II (deltamethrin and Lambda-cyhalothrin) pyrethroids but not DDT. Variability analysis identified that a single allele of each of these genes is predominantly associated with pyrethroid resistance in field populations from both countries, which is suggestive of a single origin of this resistance that has since spread across the region. Urgent resistance management strategies should be implemented in this region to limit a further spread of this resistance and minimize its impact on the success of ongoing malaria control programs.

A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector

BackgroundMetabolic resistance to insecticides is the biggest threat to the continued effectiveness of malaria vector control. However, its underlying molecular basis, crucial for successful resistance management, remains poorly characterized.ResultsHere, we demonstrate that the single amino acid change L119F in an upregulated glutathione S-transferase gene, GSTe2, confers high levels of metabolic resistance to DDT in the malaria vector Anopheles funestus. Genome-wide transcription analysis revealed that GSTe2 was the most over-expressed detoxification gene in DDT and permethrin-resistant mosquitoes from Benin. Transgenic expression of GSTe2 in Drosophila melanogaster demonstrated that over-transcription of this gene alone confers DDT resistance and cross-resistance to pyrethroids. Analysis of GSTe2 polymorphism established that the point mutation is tightly associated with metabolic resistance to DDT and its geographical distribution strongly correlates with DDT resistance patterns across Africa. Functional characterization of recombinant GSTe2 further supports the role of the L119F mutation, with the resistant allele being more efficient at metabolizing DDT than the susceptible one. Importantly, we also show that GSTe2 directly metabolizes the pyrethroid permethrin. Structural analysis reveals that the mutation confers resistance by enlarging the GSTe2 DDT-binding cavity, leading to increased DDT access and metabolism. Furthermore, we show that GSTe2 is under strong directional selection in resistant populations, and a restriction of gene flow is observed between African regions, enabling the prediction of the future spread of this resistance.ConclusionsThis first DNA-based metabolic resistance marker in mosquitoes provides an essential tool to track the evolution of resistance and to design suitable resistance management strategies.

High level of pyrethroid resistance in an Anopheles funestus population of the Chokwe District in Mozambique.

Background Although Anopheles funestus is difficult to rear, it is crucial to analyse field populations of this malaria vector in order to successfully characterise mechanisms of insecticide resistance observed in this species in Africa. In this study we carried out a large-scale field collection and rearing of An. funestus from Mozambique in order to analyse its susceptibility status to insecticides and to broadly characterise the main resistance mechanisms involved in natural populations. Methodology/Principal Findings 3,000 F1 adults were obtained through larval rearing. WHO susceptibility assays indicated a very high resistance to pyrethroids with no mortality recorded after 1h30min exposure and less than 50% mortality at 3h30min. Resistance to the carbamate, bendiocarb was also noted, with 70% mortality after 1h exposure. In contrast, no DDT resistance was observed, indicating that no kdr-type resistance was involved. The sequencing of the acetylcholinesterase gene indicated the absence of the G119S and F455W mutations associated with carbamate and organophosphate resistance. This could explain the absence of malathion resistance in this population. Both biochemical assays and quantitative PCR implicated up-regulated P450 genes in pyrethroid resistance, with GSTs playing a secondary role. The carbamate resistance observed in this population is probably conferred by the observed altered AChE with esterases also involved. Conclusion/Significance The high level of pyrethroid resistance in this population despite the cessation of pyrethroid use for IRS in 1999 is a serious concern for resistance management strategies such as rotational use of insecticides. As DDT has now been re-introduced for IRS, susceptibility to DDT needs to be closely monitored to prevent the appearance and spread of resistance to this insecticide.

Identification and distribution of a GABA receptor mutation conferring dieldrin resistance in the malaria vector Anopheles funestus in Africa.

Growing problems of pyrethroid resistance in Anopheles funestus have intensified efforts to identify alternative insecticides. Many agrochemicals target the GABA receptors, but cross-resistance from dieldrin resistance may preclude their introduction. Dieldrin resistance was detected in An. funestus populations from West (Burkina Faso) and central (Cameroon) Africa, but populations from East (Uganda) and Southern Africa (Mozambique and Malawi) were fully susceptible to this insecticide. Partial sequencing of the dieldrin target site, the γ-aminobutyric acid (GABA) receptor, identified two amino acid substitutions, A296S and V327I. The A296S mutation has been associated with dieldrin resistance in other species. The V327I mutations was detected in the resistant sample from Burkina Faso and Cameroon and consistently associated with the A296S substitution. The full-length of the An. funestus GABA-receptor gene, amplified by RT-PCR, generated a sequence of 1674 bp encoding 557 amino acid of the protein in An. funestus with 98% similarity to that of Anopheles gambiae. Two diagnostic assays were developed to genotype the A296S mutation (pyrosequencing and PCR-RFLP), and use of these assays revealed high frequency of the resistant allele in Burkina Faso (60%) and Cameroon (82%), moderate level in Benin (16%) while low frequency or absence of the mutation was observed respectively in Uganda (7.5%) or 0% in Malawi and Mozambique. The distribution of the RdlR mutation in An. funestus populations in Africa suggests extensive barriers to gene flow between populations from different regions.

Exploring Mechanisms of Multiple Insecticide Resistance in a Population of the Malaria Vector Anopheles funestus in Benin

BACKGROUND The insecticide resistance status of the malaria vector Anopheles funestus and the underlying resistance mechanisms remain uncharacterised in many parts of Africa, notably in Benin, West Africa. To fill this gap in our knowledge, we assessed the susceptibility status of a population of this species in Pahou, Southern Benin and investigated the potential resistance mechanisms. METHODOLOGY/PRINCIPAL FINDINGS WHO bioassays revealed a multiple resistance profile for An. funestus in Pahou. This population is highly resistant to DDT with no mortality in females after 1h exposure to 4%DDT. Resistance was observed against the Type I pyrethroid permethrin and the carbamate bendiocarb. A moderate resistance was detected against deltamethrin (type II pyrethroids). A total susceptibility was observed against malathion, an organophosphate. Pre-exposure to PBO did not change the mortality rates for DDT indicating that cytochrome P450s play no role in DDT resistance in Pahou. No L1014F kdr mutation was detected but a correlation between haplotypes of two fragments of the Voltage-Gated Sodium Channel gene and resistance was observed suggesting that mutations in other exons may confer the knockdown resistance in this species. Biochemical assays revealed elevated levels of GSTs and cytochrome mono-oxygenases in Pahou. No G119S mutation and no altered acetylcholinesterase gene were detected in the Pahou population. qPCR analysis of five detoxification genes revealed that the GSTe2 is associated to the DDT resistance in this population with a significantly higher expression in DDT resistant samples. A significant over-expression of CYP6P9a and CYP6P9b previously associated with pyrethroid resistance was also seen but at a lower fold change than in southern Africa. CONCLUSION The multiple insecticide resistance profile of this An. funestus population in Benin shows that more attention should be paid to this important malaria vector for the implementation and management of current and future malaria vector control programs in this country.

Dissecting the mechanisms responsible for the multiple insecticide resistance phenotype in Anopheles gambiae s.s., M form, from Vallée du Kou, Burkina Faso

With the exception of target site mutations, insecticide resistance mechanisms in the principle malaria vector Anopheles gambiae, remains largely uncharacterized in Burkina Faso. Here we detected high prevalence of resistance in Vallée du Kou (VK) to pyrethroids, DDT and dieldrin, moderate level for carbamates and full susceptibility to organophosphates. High frequencies of L1014F kdr (75%) and Rdl (87%) mutations were observed showing strong correlation with pyrethroids/DDT and dieldrin resistance. The frequency of ace1R mutation was low even in carbamate resistant mosquitoes. Microarray analysis identified genes significantly over-transcribed in VK. These include the cytochrome P450 genes, CYP6P3 and CYP6Z2, previously associated with pyrethroid resistance. Gene Ontology (GO) enrichment analysis suggested that elevated neurotransmitter activity is associated with resistance, with the over-transcription of target site resistance genes such as acetylcholinesterase and the GABA receptor. A rhodopsin receptor gene previously associated with pyrethroid resistance in Culex pipiens pallens was also over-transcribed in VK. This study highlights the complex network of mechanisms conferring multiple resistance in malaria vectors and such information should be taken into account when designing and implementing resistance control strategies.

Widespread Pyrethroid and DDT Resistance in the Major Malaria Vector Anopheles funestus in East Africa Is Driven by Metabolic Resistance Mechanisms

Background Establishing the extent, geographical distribution and mechanisms of insecticide resistance in malaria vectors is a prerequisite for resistance management. Here, we report a widespread distribution of insecticide resistance in the major malaria vector An. funestus across Uganda and western Kenya under the control of metabolic resistance mechanisms. Methodology/Principal Findings Female An. funestus collected throughout Uganda and western Kenya exhibited a Plasmodium infection rate between 4.2 to 10.4%. Widespread resistance against both type I (permethrin) and II (deltamethrin) pyrethroids and DDT was observed across Uganda and western Kenya. All populations remain highly susceptible to carbamate, organophosphate and dieldrin insecticides. Knockdown resistance plays no role in the pyrethroid and DDT resistance as no kdr mutation associated with resistance was detected despite the presence of a F1021C replacement. Additionally, no signature of selection was observed on the sodium channel gene. Synergist assays and qRT-PCR indicated that metabolic resistance plays a major role notably through elevated expression of cytochrome P450s. DDT resistance mechanisms differ from West Africa as the L119F-GSTe2 mutation only explains a small proportion of the genetic variance to DDT resistance. Conclusion The extensive distribution of pyrethroid and DDT resistance in East African An. funestus populations represents a challenge to the control of this vector. However, the observed carbamate and organophosphate susceptibility offers alternative solutions for resistance management.

A De Novo Expression Profiling of Anopheles funestus, Malaria Vector in Africa, Using 454 Pyrosequencing

Background Anopheles funestus is one of the major malaria vectors in Africa and yet there are few genomic tools available for this species compared to An. gambiae. To start to close this knowledge gap, we sequenced the An. funestus transcriptome using cDNA libraries developed from a pyrethroid resistant laboratory strain and a pyrethroid susceptible field strain from Mali. Results Using a pool of life stages (pupae, larvae, adults: females and males) for each strain, 454 sequencing generated 375,619 reads (average length of 182 bp). De novo assembly generated 18,103 contigs with average length of 253 bp. The average depth of coverage of these contigs was 8.3. In total 20.8% of all reads were novel when compared to reference databases. The sequencing of the field strain generated 204,758 reads compared to 170,861 from the insecticide resistant laboratory strain. The contigs most differentially represented in the resistant strain belong to the P450 gene family and cuticular genes which correlates with previous studies implicating both of these gene families in pyrethroid resistance. qPCR carried out on six contigs indicates that these ESTs could be suitable for gene expression studies such as microarray. 31,000 sites were estimated to contain Single Nucleotide Polymorphisms (SNPs) and analysis of SNPs from 20 contigs suggested that most of these SNPs are likely to be true SNPs. Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae. Conclusion This study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs) and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.