Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Irving H

PROVIDER: S-EPMC3499844 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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

Irving H H Riveron J M JM Ibrahim S S SS Lobo N F NF Wondji C S CS

Heredity 20120905 6

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 P45 ...[more]

PMID: 22948188

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Project description:Malaria vector control interventions rely heavily on the application of insecticides against anopheline mosquitoes, in particular the fast-acting pyrethroids that target insect voltage-gated sodium channels (VGSC). Frequent applications of pyrethroids have resulted in resistance development in the major malaria vectors including Anopheles funestus, where resistance is primarily metabolic and driven by the overexpression of microsomal cytochrome P450 monooxygenases (P450s). Here we examined the pattern of cross-resistance of the pyrethroid-resistant An. funestus strain FUMOZ-R towards transfluthrin and multi-halogenated benzyl derivatives, permethrin, cypermethrin and deltamethrin in comparison to the susceptible reference strain FANG. Transfluthrin and two multi-fluorinated derivatives exhibited micromolar potency - comparable to permethrin - to functionally expressed dipteran VGSC in a cell-based cation influx assay. The activity of transfluthrin and its derivatives on VGSC was strongly correlated with their contact efficacy against strain FUMOZ-R, although no such correlation was obtained for the other pyrethroids due to their rapid detoxification by the resistant strain. The low resistance levels for transfluthrin and derivatives in strain FUMOZ-R were only weakly synergized by known P450 inhibitors such as piperonyl butoxide (PBO), triflumizole and 1-aminobenzotriazole (1-ABT). In contrast, deltamethrin toxicity in FUMOZ-R was synergized > 100-fold by all three P450 inhibitors. The biochemical profiling of a range of fluorescent resorufin and coumarin compounds against FANG and FUMOZ-R microsomes identified 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC) as a highly sensitive probe substrate for P450 activity. BOMFC was used to develop a fluorescence-based high-throughput screening assay to measure the P450 inhibitory action of potential synergists. Azole fungicides prochloraz and triflumizole were identified as extremely potent nanomolar inhibitors of microsomal P450s, strongly synergizing deltamethrin toxicity in An. funestus. Overall, the present study contributed to the understanding of transfluthrin efficacy at the molecular and organismal level and identified azole compounds with potential to synergize pyrethroid efficacy in malaria vectors.

Project description:BackgroundThe 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 findingsA 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.ConclusionThis 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.

Dataset Information

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

Publications

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

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