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

Abstract

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 Rdl(R) mutation in An. funestus populations in Africa suggests extensive barriers to gene flow between populations from different regions.

Graphical abstract

Fig. 1

Sequence chromatograms of the fragment of exon 7 of the GABA-receptor gene for a heterozygous and homozygous dieldrin resistant An. funestus. The polymorphic site is indicated by an arrow.

Fig. 2

Alignment of amino acid sequences of the GABA-receptor gene between An. funestus (Af), An. gambiae (Ag), Ae. aegypti (Aa), Cx. quinquefasciatus (Cq) and D. melanogaster (Dm). The A296S Rdl mutation is indicated by an arrow while the V327I mutation is indicated and inverted triangle.

Fig. 3

Expected histograms and observed pyrograms of the Rdl mutation pyrosequencing assay.

Fig. 4

Agarose gel of a PCR-RFLP to detect dieldrin resistance in An. funestus. The top band is a 255 bp fragment for resistant mosquitoes while the bottom bands represent the 117 and 138 bp fragments resulting from the restriction digestion by HpyCH4V.

Fig. 5

Geographical distribution of the Rdl^R allele in six countries in Africa.

Fig. 6

Observed pyrograms of the V327I mutation for the pyrosequencing assay.

Fig. 7

Association of the V327I genotypes and those of the Rdl mutation A296S.