The spread of the Leu-Phe kdr mutation through Anopheles gambiae complex in Burkina Faso: genetic introgression and de novo phenomena

During extensive sampling in Burkina Faso and other African countries, the Leu-Phe mutation producing the kdr pyrethroid resistance phenotype was reported in both Anopheles gambiae ss and A. arabiensis. This mutation was widely distributed at high frequency in the molecular S form of A. gambiae while it has been observed at a very low frequency in both the molecular M form and A. arabiensis in Burkina Faso. While the mutation in the M form is inherited through an introgression from the S form, its occurrence is a new and independent mutation event in A. arabiensis. Three nucleotides in the upstream intron of the kdr mutation differentiated A. arabiensis from A. gambiae ss and these specific nucleotides were associated with kdr mutation in A. arabiensis. Ecological divergences which facilitated the spread of the kdr mutation within the complex of A. gambiae ss in West Africa, are discussed.     

寡核苷酸探针法(PCR-ASO)检测家蝇kdr抗性相关的L1014F点突变

为检测家蝇对拟除虫菊酯击倒抗性kdr等位基因上的L1014F点突变,采用了寡核苷酸探针法(PCR-AlleleSpecificOligonucleotideprobe,PCR-ASO),用于检测该点突变。对L1014位的上下游DNA进行PCR扩增,并将纯化产物平行点样在带正电荷的尼龙膜上,用2条地高辛标记的特异性寡核苷酸探针,1条为敏感型,1条为突变型,与之杂交,根据显影的阳性信号判断待检样品的kdr等位基因型。该方法能区分敏感纯合子、抗性纯合子及杂合子,是检测家蝇kdr抗性的有用工具。     

Absence of the kdr mutation in the molecular 'M' form suggests different pyrethroid resistance mechanisms in the malaria vector mosquito Anopheles gambiae s.s

Field tests conducted on adult Anopheles mosquitoes using standard WHO procedures, diagnostic kits and test papers in south-western Nigeria showed pyrethroid (deltamethrin and permethrin) resistance in adult populations of Anopheles gambiae sensu stricto. The knock-down resistance (kdr) mutation involved in pyrethroid resistance was only found in the molecular S form of An. gambiae s.s. even in area where both molecular M and S forms occurred in sympatry. The absence of the kdr mutation in the M form suggests an additional pyrethroid resistance mechanism in An. gambiae s.s.     

Knockdown resistance mutations (kdr) and insecticide susceptibility to DDT and pyrethroids in Anopheles gambiae from Equatorial Guinea

Objectives To determine the frequency of knockdown resistance (kdr) mutations in the malaria vector Anopheles gambiae s.s. from continental Equatorial Guinea; and to relate kdr genotypes with susceptibility to DDT and pyrethroid insecticides in this vector. Methods Female mosquitoes were collected in two villages, Miyobo and Ngonamanga, of mainland Equatorial Guinea. Insecticide susceptibility tests were performed following WHO procedures. Anopheles gambiae complex specimens were identified to species and molecular form by PCR. Genotyping of the kdr locus was performed by allele‐specific PCR and direct sequencing in a subset of samples. Results Both M and S molecular forms of A. gambiae were found in Ngonamanga whereas only the S‐form was identified in Miyobo. The two kdr mutations were detected in S‐form samples of both villages, with a higher frequency of the kdr‐e (Leu‐1014‐Ser) allele (Miyobo: 16%; Ngonamanga: 40%). The kdr‐w (Leu‐1014‐Phe) mutation was also detected in 3% of the M‐form. All individuals tested for pyrethroids were susceptible. A mortality rate of 86% was obtained for DDT. An overall kdr allele frequency (i.e. kdr‐e + kdr‐w) of 22% was detected in DDT resistant individuals, whereas susceptible individuals had a kdr frequency of 6%. Conclusion The co‐occurrence of both kdr mutations and reduced susceptibility to DDT found in A. gambiae highlights the importance of implementing efficient surveillance of insecticide resistance in Equatorial Guinea.     

两种蚊虫钠通道基因kdr突变点附近序列的研究

目的 分析白纹伊蚊和致倦库蚊钠离子通道基因kdr突变位点附近的核苷酸序列，为进行抗药性监测奠定基础。方法 根据献报道的两种蚊虫钠离子通道基因序列设计引物，分别以白纹伊蚊和致倦库蚊基因组DNA为模板进行PCR扩增，获得包含击倒抗性突变位点和一个内含子的钠通道基因片段，将其核苷酸序列与蚊虫钠通道基因cDNA序列进行比较，确定内含子的位置及序列。结果 两种蚊虫的内含子序列变异较大，它们在kdr突变位点附近的序列很保守。结论 该内含子序列的变异并不影响用PCR方法检测这两种蚊虫的拟除虫菊酯抗性。     

The pyrethroid knock-down resistance gene in the Anopheles gambiae complex in Mali and further indication of incipient speciation within An. gambiae s.s

In Mali the Anopheles gambiae complex consists of An. arabiensis and Mopti, Savanna and Bamako chromosomal forms of An. gambiae s.s. Previous chromosomal data suggests a complete reproductive isolation among these forms. Sequence analysis of rDNA regions led to the characterization of two molecular forms of An. gambiae, named M-form and S-form, which in Mali correspond to Mopti and to Savanna/Bamako, respectively, while it has failed so far to show any molecular difference between Savanna and Bamako. The population structure of An. gambiae s.l. was analysed in three villages in the Bamako and Sikasso areas of Mali and the frequency of pyrethroid resistance of the knock-down resistance (kdr) type was calculated. The results show that the kdr allele is associated only with the Savanna form populations and absent in sympatric and synchronous populations of Bamako, Mopti and An. arabiensis. This is the first molecular indication of barriers to gene flow between the Bamako and Savanna chromosomal forms. Moreover, analyses of specimens collected in the Bamako area in 1987 show that the kdr allele was already present in the Savanna population at that time, and that the frequency of this allele has gradually increased since then.     

Genetic mapping of genes conferring permethrin resistance in the malaria vector, Anopheles gambiae

Resistance to permethrin in an East African population of the major malaria vector, Anopheles gambiae is multifactorial. A mutated sodium channel allele and enhanced insecticide metabolism contribute to the resistance phenotype. We used microsatellite markers to scan the genome for quantitative trait loci (QTL) associated with permethrin resistance. Two major and one minor QTL were identified. The first QTL, rtp1, colocalizes with the sodium channel gene on chromosome 2L thus further supporting the importance of mutations in this gene in conferring permethrin resistance. The second two loci are located on the third chromosome and one of these, rtp2, flanks a large cluster of cytochrome P450 genes. Further detailed mapping of these regions will help elucidate the molecular mechanisms of metabolic resistance to insecticides.