Incipient speciation in the malaria mosquito Anopheles gambiae s.s.: Sequence analysis of intron I of the voltage-gated sodium channel gene

Incipient speciation processes within Anopheles gambiae s.s. have been revealed by polytene chromosome analysis (Coluzzi et al., 1985, Boll Zool 52: 45-63) and, more recently, by sequence analysis of different rDNA regions (della Torre et al., 2001 Insect Mol Biol, 10: 9-18; Gentile et al., 2001, Insect Moi Biol, 10: 25-32). The latter has shown the existence in West Africa of two non-panmictic molecular forms, Sand M, which in the northern part of their range of distribution coincide with chromosomal forms Savanna/Bamako and Mopti, respectively. Recently, Chandre et al. (1999, Parassitologia 41: 319-322) found that in the voltage-gated sodium channel gene, the point-mutation associated with kdr (knock-down resistance), which confers resistance to pyrethroids and DDT, shows a non-homogeneous distribution in the two molecular forms. Further, preliminary sequence analysis of the intron I upstream the kdr mutation showed two nucleotide differences respectively associated with Sand M forms (Weill et al., 2000, Insect Mol Biol, 9: 451-455). We sequenced 535 bp of intron I in more than 100 specimens from 10 West African countries from Senegal to Angola. Specimens had been previously karyotyped, assigned to a chromosomal form, identified as molecular form S or M and characterized for the presence of the kdr allele. At position 702 a T and a C were consistently associated with Sand M form, respectively. At position 896 a C was fixed in all S samples analysed, while M samples showed either a fixed C Iike S samples or an A/C polymorphism, depending on the geographical origin of the samples. In S samples in which the kdr allele is present, no difference among kds/kds, kds/kdr and kdr/kdr genotypes was recorded at both sites. Kds/kdr specimens were found in a single M sample from Benin and resulted T/C heterozygotes at sites 702: this supports the hypothesis that the kdr and the intron alleles have been transferred from S to M through introgression Weill et al. (op. cit.). Microsatellite studies carried out on Sand M forms have revealed virtually no differentiation between chromosome arms except for the 2R (Lanzara et al. 1998, Proc. NatI. Acad. Sci., USA 95:14260-14265; Taylor et al. 2001, Genetics 157:743-750) and a single microsatellite closely Iinked to the rDNA region (Wang et al. 2001, Prac. Nat. Acad. Sci. USA 98: 1 0769-1 0774). The mutation at position 702 represents the first marker in an intron DNA region that consistently correlates over a wide geographic range with the rDNA markers used to define An. gambiae molecular forms. Interestingly, the voltage-gated sodium channel gene maps on Div. 20C, on the left arm of the chromosome 2 (Ranson et al., 2000, Insect Mol Biol, 9: 491-497), a chromosomal region associated neither with rDNA (X chromosome), nor with inversions (2R) used to define the chromosomal forms.