Anopheles melas is a brackish water mosquito found along the coast of West-Africa where it can be the dominant malaria vector locally. In order to facilitate genetic studies of this species and to examine the usefulness of microsatellite markers when used in a sibling species, 45 microsatellite loci originally developed for Anopheles gambiae were sequenced in An. melas. These loci were evaluated on their suitability as polymorphic markers based on repeat structure, length, and polymorphism in wild An. melas populations. Of the 45 loci, 18 were not considered promising markers in An. melas. A total of 48 out of 90 An. gambiae primers contained at least one mismatch with the An. melas annealing site. An. melas-specific primers were designed for 27 loci, and their variability was examined in two wild populations from Equatorial Guinea. Based on a low level of polymorphism, Hardy-Weinberg disequilibrium, or poor amplification, a further 12 loci were excluded. The remaining fifteen loci were screened in four additional wild populations from a wider geographic region including Equatorial Guinea, Cameroon, The Gambia, and Guinea Bissau. These loci showed an average heterozygosity ranging from 0.18 to 0.79, with 2.5 to 15 average alleles per locus, yielding 13 highly polymorphic markers and two loci with more limited variability in a wide geographic region. To examine the effects of cross species amplification, five of the original An. gambiae markers were also amplified in the An. melas populations. Null alleles were found for one of these An. gambiae markers. We discuss the pitfalls of using microsatellite loci even in a very closely related species, and conclude that in addition to the well-known problem of null alleles associated with this practice, many loci may prove to be of very limited use as polymorphic markers even when used in a sibling species.
Fifteen An. melas-specific markers were subsequently amplified and analyzed in 11 wild An. melas populations from throughout the range of this species, including Bioko Island, Equatorial Guinea. We analyzed pair-wise population differentiation between all populations, and found that all but two comparisons were significant (p-val.<0.05), and populations clustered into three distinct groups representing Bioko Island, Central Africa, and West Africa populations. A Bayesian clustering analysis found little, if any, evidence for migration from mainland to Bioko Island populations, although there was evidence of migration from Bioko Island to the West population cluster, and from the Central to the West population cluster. Simulations of historical gene followed these same patterns and further support our predictions of unidirectional gene flow. Comparison of 1161 nucleotides amplified and sequenced from the ND4 and ND5 regions of the mtDNA showed that differentiation between An. melas population clusters is on par with levels of differentiation between member species of the An. gambiae complex, with low support for internal nodes in a maximum likelihood tree, which suggests that observed An. melas clusters are not monophyletic. From this we hypothesize that Bioko Island An. melas populations are derived from Tiko, Cameroon, and that these populations became isolated from one another when sea levels rose after the last glaciation period (?10,000-11,000 years ago), cutting off Bioko Island populations from the mainland and significantly reducing migration. Our conclusions have implications for vector control within the region, as Bioko Island is the subject of an intensive malaria control campaign, and the lack of migration from mainland West Africa to Bioko Island make it unlikely that eradicated populations of this malaria vector will be repopulated by mainland immigrants.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/ETD-TAMU-2011-12-10654 |
| Date | 2011 December 1900 |
| Creators | Deitz, Kevin |
| Contributors | Slotman, Michel A., Johnston, J. Spencer, Krutovsky, Konstantin V., Medina, Raul F. |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis, thesis, text |
| Format | application/pdf |
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