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Anopheles funestus Giles is one of the most important vectors of malaria in sub-Saharan Africa. The population structure of this mosquito in Burkina Faso, West Africa based on chromosomal inversion data led to the description of two chromosomal forms, Kiribina and Folonzo. Because both forms co-occur in the same locales yet differ significantly, both in the frequency of inverted arrangements on chromosome arms 3R and 2R and in vectorial capacity, they were hypothesized to be emerging species with at least partial barriers to gene flow. This hypothesis would be strengthened by molecular evidence of differentiation between Kiribina and Folonzo at loci outside chromosomal inversions. We surveyed molecular variation in sympatric populations of the two forms using sequences from the mitochondrial ND5 gene and genotypes at sixteen microsatellite loci distributed across the genome. Both classes of marker revealed slight but significant differentiation between the two forms (mtDNA F(ST) = 0.023, P < 0.001; microsatellite F(ST) = 0.004, P < 0.001; R(st) = 0.009, P = 0.002). Locus-by-locus analysis of the microsatellite data showed that significant differentiation was not genome-wide, but could be attributed to five loci on chromosome 3R (F(ST) = 0.010, P < 0.001; R(st) = 0.016, P = 0.002). Importantly, three of these loci are outside of, and in linkage equilibrium with, chromosomal inversions, suggesting that differentiation between chromosomal forms extends beyond the inversions themselves. The slight overall degree of differentiation indicated by both marker classes is likely an underestimate because of recent population expansion inferred for both Folonzo and Kiribina. The molecular evidence from this study is consistent with the hypothesis of incipient speciation between Kiribina and Folonzo.

Background Previous studies of Anopheles funestus chromosomal inversion polymorphisms in Burkina Faso showed large departures from Hardy-Weinberg equilibrium and linkage disequilibrium among inversions located on different chromosomes, implying the existence of two taxonomic units ("chromosomal forms") with limited genetic flow. One chromosomal form, named Folonzo, is highly polymorphic for alternative rearrangements of 3Ra, 3Rb, 2Ra, and 3La; the other, Kiribina, is predominantly characterized by the standard arrangement of these inversions. To investigate the temporal distribution of these chromosomal forms, further collections were carried out in two villages near Ouagadougou where they are found in sympatry. Methods Chromosomal karyotypes were determined from indoor-resting, half-gravid females sampled within and across six breeding seasons, from December 1998 to April 2007. Results As expected, the pattern of chromosomal polymorphism in An. funestus was consistent with assortatively mating Folonzo and Kiribina forms. When samples were assigned to each chromosomal form, their relative abundance varied within successive breeding seasons in a repeating pattern of temporal variability. Relative abundance of the Folonzo form was correlated with climatic variables related to temperature and rainfall. Conclusion The relative abundance of Folonzo and Kiribina forms of An. funestus likely reflects different larval ecologies that are linked to varying climatic conditions. Further analysis of the bionomics of these vectors is recommended in light of its relevance to vector control.

Background As Anopheles funestus is one of the principal Afro-tropical malaria vectors, a more complete understanding of its population structure is desirable. In West and Central Africa, An. funestus population structure is complicated by the coexistence of two assortatively mating chromosomal forms. Effective population size (N e ) is a key parameter in understanding patterns and levels of intraspecific variation, as it reflects the role of genetic drift. Here, N e was estimated from both chromosomal forms, Kiribina and Folonzo, in Burkina Faso. Methods Short-term N e was estimated by evaluating variation at 16 microsatellite loci across temporal samples collected annually from 2000–2002. Estimates were based on standardized variance in allele frequencies or a maximum likelihood method. Long-term N e was estimated from genetic diversity estimates using mtDNA sequences and microsatellites. Results For both forms, short-term and long-term N e estimates were on the order of 103 and 105, respectively. Long-term N e estimates were larger when based on loci from chromosome 3R (both inside and outside of inversions) than loci outside of this arm. Conclusion N e values indicate that An. funestus is not subject to seasonal bottlenecks. Though not statistically different because of large and overlapping confidence intervals, short-term N e estimates were consistently smaller for Kiribina than Folonzo, possibly due to exploitation of different breeding sites: permanent for Folonzo and intermittent for Kiribina. The higher long-term N e estimates on 3R, the arm carrying the two inversions mainly responsible for defining the chromosomal forms, give natural selection broader scope and merit further study.

Anopheles funestus is a major vector of malaria across Africa. Understanding its complex and nonequilibrium population genetic structure is an important challenge that must be overcome before vector populations can be successfully perturbed for malaria control. Here we examine the role of chromosomal inversions in structuring genetic variation and facilitating divergence in Burkina Faso, West Africa, where two incipient species (chromosomal forms) of A. funestus, defined principally by rearrangements of chromosome 3R, have been hypothesized. Sampling across an ∼300-km east–west transect largely contained within the Sudan–Savanna ecoclimatic zone, we analyzed chromosomal inversions, 16 microsatellite loci distributed genomewide, and 834 bp of the mtDNA ND5 gene. Both molecular markers revealed high genetic diversity, nearly all of which was accounted for by within-population differences among individuals, owing to recent population expansion. Across the study area there was no correlation between genetic and geographic distance. Significant genetic differentiation found between chromosomal forms on the basis of microsatellites was not genomewide but could be explained by chromosome 3R alone on the basis of loci inside and near inversions. These data are not compatible with complete reproductive isolation but are consistent with differential introgression and sympatric divergence between the chromosomal forms, facilitated by chromosome 3R inversions.

The analysis of chromosomal polymorphism of paracentric inversions in anopheline mosquitoes has often been instrumental to the discovery of sibling species complexes and intraspecific genetic heterogeneities associated with incipient speciation processes. To investigate the population structure of Anopheles funestus Giles (Diptera: Culicidae), one of the three most important vectors of human malaria in sub-Saharan Africa, a three-year survey of chromosomal polymorphism was carried out on 4,638 karyotyped females collected indoors and outdoors from two villages of central Burkina Faso. Large and temporally stable departures from Hardy-Weinberg equilibrium due to significant deficits of heterokaryotypes were found irrespective of the place of capture, and of the spatial and temporal units chosen for the analysis. Significant linkage disequilibrium was observed among inversion systems on independently assorting chromosomal arms, indicating the existence of assortative mating phenomena. Results were consistent with the existence of two chromosomal forms characterized by contrasting degrees of inversion polymorphism maintained by limitations to gene flow. This hypothesis was supported by the reestablishment of Hardy-Weinberg and linkage equilibria when individual specimens were assigned to each chromosomal form according to two different algorithms. This pattern of chromosomal variability is suggestive of an incipient speciation process in An. funestus populations from Burkina Faso.

In tropical Africa, Anopheles funestus is one of the three most important malaria vectors. We physically mapped 157 A. funestus complementary DNAs (cDNAs) to the polytene chromosomes of this species. Sequences of the cDNAs were mapped in silico to the A. gambiae genome as part of a comparative genomic study of synteny, gene order, and sequence conservation between A. funestus and A. gambiae . These species are in the same subgenus and diverged about as recently as humans and chimpanzees. Despite nearly perfect preservation of synteny, we found substantial shuffling of gene order along corresponding chromosome arms. Since the divergence of these species, at least 70 chromosomal inversions have been fixed, the highest rate of rearrangement of any eukaryote studied to date. The high incidence of paracentric inversions and limited colinearity suggests that locating genes in one anopheline species based on gene order in another may be limited to closely related taxa.

The African malaria vector Anopheles gambiae is polymorphic for alternative arrangements on the left arm of chromosome 2 (2La and 2L+ a ) that are non-randomly distributed with respect to degree of aridity. Detailed studies on the ecological role of inversion 2La have been hindered by the technical demands of traditional karyotype analysis and by sex- and stage-specific limitations on the availability of polytene chromosomes favorable for analysis. Recent molecular characterization of both inversion breakpoints presented the opportunity to develop a polymerase chain reaction (PCR)-based method for karyotype analysis. Here we report the development of this molecular diagnostic assay and the results of extensive field validation. When tested on 765 An. gambiae specimens sampled across Africa, the molecular approach compared favorably with traditional cytologic methods, correctly scoring > 94% of these specimens. By providing ready access to the 2La karyotype, this tool lays groundwork for future studies of the ecological genomics of this medically important species.