Anopheles gambiae, An. arabiensis, An. funestus, and An. nili are the major malaria vectors in sub-Saharan Africa because they are anthropophilic and susceptible to Plasmodium falciparum[1–3]. These species belong to species complexes or groups, and members within these complexes/groups vary significantly in their vectorial capacity. Moreover, species can be further sub-divided into populations adapted to different environments. Some malaria control initiatives have failed because they targeted the wrong species or population [4, 5]. Understanding and targeting the heterogeneity and complexity of all major vector species and populations is necessary for effective vector control and malaria eradication .
Most studies of African malaria vectors have involved only An. gambiae, An. arabiensis, and An. funestus, while research on other important malaria vectors has critically lagged behind. For An. nili, this is partly because molecular and cytogenetic tools for characterizing population structure, ecological adaptation, and taxonomic status have been lacking. Anopheles nili is widely distributed and contributes substantially to malaria transmission in the African savannah and forested areas, where it breeds in lotic streams and rivers [7, 8]. Sporozoite rates in this species can reach 3%, and the annual entomological inoculation rates can be over 100 . For example, An. nili is highly anthropophagous and responsible for 10.2% of malaria transmission in the densely populated area surrounding Yaounde, the capital of Cameroon . Gaps in our knowledge of this vector represent a critical barrier to progress in the field of vector biology. Recent findings of circulation of P. falciparum and other Plasmodium species in great apes and other primates [11–13] raise concerns about pathogen transfer between humans and primates, and highlight the need to improve our knowledge of malaria vectors that inhabit forested areas in Central Africa.
Multi-allelic microsatellites are informative markers for inferring the population and taxonomic status of disease vectors and parasites [1, 14–26]. Microsatellites are hyper-variable markers that tend to evolve neutrally. Eleven polymorphic microsatellite markers have been developed for An. nili. Recently, the level of genetic variability and differentiation has been explored among nine populations of An. nili from Senegal, Ivory Coast, Burkina Faso, Nigeria, Cameroon, and The Democratic Republic of Congo (DRC) . Genetic variability was determined by assessing polymorphisms at these 11 microsatellite markers, together with sequence variations in four genes within the ITS2, 28S rDNA subunit D3, and mitochondrial DNA. High F
estimates based on microsatellites (F
> 0.118, P < 0.001) were observed in all comparisons between Kenge in the DRC, and all other populations sampled from Senegal to Cameroon. Sequence variation in mtDNA genes matched these results; however, low polymorphism in rDNA genes prevented detection of any population substructure at this geographical scale. Both local adaptation and geographic isolation could cause this differentiation. Geographic isolation should affect all markers, even if they are unlinked (i.e. located in different chromosomes). However, chromosomal locations of the microsatellite markers and, therefore, the degree of their physical independence in the genome were unknown. Furthermore, because reduced recombination and increased selection within or near polymorphic inversions can result in estimates of gene flow that may differ significantly from those based on loci elsewhere in the genome [28, 29], it would also be important to know the location of microsatellite markers with respect to polymorphic inversions in An. nili when performing population genetic analyses.
Polymorphic chromosomal inversions are usually under selection and, thus, are useful markers for studying ecological adaptations of malaria mosquitoes [30–32]. The polymorphic inversions of chromosome 2 of An. gambiae have been associated with the arid Sahel Savanna [33–37] and with tolerance to desiccation and heat [38, 39]. Moreover, frequencies of these inversions are higher indoors where the nocturnal saturation deficit is higher than outdoors . Such ecological heterogeneity has important consequences for vector control. For example, indoor residual spraying of insecticides affected only indoor populations of An. gambiae in the Garki malaria control project in Nigeria . Our previous cytogenetic analysis demonstrated that two polymorphic inversions, 2Rb and 2Rc, are present simultaneously in an An. nili mosquito. However, they display very different patterns of polymorphism. Frequencies of inverted and standard 2Rb variants were almost equal (with a deficiency of heterozygotes) in Burkina Faso, whereas only the standard arrangement was found in Cameroon. In contrast, inversion 2Rc occurred at higher frequency (without a deficiency of heterozygotes) in the dry savannah of Burkina Faso (83%) and at lower frequency in the humid rainforest of Cameroon (0.6%) . Moreover, inversion 2Rc was found in the mountainous area (Magba), but not in the forested area (Mbebe) of Cameroon. These observations suggest the involvement of inversions in local adaptation (2Rb) or in an ecogeographic adaptive cline from dry to more humid environments (2Rc). Because An. nili is a forest-savannah transition species, polymorphic inversions could provide genetic plasticity that allows this species to expand its range from dry savannah to deforested areas of Central Africa, where most of the human population is present. The relationship between these two inversions has not been studied. For example, it would be useful to know if inversions 2Rb and 2Rc are in linkage disequilibrium (LD) in natural populations of An. nili.
In this study, we mapped nine microsatellite markers to polytene chromosomes of An. nili using fluorescent in situ hybridization (FISH). Plasmid clones of the An. nili microsatellites and/or ad hoc DNA fragments amplified from a low coverage assembly of the An. nili genome were used as probes. The microsatellites hybridized to unique locations on all chromosomes both inside and outside polymorphic inversions. We further demonstrated highly significant linkage disequilibrium between inversions 2Rb and 2Rc. This knowledge about polymorphic inversions and chromosomal locations of microsatellite loci helped us to better understand genetic variations and differentiation in natural populations of An. nili.