Polymorphic chromosomal inversions in Anopheles moucheti, a major malaria vector in Central Africa

Abstract

Anopheles moucheti Evans (Diptera, Culicidae) is a major vector of malaria in forested areas of Central Africa. However, few genetic tools are available for this species. The present study represents the first attempt to characterize chromosomes in An. moucheti females collected in Cameroon. Ovarian nurse cells contained polytene chromosomes, which were suitable for standard cytogenetic applications. The presence of three polymorphic chromosomal inversions in An. moucheti was revealed. Two of these inversions were located on 2R chromosomal arm. The homology between 2R chromosomal arms of An. moucheti and An. gambiae Giles was established by fluorescent in situ hybridization of six An. gambiae genic sequences. Mapping of the probes on chromosomes of An. moucheti detected substantial gene order reshuffling between the two species. The presence of polytene chromosomes and polymorphic inversions in An. moucheti provides a new tool for population genetic, taxonomic, and ecological studies of this neglected malaria vector.

Mosquitoes of the subgenus Cellia, Anopheles gambiae, An. arabiensis Patton, An. funestus Giles, An. nili Theobald, and An. moucheti, are the major malaria vectors in Africa. Anopheles gambiae, An. arabiensis, An. funestus, and An. nili have wide geographic distributions, spreading across most of West, Central and East Africa. In contrast, An. moucheti is restricted to the evergreen forest areas of Central Africa, where it breeds in slowly moving streams and rivers (Antonio-Nkondjio et al., 2009; Ayala et al., 2009). In this ecological setting, An. moucheti is the most abundant mosquito ensuring malaria transmission throughout the year. For instance, in the village of Simbock situated close to Yaounde, An. moucheti accounts for >54% of total anophelines caught, and is responsible for 39.2% of malaria transmission (Antonio-Nkondjio, Awono-Ambene, et al., 2002). Similarly, An. moucheti is the most abundant mosquito in the rural village of Olama within the equatorial forest zone of Cameroon (Antonio-Nkondjio et al., 2005). Moreover, a recent study conducted in Gabon has discovered that An. moucheti is not only responsible for the natural transmission of Plasmodium falciparum to humans, but it is also involved in the transmission of P. praefalciparum among great apes and therefore constitutes a main bridge vector candidate for transferring the malaria parasite from apes to humans (Paupy et al., 2013).

Differential ecological adaptations and behaviours of mosquitoes are often associated with dramatic changes in composition and frequency of polymorphic chromosomal inversions. For example, inversions in An. gambiae are non-randomly distributed temporally and spatially with respect to degree of aridity increasing the epidemiological significance of the vector (Coluzzi et al., 2002). Despite the importance of An. moucheti in malaria transmission, population cytogenetic studies have not been performed on this vector. Here, polytene chromosomes in An. moucheti are described and an evaluation of their suitability for the analysis of inversion polymorphism and physical genome mapping is made.

Females of An. moucheti were collected by pyrethrum spraying and bednet traps in three localities of Cameroon: Lepse (03°52′N; 11°25′E), Moloundou (02°08′N, 15°23′E), and Olama (03°24′N, 11°18′E), which are situated along different river systems. Specimens were identified in the field as members of the An. moucheti complex by using morphological identification keys (Gillies & Coetzee, 1987) and were further characterized by molecular assays as An. m. moucheti (hereafter An. moucheti) (Kengne et al., 2007). Immediately after collection, semi gravid females were dissected under a binocular and their ovaries were isolated and fixed in Carnoy's solution. Chromosomal preparations were made from Carnoy-fixed ovaries as described elsewhere (Sharakhova et al., 2011). The in situ hybridization procedure was conducted as previously described, using probes and primers from the An. gambiae genome assembly (Sharakhova et al., 2011).

The cytogenetic study was performed on chromosomal preparations obtained from 40 An. moucheti females collected in the three localities. Seventeen out of these females had ovarian nurse cells containing polytene chromosomes of sufficient quality for standard cytogenetic applications. The polytene chromosome complement of An. moucheti consisted of one short sex chromosome and four autosomal arms (Fig. 1A). The assignment of chromosomal arms was done based on their relative length and associations. Three polymorphic paracentric chromosomal inversions were found in An. moucheti. Two inversions located on chromosome 2 were named 2Ra and 2Rc (Fig. 1B, C). One inversion found on chromosome 3 was named 3Rb (Fig. 1A). Naming of the inversions was made according to the chronological order of their discovery. No inversions were found on chromosome X. Five of seven analyzed females from Lepse had one, two or three heterozygous inversions. Four of seven females from Olama had at least one heterozygous inversion. Three females from Moloundou were chromosomally monomorphic. To our knowledge, this is the first description of polymorphic chromosomal inversions in An. moucheti. Populations of other major malaria vectors, An. gambiae, An. arabiensis, An. funestus, and An. nili have significantly reduced inversion polymorphism in forested areas of Central Africa as compared with West Africa (Coluzzi et al., 2002; Cohuet et al., 2005; Sharakhova et al., 2011). The presence of inversions in An. moucheti urges further exploration of a role of chromosomal polymorphism in ecological adaptation and behavior of this mosquito.

Figure 1. Polytene chromosomes in ovarian nurse cells of An. moucheti from Cameroon.

A) The complete chromosome set from one nucleus. A heterozygote inversion b/+ is shown on chromosome 3. B) The heterozygote inversion 2Ra/+. C) The heterozygote inversion 2Rc/+. The arm names are shown near the telomeric regions. C—centromeric regions.

Polymorphic inversions in An. gambiae, An. funestus, and An. nili have been primarily found on 2R chromosomal arm, which is homologous in these species (Sharakhov et al., 2002; Sharakhova et al., 2011). Similarly, two of three polymorphic inversions were found on 2R in An. moucheti. To confirm the 2R arm homology between An. moucheti and An. gambiae, we successfully mapped six DNA probes designed based on An. gambiae genes AGAP001759, AGAP001763, AGAP001983, AGAP001984, AGAP002934, and AGAP002935 to the 2R arm of An. moucheti. The physical locations were compared with the positions of the homologous sequences in the An. gambiae genome. We observed substantial gene order reshuffling between the two species. For example, genes AGAP001759 and AGAP002935 were located in close proximity to each other in An. moucheti (Fig. 2A) but far away from each other in An. gambiae. Genes AGAP001759 and AGAP001763 were close neighbours in the An. gambiae genome but they mapped far apart on the An. moucheti chromosome (Fig. 2B). High rates of gene order reshuffling have been observed among other malaria vectors (Sharakhov et al., 2002; Sharakhova et al., 2011) indicating that paracentric inversions have been the major type of chromosomal rearrangements in the evolution of mosquitoes.

Figure 2. Mapping of An. gambiae genes to polytene chromosomes of An. moucheti.

Three DNA probes, AGAP002935 (A), AGAP001759 (A, B), and AGAP001763 (B), hybridized to euchromatic regions on the 2R chromosomal arm of An. moucheti. Arrows indicate signals of hybridization.

The present study calls for a more detailed characterization of chromosomes in the An. moucheti complex in order to facilitate population genetic, taxonomic, and genomic studies of this neglected malaria vector. The taxonomic relationships within the An. moucheti complex are unclear. Morphological and behavioural variations observed among natural populations suggest that several taxa may belong to the An. moucheti complex: An. moucheti, A. m. nigeriensis, and An. m. bervoetsi (Gillies & Coetzee, 1987). Contrary to these observations, a study of the diversity of isoenzyme markers and the inheritance of morphological characters in F1 progenies obtained from field-collected females has suggested that all three forms belong to a single species, at least in Cameroon (Antonio-Nkondjio, Simard, et al., 2002). Similar results have been obtained using ten microsatellite markers, revealing a low level of genetic differentiation among four An. moucheti populations from South Cameroon (Antonio-Nkondjio et al., 2007). However, when mosquitoes were collected from their type localities (in the Democratic Republic of the Congo (DRC) and Nigeria for An. m. bervoetsi and An. m. nigeriensis, respectively), sequence differences were revealed in the gene encoding for mitochondrial cytochrome oxidase b (CytB) and in the ribosomal internal transcribed spacers (ITS1 and ITS2) between the three morphological forms. These differences were similar in degree to the differences shown previously for members of other anopheline species groups or complexes (Kengne et al., 2007). Microsatellite analysis has further demonstrated significant genetic differentiation between An. m. bervoetsi populations from DRC and An. moucheti populations from Cameroon, suggesting that they may represent two different species (Antonio-Nkondjio et al., 2008). Cytogenetic studies have been useful for understanding population genetics and taxonomy of malaria mosquitoes (Coluzzi et al., 2002). A recent analysis of polytene chromosomes in another neglected malaria vector, An. ovengensis, has revealed high karyotypic divergence within the An. nili group (Sharakhova et al., 2013). Future studies will aim at understanding the role of inversion polymorphism in ecological adaptation, population differentiation, and speciation in the An. moucheti complex and will open the way for comprehensive comparative cytogenetic analyses across major African vector species.