Background
The malaria vectorial system in tropical Africa is dominated by four species of major importance,
Anopheles gambiae,
Anopheles coluzzii,
Anopheles arabiensis and
Anopheles funestus, which are broadly codistributed across much of tropical Africa in close association with humans[
1,
2]. The first three species belong to the same cryptic species complex (the
An. gambiae complex) whose members cannot be distinguished morphologically at any developmental stage, although they differ in aquatic larval ecology and adult behaviours relevant to malaria transmission and control (e.g., degree of anthropophily and tendency to blood-feed or rest indoors)[
3,
4].
Anopheles funestus and its presently recognized closest relatives are classified into a group and subgroup[
5,
6] rather than a species complex, owing to slight morphological distinctions mainly at immature stages. However, further cryptic taxonomic complexities within the group have recently come to light and more can be anticipated as
An. funestus research emerges from a period of neglect[
7‐
11]. Malaria transmission by the Funestus Subgroup is overwhelmingly attributed to
An. funestus sensu stricto, owing to its strong preference for human blood meals (see reviews by[
7,
12]).
Anopheles funestus s.s. is characterized by abundant genetic polymorphism, exemplified by at least 17 chromosomal re-arrangements segregating within and among populations across Africa[
13,
14]. Although this species is generally considered to be uniformly anthropophilic and endophilic throughout its range, complex and incompletely understood patterns of population structure based on cytogenetic and DNA markers have been detected[
15‐
20]. In particular, two chromosomal forms designated “Folonzo” and “Kiribina” have been described in West Africa[
16]. First discovered in Burkina Faso and most intensively characterized in that country, Folonzo and Kiribina populations carry markedly different frequencies of shared polymorphic chromosomal inversions, mainly involving arm 3R[
16,
17]. In localities where the chromosomal forms are synchronous and stably sympatric across successive breeding seasons and years, there are highly significant departures from Hardy Weinberg equilibrium and significant genetic associations among physically unlinked inversion systems; alternative homokaryotypes are more frequent than expected under random mating, and there are significant deficits of heterozygotes in virtually all population samples, consistent with assortative mating by form[
16,
17].
Neither inversions nor inversion combinations are diagnostic taxonomic characters. However, the Kiribina form is predominantly homokaryotypic for the standard chromosomal arrangements, while Folonzo, the more chromosomally polymorphic taxon, carries high frequencies of inversions 3Ra and 3Rb, and presumably corresponds to
An. funestus from East Africa, where Kiribina has not been recorded[
16]. Strongly reminiscent of the chromosomal forms of
An. gambiae[
21], these alternative karyotypes show cyclical patterns of seasonal variation in relative abundance linked to temperature and rainfall, likely reflecting differences in geographic distribution modulated by larval habitat utilization[
22]. Although direct evidence is lacking, the Folonzo form is associated with natural larval habitats such as marshes, while Kiribina is associated with larval habitats created by the practice of agriculture, notably rice fields. Molecular genetic studies using mtDNA and microsatellite markers revealed very slight but significant divergence between sympatric samples of Folonzo and Kiribina across Burkina Faso, although nuclear divergence was not genome-wide and could be explained by loci on chromosome 3R inside and outside inversions[
23,
24]. These data are suggestive of an incipient process of ecological divergence and lineage splitting, similar to, but less advanced than, that responsible for the divergence of
An. coluzzii and
An. gambiae (formerly recognized as chromosomal or molecular forms[
25,
26]).
Previous studies in Burkina Faso and Senegal have reported similarly high rates of anthropophily and comparable
Plasmodium falciparum infection rates in sympatric Folonzo and Kiribina populations[
23,
24,
27]. However, there were indications of differences in indoor resting behaviour, leading to the suggestion that the Kiribina form may be more easily diverted to outdoor resting and biting, particularly in localities where alternative hosts such as cattle outnumber the local human population[
16]. If the ecological and genetic heterogeneities between Folonzo and Kiribina indeed extend to behavioural differences of importance to malaria epidemiology and control, these vectorial differences must be understood more deeply. Toward that end, resting and biting behaviour were assessed separately for sympatric and synchronous Folonzo and Kiribina populations in the rural villages of Kuiti and Koubri near Ouagadougou, Burkina Faso. Observations spanned six breeding seasons and 8,235 fully karyotyped Folonzo and Kiribina adult half-gravid females.
Results
Resting behaviour by sympatric populations of the two chromosomal forms of
An. funestus was assessed by parallel indoor/outdoor collections across six breeding seasons in two adjacent rural villages located in the dry savanna of central Burkina Faso. The results from the 8,235 fully karyotyped samples are presented in Table
1. Samples of the Kiribina form were generally larger than corresponding samples of the Folonzo form both indoors and out, particularly for the outdoor collections. However, during most breeding seasons and for the pooled samples, the Folonzo form was more likely than Kiribina to rest inside human dwellings rather than outside in pit-shelters. For 50% of the seasons, particularly when the numbers of outdoor-resting mosquitoes were sufficiently large, a Chi-square test of association for a 2x2 contingency table indicated that the stronger indoor resting tendency of Folonzo relative to Kiribina was statistically significant (Table
1).
Table 1
Resting behaviour of
Anopheles funestus
chromosomal forms in Burkina Faso
1999-2000 | ISC/indoor | 1154 | 377 | 777 | 1.94 | 1.29-2.93 | 0.001 |
| PIT/outdoor | 155 | 31 | 124 | | | |
2000-2001 | ISC/indoor | 1164 | 208 | 956 | 1.58 | 0.55-4.54 | NS |
| PIT/outdoor | 33 | 4 | 29 | | | |
2001-2002 | ISC/indoor | 2733 | 659 | 2074 | 0.95 | 0.49-1.84 | NS |
| PIT/outdoor | 48 | 12 | 36 | | | |
2002-2003 | ISC/indoor | 485 | 301 | 184 | 3.03 | 1.70-5.37 | <0.0001 |
| PIT/outdoor | 57 | 20 | 37 | | | |
2005-2006 | ISC/indoor | 99 | 63 | 36 | 2.33 | 0.49-11.02 | NS |
| PIT/outdoor | 7 | 3 | 4 | | | |
2006-2007 | ISC/indoor | 932 | 343 | 589 | 11.87 | 8.93-15.76 | <0.0001 |
| PIT/outdoor | 1368 | 64 | 1304 | | | |
Pooled | ISC/indoor | 6567 | 1951 | 4616 | 4.84 | 4.02-5.82 | <0.0001 |
| PIT/outdoor | 1668 | 134 | 1534 | | | |
A measure of post-prandial resting behaviour by outdoor-feeding
An. funestus was estimated by focusing on those mosquitoes with exclusively bovine-derived blood meals and comparing their numbers between indoor-resting and outdoor-resting samples of each chromosomal form. Based on this measure, the Folonzo form was significantly more likely than Kiribina to rest indoors following a bovine blood meal taken outdoors (Table
2).
Table 2
Post-prandial resting behaviour of outdoor-feeding
Anopheles funestus
chromosomal forms in Burkina Faso (2005–2007)
ISC/indoor | 38 | 9 | 29 | 5.64 | 2.43-13.10 | 0.0003 |
PIT/outdoor | 518 | 27 | 491 | | | |
HLC, conducted in parallel indoors and outdoors during the 2002–2003 breeding season, were used to compare human-biting behaviour between the chromosomal forms. Based on the >1,000 female mosquitoes captured, karyotyped and assigned to chromosomal form in 2002–3, human biting behaviour indoors
versus outdoors was indistinguishable between chromosomal forms (Table
3). For both, the proportion of mosquitoes seeking human blood meals indoors
versus outdoors was higher and of a similar magnitude. Importantly, the absolute numbers of the Folonzo form captured by HLC, both indoors and out, were larger than those from the corresponding Kiribina samples. These observations suggest that the Kiribina form may be more opportunistic, and the Folonzo form more anthropophilic, in host-seeking behaviour.
Table 3
Human biting behaviour of
Anopheles funestus
chromosomal forms in Burkina Faso (2002–2003)
October | HLC/indoor | 363 | 263 | 100 | 0.87 | 0.54-1.40 | NS |
| HLC/outdoor | 117 | 88 | 29 | | | |
November | HLC/indoor | 218 | 151 | 67 | 1.18 | 0.74-1.87 | NS |
| HLC/outdoor | 131 | 86 | 45 | | | |
December | HLC/indoor | 70 | 42 | 28 | 0.62 | 0.30-1.30 | NS |
| HLC/outdoor | 58 | 41 | 17 | | | |
January | HLC/indoor | 80 | 45 | 35 | 1.63 | 0.73-3.65 | NS |
| HLC/outdoor | 34 | 15 | 19 | | | |
Pooled | HLC/indoor | 731 | 501 | 230 | 1.04 | 0.79-1.37 | NS |
| HLC/outdoor | 340 | 230 | 110 | | | |
Host selection was assessed by blood meal identification during the 2005–2006 and 2006–2007 breeding seasons. The indoor resting samples of Folonzo and Kiribina both had a relatively high human blood index, 95.9 and 89.3%, respectively (Table
4). Folonzo was the form more likely to have fed on humans in whole or in part, rather than solely on cattle (
P <0.006), but this trend may reflect differences between forms in post-prandial resting behaviour rather than differences in the intrinsic preference for human hosts (i.e., host choice). While the size of indoor samples was balanced between Folonzo and Kiribina, there was a large skew in outdoor resting sample size between the forms, only 30 for Folonzo compared to 529 for Kiribina, reflecting the greater tendency for the latter form to rest outdoors. Both outdoor resting samples had drastically lower human blood indices, 10 and 7% for Folonzo and Kiribina, respectively. Folonzo remained the form more likely to have fed on humans than cattle (OR, 1.44), although this trend was not statistically significant.
Table 4
Host selection of
Anopheles funestus
chromosomal forms in Burkina Faso (2005–2007)
ISC/indoors | Human + Mixed | 191 + 21 (212) | 232 + 11 (243) | 2.81 | 1.30-6.07 | 0.006 |
| Bovine | 9 | 29 | | | |
| Total | 221 | 272 | | | |
| HBI | 95.9% | 89.3% | | | |
PIT/outdoors | Human + Mixed | 2 + 1 (3) | 13 + 25 (38) | 1.44 | 0.42-4.95 | NS* |
| Bovine | 27 | 491 | | | |
| Total | 30 | 529 | | | |
| HBI | 10.0% | 7.2% | | | |
During the same 2005–2007 seasons that host selection was evaluated, samples were analysed for
P. falciparum infection by testing for the presence of the CSP in indoor and outdoor resting samples of the two chromosomal forms. Infection rates in the indoor resting samples did not differ significantly between forms, being similarly high in both (8.5-8.8%; Table
5). Among the outdoor resting mosquitoes, the small Folonzo sample contained no sporozoite-positives, while the much larger Kiribina sample contained 32 (3%) sporozoite positives.
Table 5
Plasmodium falciparum
sporozoite rate of
Anopheles funestus
chromosomal forms in Burkina Faso (2005–2007)
ISC/indoor | + | 27 | 45 | 0.97 | 0.59-1.59 | NS |
| - | 291 | 469 | | | |
| Total | 318 | 514 | | | |
| %CSP+ | 8.5% | 8.8% | | | |
PIT/outdoor | + | 0 | 32 | 0.00 | 0.00-2.14 | NS* |
| - | 61 | 1035 | | | |
| Total | 61 | 1067 | | | |
| %CSP+ | 0.0% | 3.0% | | | |
Discussion
Intensive longitudinal sampling of
An. funestus from adjacent villages in the Sudan savanna of Burkina Faso, West Africa, affirms and extends the previous findings by Costantini
et al.[
16] of behavioural divergence between sympatric and synchronous chromosomal forms known as Folonzo and Kiribina. The high rate of anthropophagy by both forms (>89% of indoor samples), coupled with comparably high rates of
P. falciparum infection (>8% of indoor samples) emphasize the fact that Folonzo and Kiribina both are formidable malaria vectors in this part of Africa. The Kiribina form often outnumbered Folonzo. Yet, Folonzo was disproportionately represented in indoor
versus outdoor resting samples and was more inclined to post-prandial endophily, while Kiribina was over-represented outdoors in pit shelters. This suggests that the overall
An. funestus population is not uniformly exposed to indoor-based malaria interventions such as insecticide-treated nets and house spraying by residual insecticides, and that those indoor interventions are less effective against the Kiribina form.
There is precedence for chromosomal inversion-associated heterogeneity in mosquito resting behaviour in the West African savanna, uncovered by Coluzzi and colleagues through polytene chromosome analysis of
An. gambiae and
An. arabiensis populations during the Garki Project in Nigeria[
3,
32]. Such behavioural heterogeneity was responsible for the failure to interrupt malaria transmission during the course of the Project, despite rigorous insecticide applications and simultaneous administration of anti-malarial drugs to the human population[
33]. Indeed, there are hints that this same phenomenon has been witnessed previously with respect to
An. funestus in the West African savanna, where Kiribina co-exists with Folonzo. In the absence of Kiribina in eastern and southern Africa, historical house spraying campaigns not only locally eliminated
An. funestus, but the effect was maintained for several years following the cessation of spraying, due to the apparent inability of
An. funestus to recolonize some areas[
34]. Likewise,
An. funestus was eliminated from humid forest and degraded forest areas in West Africa where malaria is meso- or hypo-endemic[
34], an environment where Folonzo is predicted to dominate[
16,
35‐
37]. However, in the savannas of West Africa where malaria is holo- or hyperendemic, similar historical indoor spraying campaigns failed to eliminate the species[
34]. Exophilic populations persisted which, despite marked anthropophily, continued to feed outdoors on cattle as well as humans, and also entered sprayed houses to bite humans, but escaped unharmed to rest outdoors. These exophilic populations likely represented what would now be recognized as the Kiribina form of
An. funestus.
More recently, further epidemiologically significant behavioural heterogeneities in
An. funestus from the same biogeographical area have been recognized following large-scale implementation of indoor-based vector control interventions. After mass deployment of insecticide-treated bed nets, the biting cycle of
An. funestus shifted from its usual peak between 02:00 and 04:00 toward a later peak between dawn and early morning hours, when human hosts are less likely to be protected by nets[
38]. Unfortunately, it is not known whether this behavioural shift was associated with a change in the chromosomal composition of the local
An. funestus population.
The Folonzo and Kiribina chromosomal forms have been well characterized across several hundred kilometres and all ecozones of Burkina Faso[
16,
23]. However, their broader geographical distribution in Africa is poorly known. Certainly, they occur as far west as Senegal[
15,
27,
39]. A recent study of sympatric populations of these forms, the first of its kind in Senegal, found stable co-existence of the forms across three successive breeding seasons and concluded, in accord with the present study, that Kiribina predominated, and rates of anthropophagy and sporozoite infection were comparable between forms, although both metrics were considerably lower in Senegal (~30 and ~3%, respectively) than they were in Burkina Faso[
27]. Unfortunately, due to very low outdoor resting sample size (five total, of which only three could be identified chromosomally as Kiribina), indoor/outdoor resting behaviour was difficult to compare between forms, and thus, between studies. Cameroon is the most easterly country in which
An. funestus chromosomal forms have been reported[
36], but their vectorial heterogeneities (if any) are essentially uncharacterized. Available cytogenetic data suggest that these forms are largely allopatric in Cameroon, with Folonzo occurring in the mesic, forested south and Kiribina to the north in the dry savannas, except for a central contact zone at the forest-savanna transition, where stable sympatric co-existence of the two forms has not been clearly resolved[
35‐
37]. In another parallel with the
An. gambiae chromosomal forms, there is no evidence for the co-occurrence of
An. funestus chromosomal forms in East Africa[
40]; existing populations of
An. funestus in eastern Africa are hypothesized to be allied with the Folonzo form[
16], although that proposal has yet to be tested genetically.
Ample indication now exists of the practical importance of population structure and behavioural heterogeneities hidden within
An. funestus, for malaria epidemiology and control in West Africa, if not beyond. In this light, the dearth of information about the wider geographic distribution and associated bionomics and vectorial parameters of the Folonzo and Kiribina forms is a problem that must be remedied as a matter of priority. The polytene chromosomes of
An. funestus are considerably more difficult to spread and analyse than those of
An. gambiae, a factor that has impeded past research on
An. funestus. The demanding and specialized task of polytene chromosome-based identification, the restrictive sex and life stage from which favourable chromosomes are obtained, and the absence of any known DNA-based diagnostics to distinguish the chromosomal forms, all but prohibit deeper field investigation of Folonzo and Kiribina, particularly studies of their larval biology which is presumed to be a driver of their ecological and behavioural divergence. Genome sequencing of
An. gambiae in 2002[
41], and the discovery of molecular forms of
An. gambiae detectable by a simple PCR assay[
26], greatly transformed understanding of the complexities of
An. gambiae population structure and its impacts on malaria transmission. Recent whole genome sequencing and a newly available reference assembly for
An. funestus[
42] offer a platform that will support a more detailed understanding of
An. funestus population structure across Africa, as well as an efficient means to discover genomic sequences potentially useful for molecular taxonomy of Folonzo and Kiribina.
For decades, patterns of chromosomal inversion polymorphism have guided discovery of population structure and even species boundaries hidden inside otherwise morphologically indistinguishable groups of anopheline mosquitoes i.e.,[
16,
43‐
46]. Such an association of inversions with population substructure could be an incidental consequence of genetic drift owing to reduced gene flow, or the result of demographic history, but the observation that polymorphic inversions are often clinally distributed with respect to environmental gradients and subject to repeating seasonal fluctuations in frequency suggests that they are subject to strong selective forces[
47]. In anopheline mosquitoes, as in many animal and plant species, chromosomal inversions are implicated in local adaptation to environmental heterogeneities[
35,
48‐
51]. To the extent that speciation may occur as a by-product of adaptive divergence, chromosomal inversions may also be instrumental in lineage splitting, as proposed by Coluzzi for anopheline mosquitoes[
52]. That Kiribina and Folonzo forms are characterized by alternative arrangements of chromosomal inversions, and that these alternative arrangements shift in relative frequency according to geography, season, and larval habitat availability, suggests a direct role for chromosomal rearrangements in adaptation to heterogeneous and changing environments (see also[
35,
50]). Thus, beyond simply serving as markers for epidemiologically relevant population structure, alternative chromosomal arrangements some how condition different physiological and behavioural responses to the environment. A mechanistic understanding of what the adaptations are and how they evolved could prove instrumental in predicting how
An. funestus may be capable of responding to future environmental challenges, including anthropogenic changes to climate and landscape, and exposure to new means of vector control.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CC, NFS and NB conceived the study. WG performed the field collections. NFS provided logistical support throughout the study. WG performed the karyotyping. WG, CC and FL analysed results. WG, CC and NB wrote the manuscript. All authors read and approved the final manuscript.