Predation of aquatic immature stages has been identified as a major evolutionary force driving habitat segregation and niche partitioning in the malaria mosquito Anopheles gambiae sensu stricto in the humid savannahs of Burkina Faso, West Africa. Here, we explored behavioural responses to the presence of a predator in wild populations of the M and S molecular forms of An. gambiae that typically breed in permanent (e.g., rice field paddies) and temporary (e.g., road ruts) water collections.
Larvae used in these experiments were obtained from eggs laid by wild female An. gambiae collected from two localities in south-western Burkina Faso during the 2008 rainy season. Single larvae were observed in an experimental arena, and behavioural traits were recorded and quantified a) in the absence of a predator and b) in the presence of a widespread mosquito predator, the backswimmer Anisops jaczewskii. Differences in the proportion of time allocated to each behaviour were assessed using Principal Component Analysis and Multivariate Analysis of Variance.
The behaviour of M and S form larvae was found to differ significantly; although both forms mainly foraged at the water surface, spending 60-90% of their time filtering water at the surface or along the wall of the container, M form larvae spent on average significantly more time browsing at the bottom of the container than S form larvae (4.5 vs. 1.3% of their overall time, respectively; P < 0.05). In the presence of a predator, larvae of both forms modified their behaviour, spending significantly more time resting along the container wall (P < 0.001). This change in behaviour was at least twice as great in the M form (from 38.6 to 66.6% of the time at the wall in the absence and presence of the predator, respectively) than in the S form (from 48.3 to 64.1%). Thrashing at the water surface exposed larvae to a significantly greater risk of predation by the notonectid (P < 0.01), whereas predation occurred significantly less often when larvae were at the container wall (P < 0.05) and might reflect predator vigilance.
Behavioural differences between larvae of the M and S form of An. gambiae in response to an acute predation risk is likely to be a reflection of different trade-offs between foraging and predator vigilance that might be of adaptive value in contrasting aquatic ecosystems. Future studies should explore the relevance of these findings under the wide range of natural settings where both forms co-exist in Africa.
In West Africa, lineage splitting between the M and S molecular forms of the major Afro-tropical malaria mosquito, Anopheles gambiae is thought to be driven by ecological divergence, occurring mainly at the larval stage. Here, we present evidences for habitat segregation between the two molecular forms in and around irrigated rice-fields located within the humid savannahs background of western Burkina Faso. Longitudinal sampling of adult mosquitoes emerging from a range of breeding sites distributed along a transect extending from the heart of the rice-fields area into the surrounding savannahs was conducted from June to November 2009. Analysis revealed that the two molecular forms and their sibling species An. arabiensis are not randomly distributed in the area. A major ecological gradient was extracted, in relation to the rice-fields perimeter. The M form was associated with larger breeding sites, which were mainly represented by rice field paddies whereas the S form and An. arabiensis were found to depend upon temporary, rain-filled breeding sites. These results support hypotheses about larval habitat segregation and confirm that both forms have different larval habitat requirement. Segregation appears clearly linked to anthropogenic permanent habitats and the community structure they support.
Anopheles gambiae; segregation; larval habitat; hydro-periodicity; community structure; niche partitioning
Ongoing lineage splitting within the African malaria mosquito Anopheles gambiae is compatible with ecological speciation, the evolution of reproductive isolation by divergent natural selection acting on two populations exploiting alternative resources. Divergence between two molecular forms (M and S) identified by fixed differences in rDNA, and characterized by marked, although incomplete, reproductive isolation is occurring in West and Central Africa. To elucidate the role that ecology and geography play in speciation, we carried out a countrywide analysis of An. gambiae M and S habitat requirements, and that of their chromosomal variants, across Burkina Faso.
Maps of relative abundance by geostatistical interpolators produced a distinct pattern of distribution: the M-form dominated in the northernmost arid zones, the S-form in the more humid southern regions. Maps of habitat suitability, quantified by Ecological Niche Factor Analysis based on 15 eco-geographical variables revealed less contrast among forms. M was peculiar as it occurred proportionally more in habitat of marginal quality. Measures of ecological niche breadth and overlap confirmed the mismatch between the fundamental and realized patterns of habitat occupation: forms segregated more than expected from the extent of divergence of their environmental envelope – a signature of niche expansion. Classification of chromosomal arm 2R karyotypes by multilocus genetic clustering identified two clusters loosely corresponding to molecular forms, with 'mismatches' representing admixed individuals due to shared ancestral polymorphism and/or residual hybridization. In multivariate ordination space, these karyotypes plotted in habitat of more marginal quality compared to non-admixed, 'typical', karyotypes. The distribution of 'typical' karyotypes along the main eco-climatic gradient followed a consistent pattern within and between forms, indicating an adaptive role of inversions at this geographical scale.
Ecological segregation between M and S is consistent with niche expansion into marginal habitats by chromosomal inversion variants during early lineage divergence; presumably, this process is promoted by inter-karyotype competition in the higher-quality core habitat. We propose that the appearance of favourable allelic combinations in other regions of suppressed recombination (e.g. pericentromeric portions defining speciation islands in An. gambiae) fosters development of reproductive isolation to protect linkage between separate chromosomal regions.
The molecular forms of Anopheles gambiae are undergoing speciation. They are characterized by a strong assortative mating and they display partial habitat segregation. The M form is mostly found in flooded/irrigated areas whereas the S form dominates in the surrounding areas, but the ecological factors that shape this habitat segregation are not known. Resource competition has been demonstrated between species undergoing divergent selection, but resource competition is not the only factor that can lead to divergence.
In a field experiment using transplantation of first instar larvae, we evaluated the role of larval predators in mediating habitat segregation between the forms. We found a significant difference in the ability of the molecular forms to exploit the different larval sites conditioned on the presence of predators. In absence of predation, the molecular forms outcompeted each other in their respective natural habitats however, the developmental success of the M form was significantly higher than that of the S form in both habitats under predator pressure.
Our results provide the first empirical evidence for specific adaptive differences between the molecular forms and stress the role of larval predation as one of the mechanisms contributing to their divergence.
The African malaria mosquito Anopheles gambiae is undergoing speciation, being split into the M and S molecular forms. Speciation is the main process promoting biological diversity, thus, new vector species might complicate disease transmission. Genetic differentiation between the molecular forms has been extensively studied, but phenotypic differences between them, the evolutionary forces that generated divergence, and the mechanisms that maintain their genetic isolation have only recently been addressed. Here, we review recent studies suggesting that selection mediated by larval predation and competition promoted divergence between temporary and permanent freshwater habitats. These differences explain the sharp discontinuity in distribution of the molecular forms between rice fields and surrounding savanna, but they can also explain the concurrent cline between humid and arid environments due to the dependence on permanent habitats in the latter. Although less pronounced, differences in adult body size, reproductive output, and longevity also suggest that the molecular forms have adapted to distinct niches. Reproductive isolation between the molecular forms is achieved by spatial swarm segregation, although within-swarm mate recognition appears to play a role in certain locations. The implications of these results to disease transmission and control are discussed and many of the gaps in our understanding are highlighted.
The interactions between predator diversity and primary consumer abundance can include direct effects and indirect, cascading effects. Understanding these effects on immature Anopheles mosquitoes is important in sub-Saharan Africa, where most cases of malaria occur. Aquatic predators and immature mosquitoes were collected from shallow pools of varying age previously excavated by brickmakers in the western highlands of Kenya. Path analysis showed an indirect negative effect of habitat age on An. gambiae (Giles, 1902) mediated by effects on predator diversity. Disturbance resets habitats to an earlier successional stage, diminishing predator diversity and increasing An. gambiae populations. The increase in vector abundance as a result of reduced predator diversity highlights the public health value in conserving native insect diversity.
trophic cascades; diversity; Anopheles gambiae; malaria; Kenya
The current status of insecticide resistance in mosquitoes and the effects of insecticides on non-target insect species have raised the need for alternative control methods for malaria vectors. Predation has been suggested as one of the important regulation mechanisms for malaria vectors in long-lasting aquatic habitats, but the predation efficiency of the potential predators is largely unknown in the highlands of western Kenya. In the current study, we examined the predation efficiency of five predators on Anopheles gambiae s.s larvae in 24 hour and semi- field evaluations.
Predators were collected from natural habitats and starved for 12 hours prior to starting experiments. Preliminary experiments were conducted to ascertain the larval stage most predated by each predator species. When each larval instar was subjected to predation, third instar larvae were predated at the highest rate. Third instar larvae of An. gambiae were introduced into artificial habitats with and without refugia at various larval densities. The numbers of surviving larvae were counted after 24 hours in 24. In semi-field experiments, the larvae were counted daily until they were all either consumed or had developed to the pupal stage. Polymerase chain reaction was used to confirm the presence of An. gambiae DNA in predator guts.
Experiments found that habitat type (P < 0.0001) and predator species (P < 0.0001) had a significant impact on the predation rate in the 24 hour evaluations. In semi-field experiments, predator species (P < 0.0001) and habitat type (P < 0.0001) were significant factors in both the daily survival and the overall developmental time of larvae. Pupation rates took significantly longer in habitats with refugia. An. gambiae DNA was found in at least three out of ten midguts for all predator species. Gambusia affins was the most efficient, being three times more efficient than tadpoles.
These experiments provide insight into the efficiency of specific natural predators against mosquito larvae. These naturally occurring predators may be useful in biocontrol strategies for aquatic stage An. gambiae mosquitoes. Further investigations should be done in complex natural habitats for these predators.
Limitations in the ability of organisms to tolerate environmental stressors affect their fundamental ecological niche and constrain their distribution to specific habitats. Evolution of tolerance, therefore, can engender ecological niche dynamics. Forest populations of the afro-tropical malaria mosquito Anopheles gambiae have been shown to adapt to historically unsuitable larval habitats polluted with decaying organic matter that are found in densely populated urban agglomerates of Cameroon. This process has resulted in niche expansion from rural to urban environments that is associated with cryptic speciation and ecological divergence of two evolutionarily significant units within this taxon, the molecular forms M and S, among which reproductive isolation is significant but still incomplete. Habitat segregation between the two forms results in a mosaic distribution of clinally parapatric patches, with the M form predominating in the centre of urban agglomerates and the S form in the surrounding rural localities. We hypothesized that development of tolerance to nitrogenous pollutants derived from the decomposition of organic matter, among which ammonia is the most toxic to aquatic organisms, may affect this pattern of distribution and process of niche expansion by the M form.
Acute toxicity bioassays indicated that populations of the two molecular forms occurring at the extremes of an urbanization gradient in Yaounde, the capital of Cameroon, differed in their response to ammonia. The regression lines best describing the dose-mortality profile differed in the scale of the explanatory variable (ammonia concentration log-transformed for the S form and linear for the M form), and in slope (steeper for the S form and shallower for the M form). These features reflected differences in the frequency distribution of individual tolerance thresholds in the two populations as assessed by probit analysis, with the M form exhibiting a greater mean and variance compared to the S form.
In agreement with expectations based on the pattern of habitat partitioning and exposure to ammonia in larval habitats in Yaounde, the M form showed greater tolerance to ammonia compared to the S form. This trait may be part of the physiological machinery allowing forest populations of the M form to colonize polluted larval habitats, which is at the heart of its niche expansion in densely populated human settlements in Cameroon.
Local adaptation; Fundamental ecological niche; Environmental stressor; Evolution of tolerance; Urbanization; Malaria; Mosquito
The Afrotropical mosquito Anopheles gambiae sensu stricto (A. gambiae), a major vector of malaria, is currently undergoing speciation into the M and S molecular forms. These forms have diverged in larval ecology and reproductive behavior through unknown genetic mechanisms, despite considerable levels of hybridization. Previous genome-wide scans using gene-based microarrays uncovered divergence between M and S that was largely confined to gene-poor pericentromeric regions, prompting a speciation-with-ongoing-gene-flow model that implicated only ~3% of the genome near centromeres in the speciation process. Here, based on the complete M and S genome sequences, we report widespread and heterogeneous genomic divergence inconsistent with appreciable levels of inter-form gene flow, suggesting a more advanced speciation process and greater challenges to identify genes critical to initiating that process.
Polymerase chain reaction analysis was performed to determine whether mosquito predators in wetland habitats feed on Anopheles gambiae sensu lato (s.l.) larvae. Aquatic mosquito predators were collected from six wetlands near Lake Victoria in Mbita, Western Kenya. This study revealed that the whole positive rate of An. gambiae s.l. from 330 predators was 54.2%. The order of positive rate was the highest in Odonata (70.2%), followed by Hemiptera (62.8%), Amphibia (41.7%), and Coleoptera (18%). This study demonstrates that the polymerase chain reaction method can determine whether aquatic mosquito predators feed on An. gambiae s.l. larvae if the predators have undigested An. gambiae s.l. in their midgut or stomach.
Anura; aquatic insect; Coleoptera; Hemiptera; Odonata
Anthropogenic habitat disturbance is a prime cause in the current trend of the Earth’s reduction in biodiversity. Here we show that the human footprint on the Central African rainforest, which is resulting in deforestation and growth of densely populated urban agglomerates, is associated to ecological divergence and cryptic speciation leading to adaptive radiation within the major malaria mosquito Anopheles gambiae.
In southern Cameroon, the frequency of two molecular forms–M and S–among which reproductive isolation is strong but still incomplete, was correlated to an index of urbanisation extracted from remotely sensed data, expressed as the proportion of built-up surface in each sampling unit. The two forms markedly segregated along an urbanisation gradient forming a bimodal cline of ∼6-km width: the S form was exclusive to the rural habitat, whereas only the M form was present in the core of densely urbanised settings, co-occurring at times in the same polluted larval habitats of the southern house mosquito Culex quinquefasciatus–a species association that was not historically recorded before.
Our results indicate that when humans create novel habitats and ecological heterogeneities, they can provide evolutionary opportunities for rapid adaptive niche shifts associated with lineage divergence, whose consequences upon malaria transmission might be significant.
Anopheles gambiae, a major vector of malaria, is widely distributed throughout sub-Saharan Africa. In an attempt to eliminate infective mosquitoes, researchers are trying to develop transgenic strains that are refractory to the Plasmodium parasite. Before any release of transgenic mosquitoes can be envisaged, we need an accurate picture of the differentiation between the two molecular forms of An. gambiae, termed M and S, which are of uncertain taxonomic status.
Insertion patterns of three transposable elements (TEs) were determined in populations from Benin, Burkina Faso, Cameroon, Ghana, Ivory Coast, Madagascar, Mali, Mozambique, Niger, and Tanzania, using Transposon Display, a TE-anchored strategy based on Amplified Fragment Length Polymorphism. The results reveal a clear differentiation between the M and S forms, whatever their geographical origin, suggesting an incipient speciation process.
Any attempt to control the transmission of malaria by An. gambiae using either conventional or novel technologies must take the M/S genetic differentiation into account. In addition, we localized three TE insertion sites that were present either in every individual or at a high frequency in the M molecular form. These sites were found to be located outside the chromosomal regions that are suspected of involvement in the speciation event between the two forms. This suggests that these chromosomal regions are either larger than previously thought, or there are additional differentiated genomic regions interspersed with undifferentiated regions.
Insecticide resistance of the main malaria vector, Anopheles gambiae, has been reported in south-western Burkina Faso, West Africa. Cross-resistance to DDT and pyrethroids was conferred by alterations at site of action in the sodium channel, the Leu-Phe kdr mutation; resistance to organophosphates and carbamates resulted from a single point mutation in the oxyanion hole of the acetylcholinesterase enzyme designed as ace-1R.
An entomological survey was carried out during the rainy season of 2005 at Vallée du Kou, a rice growing area in south-western Burkina Faso. At the Vallée du Kou, both insecticide resistance mechanisms have been previously described in the M and S molecular forms of An. gambiae. This survey aimed i) to update the temporal dynamics and the circumsporozoite infection rate of the two molecular forms M and S of An. gambiae ii) to update the frequency of the Leu-Phe kdr mutation within these forms and finally iii) to investigate the occurrence of the ace-1R mutation.
Mosquitoes collected by indoor residual collection and by human landing catches were counted and morphologically identified. Species and molecular forms of An. gambiae, ace-1R and Leu-Phe kdr mutations were determined using PCR techniques. The presence of the circumsporozoite protein of Plasmodium falciparum was determined using ELISA.
Anopheles gambiae populations were dominated by the M form. However the S form occurred in relative important proportion towards the end of the rainy season with a maximum peak in October at 51%. Sporozoite rates were similar in both forms. The frequency of the Leu-Phe kdr mutation in the S form reached a fixation level while it is still spreading in the M form. Furthermore, the ace-1R mutation prevailed predominately in the S form and has just started spreading in the M form. The two mutations occurred concomitantly both in M and S populations.
These results showed that the Vallée du Kou, a rice growing area formerly occupied mainly by M susceptible populations, is progressively colonized by S resistant populations living in sympatry with the former. As a result, the distribution pattern of insecticide resistance mutations shows the occurrence of both resistance mechanisms concomitantly in the same populations. The impact of multiple resistance mechanisms in M and S populations of An. gambiae on vector control measures against malaria transmission, such as insecticide-treated nets (ITNs) and indoor residual spraying (IRS), in this area is discussed.
Urban malaria cases are becoming common in Africa as more people move into cities and industrialization proceeds. While many species of Anopheles mosquitoes vector malaria in rural areas, only a few are found within cities. The success of anthropophilic species in cities, such as members of the An. gambiae complex, may be explained by limitations on colonization by predator species in urban environments. Habitats that are temporal or structurally simple have lower predator survivorship in a variety of ecosystems, but these have not been investigated previously in an urban area. Areas within and around the Kenyan coastal town of Malindi were previously sampled for the presence of standing water using a geographic sampling strategy with probability proportional to size sampling of planned well-drained, unplanned poorly-drained, planned poorly-drained, unplanned well-drained, and peri-urban locations. Standing aquatic habitats in these areas were reassessed. During monthly sampling, presence/absence of mosquitoes and predator taxa were noted, as were ecological habitat variables: structural complexity and presence of water. Lambda statistics were calculated to associate predator guilds, habitat types, location variables, and ecological variables. All predator guilds found in habitats were strongly associated with habitat type, as were the structural complexity and temporal nature of the habitats. Types of habitat were heterogeneously distributed throughout Malindi, with swimming pools as a common habitat type in planned urban areas and tire track pools a common habitat type in peri-urban areas of Malindi. Predator colonization of aquatic habitats in Malindi was strongly influenced by habitat type, and not associated with location characteristics. Ecological variables were affected by the type of habitats, which are co-associated with planning and drainage in Malindi. While habitat types are distributed heterogeneously within Malindi, habitats with low predation pressure are available for mosquito colonization in both urban and peri-urban areas. The temporal, peri-urban tire track pools and the structural simplicity of urban swimming pools may discourage predator colonization, thereby increasing the probability of malaria vectors in these areas of Malindi. Future studies should evaluate habitats for use in malaria surveillance and experimentally test the effects of structural complexity and temporal nature of urban habitats on the densities of mosquito larvae and their aquatic predators.
Anopheles gambiae; predation; urban; Malindi
The development of mosquito nets pre-treated with insecticide, Long Lasting Impregnated Nets (LLINs) that last the life span of the net, is a solution to the difficulty of the re-impregnation of conventional nets. Even if they showed a good efficacy in control conditions, their efficacy in the field, particularly in areas with resistance of Anopheles gambiae to pyrethroids, is not well documented. This study compares wide (Olyset®) and small (Permanet®) mesh LLINs in field conditions, using entomological parameters.
The two LLINs were tested in a rice-growing area of south-western Burkina Faso (West Africa) with year around high density of the main malaria vector An. gambiae s.s. In the study village (VK6), there is a mixed population of two molecular forms of An. gambiae, the S-form which dominates during the rainy season and the M-form which dominates the rest of the year. The two LLINs Olyset® and Permanet® were distributed in the village and 20 matched houses were selected for comparison with four houses without treated nets.
Mosquito entrance rate was ten fold higher in control houses than in houses with LLINs and there was no difference between the two net types. Among mosquitoes found in the houses, 36 % were dead in LLIN houses compared to 0% in control houses. Blood feeding rate was 80 % in control houses compared to 43 % in LLIN houses. The type of net did not significantly impact any of these parameters. No mosquitoes were found inside Permanet®, whereas dead or dying mosquitoes were collected inside the Olyset®. More than 60% of mosquitoes found on top or inside the nets had had blood meals from cattle, as shown by ELISA analysis.
The percentage of blood-fed mosquitoes in a bed net study does not necessarily determine net success. The efficacy of the two types of LLINs was comparable, during a period when the S-form of An. gambiae was carrying the kdr gene. Significantly higher numbers of mosquitoes were collected in control houses compared to intervention houses, indicating that the LLINs provided an additional deterrent effect, which enhanced their expected prevention capacity.
Studies in aquatic systems have shown that habitat complexity may provide refuge or reduce the number of encounters prey have with actively searching predators. For ambush predators, habitat complexity may enhance or have no effect on predation rates because it conceals predators, reduces prey detection by predators, or visually impairs both predators and prey. We investigated the effects of habitat complexity and predation by the ambush predators Toxorhynchites rutilus and Corethrella appendiculata on their mosquito prey Aedes albopictus and Ochlerotatus triseriatus in container analogs of treeholes. As in other ambush predator-prey systems, habitat complexity did not alter the effects of T. rutilus or C. appendiculata whose presence decreased prey survivorship, shortened development time, and increased adult size compared to treatments where predators were absent. Faster growth and larger size were due to predator-mediated release from competition among surviving prey. Male and female prey survivorship were similar in the absence of predators, however when predators were present, survivorship of both prey species was skewed in favor of males. We conclude that habitat complexity is relatively unimportant in shaping predator-prey interactions in this treehole community, where predation risk differs between prey sexes.
Container mosquitoes; Population growth measurements; Predator–prey interactions
Malaria control programs are being jeopardized by the spread of insecticide resistance in mosquito vector populations. The situation in Burkina Faso is emblematic with Anopheles gambiae populations showing high levels of resistance to most available compounds. Although the frequency of insecticide target-site mutations including knockdown resistance (kdr) and insensitive acetylcholinesterase (Ace-1R) alleles has been regularly monitored in the area, it is not known whether detoxifying enzymes contribute to the diversity of resistance phenotypes observed in the field. Here, we propose an update on the phenotypic diversity of insecticide resistance in An. gambiae populations sampled from 10 sites in Burkina Faso in 2010. Susceptibility to deltamethrin, permethrin, DDT, bendiocarb and fenithrotion was assessed. Test specimens (N = 30 per locality) were identified to species and molecular form and their genotype at the kdr and Ace-1 loci was determined. Detoxifying enzymes activities including non-specific esterases (NSEs), oxydases (cytochrome P450) and Glutathione S-Transferases (GSTs) were measured on single mosquitoes (N = 50) from each test locality and compared with the An. gambiae Kisumu susceptible reference strain. In all sites, mosquitoes demonstrated multiple resistance phenotypes, showing reduced mortality to several insecticidal compounds at the same time, although with considerable site-to-site variation. Both the kdr 1014L and Ace-1R 119S resistant alleles were detected in the M and the S forms of An. gambiae, and were found together in specimens of the S form. Variation in detoxifying enzyme activities was observed within and between vector populations. Elevated levels of NSEs and GSTs were widespread, suggesting multiple resistance mechanisms segregate within An. gambiae populations from this country. By documenting the extent and diversity of insecticide resistance phenotypes and the putative combination of their underlying mechanisms in An. gambiae mosquitoes, our work prompts for new alternative strategies to be urgently developed for the control of major malaria vectors in Burkina Faso.
Population subgroups of the African malaria vector Anopheles gambiae have not been comprehensively characterized owing to the lack of unbiased sampling methods. In the arid savanna zone of West Africa, where potential oviposition sites are scarce, widespread collection from larval pools in the peridomestic human habitat yielded a comprehensive genetic survey of local A. gambiae population subgroups, independent of adult resting behavior and ecological preference. A previously unknown subgroup of exophilic A. gambiae is sympatric with the known endophilic A. gambiae in this region. The exophilic subgroup is abundant, lacks differentiation into M and S molecular forms, and is highly susceptible to infection with wild Plasmodium falciparum. These findings might have implications for the epidemiology of malaria transmission and control.
The Anopheles gambiae sensu lato (s.l.) species complex in Burkina Faso consists of Anopheles arabiensis, and molecular forms M and S of Anopheles gambiae sensu stricto (s.s.). Previous studies comparing the M and S forms for level of infection with Plasmodium falciparum have yielded conflicting results.
Mosquito larvae were sampled from natural pools, reared to adulthood under controlled conditions, and challenged with natural P. falciparum by experimental feeding with blood from gametocyte carriers. Oocyst infection prevalence and intensity was determined one week after infection. DNA from carcasses was genotyped to identify species and molecular form.
In total, 7,400 adult mosquitoes grown from wild-caught larvae were challenged with gametocytes in 29 experimental infections spanning four transmission seasons. The overall infection prevalence averaged 40.7% for A. gambiae M form, 41.4% for A. gambiae S form, and 40.1% for A. arabiensis. There was no significant difference in infection prevalence or intensity between the three population groups. Notably, infection experiments in which the population groups were challenged in parallel on the same infective blood displayed less infection difference between population groups, while infections with less balanced composition of population groups had lower statistical power and displayed apparent differences that fluctuated more often from the null average.
The study clearly establishes that, at the study site in Burkina Faso, there is no difference in genetic susceptibility to P. falciparum infection between three sympatric population groups of the A. gambiae s.l. complex. Feeding the mosquito groups on the same infective blood meal greatly increases statistical power. Conversely, comparison of the different mosquito groups between, rather than within, infections yields larger apparent difference between mosquito groups, resulting from lower statistical power and greater noise, and could lead to false-positive results. In making infection comparisons between population groups, it is more accurate to compare the different groups after feeding simultaneously upon the same infective blood.
Anopheles gambiae; Anopheles arabiensis; Plasmodium falciparum; Malaria vector; Genetic susceptibility; Molecular form; Infection prevalence; Infection intensity
Previous efforts to uncover the genetic underpinnings of ongoing ecological speciation of the M and S forms of the African malaria vector Anopheles gambiae revealed two centromere-proximal islands of genetic divergence on X and chromosome 2. Under the assumption of considerable ongoing gene flow between M and S, these persistently divergent genomic islands were widely considered to be “speciation islands”. In the course of microarray-based divergence mapping, we discovered a third centromere-associated island of divergence on chromosome 3, which was validated by targeted re-sequencing. To test for genetic association between the divergence islands on all three chromosomes, SNP-based assays were applied in four natural populations of M and S spanning West, Central and East Africa. Genotyping of 517 female M and S mosquitoes revealed nearly complete linkage disequilibrium between the centromeres of the three independently assorting chromosomes. These results suggest that despite the potential for inter-form gene flow through hybridization, actual (realized) gene flow between M and S may be substantially less than commonly assumed, and may not explain most shared variation. Moreover, the possibility of very low gene flow calls into question whether diverged pericentromeric regions-- characterized by reduced levels of variation and recombination-- are in fact instrumental rather than merely incidental to the speciation process.
centromeres; divergent adaptation; ecological speciation; malaria vector; reproductive isolation; speciation islands
In the city of Bobo-Dioulasso in Burkina Faso, Anopheles arabiensis has superseded Anopheles gambiae s.s. as the major malaria vector and the larvae are found in highly polluted habitats normally considered unsuitable for Anopheles mosquitoes. Here we show that An. gambiae s.l. adults emerging from a highly polluted site in the city centre (Dioulassoba) have a high prevalence of DDT resistance (percentage mortality after exposure to diagnostic dose = 65.8% in the dry season and 70.4% in the rainy season, respectively). An investigation into the mechanisms responsible found an unexpectedly high frequency of the 1014S kdr mutation (allele frequency = 0.4), which is found at very low frequencies in An. arabiensis in the surrounding rural areas, and an increase in transcript levels of several detoxification genes, notably from the glutathione transferase and cytochrome P450 gene families. A number of ABC transporter genes were also expressed at elevated levels in the DDT resistant An. arabiensis. Unplanned urbanisation provides numerous breeding grounds for mosquitoes. The finding that Anopheles mosquitoes adapted to these urban breeding sites have a high prevalence of insecticide resistance has important implications for our understanding of the selective forces responsible for the rapid spread of insecticide resistant populations of malaria vectors in Africa.
The primary Afrotropical malaria mosquito vector Anopheles gambiae sensu stricto has a complex population structure. In west Africa, this species is split into two molecular forms and displays local and regional variation in chromosomal arrangements and behaviors. To investigate patterns of macrogeographic population substructure, 25 An. gambiae samples from 12 African countries were genotyped at 13 microsatellite loci. This analysis detected the presence of additional population structuring, with the M-form being subdivided into distinct west, central, and southern African genetic clusters. These clusters are coincident with the central African rainforest belt and northern and southern savannah biomes, which suggests restrictions to gene flow associated with the transition between these biomes. By contrast, geographically patterned population substructure appears much weaker within the S-form.
Anopheles gambiae; geographic regions; microsatellites; molecular forms; population structure
Vector control is one of the most effective measures to prevent the transmission of malaria, a disease that causes over 600,000 deaths annually. Around 30–40 Anopheles mosquito species are natural vectors of malaria parasites. Some of these species cannot be morphologically distinguished, but have behavioral and ecological differences. Emblematic of this is the Anopheles gambiae species complex. The correct identification of vector species is fundamental to the development of control strategies and epidemiological studies of disease transmission.
An inexpensive, disposable, field-deployable, sample-to-answer, microfluidic chip was designed, constructed, and tested for rapid molecular identification of Anopheles gambiae and Anopheles arabiensis. The chip contains three isothermal amplification reactors. One test reactor operates with specific primers to amplify Anopheles gambiae DNA, another with specific primers for Anopheles arabiensis DNA, and the third serves as a negative control. A mosquito leg was crushed on an isolation membrane. Two discs, laden with mosquito tissue, were punched out of the membrane and inserted into the two test chambers. The isolated, disc-bound DNA served as a template in the amplification processes. The amplification products were detected with intercalating fluorescent dye that was excited with a blue light-emitting diode. The emitted light was observed by eye and recorded with a cell-phone camera. When the target consisted of Anopheles gambiae, the reactor containing primers specific to An. gambiae lit up while the other two reactors remained dark. When the target consisted of Anopheles arabiensis, the reactor containing primers specific to An. arabiensis lit up while the other two reactors remained dark.
The microfluidic chip provides a means to identify mosquito type through molecular analysis. It is suitable for field work, allowing one to track the geographical distribution of mosquito populations and community structure alterations due to environmental changes and malaria intervention measures.
Understanding genetic causes and effects of speciation in sympatric populations of sexually reproducing eukaryotes is challenging, controversial, and of practical importance for controlling rapidly evolving pests and pathogens. The major African malaria vector mosquito Anopheles gambiae sensu stricto (s.s.) is considered to contain two incipient species with strong reproductive isolation, hybrids between the M and S molecular forms being very rare. Following recent observations of higher proportions of hybrid forms at a few sites in West Africa, we conducted new surveys of 12 sites in four contiguous countries (The Gambia, Senegal, Guinea-Bissau, and Republic of Guinea). Identification and genotyping of 3499 A. gambiae s.s. revealed high frequencies of M/S hybrid forms at each site, ranging from 5 to 42%, and a large spectrum of inbreeding coefficient values from 0.11 to 0.76, spanning most of the range expected between the alternative extremes of panmixia and assortative mating. Year-round sampling over 2 years at one of the sites in The Gambia showed that M/S hybrid forms had similar relative frequencies throughout periods of marked seasonal variation in mosquito breeding and abundance. Genome-wide scans with an Affymetrix high-density single-nucleotide polymorphism (SNP) microarray enabled replicate comparisons of pools of different molecular forms, in three separate populations. These showed strong differentiation between M and S forms only in the pericentromeric region of the X chromosome that contains the molecular form-specific marker locus, with only a few other loci showing minor differences. In the X chromosome, the M/S hybrid forms were more differentiated from M than from S forms, supporting a hypothesis of asymmetric introgression and backcrossing.
Genomics; speciation; hybridization; introgression; mosquito
Plasmodium falciparum is the causative agent of malaria, a disease that kills almost one million persons each year, mainly in sub-Saharan Africa. P. falciparum is transmitted to the human host by the bite of an Anopheles female mosquito, and Anopheles gambiae sensus stricto is the most tremendous malaria vector in Africa, widespread throughout the afro-tropical belt. An. gambiae s.s. is subdivided into two distinct molecular forms, namely M and S forms. The two molecular forms are morphologically identical but they are distinct genetically, and differ by their distribution and their ecological preferences. The epidemiological importance of the two molecular forms in malaria transmission has been poorly investigated so far and gave distinct results in different areas. We have developed a real-time quantitative PCR (qPCR) assay, and used it to detect P. falciparum at the oocyst stage in wild An. gambiae s.s. mosquitoes experimentally infected with natural isolates of parasites. Mosquitoes were collected at immature stages in sympatric and allopatric breeding sites and further infected at the adult stage. We next measured the infection prevalence and intensity in female mosquitoes using the qPCR assay and correlated the infection success with the mosquito molecular forms. Our results revealed different prevalence of infection between the M and S molecular forms of An. gambiae s.s. in Cameroon, for both sympatric and allopatric populations of mosquitoes. However, no difference in the infection intensity was observed. Thus, the distribution of the molecular forms of An. gambiae s.s. may impact on the malaria epidemiology, and it will be important to monitor the efficiency of malaria control interventions on the two M and S forms.