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1.  Cryptic Genetic Diversity within the Anopheles nili group of Malaria Vectors in the Equatorial Forest Area of Cameroon (Central Africa) 
PLoS ONE  2013;8(3):e58862.
Background
The Anopheles nili group of mosquitoes includes important vectors of human malaria in equatorial forest and humid savannah regions of sub-Saharan Africa. However, it remains largely understudied, and data on its populations’ bionomics and genetic structure are crucially lacking. Here, we used a combination of nuclear (i.e. microsatellite and ribosomal DNA) and mitochondrial DNA markers to explore and compare the level of genetic polymorphism and divergence among populations and species of the group in the savannah and forested areas of Cameroon, Central Africa.
Principal Findings
All the markers provided support for the current classification within the An. nili group. However, they revealed high genetic heterogeneity within An. nili s.s. in deep equatorial forest environment. Nuclear markers showed the species to be composed of five highly divergent genetic lineages that differed by 1.8 to 12.9% of their Internal Transcribed Spacer 2 (ITS2) sequences, implying approximate divergence time of 0.82 to 5.86 million years. However, mitochondrial data only detected three major subdivisions, suggesting different evolutionary histories of the markers.
Conclusions/Significance
This study enlightened additional cryptic genetic diversity within An. nili s.s. in the deep equatorial forest environment of South Cameroon, reflecting a complex demographic history for this major vector of malaria in this environment. These preliminary results should be complemented by further studies which will shed light on the distribution, epidemiological importance and evolutionary history of this species group in the African rainforest, providing opportunities for in-depth comparative studies of local adaptation and speciation in major African malaria vectors.
doi:10.1371/journal.pone.0058862
PMCID: PMC3597579  PMID: 23516565
2.  Identification of Cryptic Anopheles Mosquito Species by Molecular Protein Profiling 
PLoS ONE  2013;8(2):e57486.
Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.
doi:10.1371/journal.pone.0057486
PMCID: PMC3585343  PMID: 23469000
3.  Multiple insecticide resistance mechanisms in Anopheles gambiae s.l. populations from Cameroon, Central Africa 
Parasites & Vectors  2013;6:41.
Background
Increasing incidence of DDT and pyrethroid resistance in Anopheles mosquitoes is seen as a limiting factor for malaria vector control. The current study aimed at an in-depth characterization of An. gambiae s.l. resistance to insecticides in Cameroon, in order to guide malaria vector control interventions.
Methods
Anopheles gambiae s.l. mosquitoes were collected as larvae and pupae from six localities spread throughout the four main biogeographical domains of Cameroon and reared to adults in insectaries. Standard WHO insecticide susceptibility tests were carried out with 4% DDT, 0.75% permethrin and 0.05% deltamethrin. Mortality rates and knockdown times (kdt50 and kdt95) were determined and the effect of pre-exposure to the synergists DEF, DEM and PBO was assessed. Tested mosquitoes were identified to species and molecular forms (M or S) using PCR-RFLP. The hot ligation method was used to depict kdr mutations and biochemical assays were conducted to assess detoxifying enzyme activities.
Results
The An. arabiensis population from Pitoa was fully susceptible to DDT and permethrin (mortality rates > 98%) and showed reduced susceptibility to deltamethrin. Resistance to DDT was widespread in An. gambiae s.s. populations and heterogeneous levels of susceptibility to permethrin and deltamethrin were observed. In many cases, prior exposure to synergists partially restored insecticide knockdown effect and increased mortality rates, suggesting a role of detoxifying enzymes in increasing mosquito survival upon challenge by pyrethroids and, to a lower extent DDT. The distribution of kdr alleles suggested a major role of kdr-based resistance in the S form of An. gambiae. In biochemical tests, all but one mosquito population overexpressed P450 activity, whereas baseline GST activity was low and similar in all field mosquito populations and in the control.
Conclusion
In Cameroon, multiple resistance mechanisms segregate in the S form of An. gambiae resulting in heterogeneous resistance profiles, whereas in the M form and An. arabiensis insecticide tolerance seems to be essentially mediated by enzyme-based detoxification. Synergists partially restored susceptibility to pyrethroid insecticides, and might help mitigate the impact of vector resistance in the field. However, additional vector control tools are needed to further impact on malaria transmission in such settings.
doi:10.1186/1756-3305-6-41
PMCID: PMC3583743  PMID: 23433176
4.  Multiple Insecticide Resistance in Anopheles gambiae s.l. Populations from Burkina Faso, West Africa 
PLoS ONE  2012;7(11):e48412.
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.
doi:10.1371/journal.pone.0048412
PMCID: PMC3506617  PMID: 23189131
5.  Patterns of Genomic Differentiation between Ecologically Differentiated M and S Forms of Anopheles gambiae in West and Central Africa 
Genome Biology and Evolution  2012;4(12):1202-1212.
Anopheles gambiae M and S are thought to be undergoing ecological speciation by adapting to different larval habitats. Toward an improved understanding of the genetic determinants and evolutionary processes shaping their divergence, we used a 400,000 single-nucleotide polymorphism (SNP) genotyping array to characterize patterns of genomic differentiation between four geographically paired M and S population samples from West and Central Africa. In keeping with recent studies based on more limited genomic or geographic sampling, divergence was not confined to a few isolated “speciation islands.” Divergence was both widespread across the genome and heterogeneous. Moreover, we find consistent patterns of genomic divergence across sampling sites and mutually exclusive clustering of M and S populations using genetic distances based on all 400,000 SNPs, implying that M and S are evolving collectively across the study area. Nevertheless, the clustering of local M and S populations using genetic distances based on SNPs from genomic regions of low differentiation is consistent with recent gene flow and introgression. To account for these data and reconcile apparent paradoxes in reported patterns of M–S genomic divergence and hybridization, we propose that extrinsic ecologically based postmating barriers vary in strength as environmental conditions fluctuate or change.
doi:10.1093/gbe/evs095
PMCID: PMC3542583  PMID: 23132896
divergent selection; genome scan; introgression; population genomics; SNP genotyping; speciation islands
6.  Anthropogenic Habitat Disturbance and Ecological Divergence between Incipient Species of the Malaria Mosquito Anopheles gambiae 
PLoS ONE  2012;7(6):e39453.
Background
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.
Methodology/Principal Findings
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.
Conclusions/Significance
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.
doi:10.1371/journal.pone.0039453
PMCID: PMC3382172  PMID: 22745756
7.  Population dynamics of Anopheles gambiae s.l. in Bobo-Dioulasso city: bionomics, infection rate and susceptibility to insecticides 
Parasites & Vectors  2012;5:127.
Background
Historical studies have indicated that An. gambiae s.s. is the predominant malaria vector species in Bobo-Dioulasso the second biggest city of Burkina Faso (West Africa). However, over the last decade, An. arabiensis appears to be replacing An. gambiae s.s. as the most prevalent malaria vector in this urban setting. To investigate this species transition in more detail the present study aims to provide an update on the malaria vector composition in Bobo-Dioulasso, and also the Plasmodium infection rates and susceptibility to insecticides of the local An. gambiae s.l. population.
Methods
An entomological survey was carried out from May to December 2008 in Dioulassoba and Kodeni (central and peripheral districts respectively), which are representative of the main ecological features of the city. Sampling consisted of the collection of larval stages from water bodies, and adults by monthly indoor residual spraying (IRS) using aerosol insecticides. Insecticide susceptibility tests were performed using the WHO filter paper protocol on adults emerged from larvae. PCR was used to determine vector species and to identify resistance mechanisms (kdr and ace-1R). The Plasmodium infection rate was estimated by ELISA performed on female mosquitoes collected indoors by IRS.
Results
An. arabiensis was found to be the major malaria vector in Bobo-Dioulasso, comprising 50 to 100% of the vector population. The sporozoite infection rate for An. arabiensis was higher than An. gambiae s.s. at both Dioulassoba and Kodeni. An. gambiae s.l. was resistant to DDT and cross-resistant to pyrethroids at the two sites with higher levels of resistance observed in An. gambiae s.s. than An. arabiensis. Resistance to 0.1% bendiocarb was observed in the An. gambiae s.s. S form but not the M form or in An. arabiensis. The L1014F kdr mutation was detected in the two molecular forms of An. gambiae s.s. at varying frequencies (0.45 to 0.92), but was not detected in An. arabiensis, suggesting that other mechanisms are involved in DDT resistance in this species. The ace-1R mutation was only detected in the S molecular form and was observed at the two sites at similar frequency (0.3).
Conclusions
Over the last ten years, An. arabiensis has become the major malaria vector in Bobo-Dioulasso city where it was formerly present only at low frequency. However, the ecological determinant that enhances the settlement of this species into urban and peri-urban areas of Bobo-Dioulasso remains to be clarified. The impact of the changing An. gambiae s.l. population in this region for vector control including resistance management strategies is discussed.
doi:10.1186/1756-3305-5-127
PMCID: PMC3424103  PMID: 22721002
Malaria; Anopheles gambiae s.l.; An. arabiensis; Insecticide resistance; Bobo-Dioulasso; Burkina Faso
8.  Behavioural responses of Anopheles gambiae sensu stricto M and S molecular form larvae to an aquatic predator in Burkina Faso 
Parasites & Vectors  2012;5:65.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1756-3305-5-65
PMCID: PMC3352179  PMID: 22463735
9.  Notes on the blood-feeding behavior of Aedes albopictus (Diptera: Culicidae) in Cameroon 
Parasites & Vectors  2012;5:57.
Background
The invasive mosquito Aedes albopictus is often considered a poor vector of human pathogens, owing to its catholic feeding behavior. However, it was recently incriminated as a major vector in several Chikungunya epidemics, outside of its native range. Here we assessed two key elements of feeding behavior by Ae. albopictus females in Yaoundé, Cameroon, Central Africa. Host preference was explored and the human-biting activity of females was monitored over 24 h to determine periods of maximum bite exposure.
Findings
Analysis of ingested blood in outdoor-resting females showed that Ae. albopictus preferentially fed on humans rather than on available domestic animals (95% of the blood meals contained human blood). Our results further showed that Ae. albopictus is a day-biting species in Yaoundé, with a main peak of activity in the late afternoon.
Conclusion
This is the first report on the feeding behavior of Ae. albopictus in Central Africa. The species is highly aggressive to humans and might therefore be involved in human-human virus transmission in this setting.
doi:10.1186/1756-3305-5-57
PMCID: PMC3342132  PMID: 22433236
Aedes albopictus; Feeding behavior; Host preference; Nychthemeral activity; Cameroon
10.  Spatially Explicit Analyses of Anopheline Mosquitoes Indoor Resting Density: Implications for Malaria Control 
PLoS ONE  2012;7(2):e31843.
Background
The question of sampling and spatial aggregation of malaria vectors is central to vector control efforts and estimates of transmission. Spatial patterns of anopheline populations are complex because mosquitoes' habitats and behaviors are strongly heterogeneous. Analyses of spatially referenced counts provide a powerful approach to delineate complex distribution patterns, and contributions of these methods in the study and control of malaria vectors must be carefully evaluated.
Methodology/Principal Findings
We used correlograms, directional variograms, Local Indicators of Spatial Association (LISA) and the Spatial Analysis by Distance IndicEs (SADIE) to examine spatial patterns of Indoor Resting Densities (IRD) in two dominant malaria vectors sampled with a 5×5 km grid over a 2500 km2 area in the forest domain of Cameroon. SADIE analyses revealed that the distribution of Anopheles gambiae was different from regular or random, whereas there was no evidence of spatial pattern in Anopheles funestus (Ia = 1.644, Pa<0.05 and Ia = 1.464, Pa>0.05, respectively). Correlograms and variograms showed significant spatial autocorrelations at small distance lags, and indicated the presence of large clusters of similar values of abundance in An. gambiae while An. funestus was characterized by smaller clusters. The examination of spatial patterns at a finer spatial scale with SADIE and LISA identified several patches of higher than average IRD (hot spots) and clusters of lower than average IRD (cold spots) for the two species. Significant changes occurred in the overall spatial pattern, spatial trends and clusters when IRDs were aggregated at the house level rather than the locality level. All spatial analyses unveiled scale-dependent patterns that could not be identified by traditional aggregation indices.
Conclusions/Significance
Our study illustrates the importance of spatial analyses in unraveling the complex spatial patterns of malaria vectors, and highlights the potential contributions of these methods in malaria control.
doi:10.1371/journal.pone.0031843
PMCID: PMC3279417  PMID: 22348131
11.  Chromosomal Inversions, Natural Selection and Adaptation in the Malaria Vector Anopheles funestus 
Molecular Biology and Evolution  2010;28(1):745-758.
Chromosomal polymorphisms, such as inversions, are presumably involved in the rapid adaptation of populations to local environmental conditions. Reduced recombination between alternative arrangements in heterozygotes may protect sets of locally adapted genes, promoting ecological divergence and potentially leading to reproductive isolation and speciation. Through a comparative analysis of chromosomal inversions and microsatellite marker polymorphisms, we hereby present biological evidence that strengthens this view in the mosquito Anopheles funestus s.s, one of the most important and widespread malaria vectors in Africa. Specimens were collected across a wide range of geographical, ecological, and climatic conditions in Cameroon. We observed a sharp contrast between population structure measured at neutral microsatellite markers and at chromosomal inversions. Microsatellite data detected only a weak signal for population structuring among geographical zones (FST < 0.013, P < 0.01). By contrast, strong differentiation among ecological zones was revealed by chromosomal inversions (FST > 0.190, P < 0.01). Using standardized estimates of FST, we show that inversions behave at odds with neutral expectations strongly suggesting a role of environmental selection in shaping their distribution. We further demonstrate through canonical correspondence analysis that heterogeneity in eco-geographical variables measured at specimen sampling sites explained 89% of chromosomal variance in A. funestus. These results are in agreement with a role of chromosomal inversions in ecotypic adaptation in this species. We argue that this widespread mosquito represents an interesting model system for the study of chromosomal speciation mechanisms and should provide ample opportunity for comparative studies on the evolution of reproductive isolation and speciation in major human malaria vectors.
doi:10.1093/molbev/msq248
PMCID: PMC3002248  PMID: 20837604
chromosome inversions; Anopheles funestus; natural selection; local adaptation; malaria; Cameroon
12.  Kdr-based insecticide resistance in Anopheles gambiae s.s populations in 
BMC Research Notes  2011;4:463.
Background
The spread of insecticide resistance in the malaria mosquito, Anopheles gambiae is a serious threat for current vector control strategies which rely on the use of insecticides. Two mutations at position 1014 of the S6 transmembrane segment of domain II in the voltage gated sodium channel, known as kdr (knockdown resistance) mutations leading to a change of a Leucine to a Phenylalanine (L1014F) or to a Serine (L1014S) confer resistance to DDT and pyrethroid insecticides in the insect. This paper presents the current distribution of the kdr alleles in wild Anopheles gambiae populations in Cameroon.
Results
A total of 1,405 anopheline mosquitoes were collected from 21 localities throughout Cameroon and identified as An. gambiae (N = 1,248; 88.8%), An. arabiensis (N = 120; 8.5%) and An. melas (N = 37; 2.6%). Both kdr alleles 1014F and 1014S were identified in the M and S molecular forms of An. gambiae s.s. The frequency of the 1014F allele ranged from 1.7 to 18% in the M-form, and from 2 to 90% in the S-form. The 1014S allele ranged from 3-15% in the S-form and in the M-form its value was below 3%. Some specimens were found to carry both resistant kdr alleles.
Conclusion
This study provides an updated distribution map of the kdr alleles in wild An. gambiae populations in Cameroon. The co-occurrence of both alleles in malaria mosquito vectors in diverse ecological zones of the country may be critical for the planning and implementation of malaria vector control interventions based on IRS and ITNs, as currently ongoing in Cameroon.
doi:10.1186/1756-0500-4-463
PMCID: PMC3221647  PMID: 22035176
13.  A behavioral mechanism underlying ecological divergence in the malaria mosquito Anopheles gambiae 
Behavioral Ecology  2010;21(5):1087-1092.
Disruptive selection mediated by predation on aquatic immature stages has been proposed as a major force driving ecological divergence and fostering speciation between the M and S molecular forms of the African malaria mosquito, Anopheles gambiae. In the dry savannahs of West Africa where both molecular forms co-occur, the S form thrives in temporary pools filled with rainwater, whereas the M form preferentially breeds in permanent freshwater habitats where predator pressure is higher. Here, we explored the proximal mechanisms by which predation may contribute to habitat segregation between molecular forms using progeny of female mosquitoes captured in Burkina Faso. We show that the S form suffers higher predation rates than the M form when simultaneously exposed to the widespread predator, Anisops jaczewskii in an experimental arena. Furthermore, behavioral plasticity induced by exposure to the predator was observed in the M form, but not in the S form, and may partially explain its habitat use and ecological divergence from the S form. We discuss the role of adaptive phenotypic plasticity in allowing successful colonization of a new ecological niche by the M form and highlight further research areas that need to be addressed for a better understanding of the ultimate mechanisms underlying ecological speciation in this pest of major medical importance.
doi:10.1093/beheco/arq114
PMCID: PMC2920295  PMID: 22476108
adaptation; Anopheles gambiae; behavior; habitat divergence; mosquito; notonectidae; phenotypic plasticity, predation; speciation
14.  Genetic Structure of the Tiger Mosquito, Aedes albopictus, in Cameroon (Central Africa) 
PLoS ONE  2011;6(5):e20257.
Background
Aedes albopictus (Skuse, 1884) (Diptera: Culicidae), a mosquito native to Asia, has recently invaded all five continents. In Central Africa it was first reported in the early 2000s, and has since been implicated in the emergence of arboviruses such as dengue and chikungunya in this region. Recent genetic studies of invasive species have shown that multiple introductions are a key factor for successful expansion in new areas. As a result, phenotypic characters such as vector competence and insecticide susceptibility may vary within invasive pest species, potentially affecting vector efficiency and pest management. Here we assessed the genetic variability and population genetics of Ae. albopictus isolates in Cameroon (Central Africa), thereby deducing their likely geographic origin.
Methods and Results
Mosquitoes were sampled in 2007 in 12 localities in southern Cameroon and analyzed for polymorphism at six microsatellite loci and in two mitochondrial DNA regions (ND5 and COI). All the microsatellite markers were successfully amplified and were polymorphic, showing moderate genetic structureamong geographic populations (FST = 0.068, P<0.0001). Analysis of mtDNA sequences revealed four haplotypes each for the COI and ND5 genes, with a dominant haplotype shared by all Cameroonian samples. The weak genetic variation estimated from the mtDNA genes is consistent with the recent arrival of Ae. albopictus in Cameroon. Phylogeographic analysis based on COI polymorphism indicated that Ae. albopictus populations from Cameroon are related to tropical rather than temperate or subtropical outgroups.
Conclusion
The moderate genetic diversity observed among Cameroonian Ae. albopictus isolates is in keeping with recent introduction and spread in this country. The genetic structure of natural populations points to multiple introductions from tropical regions.
doi:10.1371/journal.pone.0020257
PMCID: PMC3101236  PMID: 21629655
15.  Association Mapping of Insecticide Resistance in Wild Anopheles gambiae Populations: Major Variants Identified in a Low-Linkage Disequilbrium Genome 
PLoS ONE  2010;5(10):e13140.
Background
Association studies are a promising way to uncover the genetic basis of complex traits in wild populations. Data on population stratification, linkage disequilibrium and distribution of variant effect-sizes for different trait-types are required to predict study success but are lacking for most taxa. We quantified and investigated the impacts of these key variables in a large-scale association study of a strongly selected trait of medical importance: pyrethroid resistance in the African malaria vector Anopheles gambiae.
Methodology/Principal Findings
We genotyped ≈1500 resistance-phenotyped wild mosquitoes from Ghana and Cameroon using a 1536-SNP array enriched for candidate insecticide resistance gene SNPs. Three factors greatly impacted study power. (1) Population stratification, which was attributable to co-occurrence of molecular forms (M and S), and cryptic within-form stratification necessitating both a partitioned analysis and genomic control. (2) All SNPs of substantial effect (odds ratio, OR>2) were rare (minor allele frequency, MAF<0.05). (3) Linkage disequilibrium (LD) was very low throughout most of the genome. Nevertheless, locally high LD, consistent with a recent selective sweep, and uniformly high ORs in each subsample facilitated significant direct and indirect detection of the known insecticide target site mutation kdr L1014F (OR≈6; P<10−6), but with resistance level modified by local haplotypic background.
Conclusion
Primarily as a result of very low LD in wild A. Gambiae, LD-based association mapping is challenging, but is feasible at least for major effect variants, especially where LD is enhanced by selective sweeps. Such variants will be of greatest importance for predictive diagnostic screening.
doi:10.1371/journal.pone.0013140
PMCID: PMC2956759  PMID: 20976111
16.  Population genetic structure of the malaria vector Anopheles nili in sub-Saharan Africa 
Malaria Journal  2010;9:161.
Background
Anopheles nili is a widespread efficient vector of human malaria parasites in the humid savannas and forested areas of sub-Saharan Africa. Understanding An. nili population structure and gene flow patterns could be useful for the development of locally-adapted vector control measures.
Methods
Polymorphism at eleven recently developed microsatelitte markers, and sequence variation in four genes within the 28s rDNA subunit (ITS2 and D3) and mtDNA (COII and ND4) were assessed to explore the level of genetic variability and differentiation among nine populations of An. nili from Senegal, Ivory Coast, Burkina Faso, Nigeria, Cameroon and the Democratic Republic of Congo (DRC).
Results
All microsatellite loci successfully amplified in all populations, showing high and very similar levels of genetic diversity in populations from West Africa and Cameroon (mean Rs = 8.10-8.88, mean He = 0.805-0.849) and much lower diversity in the Kenge population from DRC (mean Rs = 5.43, mean He = 0.594). Bayesian clustering analysis of microsatellite allelic frequencies revealed two main genetic clusters in the dataset. The first one included only the Kenge population and the second grouped together all other populations. High Fst estimates based on microsatellites (Fst > 0.118, P < 0.001) were observed in all comparisons between Kenge and all other populations. By contrast, low Fst estimates (Fst < 0.022, P < 0.05) were observed between populations within the second cluster. The correlation between genetic and geographic distances was weak and possibly obscured by demographic instability. Sequence variation in mtDNA genes matched these results, whereas low polymorphism in rDNA genes prevented detection of any population substructure at this geographical scale.
Conclusion
Overall, high genetic homogeneity of the An. nili gene pool was found across its distribution range in West and Central Africa, although demographic events probably resulted in a higher level of genetic isolation in the marginal population of Kenge (DRC). The role of the equatorial forest block as a barrier to gene flow and the implication of such findings for vector control are discussed.
doi:10.1186/1475-2875-9-161
PMCID: PMC2898787  PMID: 20540796
17.  Bionomics of Anopheline species and malaria transmission dynamics along an altitudinal transect in Western Cameroon 
BMC Infectious Diseases  2010;10:119.
Background
Highland areas of Africa are mostly malaria hypoendemic, due to climate which is not appropriate for anophelines development and their reproductive fitness. In view of designing a malaria control strategy in Western Cameroon highlands, baseline data on anopheline species bionomics were collected.
Methods
Longitudinal entomological surveys were conducted in three localities at different altitudinal levels. Mosquitoes were captured when landing on human volunteers and by pyrethrum spray catches. Sampled Anopheles were tested for the presence of Plasmodium circumsporozoite proteins and their blood meal origin with ELISA. Entomological parameters of malaria epidemiology were assessed using Mac Donald's formula.
Results
Anopheline species diversity and density decreased globally from lowland to highland. The most aggressive species along the altitudinal transect was Anopheles gambiae s.s. of S molecular form, followed in the lowland and on the plateau by An. funestus, but uphill by An. hancocki. An. gambiae and An. ziemanni exhibited similar seasonal biting patterns at the different levels, whereas different features were observed for An. funestus. Only indoor resting species could be captured uphill; it is therefore likely that endophilic behaviour is necessary for anophelines to climb above a certain threshold. Of the ten species collected along the transect, only An. gambiae and An. funestus were responsible for malaria transmission, with entomological inoculation rates (EIR) of 90.5, 62.8 and zero infective bites/human/year in the lowland, on the plateau and uphill respectively. The duration of gonotrophic cycle was consistently one day shorter for An. gambiae as compared to An. funestus at equal altitude. Altitudinal climate variations had no effect on the survivorship and the subsequent life expectancy of the adult stage of these malaria vectors, but most probably on aquatic stages. On the contrary increasing altitude significantly extended the duration of gonotrophic cycle and reduced: the EIR, their preference to human blood and consequently the malaria stability index.
Conclusion
Malaria epidemiological rooting in the outskirts of Western Cameroon highlands evolves with increasing altitude, gradually from stable to unstable settings. This suggests a potential risk of malaria epidemic in highlands, and the need for a continuous epidemiological surveillance.
doi:10.1186/1471-2334-10-119
PMCID: PMC2889998  PMID: 20482824
18.  Habitat suitability and ecological niche profile of major malaria vectors in Cameroon 
Malaria Journal  2009;8:307.
Background
Suitability of environmental conditions determines a species distribution in space and time. Understanding and modelling the ecological niche of mosquito disease vectors can, therefore, be a powerful predictor of the risk of exposure to the pathogens they transmit. In Africa, five anophelines are responsible for over 95% of total malaria transmission. However, detailed knowledge of the geographic distribution and ecological requirements of these species is to date still inadequate.
Methods
Indoor-resting mosquitoes were sampled from 386 villages covering the full range of ecological settings available in Cameroon, Central Africa. Using a predictive species distribution modeling approach based only on presence records, habitat suitability maps were constructed for the five major malaria vectors Anopheles gambiae, Anopheles funestus, Anopheles arabiensis, Anopheles nili and Anopheles moucheti. The influence of 17 climatic, topographic, and land use variables on mosquito geographic distribution was assessed by multivariate regression and ordination techniques.
Results
Twenty-four anopheline species were collected, of which 17 are known to transmit malaria in Africa. Ecological Niche Factor Analysis, Habitat Suitability modeling and Canonical Correspondence Analysis revealed marked differences among the five major malaria vector species, both in terms of ecological requirements and niche breadth. Eco-geographical variables (EGVs) related to human activity had the highest impact on habitat suitability for the five major malaria vectors, with areas of low population density being of marginal or unsuitable habitat quality. Sunlight exposure, rainfall, evapo-transpiration, relative humidity, and wind speed were among the most discriminative EGVs separating "forest" from "savanna" species.
Conclusions
The distribution of major malaria vectors in Cameroon is strongly affected by the impact of humans on the environment, with variables related to proximity to human settings being among the best predictors of habitat suitability. The ecologically more tolerant species An. gambiae and An. funestus were recorded in a wide range of eco-climatic settings. The other three major vectors, An. arabiensis, An. moucheti, and An. nili, were more specialized. Ecological niche and species distribution modelling should help improve malaria vector control interventions by targeting places and times where the impact on vector populations and disease transmission can be optimized.
doi:10.1186/1475-2875-8-307
PMCID: PMC2805691  PMID: 20028559
19.  Insecticide resistance in Anopheles gambiae: data from the first year of a multi-country study highlight the extent of the problem 
Malaria Journal  2009;8:299.
Background
Insecticide resistance in malaria vectors is a growing concern in many countries which requires immediate attention because of the limited chemical arsenal available for vector control. The current extent and distribution of this resistance in many parts of the continent is unknown and yet such information is essential for the planning of effective malaria control interventions.
Methods
In 2008, a network was established, with financial support from WHO/TDR, to investigate the extent of insecticide resistance in malaria vectors in five African countries. Here, the results of bioassays on Anopheles gambiae sensu lato from two rounds of monitoring from 12 sentinel sites in three of the partner countries are reported.
Results
Resistance is very heterogeneous even over relatively small distances. Furthermore, in some sites, large differences in mortality rates were observed during the course of the malaria transmission season. Using WHO diagnostic doses, all populations from Burkina Faso and Chad and two of the four populations from Sudan were classified as resistant to permethrin and/or deltamethrin. Very high frequencies of DDT resistance were found in urban areas in Burkina Faso and Sudan and in a cotton-growing district in Chad. In areas where both An. gambiae s.s. and Anopheles arabiensis were present, resistance was found in both species, although generally at a higher frequency in An gambiae s.s. Anopheles gambiae s.l. remains largely susceptible to the organophosphate fenitrothion and the carbamate bendiocarb in the majority of the sentinel sites with the exception of two sites in Burkina Faso. In the cotton-growing region of Soumousso in Burkina Faso, the vector population is resistant to all four classes of insecticide available for malaria control.
Conclusions
Possible factors influencing the frequency of resistant individuals observed in the sentinel sites are discussed. The results of this study highlight the importance of standardized longitudinal insecticide resistance monitoring and the urgent need for studies to monitor the impact of this resistance on malaria vector control activities.
doi:10.1186/1475-2875-8-299
PMCID: PMC2804687  PMID: 20015411
20.  Trends in DDT and pyrethroid resistance in Anopheles gambiae s.s. populations from urban and agro-industrial settings in southern Cameroon 
Background
Pyrethroid insecticides are widely used for insect pest control in Cameroon. In certain insect species, particularly the malaria vector Anopheles gambiae, resistance to this class of insecticides is a source of great concern and needs to be monitored in order to sustain the efficacy of vector control operations in the fields. This study highlights trends in DDT and pyrethroid resistance in wild An. gambiae populations from South Cameroon.
Methods
Mosquitoes were collected between 2001 and 2007 in four sites in South Cameroon, where insecticides are used for agricultural or personal protection purposes. Insecticide use was documented in each site by interviewing residents. Batches of 2-4 days old adult female mosquitoes reared from larval collections were tested for susceptibility to DDT, permethrin and deltamethrin using standard WHO procedures. Control, dead and survivors mosquitoes from bioassays were identified by PCR-RFLP and characterized for the kdr mutations using either the AS-PCR or the HOLA method.
Results
Four chemical insecticide groups were cited in the study sites: organochlorines, organophosphates, carbamates and pyrethroids. These chemicals were used for personal, crop or wood protection. In the four An. gambiae populations tested, significant variation in resistance levels, molecular forms composition and kdr frequencies were recorded in the time span of the study. Increases in DDT and pyrethroid resistance, as observed in most areas, were generally associated with an increase in the relative frequency of the S molecular form carrying the kdr mutations at higher frequencies. In Mangoum, however, where only the S form was present, a significant increase in the frequency of kdr alleles between 2003 to 2007 diverged with a decrease of the level of resistance to DDT and pyrethroids. Analyses of the kdr frequencies in dead and surviving mosquitoes showed partial correlation between the kdr genotypes and resistance phenotypes, suggesting that the kdr mechanism may act with certain co-factors to be identified.
Conclusion
These results demonstrate the ongoing spread of kdr alleles in An. gambiae in Central Africa. The rapid evolution of insecticide resistance in this highly dynamic and genetically polymorphic species remains a challenge for its control.
doi:10.1186/1471-2334-9-163
PMCID: PMC2764715  PMID: 19793389
21.  Malaria vectors and transmission dynamics in Goulmoun, a rural city in south-western Chad 
Background
Knowledge of some baseline entomological data such as Entomological Inoculation Rates (EIR) is crucially needed to assess the epidemiological impact of malaria control activities directed either against parasites or vectors. In Chad, most published surveys date back to the 1960's. In this study, anopheline species composition and their relation to malaria transmission were investigated in a dry Sudanian savannas area of Chad.
Methods
A 12-month longitudinal survey was conducted in the irrigated rice-fields area of Goulmoun in south western Chad. Human landing catches were performed each month from July 2006 to June 2007 in three compounds (indoors and outdoors) and pyrethrum spray collections were conducted in July, August and October 2006 in 10 randomly selected rooms. Mosquitoes belonging to the Anopheles gambiae complex and to the An. funestus group were identified by molecular diagnostic tools. Plasmodium falciparum infection and blood meal sources were detected by ELISA.
Results
Nine anopheline species were collected by the two sampling methods. The most aggressive species were An. arabiensis (51 bites/human/night), An. pharoensis (12.5 b/h/n), An. funestus (1.5 b/h/n) and An. ziemanni (1.3 b/h/n). The circumsporozoite protein rate was 1.4% for An. arabiensis, 1.4% for An. funestus, 0.8% for An. pharoensis and 0.5% for An. ziemanni. Malaria transmission is seasonal, lasting from April to December. However, more than 80% of the total EIR was concentrated in the period from August to October. The overall annual EIR was estimated at 311 bites of infected anophelines/human/year, contributed mostly by An. arabiensis (84.5%) and An. pharoensis (12.2%). Anopheles funestus and An. ziemanni played a minor role. Parasite inoculation occurred mostly after 22:00 hours but around 20% of bites of infected anophelines were distributed earlier in the evening.
Conclusion
The present study revealed the implication of An. pharoensis in malaria transmission in the irrigated rice fields of Goulmoun, complementing the major role played by An. arabiensis. The transmission period did not depend upon irrigation. Correct use of insecticide treated nets in this area may be effective for vector control although additional protective measures are needed to prevent pre-bedtime exposure to the bites of infected anophelines.
doi:10.1186/1471-2334-9-71
PMCID: PMC2697161  PMID: 19463189
22.  Ecological niche partitioning between Anopheles gambiae molecular forms in Cameroon: the ecological side of speciation 
BMC Ecology  2009;9:17.
Background
Speciation among members of the Anopheles gambiae complex is thought to be promoted by disruptive selection and ecological divergence acting on sets of adaptation genes protected from recombination by polymorphic paracentric chromosomal inversions. However, shared chromosomal polymorphisms between the M and S molecular forms of An. gambiae and insufficient information about their relationship with ecological divergence challenge this view. We used Geographic Information Systems, Ecological Niche Factor Analysis, and Bayesian multilocus genetic clustering to explore the nature and extent of ecological and chromosomal differentiation of M and S across all the biogeographic domains of Cameroon in Central Africa, in order to understand the role of chromosomal arrangements in ecological specialisation within and among molecular forms.
Results
Species distribution modelling with presence-only data revealed differences in the ecological niche of both molecular forms and the sibling species, An. arabiensis. The fundamental environmental envelope of the two molecular forms, however, overlapped to a large extent in the rainforest, where they occurred in sympatry. The S form had the greatest niche breadth of all three taxa, whereas An. arabiensis and the M form had the smallest niche overlap. Correspondence analysis of M and S karyotypes confirmed that molecular forms shared similar combinations of chromosomal inversion arrangements in response to the eco-climatic gradient defining the main biogeographic domains occurring across Cameroon. Savanna karyotypes of M and S, however, segregated along the smaller-scale environmental gradient defined by the second ordination axis. Population structure analysis identified three chromosomal clusters, each containing a mixture of M and S specimens. In both M and S, alternative karyotypes were segregating in contrasted environments, in agreement with a strong ecological adaptive value of chromosomal inversions.
Conclusion
Our data suggest that inversions on the second chromosome of An. gambiae are not causal to the evolution of reproductive isolation between the M and S forms. Rather, they are involved in ecological specialization to a similar extent in both genetic backgrounds, and most probably predated lineage splitting between molecular forms. However, because chromosome-2 inversions promote ecological divergence, resulting in spatial and/or temporal isolation between ecotypes, they might favour mutations in other ecologically significant genes to accumulate in unlinked chromosomal regions. When such mutations occur in portions of the genome where recombination is suppressed, such as the pericentromeric regions known as speciation islands in An. gambiae, they would contribute further to the development of reproductive isolation.
doi:10.1186/1472-6785-9-17
PMCID: PMC2698860  PMID: 19460146
23.  Living at the edge: biogeographic patterns of habitat segregation conform to speciation by niche expansion in Anopheles gambiae 
BMC Ecology  2009;9:16.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1472-6785-9-16
PMCID: PMC2702294  PMID: 19460144
24.  Insecticide resistance in Anopheles gambiae from south-western Chad, Central Africa 
Malaria Journal  2008;7:192.
Background
Indoor residual spraying and insecticide-treated nets (ITN) are essential components of malaria vector control in Africa. Pyrethroids are the only recommended compounds for nets treatment because they are fast-acting insecticides with low mammalian toxicity. However, there is growing concern that pyrethroid resistance may threaten the sustainability of ITN scaling-up programmes. Here, insecticide susceptibility was investigated in Anopheles gambiae sensu lato from an area of large scale ITN distribution programme in south-western Chad.
Methods
Susceptibility to 4% DDT, 0.05% deltamethrin, 0.75% permethrin, 0.1% bendiocarb and 5% malathion was assessed using the WHO standard procedures for adult mosquitoes. Tests were carried out with two to four days-old, non-engorged female mosquitoes. The An. gambiae Kisumu strain was used as a reference. Knockdown effect was recorded every 5 min and mortality scored 24 h after exposure. Mosquitoes were identified to species and molecular form by PCR-RFLP and genotypes at the kdr locus were determined in surviving specimens by Hot Oligonucleotide Ligation Assay (HOLA).
Results
During this survey, full susceptibility to malathion was recorded in all samples. Reduced susceptibility to bendiocarb (mortality rate of 96.1%) was found in one sample out of nine assayed. Increased tolerance to pyrethroids was detected in most samples (8/9) with mortality rates ranging from 70.2 to 96.6% for deltamethrin and from 26.7 to 96.3% for permethrin. Pyrethroid tolerance was not associated with a significant increase of knock-down times. Anopheles arabiensis was the predominant species of the An. gambiae complex in the study area, representing 75 to 100% of the samples. Screening for kdr mutations detected the L1014F mutation in 88.6% (N = 35) of surviving An. gambiae sensu stricto S form mosquitoes. All surviving An. arabiensis (N = 49) and M form An. gambiae s.s. (N = 1) carried the susceptible allele.
Conclusion
This first investigation of malaria vector susceptibility to insecticides in Chad revealed variable levels of resistance to pyrethroid insecticides (permethrin and deltamethrin) in most An. gambiae s.l. populations. Resistance was associated with the L1014F kdr mutation in the S form of An. gambiae s.s.. Alternative mechanisms, probably of metabolic origin are involved in An. arabiensis. These results emphasize the crucial need for insecticide resistance monitoring and in-depth investigation of resistance mechanisms in malaria vectors in Chad. The impact of reduced susceptibility to pyrethroids on ITN efficacy should be further assessed.
doi:10.1186/1475-2875-7-192
PMCID: PMC2566574  PMID: 18823537
25.  Insertion polymorphisms of SINE200 retrotransposons within speciation islands of Anopheles gambiae molecular forms 
Malaria Journal  2008;7:163.
Background
SINEs (Short INterspersed Elements) are homoplasy-free and co-dominant genetic markers which are considered to represent useful tools for population genetic studies, and could help clarifying the speciation processes ongoing within the major malaria vector in Africa, Anopheles gambiae s.s. Here, we report the results of the analysis of the insertion polymorphism of a nearly 200 bp-long SINE (SINE200) within genome areas of high differentiation (i.e. "speciation islands") of M and S A. gambiae molecular forms.
Methods
A SINE-PCR approach was carried out on thirteen SINE200 insertions in M and S females collected along the whole range of distribution of A. gambiae s.s. in sub-Saharan Africa. Ten specimens each for Anopheles arabiensis, Anopheles melas, Anopheles quadriannulatus A and 15 M/S hybrids from laboratory crosses were also analysed.
Results
Eight loci were successfully amplified and were found to be specific for A. gambiae s.s.: 5 on 2L chromosome and one on X chromosome resulted monomorphic, while two loci positioned respectively on 2R (i.e. S200 2R12D) and X (i.e. S200 X6.1) chromosomes were found to be polymorphic. S200 2R12D was homozygote for the insertion in most S-form samples, while intermediate levels of polymorphism were shown in M-form, resulting in an overall high degree of genetic differentiation between molecular forms (Fst = 0.46 p < 0.001) and within M-form (Fst = 0.46 p < 0.001). The insertion of S200 X6.1 was found to be fixed in all M- and absent in all S-specimens. This led to develop a novel easy-to-use PCR approach to straightforwardly identify A. gambiae molecular forms. This novel approach allows to overcome the constraints associated with markers on the rDNA region commonly used for M and S identification. In fact, it is based on a single copy and irreversible SINE200 insertion and, thus, is not subjected to peculiar evolutionary patterns affecting rDNA markers, e.g. incomplete homogenization of the arrays through concerted evolution and/or mixtures of M and S IGS-sequences among the arrays of single chromatids.
Conclusion
The approach utilized allowed to develop new easy-to-use co-dominant markers for the analysis of genetic differentiation between M and S-forms and opens new perspectives in the study of the speciation process ongoing within A. gambiae.
doi:10.1186/1475-2875-7-163
PMCID: PMC2546427  PMID: 18724871

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