Insertion polymorphism of transposable elements and population structure of Anopheles gambiae M and S molecular forms in Cameroon

The insertion polymorphism of five transposable element (TE) families was studied by Southern blots in several populations of the M and S molecular forms of the mosquito Anopheles gambiae sensu stricto from southern Cameroon. We showed that the mean TE insertion site number and the within-population insertion site polymorphism globally differed between the M and S molecular forms. The comparison of the TE insertion profiles of the populations revealed a significant differentiation between these two molecular forms (0.163 < Phi(ST) < 0.371). We cloned several insertions of a non-LTR retrotransposon (Aara8) that were fixed in one form and absent in the other one. The only insertion that could be clearly located on a chromosome arm mapped to cytological division 6 of chromosome X, confirming the importance of this region in the ongoing speciation between the M and S molecular forms.     

Evidence for subdivision within the M molecular form of Anopheles gambiae

The principal vector of malaria in sub-Saharan Africa, Anopheles gambiae is subdivided into two molecular forms M and S. Additionally, several chromosomal forms, characterized by the presence of various inversion polymorphisms, have been described. The molecular forms M and S each contain several chromosomal forms, including the Savanna, Mopti and Forest forms. The M and S molecular forms are now considered to be the reproductive units within A. gambiae and it has recently been argued that a low recombination rate in the centromeric region of the X chromosome has facilitated isolation between these forms. The status of the chromosomal forms remains unclear however. Therefore, we studied genetic differentiation between Savanna S, Forest S, Forest M and Mopti M populations using microsatellites. Genetic differentiation between Savanna S and Forest S populations is very low (F(ST) = 0.0053 +/- 0.0049), even across large distances. In comparison, the Mopti M and Forest M populations show a relatively high degree of genetic differentiation (F(ST) = 0.0406 +/- 0.0054) indicating that the M molecular form may not be a single entity, but could be subdivided into at least two distinct chromosomal forms. Previously it was proposed that inversions have played a role in the origin of species within the A. gambiae complex. We argue that a possible subdivision within the M molecular form could be understood through this process, with the acquisition of inversions leading to the expansion of the M molecular form into new habitat, dividing it into two distinct chromosomal forms.     

Evidence for X-linked introgression between molecular forms of Anopheles gambiae from Angola

The M and S molecular forms of the African malaria vector Anopheles gambiae (Diptera: Culicidae) are morphologically identical incipient species in which reproductive isolation is incomplete, enabling low-level gene flow between forms. In an attempt to find differences between the M and S forms, sequence variation was studied at loci along the X chromosome in adult female An. gambiae from Angola. A high proportion of M form specimens from Angola (79% of the 456 X chromosomes sampled) were found to contain a 16-bp insertion in intron 4 of the X-linked GPRCCK1 locus, relative to the AgamP3 release of the An. gambiae PEST genome sequence. The insertion was in Hardy-Weinberg equilibrium in Angolan M form populations. The same insertion was found in all S form specimens examined, regardless of where in Africa they were sampled, but was absent from a sample of M form specimens collected in Ghana, Bioko and Mali. In M form specimens from Angola, there was an association between alleles at the GPRCCK1 locus and those at a microsatellite locus, AGXH678, close to the centromere of the X chromosome, with significant linkage disequilibrium between loci separated by 0.472 Mbp (P < 0.033). We show that the insertion results from introgression from the S form into the M form, rather than from the retention of an ancestral character. Gene flow from the S to M form could allow genes of adaptive value to be transferred, including those conferring insecticide resistance and others influencing ecology and behaviour, and thus malaria transmission and control. We discuss factors that may have led to this introgression event.     

Reproductive Isolation among Sympatric Molecular Forms of An. gambiae from Inland Areas of South-Eastern Senegal

The Anopheles gambiae species complex includes at least seven morphologically indistinguishable species, one of which, Anopheles gambiae sensu stricto, is the primary mosquito vector responsible for the transmission of malaria across sub-Saharan Africa. Sympatric ecological diversification of An. gambiae s.s. is in progress within this complex, leading to the emergence of at least two incipient species (the M and S molecular forms now recognized as good species and named An. coluzzii and An. gambiae respectively) that show heterogeneous levels of divergence in most parts of Africa. However, this process seems to have broken down in coastal areas of West Africa at the extreme edge of the distribution. We undertook a longitudinal study to describe An. gambiae s.s. populations collected from two inland transects with different ecological characteristics in south-eastern Senegal. Analysis of samples collected from 20 sites across these two transects showed the M and S molecular forms coexisted at almost all sampled sites. Overall, similar hybridization rates (2.16% and 1.86%) were recorded in the two transects; sites with relatively high frequencies of M/S hybrids (up to 7%) were clustered toward the north-western part of both transects, often near urban settings. Estimated inbreeding indices for this putative speciation event varied spatially (range: 0.52–1), with hybridization rates being generally lower than expected under panmictic conditions. Such observations suggest substantial reproductive isolation between the M and S molecular forms, and further support the ongoing process of speciation in these inland areas. According to a recent reclassification of the An. gambiae complex, the M and S molecular forms from this zone correspond to An. coluzzii and An. gambiae, respectively. There is considerable evidence that these molecular forms differ in their behavioural and ecological characteristics. Detailed study of these characteristics will allow the development and implementation of better insect control strategies for combating malaria.     

Morphological differentiation may mediate mate-choice between incipient species of Anopheles gambiae s.s

The M and S molecular forms of Anopheles gambiae s.s. have been considered incipient species for more than ten years, yet the mechanism underlying assortative mating of these incipient species has remained elusive. The discovery of the importance of harmonic convergence of wing beat frequency in mosquito mating and its relation to wing size have laid the foundation for exploring phenotypic divergence in wing size of wild populations of the two forms. In this study, wings from field collected mosquitoes were measured for wing length and wing width from two parts of the sympatric distribution, which differ with respect to the strength of assortative mating. In Mali, where assortative mating is strong, as evidenced by low rates of hybridization, mean wing lengths and wing widths were significantly larger than those from Guinea-Bissau. In addition, mean wing widths in Mali were significantly different between molecular forms. In Guinea-Bissau, assortative mating appears comparatively reduced and wing lengths and widths did not differ significantly between molecular forms. The data presented in this study support the hypothesis that wing beat frequency may mediate assortative mating in the incipient species of A. gambiae and represent the first documentation of a morphological difference between the M and S molecular forms.     

Ecological zones rather than molecular forms predict genetic differentiation in the malaria vector Anopheles gambiae s.s. in Ghana

The malaria mosquito Anopheles gambiae s.s. is rapidly becoming a model for studies on the evolution of reproductive isolation. Debate has centered on the taxonomic status of two forms (denoted M and S) within the nominal taxon identified by point mutations in the X-linked rDNA region. Evidence is accumulating that there are significant barriers to gene flow between these forms, but that the barriers are not complete throughout the entire range of their distribution. We sampled populations from across Ghana and southern Burkina Faso, West Africa, from areas where the molecular forms occurred in both sympatry and allopatry. Neither Bayesian clustering methods nor F(ST)-based analysis of microsatellite data found differentiation between the M and S molecular forms, but revealed strong differentiation among different ecological zones, irrespective of M/S status and with no detectable effect of geographical distance. Although no M/S hybrids were found in the samples, admixture analysis detected evidence of contemporary interform gene flow, arguably most pronounced in southern Ghana where forms occur sympatrically. Thus, in the sampled area of West Africa, lack of differentiation between M and S forms likely reflects substantial introgression, and ecological barriers appear to be of greater importance in restricting gene flow.     

Environmental Factors Associated with the Distribution of Anopheles gambiae s.s in Ghana; an Important Vector of Lymphatic Filariasis and Malaria

Anopheles gambiae s.s mosquitoes are important vectors of lymphatic filariasis (LF) and malaria in Ghana. To better understand their ecological aspects and influence on disease transmission, we examined the spatial distribution of the An. gambiae (M and S) molecular forms and associated environmental factors, and determined their relationship with disease prevalence. Published and current data available on the An. gambiae species in Ghana were collected in a database for analysis, and the study sites were georeferenced and mapped. Using the An. gambiae s.s sites, environmental data were derived from climate, vegetation and remote-sensed satellite sources, and disease prevalence data from existing LF and malaria maps in the literature. The data showed that An. gambiae M and S forms were sympatric in most locations. However, the S form predominated in the central region, while the M form predominated in the northern and coastal savanna regions. Bivariate and multiple regression analyses identified temperature as a key factor distinguishing their distributions. An. gambiae M was significantly correlated with LF, and 2.5 to 3 times more prevalent in the high LF zone than low to medium zones. There were no significant associations between high prevalence An. gambiae s.s locations and malaria. The distribution of the An. gambiae M and S forms and the diseases they transmit in Ghana appear to be distinct, driven by different environmental factors. This study provides useful baseline information for disease control, and future work on the An. gambiae s.s in Ghana.     

DNA analysis of transferred sperm reveals significant levels of gene flow between molecular forms of Anopheles gambiae

Anopheles gambiae populations in west Africa are complex, being composed of multiple, sympatric subpopulations. Recent studies have failed to reveal significant genetic differences among subpopulations, stimulating a debate regarding the levels of gene flow among them. The observed homogeneity may be the consequence of substantial contemporary gene flow or it may be that reproductive isolation is complete, but too recent for the accumulation of significant levels of genic divergence. Here, we report the results of a study estimating contemporary levels of gene flow between An. gambiae subpopulations by analysing females and transferred sperm removed from their reproductive systems. A total of 251 female and associated sperm extracts was analysed from a single site in Mali. Two molecular forms of An. gambiae, the M- and S-forms, occurred in sympatry at this site. Overall, we found very strong positive assortative mating within forms, however, we did observe significant hybridization between forms. In the M subpopulation 2/195 females (1.03%) contained sperm from S-form males and in 55 S-form females we found one female containing M-form sperm (1.82%). We also identified a mated M xS hybrid adult female. From mating frequencies, we estimate the Nem between the M- and S-form at 16.8, and from the adult hybrid frequency at 5.6. These values are consistent with our earlier estimate, based on FST for 21 microsatellite loci in which Nem = 5.8. We conclude that the general lack of genetic divergence between the M and S subpopulations of An. gambiae can be explained entirely by contemporary gene flow.     

Locus- and population-specific selection and differentiation between incipient species of Anopheles gambiae

Anopheles gambiae, the primary mosquito vector of malaria in sub-Saharan Africa, is divided into 2 sympatric incipient species known as M form and S form. Recent genomic analysis of each form revealed that differentiation between forms is clustered into 3 unlinked regions of the genome. Here, we expand the investigation of these "genomic islands of speciation" to multiple populations, including all of the genes across one of the islands. Differentiation between the M and S forms in 2 of the islands is complete across all individuals in all populations, confirming that the M and S forms are reproductively isolated taxa. Differentiation at the third island (on chromosome 2R) is limited to Cameroon populations. There is reduced variation in the M form in Cameroon at this location and increased divergence to the outgroup Anopheles arabiensis, supporting an association of adaptation with reproductive isolation.     

Reduced recombination rate and genetic differentiation between the M and S forms of Anopheles gambiae s.s

Genetic differentiation between the largely sympatric molecular forms M and S of Anopheles gambiae appears mostly limited to division 6 and part of division 5 of the X chromosome. This region is adjacent to the centromere and includes the rDNA that was used to define these forms. This localized differentiation between populations that experience gene flow strongly suggests that this region contains genes responsible for reproductive isolation. Regions adjacent to centromeres are known to experience less recombination in several species and it has recently been suggested that low recombination rates can facilitate the accumulation and maintenance of isolation genes in partially isolated populations. Therefore, we measured the recombination rate in division 5D/6 directly and estimate that it is at least 16-fold reduced across this region compared to the remainder of the X chromosome. Additionally, sequence data from four loci from field-collected mosquitoes from several West African countries show very strong differentiation between the molecular forms in division 5D/6, whereas none was observed in two loci elsewhere on the X chromosome. Furthermore, genetic variation was substantially lower in division 5D/6 compared to the two reference loci, and the inferred genealogies of the division 5D/6 genes show patterns consistent with selective sweeps. This suggests that the reduced recombination rate has increased the effect of selection on this region and that our data are consistent with the hypothesis that reduced recombination rates can play a role in the accumulation of isolation genes in the face of gene flow.     

Multilevel analyses of genetic differentiation in Anopheles gambiae s.s. reveal patterns of gene flow important for malaria-fighting mosquito projects

Malaria control projects based on the introduction and spread of transgenes into mosquito populations depend on the extent of isolation between those populations. On the basis of the distribution of paracentric inversions, Anopheles gambiae has been subdivided into five subspecific chromosomal forms. Estimating gene flow between and within these forms of An. gambiae presents a number of challenges. We compared patterns of genetic divergence (F(ST)) between sympatric populations of the Bamako and Mopti forms at five sites. We used microsatellite loci within the j inversion on chromosome 2, which is fixed in the Bamako form but absent in the Mopti form, and microsatellites on chromosome 3, a region void of inversions. Estimates of genetic diversity and F(ST)'s suggest genetic exchanges between forms for the third chromosome but little for the j inversion. These results suggest a role for the inversion in speciation. Extensive gene flow within forms among sites resulted in populations clustering according to form despite substantial gene flow between forms. These patterns underscore the low levels of current gene flow between chromosomal forms in this area of sympatry. Introducing refractoriness genes in areas of the genome void of inversions may facilitate their spread within forms but their passage between forms may prove more difficult than previously thought.     

Insecticide resistance in the Anopheles gambiae complex in Benin: a nationwide survey

Benin has embraced World Health Organization-recommended preventive strategies to control malaria. Its National Malaria Control Programme is implementing and/or coordinating various actions and conducting evaluation trials of mosquito control strategies. Mosquito control is based on the use of insecticide-treated nets and indoor residual spraying, but the efficacy of these strategies to control malaria vectors is endangered by insecticide resistance. Here, we present the results of a nationwide survey on the status of insecticide susceptibility and resistance in Anopheles gambiae s.l. (Diptera: Culicidae) carried out in Benin in 2006-2007 (i.e. before extensive vector control was undertaken). Overall, our study showed that the S molecular form of An. gambiae s.s. predominates and is widely distributed across the country, whereas the frequency of the M form shows a strong decline with increasing latitude. Susceptibility to DDT, permethrin, carbosulfan and chlorpyrifos-methyl was assessed; individual mosquitoes were identified for species and molecular forms, and genotyped for the kdr and ace-1 loci. Full susceptibility to chlorpyrifos-methyl was recorded and very few samples displayed resistance to carbosulfan. High resistance levels to permethrin were detected in most samples and almost all samples displayed resistance to DDT. The kdr-Leu-Phe mutation was present in all localities and in both molecular forms of An. gambiae s.s. Furthermore, the ace-1(R) mutation was predominant in the S form, but absent from the M form. By contrast, no target modification was observed in Anopheles arabiensis. Resistance in the An. gambiae S molecular form in this study seemed to be associated with agricultural practices. Our study showed important geographic variations which must be taken into account in the vector control strategies that will be applied in different regions of Benin. It also emphasizes the need to regularly monitor insecticide resistance across the country and to adapt measures to manage resistance.     

Larval habitat segregation between the molecular forms of the mosquito Anopheles gambiae in a rice field area of Burkina Faso, West Africa

In West Africa, lineage splitting between the M and S molecular forms of the major Afro-tropical malaria mosquito, Anopheles gambiae (Diptera: Culicidae), is thought to be driven by ecological divergence, occurring mainly at the larval stage. Here, we present evidence for habitat segregation between the two molecular forms in and around irrigated rice fields located within the humid savannahs 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 field area into the surrounding savannah was conducted from June to November 2009. Analysis revealed that the two molecular forms and their sibling species Anopheles arabiensis are not randomly distributed in the area. A major ecological gradient was extracted in relation to the perimeter of the rice fields. The M form was associated with larger breeding sites mostly consisting of rice 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 the suggestion that the forms have different larval habitat requirements. Segregation appears to be clearly linked to anthropogenic permanent habitats and the community structure they support.     

Evidence of introgression of the ace-1(R) mutation and of the ace-1 duplication in West African Anopheles gambiae s. s

Background

The role of inter-specific hybridisation is of particular importance in mosquito disease vectors for predicting the evolution of insecticide resistance. Two molecular forms of Anopheles gambiae s.s., currently recognized as S and M taxa, are considered to be incipient sibling species. Hybrid scarcity in the field was suggested that differentiation of M and S taxa is maintained by limited or absent gene flow. However, recent studies have revealed shared polymorphisms within the M and S forms, and a better understanding of the occurrence of gene flow is needed. One such shared polymorphism is the G119S mutation in the ace-1 gene (which is responsible for insecticide resistance); this mutation has been described in both the M and S forms of A. gambiae s.s.

Methods and Results

To establish whether the G119S mutation has arisen independently in each form or by genetic introgression, we analysed coding and non-coding sequences of ace-1 alleles in M and S mosquitoes from representative field populations. Our data revealed many polymorphic sites shared by S and M forms, but no diversity was associated with the G119S mutation. These results indicate that the G119S mutation was a unique event and that genetic introgression explains the observed distribution of the G119S mutation within the two forms. However, it was impossible to determine from our data whether the mutation occurred first in the S form or in the M form. Unexpectedly, sequence analysis of some resistant individuals revealed a duplication of the ace-1 gene that was observed in both A. gambiae s.s. M and S forms. Again, the distribution of this duplication in the two forms most likely occurred through introgression.

Conclusions

These results highlight the need for more research to understand the forces driving the evolution of insecticide resistance in malaria vectors and to regularly monitor resistance in mosquito populations of Africa.     

A hybrid zone between two subspecies of Chorthippus parallelus. X-chromosome variation through a contact zone

Two grasshopper subspecies, Chorthippus parallelus parallelus (Cpp) and Chorthippus parallelus erythropus (Cpe), meet along the Pyrenees where they hybridize and produce a hybrid zone. A contact zone located in the Col de Portalet has been analyzed for the distribution of chromosome markers on the sex (X) chromosome in pure and hybrid populations. C-banding allowed us to distinguish both pure subspecific forms and recombinant forms, and to demonstrate their different frequencies through the contact zone. Interestingly, the distal C-band (P) on the X chromosome that characterizes Cpp individuals occurs at very high frequency through the zone and then drops sharply, close to where pure Cpe populations are found. A novel interstitial C-band (H) has been found, probably derived from that characterizing Cpe individuals (E). This marker band is only present in hybrid populations. These data are discussed in the light of the dynamics of the hybrid zone.     

Chromosome Differentiated Populations of Cruzii: Evidence for a Third Sibling Species

Anopheles cruziiis the most common species of mosquito in Southeast Brazil and a vector of human and monkey malaria. The banding pattern of the ovarian polytene chromosomes and the frequencies of paracentric inversions of individuals from two populations were studied. A new sequence of bands on the sex chromosome, defined as form C, was disclosed. In both populations where forms A (considered as standard) and C are sympatric no heterozygotes were detected. A sequence of events that could account for the observed changes in the banding sequences of the X chromosome forms was proposed. The frequencies of 22 paracentric inversions were used to assess panmixia and the results indicated the presence of two distinct genetic pools in each population. We consider these results as evidence of another sibling species in the taxon cruzii, characterized by a distinctive form of the X chromosome and provisionally designated Anopheles cruziispecies C.     

Assortative mating in mixed swarms of the mosquito Anopheles gambiae s.s. M and S molecular forms,in Burkina Faso,West Africa

The molecular form composition of Anopheles gambiae Giles s.s. (Diptera: Culicidae) mating swarms and the associated mating pairs (copulae) were investigated during two rainy seasons (July to October, 2005 and July to November, 2006) in the villages of Soumousso and Vallée du Kou (VK7). Although the habitats of these villages differ markedly, sympatric populations of M and S molecular forms of An. gambiae s.s. occur in both places periodically. The main aim was to assess the degree to which these molecular forms mate assortatively. In Soumousso, a wooded savannah habitat, the majority of swarm samples consisted of only S‐form males (21/28), although a few M‐form males were found in mixed M‐ and S‐form swarms. In VK7, a rice growing area, the majority of swarm samples consisted of only M‐form males (38/62), until October and November 2006, when there were nearly as many mixed‐form as single‐form swarms. Overall, ～60% of M‐ and S‐form swarms were temporally or spatially segregated; the two forms were effectively prevented from encountering each other. Of the remaining 40% of swarms, however, only about half were single‐form and the rest were mixed‐form. Of the 33 copulae collected from mixed‐form swarms, only four were mixed‐form pairs, significantly fewer than expected by random pairing between forms (χ² = 10.34, d.f. = 2, P < 0.01). Finally, all specimens of inseminated females were of the same form as the sperm contained within their spermatheca (n = 91), even for the four mixed‐form copulae. These findings indicate that assortative mating occurs within mixed‐form swarms, mediated most probably by close‐range mate recognition cues.     

Species abundance and insecticide resistance of Anopheles gambiae in selected areas of Ghana and Burkina Faso

The Ghanaian National Malaria Control Programme has prioritized insecticide-treated materials as a key strategy for malaria control. We report on a survey of the distribution of the molecular forms of Anopheles gambiae Giles (Diptera: Culicidae) and insecticide resistance (the kdr mutation), carried out by sampling mosquitoes from 11 locations in Ghana and one additional site in Burkina Faso. The molecular M and S forms of An. gambiae were found to occur in sympatry in southern Ghana. The S form predominated throughout its distribution in the coastal savannah, except at one location in the strand and mangrove zone where rice was cultivated. The M form was the only form collected in northern Ghana and was the predominant form (97.5%) in Burkina Faso. No M/S hybrids were detected. The kdr mutation was observed at very high frequencies (98-100%) within the S form but reached a maximum of only 3.38% in the M form in one population at an irrigation scheme in the Ghanaian coastal savannah zone.     

Distribution of a knockdown resistance mutation (L1014S) in Anopheles gambiae s.s. and Anopheles arabiensis in western and southern Kenya

In Kenya, insecticide-treated mosquito nets (ITNs) distributed to pregnant women and children under 5 years old through various programs have resulted in a significant reduction in malaria deaths. All of the World Health Organization-recommended insecticides for mosquito nets are pyrethroids, and vector mosquito resistance to these insecticides is one of the major obstacles to an effective malaria control program. Anopheles gambiae s.s. and Anopheles arabiensis are major malaria vectors that are widely distributed in Kenya. Two point mutations in the voltage-gated sodium channel (L1014F and L1014S) are associated with knockdown resistance (kdr) to DDT and pyrethroids in An. gambiae s.s. While the same point mutations have been reported to be rare in An. arabiensis, some evidence of metabolic resistance has been reported in this species. In order to determine the distribution of the point mutation L1014S in An. gambiae s.s. and An. arabiensis in southern and western Kenya, we collected larvae and screened for the mutation by DNA sequencing. We found high allelic and homozygous frequencies of the L1014S mutation in An. gambiae s.s. The L1014S mutation was also widely distributed in An. arabiensis, although the allelic frequency was lower than in An. gambiae s.s. The same intron sequence (length: 57 base) found in both species indicated that the mutation was introgressed by hybridization. The allelic frequency of L1014S was higher in both species in western regions, demonstrating the strong selection pressure imposed by long-lasting insecticide-treated nets (LLITN)/ITN on the An. gambiae s.s. and An. arabiensis populations in those areas. The present contribution of the L1014S mutation to pyrethroid resistance in An. arabiensis may be negligible. However, the homozygous frequency could increase with continuing selection pressure due to expanded LLITN coverage in the future.     

Resistance to carbosulfan in Anopheles gambiae from Ivory Coast,based on reduced sensitivity of acetylcholinesterase

Resistance to carbosulfan, a carbamate insecticide, was detected in field populations of the malaria vector mosquito Anopheles gambiae Giles (Diptera: Culicidae) from two ecologically contrasted localities near Bouaké, Ivory Coast: rural M'bé with predominantly M form of An. gambiae susceptible to pyrethroids; suburban Yaokoffikro with predominantly S form of An. gambiae highly resistant to pyrethroids (96% kdr). The discriminating concentration of 0.4% carbosulfan (i.e. double the LC100) was determined from bioassays with the susceptible An. gambiae Kisumu strain. Following exposure to the diagnostic dosage (0.4% carbosulfan for 1 h), mortality rates of female An. gambiae adults (reared from larvae collected from ricefields) were 62% and 29% of those from M'bé and Yaokoffikro, respectively, 24 h post-exposure. Exposure for 3 min to netting impregnated with the operational dosage of carbosulfan 200 mg/m2 gave mortality rates of 88% of those from M'bé and only 12.2% for Yaokoffikro. In each case the control untreated mortality rate was insignificant. Biochemical assays to detect possible resistance mechanism(s) revealed the presence of insensitive AChE in populations of An. gambiae at both localities, more prevalent in the S form at Yaokoffikro than in M form at M'bé, as expected from bioassays results. Our study demonstrates the need to monitor carbamate resistance among populations of the An. gambiae complex in Africa, to determine its spread and anticipate vector control failure if these insecticides are employed.