Mosquitocidal effect of ivermectin-treated nettings and sprayed walls on Anopheles gambiae s.s.

Ivermectin (IVM) has been proposed as a new tool for malaria control as it is toxic on vectors feeding on treated humans or cattle. Nevertheless, IVM may have a direct mosquitocidal effect when applied on bed nets or sprayed walls. The potential for IVM application as a new insecticide for long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) was tested in this proof-of-concept study in a laboratory and semi-field environment. Laboratory-reared, insecticide-susceptible Kisumu Anopheles gambiae were exposed to IVM on impregnated netting materials and sprayed plastered- and mud walls using cone bioassays. The results showed a direct mosquitocidal effect of IVM on this mosquito strain as all mosquitoes died by 24 h after exposure to IVM. The effect was slower on the IVM-sprayed walls compared to the treated nettings. Further work to evaluate possibility of IVM as a new insecticide formulation in LLINs and IRS will be required.

Despite structural identity, ace-1 heterogenous duplication resistance alleles are quite diverse in Anopheles mosquitoes.

Anopheles gambiae s.l. has been the target of intense insecticide treatment since the mid-20th century to try and control malaria. A substitution in the ace-1 locus has been rapidly selected for, allowing resistance to organophosphate and carbamate insecticides. Since then, two types of duplication of the ace-1 locus have been found in An. gambiae s.l. populations: homogeneous duplications that are composed of several resistance copies, or heterogeneous duplications that contain both resistance and susceptible copies. The substitution induces a trade-off between resistance in the presence of insecticides and disadvantages in their absence: the heterogeneous duplications allow the fixation of the intermediate heterozygote phenotype. So far, a single heterogeneous duplication has been described in An. gambiae s.l. populations (in contrast with the multiple duplicated alleles found in Culex pipiens mosquitoes). We used a new approach, combining long and short-read sequencing with Sanger sequencing to precisely identify and describe at least nine different heterogeneous duplications, in two populations of An. gambiae s.l. We show that these alleles share the same structure as the previously identified heterogeneous and homogeneous duplications, namely 203-kb tandem amplifications with conserved breakpoints. Our study sheds new light on the origin and maintenance of these alleles in An. gambiae s.l. populations, and their role in mosquito adaptation.

Residual malaria transmission and the role of Anopheles arabiensis and Anopheles melas in central Senegal.

Understanding the behavior and ecology of local malaria vectors is essential for the effectiveness of the commonly used vector-targeted malaria control tools in areas of low malaria transmission. This study was conducted to determine species composition, biting behavior and infectivity of the major Anopheles vectors of Plasmodium falciparum in low transmission settings in central Senegal. Adult mosquitoes were collected using human landing catches during 2 consecutive nights and Pyrethrum Spray Catches in 30-40 randomly selected rooms, from July 2017 to December 2018 in 3 villages. Anopheline mosquitoes were morphologically identified using conventional keys; their reproductive status assessed by ovary dissections, and a sub-sample of Anopheles gambiae s.l. were identified to species level using polymerase chain reaction (PCR). Plasmodium sporozoite infections were detected using real-time quantitative PCR. During this study 3684 Anopheles were collected of which 97% were An. gambiae s.l., 0.6% were Anopheles funestus, and 2.4% were Anopheles pharoensis. Molecular identification of 1,877 An. gambiae s.l. revealed a predominance of Anopheles arabiensis (68.7%), followed by Anopheles melas (28.8%), and Anopheles coluzzii (2.1%). The overall human-biting rate of An. gambiae s.l. was highest in the inland site of Keur Martin with 4.92 bites per person per night, while it was similar in the deltaic site, Diofior (0.51) and the coastal site, Mbine Coly (0.67). Parity rates were similar in An. arabiensis (45%) and An. melas (42%). Sporozoite infections were detected in both An. arabiensis and An. melas with the respective infection rates of 1.39% (N = 8) and 0.41% (N = 1). Results suggest that low residual malaria in central Senegal is transmitted by An. arabiensis and An. melas. Consequently, both vectors will need to be targeted as part of malaria elimination efforts in this area of Senegal.

Distribution of Anopheles gambiae thioester-containing protein 1 alleles along malaria transmission gradients in The Gambia.

BACKGROUND: Thioester-containing protein 1 (TEP1) is a highly polymorphic gene playing an important role in mosquito immunity to parasite development and associated with Anopheles gambiae vectorial competence. Allelic variations in TEP1 could render mosquito either susceptible or resistant to parasite infection. Despite reports of TEP1 genetic variations in An. gambiae, the correlation between TEP1 allelic variants and transmission patterns in malaria endemic settings remains unclear. METHODS: TEP1 allelic variants were characterized by PCR from archived genomic DNA of > 1000 An. gambiae mosquitoes collected at 3 time points between 2009 and 2019 from eastern Gambia, where malaria transmission remains moderately high, and western regions with low transmission. RESULTS: Eight common TEP1 allelic variants were identified at varying frequencies in An. gambiae from both transmission settings. These comprised the wild type TEP1, homozygous susceptible genotype, TEP1s; homozygous resistance genotypes: TEP1rA and TEP1rB, and the heterozygous resistance genotypes: TEP1srA, TEP1srB, TEP1rArB and TEP1srArB. There was no significant disproportionate distribution of the TEP1 alleles by transmission setting and the temporal distribution of alleles was also consistent across the transmission settings. TEP1s was the most common in all vector species in both settings (allele frequencies: East = 21.4-68.4%. West = 23.5-67.2%). In Anopheles arabiensis, the frequency of wild type TEP1 and susceptible TEP1s was significantly higher in low transmission setting than in high transmission setting (TEP1: Z = - 4.831, P < 0.0001; TEP1s: Z = - 2.073, P = 0.038). CONCLUSIONS: The distribution of TEP1 allele variants does not distinctly correlate with malaria endemicity pattern in The Gambia. Further studies are needed to understand the link between genetic variations in vector population and transmission pattern in the study settings. Future studies on the implication for targeting TEP1 gene for vector control strategy such as gene drive systems in this settings is also recommended.

Anopheles gambiae s.l. swarms trapping as a complementary tool against residual malaria transmission in eastern Gambia.

Malaria remains a major health problem and vector control is an essential approach to decrease its burden, although it is threatened by insecticide resistance. New approaches for vector control are needed. The females of Anopheles gambiae s.l. mate once in their life and in the swarms formed by males. Trapping swarms of Anopheles gambiae s.l. males is a potential new intervention for vector control, alternative to the use of insecticides, as it would disrupt mating . The proof-of-concept pilot study aiming at investigating swarm trapping as a potential vector control intervention, was carried out in 6 villages as in eastern Gambia. Swarms of Anopheles gambiae s.l. were identified and their size, height, and duration determined during the baseline year. Swarm trapping by local volunteers was implemented the following transmission season in 4 villages while the other 2 villages were taken as controls. Entomological outcomes were monitored by Human Landing Catches and Pyrethrum Spray Catches. A cross-sectional survey to determine malaria prevalence was carried out at the peak of the malaria transmission season for two consecutive years. At baseline, 23 swarming sites of Anopheles gambiae s.l. were identified. Before the intervention, mean indoor resting density per house and malaria prevalence were similar between control and intervention villages. Following the intervention, Anopheles gambiae s.l. indoor resting density was 44% lower in intervention than in control villages (adj IRR: 0.0.56; 95% CI 0.47-0.68); the odds of malaria infections were 68% lower in intervention than in control villages (OR: 0.32; 95% CI 0.11-0.97). Swarm trapping seems to be a promising, community-based vector control intervention that could reduce malaria prevalence by reducing vector density. Such results should be further investigated and confirmed by larger cluster-randomized trials.

Evolution of the Ace-1 and Gste2 Mutations and Their Potential Impact on the Use of Carbamate and Organophosphates in IRS for Controlling Anopheles gambiae s.l., the Major Malaria Mosquito in Senegal.

Widespread of insecticide resistance amongst the species of the Anopheles gambiae complex continues to threaten vector control in Senegal. In this study, we investigated the presence and evolution of the Ace-1 and Gste2 resistance genes in natural populations of Anopheles gambiae s.l., the main malaria vector in Senegal. Using historical samples collected from ten sentinel health districts, this study focused on three different years (2013, 2017, and 2018) marking the periods of shift between the main public health insecticides families (pyrethroids, carbamates, organophosphates) used in IRS to track back the evolutionary history of the resistance mutations on the Ace-1 and Gste2 loci. The results revealed the presence of four members of the Anopheles gambiae complex, with the predominance of An. arabiensis followed by An. gambiae, An. coluzzii, and An. gambiae-coluzzii hybrids. The Ace-1 mutation was only detected in An. gambiae and An. gambiae-coluzzii hybrids at low frequencies varying between 0.006 and 0.02, while the Gste2 mutation was found in all the species with a frequency ranging between 0.02 and 0.25. The Ace-1 and Gste2 genes were highly diversified with twenty-two and thirty-one different haplotypes, respectively. The neutrality tests on each gene indicated a negative Tajima's D, suggesting the abundance of rare alleles. The presence and spread of the Ace-1 and Gste2 resistance mutations represent a serious threat to of the effectiveness and the sustainability of IRS-based interventions using carbamates or organophosphates to manage the widespread pyrethroids resistance in Senegal. These data are of the highest importance to support the NMCP for evidence-based vector control interventions selection and targeting.

Spatial-temporal pattern of malaria in Burkina Faso from 2013 to 2020.

Despite the implementation of different strategies to fight against malaria in Burkina Faso since 2005, it remains today the leading cause of hospitalization and death. Adapting interventions to the spatial and temporal distribution of malaria could help to reduce this burden. This study aims to determine the structure and stability of malaria hotspots in Burkina Faso, with the objective of adapting interventions at small geographical scales. Data on malaria cases from 2013 to 2020 were acquired at municipalities level. Municipality-wise malaria endemicity levels were mapped through geographical information system (GIS) tools. Spatial statistical analysis using Kulldoff sweeps were carried out to identify malaria hotspots. Then we mapped the monthly malaria risk. Malaria is endemic in all the municipalities of Burkina Faso. However, two stable main spatial clusters (South-Western and Eastern part of the country) are emerging with seasonal reinforcement. Interventions targeting the identified clusters could significantly reduce the incidence of malaria in Burkina Faso. This also prompts for further studies to identify the local determinants of this high transmission for the future success of malaria control.

Evolution of the Pyrethroids Target-Site Resistance Mechanisms in Senegal: Early Stage of the Vgsc-1014F and Vgsc-1014S Allelic Frequencies Shift.

The evolution and spread of insecticide resistance mechanisms amongst malaria vectors across the sub-Saharan Africa threaten the effectiveness and sustainability of current insecticide-based vector control interventions. However, a successful insecticide resistance management plan relies strongly on evidence of historical and contemporary mechanisms circulating. This study aims to retrospectively determine the evolution and spread of pyrethroid resistance mechanisms among natural Anopheles gambiae s.l. populations in Senegal. Samples were randomly drawn from an existing mosquito sample, collected in 2013, 2017, and 2018 from 10 sentinel sites monitored by the Senegalese National Malaria Control Programme (NMCP). Molecular species of An. gambiae s.l. and the resistance mutations at the Voltage-gated Sodium Channel 1014 (Vgsc-1014) locus were characterised using PCR-based assays. The genetic diversity of the Vgsc gene was further analyzed by sequencing. The overall species composition revealed the predominance of Anopheles arabiensis (73.08%) followed by An. gambiae s.s. (14.48%), Anopheles coluzzii (10.94%) and Anopheles gambiae-coluzii hybrids (1.48%). Both Vgsc-1014F and Vgsc-1014S mutations were found in all studied populations with a spatial variation of allele frequencies from 3% to 90%; and 7% to 41%, respectively. The two mutations have been detected since 2013 across all the selected health districts, with Vgsc-L1014S frequency increasing over the years while Vgsc-1014F decreasing. At species level, the Vgsc-1014F and Vgsc-1014S alleles were more frequent amongst An. gambiae s.s. (70%) and An. arabiensis (20%). The Vgsc gene was found to be highly diversified with eight different haplotypes shared between Vgsc-1014F and Vgsc-1014S. The observed co-occurrence of Vgsc-1014F and Vgsc-1014S mutations suggest that pyrethroid resistance is becoming a widespread phenomenon amongst malaria vector populations, and the NMCP needs to address this issue to sustain the gain made in controlling malaria.

Influence of insecticide resistance on the biting and resting preferences of malaria vectors in the Gambia.

BACKGROUND: The scale-up of indoor residual spraying and long-lasting insecticidal nets, together with other interventions have considerably reduced the malaria burden in The Gambia. This study examined the biting and resting preferences of the local insecticide-resistant vector populations few years following scale-up of anti-vector interventions. METHOD: Indoor and outdoor-resting Anopheles gambiae mosquitoes were collected between July and October 2019 from ten villages in five regions in The Gambia using pyrethrum spray collection (indoor) and prokopack aspirator from pit traps (outdoor). Polymerase chain reaction assays were performed to identify molecular species, insecticide resistance mutations, Plasmodium infection rate and host blood meal. RESULTS: A total of 844 mosquitoes were collected both indoors (421, 49.9%) and outdoors (423, 50.1%). Four main vector species were identified, including An. arabiensis (indoor: 15%, outdoor: 26%); An. coluzzii (indoor: 19%, outdoor: 6%), An. gambiae s.s. (indoor: 11%, outdoor: 16%), An. melas (indoor: 2%, outdoor: 0.1%) and hybrids of An. coluzzii-An. gambiae s.s (indoors: 3%, outdoors: 2%). A significant preference for outdoor resting was observed in An. arabiensis (Pearson X2 = 22.7, df = 4, P<0.001) and for indoor resting in An. coluzzii (Pearson X2 = 55.0, df = 4, P<0.001). Prevalence of the voltage-gated sodium channel (Vgsc)-1014S was significantly higher in the indoor-resting (allele freq. = 0.96, 95%CI: 0.78-1, P = 0.03) than outdoor-resting (allele freq. = 0.82, 95%CI: 0.76-0.87) An. arabiensis population. For An. coluzzii, the prevalence of most mutation markers was higher in the outdoor (allele freq. = 0.92, 95%CI: 0.81-0.98) than indoor-resting (allele freq. = 0.78, 95%CI: 0.56-0.86) mosquitoes. However, in An. gambiae s.s., the prevalence of Vgsc-1014F, Vgsc-1575Y and GSTe2-114T was high (allele freq. = 0.96-1), but did not vary by resting location. The overall sporozoite positivity rate was 1.3% (95% CI: 0.5-2%) in mosquito populations. Indoor-resting An. coluzzii had mainly fed on human blood while indoor-resting An. arabiensis fed on animal blood. CONCLUSION: In this study, high levels of resistance mutations were observed that could be influencing the mosquito populations to rest indoors or outdoors. The prevalent animal-biting behaviour demonstrated in the mosquito populations suggest that larval source management could be an intervention to complement vector control in this setting.

Dynamic of resistance alleles of two major insecticide targets in Anopheles gambiae (s.l.) populations from Benin, West Africa.

BACKGROUND: Insecticide resistance is a growing concern for malaria control and vector control effectiveness relies on assessing it distribution and understanding its evolution. METHODS: We assessed resistance levels and the frequencies of two major target-site mutations, L1014F-VGSC and G119S-ace-1, conferring resistance to pyrethroids (PYRs) and carbamates/organophosphates (CXs/OPs) insecticides. These data were compared to those acquired between 2006 and 2010 to follow resistance evolutionary trends over ten years. RESULTS: We report the results of a 3-year survey (2013-2015) of insecticide resistance in 13 localities across the whole country of Benin. Permethrin (PYR) resistance was found in all populations tested, L1014F-VGSC being almost fixed everywhere, while bendiocarb resistance was limited to a few localities, G119S-ace-1 remaining rare, with very limited variations during surveyed period. Interestingly, we found no effect of the type of insecticide pressure on the dynamics of these mutations. CONCLUSIONS: These results confirm both the high prevalence of PYR resistance and the potential of CXs/OPs as short- to medium-term alternatives in Benin. They also underline the need for regular resistance monitoring and informed management in their usage, as the G119S-ace-1 mutation is already present in Benin and surrounding countries. Their unwise usage would rapidly lead to its spread, which would jeopardize PYR-resistant Anopheles control.

Status of insecticide resistance in Anopheles gambiae (s.l.) of The Gambia.

BACKGROUND: Vector control activities, namely long-lasting insecticidal nets (LLIN) and indoor residual spraying (IRS), have contributed significantly to the decreasing malaria burden observed in The Gambia since 2008. Nevertheless, insecticide resistance may threaten such success; it is important to regularly assess the susceptibility of local malaria vectors to available insecticides. METHODS: In the transmission seasons of 2016 and 2017, Anopheles gambiae (s.l.) larvae were sampled in or around the nine vector surveillance sentinel sites of the Gambia National Malaria Control Programme (GNMCP) and in a few additional sampling points. Using WHO susceptibility bioassays, female adult mosquitoes were exposed to insecticide-impregnated papers. Molecular identification of sibling species and insecticide resistance molecular markers was done on a subset of 2000 female mosquitoes. RESULTS: A total of 4666 wild-caught female adult mosquitoes were exposed to either permethrin (n = 665), deltamethrin (n = 744), DDT (n = 1021), bendiocarb (n = 990) or pirimiphos-methyl (n = 630) insecticide-impregnated papers and control papers (n = 616). Among the 2000 anophelines, 1511 (80.7%) were Anopheles arabiensis, 204 (10.9%) Anopheles coluzzii, 75 (4%) Anopheles gambiae (s.s.), and 83 (4.4%) An. gambiae (s.s.) and An. coluzzii hybrids. There was a significant variation in the composition and species distribution by regions and year, P = 0.009. Deltamethrin, permethrin and DDT resistance was found in An. arabiensis, especially in the coastal region, and was mediated by Vgsc-1014F/S mutations (odds ratio = 34, P = 0.014). There was suspected resistance to pirimiphos-methyl (actellic 300CS) in the North Bank Region although only one survivor had the Ace-1-119S mutation. CONCLUSIONS: As no confirmed resistance to bendiocarb and actellic 300CS was detected, the national malaria control programme can continue using these insecticides for IRS. Nevertheless, the detection of Ace-1 119S mutation warrants extensive monitoring. The source of insecticide pressure driving insecticide resistance to pyrethroids and DDT detected at the coastal region should be further investigated in order to properly manage the spread of resistance in The Gambia.

The current distribution and characterization of the L1014F resistance allele of the kdr gene in three malaria vectors (Anopheles gambiae, Anopheles coluzzii, Anopheles arabiensis) in Benin (West Africa).

BACKGROUND: The fight against malaria faces various biological obstacles, including the resistance of parasites to anti-malarial drugs and the resistance of mosquito vectors to insecticides. The resistance of Anopheles gambiae sensu lato (s.l.) to pyrethroids, the only class of insecticides used to impregnate mosquito nets, is known in Benin; the expansion of this resistance is influenced by the existence of gene flow between species, otherwise by the presence or absence of the kdr mutation in them. The objective of this study is to determine the spatial distribution of An. gambiae and the level of expression of the pyrethroid resistance kdr gene in seven agro-ecological zones of Benin. METHODS: The study was conducted in 18 localities belonging to seven agro-ecological zones where environmental parameters varied. The sites represent the main areas of eco-epidemiological malaria in Benin. Anopheles gambiae larvae were collected in natural breeding sites using ladles and dipping method and reared under standard conditions. These larvae were reared under standard conditions of temperature and humidity (26 to 30 °C and 60 to 90%) at the insectarium of the Centre de Recherche Entomologique de Cotonou (CREC). Adult female mosquitoes having emerged are morphologically and molecularly identified. Homozygous resistant (1014F/1014F), homozygous sensitive (1014L/1014L) and heterozygous (1014F/1014L) genotypes of the L1014F kdr gene mutation are determined by PCR. RESULTS: A total of 677 An. gambiae was subjected at the PCR. The results revealed the presence of three vector species of the An. gambiae complex, of which 409 Anopheles coluzzii, 259 An. gambiae, 5 hybrids (An. coluzzii/An. gambiae) and 4 Anopheles arabiensis in the different agro-ecological zones. The four An. arabiensis were only found in Dassa, a locality in the cotton zone of central Benin. The frequency of distribution of the L1014F allele of the kdr gene varies from 84.48 to 100% in An. gambiae, from 80 to 100% in An. coluzzii and from 0 to 75% in An. arabiensis in the different agro-ecological zones. Moreover, a significant difference is generally observed in the distribution of the L1014F allele (P < 0.05). By comparing in pairs the distribution frequencies of this allele in the two species by agro-ecological zone, only a significant difference is noted in the central cotton and fishery zones (P = 0.0496). CONCLUSION: In summary, even if the data are in small portions, the An. Arabiensis species was found only in central Benin and the L1014F allele of the kdr gene is widespread and seems to fix in all the species recorded in the different agro-ecological zones. This situation amplifies the problem of resistance, which could eventually be a significant obstacle for the malaria vectors control. Similarly, a study of their genetic structure via the L1014F allele is necessary in order to put in place strategies to manage this resistance. These strategies will take into account both the ecology and the genetic diversity of the organisms involved to preserve the effectiveness of pyrethroids, the only insecticides used for the impregnation of mosquito nets.

Genetic structure of Anopheles gambiae s.s populations following the use of insecticides on several consecutive years in southern Benin.

BACKGROUND: Several studies have reported the strong resistance of Anopheles gambiae s.l. complex species to pyrethroids. The voltage-dependent sodium channel (Vgsc) gene is the main target of pyrethroids and DDT. In Benin, the frequency of the resistant allele (L1014F) of this gene varies along the north-south transect. Monitoring the evolution of resistance is necessary to better appreciate the genetic structure of vector populations in localities subject to the intensive use of chemicals associated with other control initiatives. The purpose of this study was to map the distribution of pyrethroid insecticide resistance alleles of the Kdr gene in malaria vectors in different regions and ecological facies in order to identify the evolutionary forces that might be the basis of anopheline population dynamics. METHODS: The characterization of Anopheles gambiae s.l. populations and resistance mechanisms were performed using adult mosquitoes obtained from larvae collected in the four agroecological zones in southern Benin. Genomic DNA extraction was performed on whole mosquitoes.The extracted genomic DNA from them were used for the molecular identification of species in Anopheles gambiae s.l. complex and the identification of genotypes related to pyrethroid resistance as the Kdr gene amino acid position 1014 in sodium channel. Molecular speciation and genotyping of Kdr resistant alleles (1014) were done using PCR.Genepop software version 4.2 was used to calculate allelic and genotypic frequencies in each agroecological zone. The p value of the allelic frequency was determined using the binomial test function in R version 3.3.3. The Hardy-Weinberg equilibrium was checked for each population with Genetics software version 1.3.8.1. The observed heterozygosity and the expected heterozygosity as well as the fixation index and genetic differentiation index within and between populations were calculated using Genepop software version 4.2. RESULTS: During the study period, Anopheles coluzzii was the major species in all agroecological zones while Anopheles gambiae was scarcely represented. Regardless of the species, resistant homozygote individuals (L1014F/L1014F) were dominant in all agroecological zones, showing a strong selection of the resistant allele (L1014F). All populations showed a deficit of heterozygosity. No genetic differentiation was observed between the different populations of the two species. For Anopheles coluzzii, there was a small differentiation among the populations of the central cotton and bar-lands zones. The genetic differentiation was modest among the population of the fisheries zone (Fst = 0.1295). The genetic differentiation was very high in the population of Anopheles gambiae of the bar-lands zone (Fst = 0.2408). CONCLUSION: This study revealed that the use of insecticides in Benin for years has altered the genetic structure of Anopheles gambiae s.s. populations in all agroecological zones of southern Benin. It would be desirable to orientate vector control efforts towards the use of insecticides other than pyrethroids and DDT or combinations of insecticides with different modes of action.

Adaptive deletion in resistance gene duplications in the malaria vector Anopheles gambiae.

While gene copy-number variations play major roles in long-term evolution, their early dynamics remains largely unknown. However, examples of their role in short-term adaptation are accumulating: identical repetitions of a locus (homogeneous duplications) can provide a quantitative advantage, while the association of differing alleles (heterogeneous duplications) allows carrying two functions simultaneously. Such duplications often result from rearrangements of sometimes relatively large chromosome fragments, and even when adaptive, they can be associated with deleterious side effects that should, however, be reduced by subsequent evolution. Here, we took advantage of the unique model provided by the malaria mosquito Anopheles gambiae s.l. to investigate the early evolution of several duplications, heterogeneous and homogeneous, segregating in natural populations from West Africa. These duplications encompass ~200 kb and 11 genes, including the adaptive insecticide resistance ace-1 locus. Through the survey of several populations from three countries over 3-4 years, we showed that an internal deletion of all coamplified genes except ace-1 is currently spreading in West Africa and introgressing from An. gambiae s.s. to An. coluzzii. Both observations provide evidences of its selection, most likely due to reducing the gene-dosage disturbances caused by the excessive copies of the nonadaptive genes. Our study thus provides a unique example of the early adaptive trajectory of duplications and underlines the role of the environmental conditions (insecticide treatment practices and species ecology). It also emphasizes the striking diversity of adaptive responses in these mosquitoes and reveals a worrisome process of resistance/cost trade-off evolution that could impact the control of malaria vectors in Africa.

The evolutionary fate of heterogeneous gene duplications: A precarious overdominant equilibrium between environment, sublethality and complementation.

Gene duplications occur at a high rate. Although most appear detrimental, some homogeneous duplications (identical gene copies) can be selected for beneficial increase in produced proteins. Heterogeneous duplications, which combine divergent alleles of a single locus, are seldom studied due to the paucity of empirical data. We investigated their role in an ongoing adaptive process at the ace-1 locus in Culex pipiens mosquitoes. We assessed the worldwide diversity of the ace-1 alleles (single-copy, susceptible S and insecticide-resistant R, and duplicated D that pair one S and one R copy), analysed their phylogeography and measured their fitness to understand their early dynamics using population genetics models. It provides a coherent and comprehensive evolutionary scenario. We show that D alleles are present in most resistant populations and display a higher diversity than R alleles (27 vs. 4). Most appear to result from independent unequal crossing-overs between local single-copy alleles, suggesting a recurrent process. Most duplicated alleles have a limited geographic distribution, probably resulting from their homozygous sublethality (HS phenotype). In addition, heterozygotes carrying different HS D alleles showed complementation, indicating different recessive lethal mutations. Due to mosaic insecticide control practices, balancing selection (overdominance) plays a key role in the early dynamics of heterogeneous duplicated alleles; it also favours a high local polymorphism of HS D alleles in natural populations (overdominance reinforced by complementation). Overall, our study shows that the evolutionary fate of heterogeneous duplications (and their long-term role) depends on finely balanced selective pressures due to the environment and to their genomic structure.

The ace-1 Locus Is Amplified in All Resistant Anopheles gambiae Mosquitoes: Fitness Consequences of Homogeneous and Heterogeneous Duplications.

Gene copy-number variations are widespread in natural populations, but investigating their phenotypic consequences requires contemporary duplications under selection. Such duplications have been found at the ace-1 locus (encoding the organophosphate and carbamate insecticides' target) in the mosquito Anopheles gambiae (the major malaria vector); recent studies have revealed their intriguing complexity, consistent with the involvement of various numbers and types (susceptible or resistant to insecticide) of copies. We used an integrative approach, from genome to phenotype level, to investigate the influence of duplication architecture and gene-dosage on mosquito fitness. We found that both heterogeneous (i.e., one susceptible and one resistant ace-1 copy) and homogeneous (i.e., identical resistant copies) duplications segregated in field populations. The number of copies in homogeneous duplications was variable and positively correlated with acetylcholinesterase activity and resistance level. Determining the genomic structure of the duplicated region revealed that, in both types of duplication, ace-1 and 11 other genes formed tandem 203kb amplicons. We developed a diagnostic test for duplications, which showed that ace-1 was amplified in all 173 resistant mosquitoes analyzed (field-collected in several African countries), in heterogeneous or homogeneous duplications. Each type was associated with different fitness trade-offs: heterogeneous duplications conferred an intermediate phenotype (lower resistance and fitness costs), whereas homogeneous duplications tended to increase both resistance and fitness cost, in a complex manner. The type of duplication selected seemed thus to depend on the intensity and distribution of selection pressures. This versatility of trade-offs available through gene duplication highlights the importance of large mutation events in adaptation to environmental variation. This impressive adaptability could have a major impact on vector control in Africa.

Estimation of allele-specific Ace-1 duplication in insecticide-resistant Anopheles mosquitoes from West Africa.

BACKGROUND: Identification of variation in Ace-1 copy number and G119S mutation genotype from samples of Anopheles gambiae and Anopheles coluzzii across West Africa are important diagnostics of carbamate and organophosphate resistance at population and individual levels. The most widespread and economical method, PCR-RFLP, suffers from an inability to discriminate true heterozygotes from heterozygotes with duplication. METHODS: In addition to PCR-RFLP, in this study three different molecular techniques were applied on the same mosquito specimens: TaqMan qPCR, qRTPCR and ddPCR. To group heterozygous individuals recorded from the PCR-RFLP analysis into different assumptive genotypes K-means clustering was applied on the Z-scores of data obtained from both the TaqMan and ddPCR methods. The qRTPCR analysis was used for absolute quantification of copy number variation. RESULTS: The results indicate that most heterozygotes are duplicated and that G119S mutation must now be regarded as a complex genotype ranging from primarily single-copy susceptible Glycine homozygotes to balanced and imbalanced heterozygotes, and multiply-amplified resistant Serine allele homozygotes. Whilst qRTPCR-based gene copy analysis suffers from some imprecision, it clearly illustrates differences in copy number among genotype groups identified by TaqMan or ddPCR. Based on TaqMan method properties, and by coupling TaqMan and ddPCR methods simultaneously on the same type of mosquito specimens, it demonstrated that the TaqMan genotype assays associated with the K-means clustering algorithm could provide a useful semi-quantitative estimate method to investigate the level of allele-specific duplication in mosquito populations. CONCLUSIONS: Ace-1 gene duplication is evidently far more complex in An. gambiae and An. coluzzii than the better-studied mosquito Culex quinquefasciatus, which consequently can no longer be considered an appropriate model for prediction of phenotypic consequences. These require urgent further evaluation in Anopheles. To maintain the sustained effectiveness carbamates and organophosphates as alternative products to pyrethroids for malaria vector control, monitoring of duplicated resistant alleles in natural populations is essential to guide the rational use of these insecticides.

An ace-1 gene duplication resorbs the fitness cost associated with resistance in Anopheles gambiae, the main malaria mosquito.

Widespread resistance to pyrethroids threatens malaria control in Africa. Consequently, several countries switched to carbamates and organophophates insecticides for indoor residual spraying. However, a mutation in the ace-1 gene conferring resistance to these compounds (ace-1(R) allele), is already present. Furthermore, a duplicated allele (ace-1(D)) recently appeared; characterizing its selective advantage is mandatory to evaluate the threat. Our data revealed that a unique duplication event, pairing a susceptible and a resistant copy of the ace-1 gene spread through West Africa. Further investigations revealed that, while ace-1(D) confers less resistance than ace-1(R), the high fitness cost associated with ace-1(R) is almost completely suppressed by the duplication for all traits studied. ace-1 duplication thus represents a permanent heterozygote phenotype, selected, and thus spreading, due to the mosaic nature of mosquito control. It provides malaria mosquito with a new evolutionary path that could hamper resistance management.

Phenotypic effects of concomitant insensitive acetylcholinesterase (ace-1(R)) and knockdown resistance (kdr(R)) in Anopheles gambiae: a hindrance for insecticide resistance management for malaria vector control.

BACKGROUND: Malaria is endemic in sub-Saharan Africa with considerable burden for human health. Major insecticide resistance mechanisms such as kdr(R) and ace-1(R) alleles constitute a hindrance to malaria vector control programs. Anopheles gambiae bearing both kdr and ace-1 resistant alleles are increasingly recorded in wild populations. In order to maintain the efficacy of vector control strategies, the characterization of concomitant kdr and ace-1 resistance, and their pleiotropic effects on malaria vector phenotype on insecticide efficacy are important. METHODS: Larval and adult bioassays were performed with different insecticide classes used in public health following WHO standard guidelines on four laboratory Anopheles gambiae strains, sharing the same genetic background but harboring distinct resistance status: KISUMU with no resistance allele; ACERKIS with ace-1(R) allele; KISKDR with kdr(R) allele and ACERKDRKIS with both resistance alleles' ace-1(R) and kdr(R) . RESULTS: Larval bioassays indicate that the homozygote resistant strain harboring both alleles (ACERKDRKIS) displayed slightly but significantly higher resistance level to various insecticides like carbamates (bendiocarb, p < 0.001; propoxur, p = 0.02) and organophosphates (chlorpyriphos-methyl, p = 0.002; fenitrothion, p < 0.001) when compared to ACERKIS strain. However, no differences were recorded between ACERKDRKIS and KISKDR resistance level against permethrin (Pyrethroid, p = 0.7) and DDT (Organochlorine, p = 0.24). For adult bioassays, the percentages of mosquitoes knocked down were significantly lower for ACERKDRKIS than for KISKDR with permethrin (p = 0.003) but not with deltamethrin. The percentage of mortality from adult bioassays was similar between ACERKDRKIS and ACERKIS with carbamates and organophosphates, or between ACERKDRKIS and KISKDR with pyrethroid and DDT. Concerning acetylcholinesterase enzyme, ACERKDRKIS strain showed similarAChE1 activity than that of ACERKIS. CONCLUSION: The presence of both kdr(R) and ace-1(R) alleles seems to increase the resistance levels to both carbamate and organophosphate insecticides and at operational level, may represent an important threat to malaria vector control programs in West Africa.

Characterization of swarming and mating behaviour between Anopheles coluzzii and Anopheles melas in a sympatry area of Benin.

The swarm structure of two sibling species, Anopheles gambiae coluzzii and Anopheles melas, was characterize to explore the ecological and environmental parameters associated with the formation of swarms and their spatial distribution. Swarms and breeding sites were searched and sampled between January and December 2010, and larval and adult samples were identified by PCR. During the dry season, 456 swarms of An. gambiae s.l. were sampled from 38 swarm sites yielding a total of 23,274 males and 76 females. Of these 38 swarming sites, 18 were composed exclusively of An. gambiae coluzzii and 20 exclusively of An. melas, presenting clear evidence of reproductive swarm segregation. The species makeup of couples sampled from swarms also demonstrated assortative mating. The swarm site localization was close to human dwellings in the case of the An. gambiae coluzzii and on salt production sites for An. melas. At the peak of the rainy season, swarms of An. melas were absent. These findings offer evidence that the ecological speciation of these two sibling species of mosquitoes is associated with spatial swarm segregation and assortative mating, providing strong support for the hypothesis that mate recognition is currently maintaining adaptive differentiation and promoting ecological speciation. Further studies on the swarming and mating systems of An. gambiae, with the prospect of producing a predictive model of swarm distribution, are needed to inform any future efforts to implement strategies based on the use of GMM or SIT.