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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.

The members of Anopheles gambiae species complex are ubiquitous in Afro-tropics. They have been exposed continuously to insecticides, contributing to evolution of resistance within the complex. This study used whole genome sequence data from phase 3 of the An. gambiae 1000 Genomes Project to investigate the population structure and resistance mechanisms of a newly identified species, An. gambiae Bissau molecular form (Bissau) in The Gambia. Bissau exhibited subtle divergence from sister taxa An. coluzzii (Fixation index (F ST ) of 0.013) and An. gambiae s.s. (F ST of 0.023), suggesting ongoing geneflow among them. It also displayed a low but evident level of sub-clustering correlating with geographical location, contrary to sister taxa whose populations were not spatially structured. Additionally, Bissau displayed a higher number of substitutions, though at very low frequencies, in target site regions (specifically Vgsc and Ace-1 ) of the genome compared to its sister taxa. The well-established Vgsc -L995F mutation, normally associated with dichloro-diphenyl-trichloroethane (DDT) and pyrethroid resistance, was detected in all taxa. Also present, but at a lower frequency (< 20%) was N1570Y allele, normally associated with increased level of pyrethroid resistance when it co-occurs with L995F. Additionally, variants T791M and A1746S were found to occur alongside L995F in Bissau population at an elevated linkage disequilibrium (LD r 2  = 0.7). These findings accentuate the critical role this novel species could play on the emergence and spread of insecticide resistance in The Gambia.

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. 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. 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. 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.

Background Insecticide resistance amongst vector populations is a major challenge, exacerbated by the continued use of the same active ingredients. The present study assessed the impact of long-lasting insecticidal nets (LLINs) bi-treated with chlorfenapyr-alphacypermethrin (PY-CFP LLIN) or pyriproxyfen-alphacypermethrin (PY-PPF LLIN) on the genetic structure of resistant populations of Anopheles gambiae in 60 clusters divided into three arms from three districts in southern Benin. Methods The study was conducted between September 2019 and October 2021 in 123 villages grouped in 60 clusters. Mosquitoes were collected indoors and outdoors using human landing catches (HLCs) in 4 households in each cluster every 3 months. After morphological identification, a subsample of An. gambiae sensu lato ( s.l.) was analysed by PCR to detect the molecular species and the presence of L1014F vgsc-kdr and G119S -ace-1 mutations. Results Anopheles coluzzii (56.9%) and An. gambiae sensu stricto ( s.s .) (42.8%), with a few hybrids (0.2%), were identified within 4242 samples of An. gambiae tested. The frequency of L1014F vgsc-kdr decreased in An. coluzzii collected both indoors and outdoors locations in the PY-CFP LLIN and PY-PPF LLIN arms post-intervention compared to baseline. In An. gambiae , the frequency of the L1014F allele decreased in year one but increased above baseline in year 2. In both species, the allelic frequency of G119S -ace-1 was < 10%. For L1014F vgsc-kdr , the fixation index was positive ( F IS  > 0) in both species. However, it was negative ( F IS  < 0) for the presence of G119S -ace-1 . Weak genetic differentiation, especially in the PY-PPF LLIN and PY-CFP LLIN arms ( F ST  ≤ 0.05), was observed in An. gambiae s.s. populations with L1014F vgsc-kdr , while it was generally higher for both species with G119S -ace-1 . Conclusion The frequency of the L1014F vgsc-kdr resistance allele was high, while that of the G119S- ace-1 allele was low throughout the study period. Consistent changes in allele frequencies were not observed in any of the treatment arms suggesting that the pyrethroid component of dual AI (active ingredients) nets continues to select for the resistant allele and there is little if any evidence that the non-pyrethroid insecticide selects for the wild-type kdr allele.

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.

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.

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.

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.

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.

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.