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Although malaria is endemic in coastal Côte d’Ivoire, updated data on the resistance profile of the main vector, Anopheles gambiae sensu lato (s.l.), are still lacking, thus compromising decision-making for an effective vector control intervention. This study investigated the complex members and the insecticide resistance in the Anopheles gambiae s.l. populations in coastal Côte d’Ivoire. Between 2018 and 2020, cross sectional survey bioassays were conducted on female An . gambiae s.l. mosquitoes in three coastal health districts (Aboisso, Jacqueville and San Pedro) of Côte d’Ivoire. Pyrethroids deltamethrin, permethrin and alphacypermethrin (1X, 5X and 10X), clothianidin and synergist piperonyl butoxide (PBO) combined with pyrethroid 1X were tested using WHO tube bioassays. Chlorfenapyr was evaluated using CDC bottle bioassays. An . gambiae complex members and kdr 995F, kdr 995S and Ace-1 280S mutations were identified using polymerase chain reaction (PCR) technique. Overall, An . gambiae s.l. populations were primarily composed of Anopheles coluzzii (88.24%, n = 312), followed by Anopheles gambiae sensu stricto (7.56%) and hybrids (4.17%). These populations displayed strong resistance to pyrethroids at standard diagnostic doses, with mortality remaining below 98% even at 10X doses, except for alphacypermethrin in Aboisso. Pre-exposure to PBO significantly increased mortality but did not induce susceptibility, except for alphacypermethrin in Jacqueville. Clothianidin induced full susceptibility in Jacqueville and San Pedro, while chlorfenapyr induced susceptibility in Aboisso at 100 μg ai/bottle and all three districts at 200 μg ai/bottle. kdr 995F mutation dominated, with frequencies varying from 71.2% to 79.3%. kdr 995S had low, rates with frequencies ranging from 2.3% to 5.7%. Ace-1 280S prevalence varied between 4.2% and 42.9%. Coastal Côte d’Ivoire’s An . gambiae s.l. populations were mainly composed of An . coluzzii and showed high resistance to pyrethroids. Clothianidin, chlorfenapyr, and PBO with pyrethroids increased mortality, indicating their potential use as an alternative for malaria vector control.

Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassalé, Côte d'Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassalé. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassalé An. gambiae results from coupling of over-expressed target site allelic variants with heightened CYP6 P450 expression, which also provides resistance across contrasting insecticides. Mosquito populations displaying such a diverse basis of extreme and cross-resistance are likely to be unresponsive to standard insecticide resistance management practices.

Background Before implementing vector control interventions, it is important to assess the mosquito species composition and their biting and resting behaviour. In preparation for an indoor residual spraying campaign for malaria control in Sakassou, Central Côte d’Ivoire, baseline entomological data were collected to characterize the local vector species composition and their biting behaviour. Methods A longitudinal entomological survey was carried out from November 2018 to July 2020 with monthly assessments, except in April and May 2020 due to lockdown restriction of the COVID-19 pandemic. Mosquitoes were collected with the human landing catch method outside and inside of four houses, with two houses located in an urban setting and two in a rural setting. Additionally, in each setting, 15 houses were sampled using the pyrethrum spray catch method and two houses with Centers for Disease Control and Prevention light traps. Mosquitoes were identified morphologically to the lowest possible taxonomic level, while a subset of Anopheles gambiae sensu lato ( s.l .) specimens were further determined to species level by diagnostic polymerase chain reaction (PCR). Parity and Plasmodium sporozoite rates were estimated from a subsample of the caught specimens by dissecting the ovaries and screening heads and thoraces using enzyme-linked immunosorbent assays. Results Overall, 98,346 mosquitoes, including 91,799 anophelines and 6,547 culicines, were collected with counts following the rain pattern. Anopheles gambiae s.l. was the predominant taxon (90%). Out of 826 specimens successfully screened with a diagnostic PCR, all were identified as Anopheles coluzzii . The estimated biting rates of An.   coluzzii were high, peaking shortly after midnight. The overall Plasmodium falciparum infection rate in An. coluzzii was 0.02 (95% confidence interval: 0.01–0.03). Conclusions Anopheles coluzzii is the main malaria vector in Sakassou, exhibiting indoor resting and biting behaviours, with the peak biting rate after midnight when people are likely to be asleep. Given these characteristics, both insecticide-treated nets and indoor residual spraying hold promise for malaria vector control. As biting started early in the evening when people tend to be outside, outdoor interventions should also be considered.

Background Anopheles mosquito resistance to insecticide remains a serious threat to malaria vector control affecting several sub-Sahara African countries, including Côte d’Ivoire, where high pyrethroid, carbamate and organophosphate resistance have been reported. Since 2017, new insecticides, namely neonicotinoids (e.g.; clothianidin) and pyrroles (e.g.; chlorfenapyr) have been pre-qualified by the World Health Organization (WHO) for use in public health to manage insecticide resistance for disease vector control. Methods Clothianidin and chlorfenapyr were tested against the field-collected Anopheles gambiae populations from Gagnoa, Daloa and Abengourou using the WHO standard insecticide susceptibility biossays. Anopheles gambiae larvae were collected from several larval habitats, pooled and reared to adulthood in each site in July 2020. Non-blood-fed adult female mosquitoes aged 2 to 5 days were exposed to diagnostic concentration deltamethrin, permethrin, alpha-cypermethrin, bendiocarb, and pirimiphos-methyl. Clothianidin 2% treated papers were locally made and tested using WHO tube bioassay while chlorfenapyr (100 µg/bottle) was evaluated using WHO bottle assays. Furthermore, subsamples of exposed mosquitoes were identified to species and genotyped for insecticide resistance markers including the knock-down resistance ( kdr ) west and east, and acetylcholinesterase ( Ace -1) using molecular techniques. Results High pyrethroid resistance was recorded with diagnostic dose in Abengourou (1.1 to 3.4% mortality), in Daloa (15.5 to 33.8%) and in Gagnoa (10.3 to 41.6%). With bendiocarb, mortality rates ranged from 49.5 to 62.3%. Complete mortality (100% mortality) was recorded with clothianidin in Gagnoa, 94.9% in Daloa and 96.6% in Abengourou, while susceptibility (mortality > 98%) to chlorfenapyr 100 µg/bottle was recorded at all sites and to pirimiphos-methyl in Gagnoa and Abengourou. Kdr-west mutation was present at high frequency (0.58 to 0.73) in the three sites and Kdr - east mutation frequency was recorded at a very low frequency of 0.02 in both Abengourou and Daloa samples and absent in Gagnoa. The Ace -1 mutation was present at frequencies between 0.19 and 0.29 in these areas. Anopheles coluzzii  represented 100% of mosquitoes collected in Daloa and Gagnoa, and 72% in Abengourou. Conclusions This study showed that clothianidin and chlorfenapyr insecticides induce high mortality in the natural and pyrethroid-resistant An. gambiae populations in Côte d’Ivoire. These results could support a resistance management plan by proposing an insecticide rotation strategy for vector control interventions.

Background Malaria remains a threat in sub-Saharan Africa, particularly in Côte d'Ivoire, where it is endemic and represents the leading cause of hospital consultations, morbidity and mortality. The strong climatic variations that exist between coastal and savannah areas of Côte d’Ivoire suggest that vector control interventions should be scheduled according to the eco-epidemiological diversity. This study evaluates bioecological parameters of vectors and malaria transmission in two health districts, one coastal and one central of Côte d'Ivoire. Methods The study was conducted in the coastal (Jacqueville) and savannah (Béoumi) areas of Côte d’Ivoire from November 2018 to March 2019. Human Landing Catches (HLC) were conducted monthly at the study sites to determine Anopheles vector species composition, biting behaviour as well as entomological parameters of malaria transmission. Mosquitoes were collected over 12 h, from 6:00 pm to 6:00 am during 2 days per month. Mosquitoes infectivity was revealed by enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum circumsporozoite protein. A random sample of 100 Anopheles gambiae sensu lato ( s.l. ) including all CSP-positive females, were further classified by polymerase chain reaction (PCR) at the species and molecular form levels. Results In Jacqueville, 853 (99.7%) An. gambiae s.l ., and 3 (0.35%) Anopheles pharoensis were collected. In Béoumi, 811 (96.3%) An. gambiae s.l ., 23 (2.73%) Anopheles funestus and 8 (0. 95%) An. pharoensis have been found. Anopheles coluzzii represented the only species of the An. gambiae complex in Jacqueville. Among the An. gambiae s.l. samples tested in Beoumi, 29 (58%) were An. coluzzii and the rest 21 (42%) was An. gambiae sensu stricto . The human biting rate (HBR) in Jacqueville increased from 5.7 (b/p/n) in November to 17.3 (b/p/n) in March. Conversely in Béoumi the HBR decreased from 16.4 (b/p/n) in November to 0.69 (b/p/n) in March. In Jacqueville, the entomological inoculation rate (EIR) varies from 0.21 to 0.56 (ib/p/n) with the pic of 0.56 (ib/p/n) in February. In Béoumi no infection was detected in the parous An. gambiae s.l . samples tested during the study period. Conclusions This study evaluates bioecological parameters of vectors and malaria transmission in two health districts, one coastal and one central of Côte d'Ivoire.

From August 2020 to June 2021, we assessed the efficacy of SumiShield 50WG (clothianidin), Fludora Fusion 56.25WP-SB (mixture of clothianidin and deltamethrin) and Actellic 300CS (pirimiphos-methyl) in experimental huts when partially sprayed against wild, free-flying populations of Anopheles gambiae s.l. in Tiassalé, Côte d'Ivoire. A one-month baseline period of mosquito collections was conducted to determine mosquito density and resting behavior in unsprayed huts, after which two treatments of partial indoor residual spraying (IRS) were tested: spraying only the top half of walls + ceilings or only the bottom half of walls + ceilings. These were compared to fully sprayed applications using the three IRS insecticide formulations, during twenty nights per month of collection for nine consecutive months. Mortality was assessed at the time of collection, and after a 24 h holding period (Actellic) or up to 120 h (SumiShield and Fludora Fusion). Unsprayed huts were used as a negative control. The efficacy of each partially sprayed treatment of each insecticide was compared monthly to the fully sprayed huts over the study period with a non-inferiority margin set at 10%. The residual efficacy of each insecticide sprayed was also monitored. A total of 2197 Anopheles gambiae s.l. were collected during the baseline and 17,835 during the 9-month period after spraying. During baseline, 42.6% were collected on the bottom half versus 24.3% collected on the top half of the walls, and 33.1% on the ceilings. Over the nine-month post treatment period, 73.5% were collected on the bottom half of the wall, 11.6% collected on the top half and 14.8% on the ceilings. For Actellic, the mean mortality over the nine-month period was 88.5% [87.7, 89.3] for fully sprayed huts, 88.3% [85.1, 91.4] for bottom half + ceiling sprayed walls and 80.8% [74.5, 87.1] for the top half + ceiling sprayed huts. For Fludora Fusion an overall mean mortality of 85.6% [81.5, 89.7] was recorded for fully sprayed huts, 83.7% [82.9, 84.5] for bottom half + ceiling sprayed huts and 81.3% [79.6, 83.0] for the top half + ceiling sprayed huts. For SumiShield, the overall mean mortality was 86.7% [85.3, 88.1] for fully sprayed huts, 85.6% [85.4, 85.8] for the bottom half + ceiling sprayed huts and 76.9% [76.6, 77.3] for the top half + ceiling sprayed huts. For Fludora Fusion, both iterations of partial IRS were non-inferior to full spraying. However, for SumiShield and Actellic, this was true only for the huts with the bottom half + ceiling, reflecting the resting site preference of the local vectors. The results of this study suggest that partial spraying may be a way to reduce the cost of IRS without substantially compromising IRS efficacy.

Background Insecticide-based interventions, including insecticide-treated nets (ITNs) and indoor residual spraying (IRS), are central to malaria vector control in sub-Saharan Africa. In Côte d’Ivoire, increasing insecticide resistance in the key malaria vector, Anopheles gambiae sensu lato ( An. gambiae s.l.), has been reported across the country, potentially compromising the current ITN-based control strategy. To assess the feasibility of supplementing control efforts with IRS using clothianidin as an alternative insecticide in areas with high malaria prevalence, we have examined the intensity and molecular mechanisms of insecticide resistance in wild An. gambiae s.l. populations with a focus on pyrethroids and the neonicotinoid clothianidin. Methods Using the World Health Organization (WHO) insecticide susceptibility test, we assessed the intensity of insecticide resistance in 2- to 5-day-old female An. gambiae s.l. mosquitoes from Sakassou, Central Côte d’Ivoire, collected locally at the larval stage, against the pyrethroids alpha-cypermethrin, deltamethrin and permethrin, the neonicotinoid clothianidin and the organophosphate pirimiphos-methyl. To characterise the mechanisms underlying insecticide resistance, we conducted synergist assays using piperonyl butoxide (PBO) in combination with pyrethroids. Additionally, we performed diagnostic PCR to determine the An. gambiae s.l. sibling species and to characterise resistance mechanisms targeting the knockdown resistance ( kdr ) markers L995F and L995S and the insensitive acetylcholinesterase ( Ace-1 R ) G280S resistance marker. We also compared expression levels of the key cytochrome P450 monooxygenases (P450s) CYP6M2, CYP6P3, CYP6P4 and CYP6P5 between field-collected mosquitoes and those from a laboratory insecticide-susceptible colony. Results The diagnostic PCR identified all An. gambiae s.l. specimens that yielded a positive result as Anopheles coluzzii. Anopheles coluzzii individuals showed resistance to all pyrethroids tested, with very low mortality rates for alpha-cypermethrin and deltamethrin (range: 0% to 4%) and permethrin (range: 0% to 1%). In contrast, mortality rates against the organophosphate pirimiphos-methyl ranged from 52% to 97%. However, all mosquitoes remained fully susceptible to the neonicotinoid clothianidin (100% mortality). Pre-exposure to PBO increased the mortality rates following exposure to pyrethroids but did not restore susceptibility completely, with mortality rates of between 4.9% and 30.7% for alpha-cypermethrin, between 5.4% and 68.2% for deltamethrin and between 1.9% and 18.7% for permethrin. Among the four key resistance genes assessed in pyrethroid resistance, only the CYP6M2 gene showed significant overexpression (fold change 2.1, p -value = 0.025) in the field-sampled An. coluzzii compared to the susceptible laboratory colony. Taken together, our results suggest the involvement of a metabolic resistance mechanism. In addition to metabolic resistance, we detected all three target-site resistance alleles (i.e. kdr -L995F, kdr -L995S and Ace.1 -G280S), with kdr -L995F predominating, with allelic frequencies ranging from 53% to 71% across years. Conclusions Anopheles coluzzii mosquitoes from Sakassou show high levels of resistance to pyrethroids but remain susceptible to clothianidin, indicating that clothianidin-based IRS may serve as an effective complementary strategy for malaria control. Graphical Abstract

Background Due to the rapid expansion of pyrethroid-resistance in malaria vectors in Africa, Global Plan for Insecticide Resistance Management (GPIRM) has recommended the development of long-lasting insecticidal nets (LLINs), containing insecticide mixtures of active ingredients with different modes of action to mitigate resistance and improve LLIN efficacy. This good laboratory practice (GLP) study evaluated the efficacy of the chlorfenapyr and deltamethrin-coated PermaNet ® Dual, in comparison with the deltamethrin and synergist piperonyl butoxide (PBO)-treated PermaNet ® 3.0 and the deltamethrin-coated PermaNet ® 2.0, against wild free-flying pyrethroid-resistant Anopheles gambiae sensu lato ( s.l .), in experimental huts in Tiassalé, Côte d’Ivoire (West Africa). Methods PermaNet ® Dual, PermaNet ® 3.0 and PermaNet ® 2.0, unwashed and washed (20 washes), were tested against free-flying pyrethroid-resistant An. gambiae s.l. in the experimental huts in Tiassalé, Côte d’Ivoire from March to August 2020. Complementary laboratory cone bioassays (daytime and 3-min exposure) and tunnel tests (nightly and 15-h exposure) were performed against pyrethroid-susceptible An. gambiae sensu stricto ( s.s .) (Kisumu strain) and pyrethroid-resistant An. gambiae s.l. (Tiassalé strain). Results PermaNet ® Dual demonstrated significantly improved efficacy, compared to PermaNet ® 3.0 and PermaNet ® 2.0, against the pyrethroid-resistant An. gambiae s.l. Indeed, the experimental hut trial data showed that the mortality and blood-feeding inhibition in the wild pyrethroid-resistant An. gambiae s.l. were overall significantly higher with PermaNet ® Dual compared with PermaNet ® 3.0 and PermaNet ® 2.0, for both unwashed and washed samples. The mortality with unwashed and washed samples were 93.6 ± 0.2% and 83.2 ± 0.9% for PermaNet ® Dual, 37.5 ± 2.9% and 14.4 ± 3.9% for PermaNet ® 3.0, and 7.4 ± 5.1% and 11.7 ± 3.4% for PermaNet ® 2.0, respectively. Moreover, unwashed and washed samples produced the respective percentage blood-feeding inhibition of 41.4 ± 6.9% and 43.7 ± 4.8% with PermaNet ® Dual, 51.0 ± 5.7% and 9.8 ± 3.6% with PermaNet ® 3.0, and 12.8 ± 4.3% and − 13.0 ± 3.6% with PermaNet ® 2.0. Overall, PermaNet ® Dual also induced higher or similar deterrence, exophily and personal protection when compared with the standard PermaNet ® 3.0 and PermaNet ® 2.0 reference nets, with both unwashed and washed net samples. In contrast to cone bioassays, tunnel tests predicted the efficacy of PermaNet ® Dual seen in the current experimental hut trial. Conclusion The deltamethrin-chlorfenapyr-coated PermaNet ® Dual induced a high efficacy and performed better than the deltamethrin-PBO PermaNet ® 3.0 and the deltamethrin-only PermaNet ® 2.0, testing both unwashed and 20 times washed samples against the pyrethroid-susceptible and resistant strains of An. gambiae s.l. The inclusion of chlorfenapyr with deltamethrin in PermaNet ® Dual net greatly improved protection and control of pyrethroid-resistant An. gambiae populations. PermaNet ® Dual thus represents a promising tool, with a high potential to reduce malaria transmission and provide community protection in areas compromised by mosquito vector resistance to pyrethroids.

Resistance to insecticides in Anopheles mosquitoes threatens the effectiveness of malaria control, but the genetics of resistance are only partially understood. We performed a large scale multi-country genome-wide association study of resistance to two widely used insecticides: deltamethrin and pirimiphos-methyl, using sequencing data from An. gambiae and An. coluzzii from ten locations in West Africa. Resistance was highly multi-genic, multi-allelic and variable between populations. While the strongest and most consistent association with deltamethrin resistance came from Cyp6aa1 , this was based on several independent copy number variants (CNVs) in An. coluzzii , and on a non-CNV haplotype in An. gambiae . For pirimiphos-methyl, signals included Ace1 , cytochrome P450s, glutathione S-transferases and the nAChR target site of neonicotinoid insecticides. The regions around Cyp9k1 and the Tep family of immune genes showed evidence of cross-resistance to both insecticides. These locally-varying, multi-allelic patterns highlight the challenges involved in genomic monitoring of resistance, and may form the basis for improved surveillance methods. Insecticide resistance in mosquitoes threatens the success of malaria control programmes. This study found that in different populations of a malaria mosquito species in West Africa, resistance is associated with different genes or different mutations in the same set of genes.

Abstract The primary control methods for the African malaria mosquito, Anopheles gambiae, are based on insecticidal interventions. Emerging resistance to these compounds is therefore of major concern to malaria control programs. The organophosphate (OP), pirimiphos-methyl, is a relatively new chemical in the vector control armory but is now widely used in indoor-residual spray campaigns. While generally effective, phenotypic resistance has developed in some areas in malaria vectors. Here, we used a population genomic approach to identify novel mechanisms of resistance to pirimiphos-methyl in A. gambiae s.l mosquitoes. In multiple populations, we found large and repeated signals of selection at a locus containing a cluster of detoxification enzymes, some of whose orthologs are known to confer resistance to OPs in Culex pipiens. Close examination revealed a pair of alpha-esterases, Coeae1f and Coeae2f, and a complex and diverse pattern of haplotypes under selection in A. gambiae, A. coluzzii and A. arabiensis. As in C. pipiens, copy number variants have arisen at this locus. We used diplotype clustering to examine whether these signals arise from parallel evolution or adaptive introgression. Using whole-genome sequenced phenotyped samples, we found that in West Africa, a copy number variant in A. gambiae is associated with resistance to pirimiphos-methyl. Overall, we demonstrate a striking example of contemporary parallel evolution which has important implications for malaria control programs.