Steve Zinsou Hougbe

The emergence and widespread distribution of insecticide resistance in Anopheles gambiae s.l. raise serious concerns regarding the long-term effectiveness of vector control tools. Assessing resistance intensity provides more operationally relevant information than diagnostic-dose bioassays alone. The phenotypic intensity of resistance to pyrethroids (permethrin and deltamethrin), bendiocarb, chlorfenapyr and clothianidin in Anopheles gambiae s.l. was evaluated along a south-north transect in Benin using WHO tube tests and CDC bottle bioassays. Mortality was recorded at diagnostic doses and at higher concentrations, in accordance with WHO guidelines. At diagnostic doses, mortality rates observed with permethrin and deltamethrin were low, ranging approximately from 5-30% to 10-45%, respectively, depending on the study sites. Even at 5 × and 10 × concentrations, pyrethroid-induced mortality often remained below 90%, indicating high-intensity resistance. Resistance levels were consistently higher for permethrin than for deltamethrin, in both WHO tube tests and CDC bottle bioassays. In contrast, bendiocarb induced high mortality rates, generally exceeding 90-98% in most localities, with low to moderate resistance detected in only a few sites. Complete susceptibility of Anopheles gambiae s.l. was observed to chlorfenapyr and clothianidin, with mortality rates ≥ 98% across all communes. The intense and widespread pyrethroid resistance observed in Benin represents a major threat to interventions relying on this insecticide class. WHO tube tests and CDC bottle bioassays showed similar qualitative trends, although differences in mortality levels were observed between the two methods. The relatively preserved susceptibility to bendiocarb, as well as to chlorfenapyr and clothianidin, supports their potential use in insecticide rotation strategies, including bi-treated insecticidal nets (e.g., Interceptor^® G2, PermaNet^® Dual, Yorkool^® G5, and Mont Inari) as well as indoor residual spraying campaigns using bendiocarb or clothianidin.

Pyrethroid resistance in Anopheles gambiae s.l. compromises malaria vector control in sub-Saharan Africa. In Benin, resistance has been documented for over

Pyrethroid resistance in Anopheles gambiae sensu lato threatens the efficacy of long-lasting insecticidal nets (LLINs) in Benin. District-level resistance

Malaria transmission in Benin is shaped by ecological heterogeneity, vector biting behavior, and increasing insecticide resistance. However, integrated multi-season data combining vector behavior, infection, and resistance remain limited in southern and central Benin. From December 2023 to October 2024, an entomological and molecular survey was conducted in six districts across southern and central Benin over dry and rainy season collection rounds. Adult mosquitoes were collected using human landing catches (HLC) and pyrethrum spray catches (PSC), identified morphologically and molecularly, and tested for Plasmodium falciparum infection by qPCR. WHO insecticide susceptibility tests were performed, complemented by molecular analyses of resistance mutations (kdr L1014F and ace-1R G119S). Anopheles gambiae s.l. was the predominant vector (> 98%), exhibiting both indoor and outdoor biting with site- and season-specific heterogeneity. Among indoor-resting females collected by PSC, human blood meals predominated, indicating high anthropophagy in this subset. EIRs were highest in Ifangni and Toffo, including during the dry season. Pyrethroid resistance was widespread, with kdr allele frequencies exceeding 65% in all sites; susceptibility to pirimiphos-methyl was generally higher but reduced mortality was observed in some districts. These findings support stratified, locally tailored vector control in southern and central Benin, including deployment of next-generation nets where pyrethroid resistance is intense, IRS guided by site-specific susceptibility profiles and rotation, and larval source management in areas with stable breeding habitats. Not applicable.

Insecticide resistance in Anopheles gambiae s.l. threatens malaria vector control strategies in sub-Saharan Africa. Organophosphates such as pirimiphos-methyl have been deployed in Benin through Indoor Residual Spraying (IRS) since 2013 as alternatives to pyrethroids. However, no published evidence had documented resistance to this compound. This study provides the first confirmation of reduced susceptibility to pirimiphos-methyl in Benin and investigates potential resistance mechanisms. The study was conducted from January 2022 to December 2024. Larvae of An. gambiae s.l. were collected from 20 districts along a north–south transect of Benin and reared to adults under insectary conditions. Susceptibility tests were performed according to WHO protocols using 0.25% pirimiphos-methyl-impregnated papers, and 24-h mortality was recorded. Molecular assays were conducted for species identification and detection of the Ace-1R (G119S) mutation. A total of 1,744 females were tested. Full susceptibility was observed in eight districts. Suspected and confirmed resistance was detected in eight and four districts, respectively, including key IRS-treated areas. Molecular analysis identified An. coluzzii (47.7%), An. gambiae s.s. (47.9%), and An. arabiensis (4.4%). The Ace-1R mutation occurred at low frequencies (< 5%), suggesting a limited role of target-site resistance. This study provides the first evidence of pirimiphos-methyl resistance in An. gambiae s.l. populations in Benin. The findings underline the need for: (i) strengthened and geographically expanded resistance surveillance, (ii) rotation or replacement of pirimiphos-methyl with new chemistries such as clothianidin or chlorfenapyr, and (iii) reinforcement of integrated resistance management strategies to preserve the effectiveness of IRS and other vector control interventions. These recommendations are essential for supporting national malaria control programs in evidence-based decision-making.

The Plasmodium transmission-blocking endosymbiont Microsporidia MB was previously identified in Anopheles gambiae s.l., but its association with the carria

Background: The intensity and spread of resistance of Anopheles populations to traditional neurotoxic insecticides pose a considerable threat to vector control tools. This resistance has led to the search for new classes of insecticides with a different mode of action. Thus, this study aimed to collect data on the susceptibility of An. gambiae s.l. to chlorfenapyr, a pyrrole insecticide in several communes of the south–north transect of Benin. Methods: An. gambiae s.l. were collected as larvae and pupae from eight communes located along the south–north transect of Benin and reared to adulthood. Female mosquitoes aged 3–5 days were exposed to pyrethroid-impregnated papers (permethrin, deltamethrin, alphacypermethrin) using WHO tube assays and to chlorfenapyr using the CDC bottle assay. Polymerase chain reaction was used to assess the molecular species composition in the An. gambiae s.l. and the frequency of knockdown resistance (kdr) mutations. Results: A high level of resistance to pyrethroids was observed at all sites. At all sites, mean mortality was 36.51% with deltamethrin, compared with 83.75% with pre-exposed delthamethrin, representing a 2.29-fold increase in mortality (P < 0.0001). Nevertheless, the different mosquito populations in each commune showed susceptibility to chlorfenapyr with 100% mortality already after 48 h of exposure. Furthermore, An. gambiae s.s., An. coluzzii and An. arabiensis were species of the Anopheles gambiae s.l. identified in sites, with An. coluzzii (60%) being the most represented, followed by An. gambiae s.s. (27%). Moreover, the west kdr frequency was very high (62 to 100%) in all sites, regardless of the species of the Anopheles gambiae s.l. Conclusion: The Anopheles gambiae s.l. populations tested were susceptibility to chlorfenapyr in the north–south transect of Benin despite their high level of resistance to pyrethroids. These results will be very useful for the National Malaria Control Program (NMCP) of Benin in the choice of insecticides according to the control strategy to be implemented.

Several studies have been conducted in Benin over the past two decades on insecticide resistance in mosquito vectors. These studies, by various authors, lead to diverse and scattered data. The present paper provides a retrospective analysis of these data to assess the current state of insecticide resistance and its evolution over two decades. Phenotypic trends were compared to mechanisms of insecticide resistance, focusing on the pyrethroids target-site mutation at codon 1014 of the voltage-gated sodium channel and the overproduction of detoxification enzymes capable of neutralizing insecticides before reaching their target. Data were collected from studies between 1996 and 1998 and from 2010 to 2024. For each selected study, the following information were extracted and organized in a Microsoft Excel spreadsheet: study year, adherence to WHO insecticide susceptibility testing protocols, mosquito species tested, study site characteristics, insecticides assessed, data source, and resistance mechanisms identified. Municipalities with data gaps exceeding five consecutive years were excluded. The earliest reported cases of insecticide resistance in Benin date back to 1963, involving organochlorines. Resistance to pyrethroids was first observed in 1999, initially limited in scope. However, from 2010 to 2024, resistance to all pyrethroids spread across all regions of Benin, reaching high levels. In some municipalities, mortality rates in Anopheles gambiae sensu lato (s.l.) populations exposed to permethrin-treated papers fell below 10%. The frequency of the kdr L1014F mutation has mirrored phenotypic resistance trends, increasing from 10% homozygous resistant (kdr/kdr) individuals in 2011 to 90% in 2024 in the municipality of Allada. Detoxification enzymes, such as α-esterase, β-esterase, monooxygenase and glutathione S-transferase showed low, but steadily increasing activity between 2015 and 2024. Resistance to bendiocarb, first reported in 2012, has shown minimal progression, while resistance to pirimiphos methyl has been observed in some municipalities since 2022. First observed with organochlorines around the 1960s, and later with pyrethroids in 1999, insecticide resistance in mosquito vectors has continued to intensify. Over the last 20 years, it has gradually expanded, now affecting all ecological environments in Benin. In this context, the National Malaria Control Programme should prioritize the use of new mosquito nets for future vector control campaigns in Benin.