Phenotypic intensity of insecticide resistance in Anopheles gambiae sensu lato along the south–north transect in Benin

Phenotypic intensity of insecticide resistance in Anopheles gambiae sensu lato along the south–north transect in Benin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Phenotypic intensity of insecticide resistance in Anopheles gambiae sensu lato along the south–north transect in Benin Casimir Dossou Kpanou, Albert Sourou Salako, Mohammed N. A. Fagbemi, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8918876/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The emergence and widespread distribution of insecticide resistance in Anopheles gambiae s.l. raise serious concerns about the long-term effectiveness of vector control tools. Assessing resistance intensity provides more operationally relevant information than diagnostic-dose bioassays alone. Phenotypic resistance intensity to pyrethroids (permethrin and deltamethrin) and bendiocarb in Anopheles gambiae s.l. was assessed 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% and 10–45%, respectively, depending on the study site. Even at 5× and 10× concentrations, pyrethroid-induced mortality frequently remained below 90%, indicating high-intensity resistance. Resistance levels were consistently higher for permethrin than for deltamethrin in both WHO and CDC assays. In contrast, bendiocarb induced high mortality rates, generally exceeding 90–98% in most localities, with low to moderate resistance detected at only a few sites. Results obtained using both bioassay methods were concordant. The intense and widespread resistance to pyrethroids observed in Benin represents a major threat to interventions relying on this insecticide class. The relatively preserved susceptibility to bendiocarb supports its potential use within insecticide rotation strategies. Integrating resistance intensity data into national surveillance systems is essential to inform evidence-based vector control decision making. Health sciences/Diseases Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Zoology Anopheles gambiae s.l. resistance intensity pyrethroids bendiocarb Benin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Malaria control in sub-Saharan Africa relies primarily on insecticide-based interventions, particularly long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). These strategies have contributed substantially to reductions in malaria morbidity and mortality over the past two decades [1]. However, their effectiveness is increasingly threatened by the rapid spread and intensification of insecticide resistance in Anopheles mosquito populations[2–3]. In Benin, resistance to pyrethroids, the only insecticide class currently approved for LLIN impregnation was first reported in the late 1990s and has since expanded across all ecological zones of the country [4–6]. Continuous selection pressure exerted by large-scale LLIN deployment and IRS campaigns has likely contributed to the maintenance and intensification of resistance phenotypes [7–9]. Traditionally, resistance surveillance has relied on mortality assessment at diagnostic doses. However, this binary approach does not fully capture the operational significance of resistance. In response, the World Health Organization (WHO) introduced resistance intensity bioassays in 2016, recommending the use of multiple concentrations (5× and 10× the diagnostic dose) to better characterize resistance levels and their potential impact on vector control interventions [10]. Several studies conducted in West Africa have reported high-intensity pyrethroid resistance in Anopheles gambiae s.l., often associated with reduced effectiveness of insecticide-treated nets under experimental and operational conditions [11–12]. Nevertheless, data on the spatial distribution and national-scale patterns of resistance intensity in Benin remain limited, particularly along ecological gradients. This study aimed to assess and map the phenotypic intensity of insecticide resistance in Anopheles gambiae s.l. along a south–north transect in Benin using WHO tube tests and CDC bottle bioassays, in order to support resistance management strategies and evidence-based decision-making in vector control. Methods Study sites The study was conducted from September 2017 to September 2019 in 13 municipalities selected along a north–south transect in Benin in order to capture the full range of the country’s geo-climatic, ecological, and epidemiological settings (Fig. 1 ): Cotton-growing zone (Kandi, N’Dali, and Parakou): This zone is characterized by intensive cotton cultivation and heavy use of pesticides for cotton pest control. Multiple classes of insecticides are routinely applied, resulting in strong and sustained selection pressure on mosquito populations. Rice-growing zone (Malanville): The Malanville area includes a 70-hectare irrigated rice perimeter where two rice cropping cycles are practiced annually, including one during the dry season. This corresponds to off-season rice cultivation with prolonged availability of aquatic breeding habitats. Urban market-gardening zone: This zone includes the cities of Cotonou and Porto-Novo. Although no agricultural insecticide treatments are applied, vector control relies heavily on long-lasting insecticidal nets, aerosol insecticide sprays, and mosquito coils used by households. Cereal-growing zone: This zone comprises the localities of Missérété, Bantè, Ouidah, and Allada, located in the Ouémé, Collines, and Atlantique departments. Agricultural activities are dominated by cereal production, with moderate use of agrochemicals. Forest zone: This zone is represented by Bohicon, a municipality located in the Zou department. Agricultural activities mainly include millet and maize cultivation. Hilly zone: Located in central Benin, this zone is characterized by rocky outcrops and heterogeneous landscapes. It is represented by the municipalities of Dassa and Savè. Collection and rearing of immature mosquitoes During the rainy seasons, mosquito larvae and pupae were collected from breeding sites using a larval dipper. The larvae and pupae were then transported to the insectary and reared to adulthood. Adult mosquitoes were morphologically identified under a binocular microscope using the identification key of [13]. Unfed female Anopheles gambiae s.l. aged 2–5 days were used for clothianidin susceptibility tests in WHO tubes. Resistance intensity testing WHO susceptibility tube tests WHO susceptibility tube tests were conducted in accordance with WHO protocols [10] using 2–5-day-old female Anopheles gambiae s.l. Mosquitoes were exposed to WHO test papers impregnated with permethrin, deltamethrin, and bendiocarb at 1× (diagnostic dose), 5×, and 10× concentrations, respectively. Approximately 20–25 mosquitoes were introduced into each of four tubes lined with insecticide-treated papers for a 1-hour exposure period. In parallel, the same number of mosquitoes was introduced into two tubes lined with untreated papers serving as controls. During exposure, the number of knocked-down mosquitoes was recorded at regular time intervals (0, 10, 15, 20, 30, 45, and 60 min). After exposure, mosquitoes were transferred to holding tubes and maintained at 27 ± 2°C and 75 ± 5% relative humidity, with free access to a 10% sugar solution. Mortality was recorded 24 h post-exposure and interpreted according to WHO criteria [10]. Following bioassays, a subset of mosquitoes was preserved for molecular analyses. CDC bottle bioassay Insecticide resistance intensity was also assessed using the CDC bottle bioassay method. Female Anopheles gambiae s.l. aged 2–5 days were exposed for 30 min to 250-ml Wheaton glass bottles coated with pyrethroid insecticides (permethrin and deltamethrin) and a carbamate (bendiocarb) at 1×, 5×, and 10× diagnostic doses. For each insecticide dose, 20–25 mosquitoes were introduced into each of four coated bottles. A bottle coated with acetone only was used as a control. Mortality was recorded at the diagnostic time of 30 min in accordance with CDC guidelines [10]. Data analysis To determine the level of resistance intensity to pyrethroids, mortality rates recorded 24 h after WHO tube tests and at the 30-min diagnostic time for CDC bottle bioassays were interpreted according to WHO criteria [10]: Mortality ≥ 98%: the mosquito population was considered susceptible. Mortality between 90 and 97%: suspected resistance. Mortality < 90%: confirmed resistance. For resistance intensity assays, the following criteria were applied: 5× diagnostic dose: mortality ≥ 98% indicates low-intensity resistance. 10× diagnostic dose: mortality ≥ 98% indicates moderate-intensity resistance. Results Response of Anopheles gambiae s.l. populations to increasing doses of deltamethrin Using WHO tube assays, Anopheles gambiae s.l. populations from all thirteen localities exhibited very low mortality rates (< 50%) 24 h after exposure to the diagnostic dose of deltamethrin (0.05%), indicating widespread resistance across all study sites. Increasing the deltamethrin concentration to 5× resulted in higher mortality rates; however, mortality remained low, ranging from 40% to 85%, suggesting sustained resistance. At the 10× concentration, mortality increased further but resistance was not fully overcome, with mortality rates ranging from 66% to 98%. According to WHO criteria (WHO, 2016), high-intensity resistance was observed in all localities except Malanville, Kandi, and N’Dali, where moderate resistance intensity was recorded (Fig. 2 ; Supplementary Files, Table S1). CDC bottle bioassays similarly revealed low mortality rates (50–88%) at the diagnostic dose of deltamethrin (12.5 µg per bottle) at the 30-min diagnostic time, confirming generalized resistance across all study sites. Exposure to 5× and 10× doses resulted in increased mortality levels, which were generally higher than those observed using WHO tube assays; however, complete knockdown was not achieved within the diagnostic time. Consequently, mosquito populations from Bohicon, Porto-Novo, Cotonou, Kandi, Savè, Bantè, Ouidah, Missérété, Allada, and Dassa exhibited high-intensity resistance, whereas populations from Parakou, Malanville, and N’Dali showed moderate resistance intensity (Fig. 3 ; Supplementary Files, Table S2). Response of Anopheles gambiae s.l. populations to increasing doses of permethrin Using the WHO tube assay, Anopheles gambiae s.l. populations from the thirteen study localities exhibited very low mortality rates (14–52%) 24 h after exposure to the diagnostic dose of permethrin (0.75%), indicating generalized resistance across all sites. Increasing the permethrin concentration to 5× resulted in higher mortality rates; however, mortality remained relatively low, ranging from 46% to 91% (Fig. 4 ; Supplementary Files, Table S1), suggesting sustained resistance. At the 10× concentration, mortality increased further but resistance was not fully overcome, with mortality rates ranging from 77% to 99% (Fig. 4 ; Supplementary Files, Table S1). According to WHO criteria (WHO, 2016), high-intensity resistance was observed in all localities except Parakou and Kandi, where moderate resistance intensity was recorded. Using CDC bottle bioassays, Anopheles gambiae s.l. populations from all thirteen localities also showed low mortality rates (34–71%) at the diagnostic time of 30 min following exposure to the diagnostic dose of permethrin (21.5 µg per bottle), indicating widespread resistance (Fig. 5 ; Supplementary Files, Table S2). Exposure to 5× and 10× concentrations resulted in increased mortality; however, complete knockdown was not achieved within the diagnostic time. Mortality rates ranged from 76% to 97% at the 5× dose and from 91% to 100% at the 10× dose. Response of Anopheles gambiae s.l. populations to increasing doses of bendiocarb Using the WHO tube assay, only Anopheles gambiae s.l. populations from Dassa and N’Dali exhibited mortality rates below 90% (83% and 87%, respectively) 24 h after exposure to the diagnostic dose of bendiocarb (0.1%), indicating resistance in these localities. In contrast, populations from Cotonou, Porto-Novo, Bohicon, Savè, Parakou, Malanville, Bantè, and Ouidah showed mortality rates ranging from 90% to 97%, suggesting suspected resistance. However, populations from Missérété and Allada exhibited mortality rates between 98% and 100%, indicating full susceptibility to bendiocarb at 0.1% (Fig. 6 ; Supplementary Files, Table S1). Using the CDC bottle bioassay, Anopheles gambiae s.l. populations from Dassa, Savè, Parakou, Kandi, Bantè, Malanville, and N’Dali showed mortality rates below 98% (ranging from 93% to 97.5%), indicating reduced susceptibility. In contrast, all other populations (Cotonou, Porto-Novo, Bohicon, Allada, Missérété, and Ouidah) exhibited mortality rates between 98% and 100%, suggesting susceptibility of vectors from these localities to bendiocarb (Fig. 7 ; Supplementary Files, Table S2). Mapping of insecticide resistance intensity in malaria vectors The mapping of resistance intensity (Fig. 8 ) revealed a moderate to high distribution of resistance to pyrethroids (deltamethrin and permethrin) from southern to northern Benin. In contrast, resistance intensity to bendiocarb was low to moderate along the south–north transect, with complete susceptibility observed in the municipalities of Missérété, Ouidah, and Allada. Discussion This study demonstrates widespread and high-intensity phenotypic resistance to insecticides in Anopheles gambiae sensu lato along the south–north transect in Benin, as evidenced by the concordant results obtained using both WHO tube assays and CDC bottle bioassays. At diagnostic doses, mortality rates associated with pyrethroids were consistently low, ranging from approximately 5–30% for permethrin and 10–45% for deltamethrin, confirming pronounced resistance phenotypes across all ecological zones. Even at elevated concentrations (5× and 10×), pyrethroid-induced mortality frequently remained below 90%, with some populations exhibiting mortality rates as low as 60–75%, reflecting high-intensity resistance. Resistance levels were consistently higher for permethrin than for deltamethrin in both WHO and CDC assays, suggesting differential expression of resistance within the pyrethroid class. This pattern has been previously reported in Benin and other West African settings [5;11–12;14] In contrast, bendiocarb induced substantially higher mortality rates in both bioassays, generally exceeding 90–98% in most study sites. Only a limited number of localities showed reduced susceptibility, with mortality rates ranging from 80–89%, indicating low to moderate resistance intensity. These findings are consistent with previous studies reporting sustained efficacy of bendiocarb in areas with limited historical use of carbamates [7–8;15]. The strong concordance observed between WHO tube tests and CDC bottle bioassays reinforces the reliability of the resistance intensity profiles reported in this study. While WHO assays provide standardized thresholds for resistance classification, CDC assays offer complementary confirmation of resistant phenotypes under flexible exposure conditions [10;16]. Nevertheless, several limitations should be acknowledged. The study was conducted at discrete time points and did not account for temporal or seasonal variation in mortality rates. In addition, resistance mechanisms were not investigated. Although the transect encompassed the main ecological zones of the country, fine-scale spatial heterogeneity may not have been fully captured. Finally, the study focused on a limited number of insecticides and did not include newer compounds recently introduced into vector control programs. Despite these limitations, the consistency of mortality patterns observed across methods and insecticides provides robust evidence of widespread and high-intensity resistance in Benin. Conclusion This study highlights widespread and high-intensity phenotypic resistance to pyrethroids in Anopheles gambiae s.l. in Benin, with marked spatial variation along the south–north transect. The persistence of resistance even at elevated insecticide concentrations represents a major threat to the effectiveness of pyrethroid-based interventions. Conversely, the relatively preserved susceptibility to bendiocarb supports its continued relevance for indoor residual spraying. These findings underscore the importance of integrating resistance intensity data into national vector control strategies and promoting insecticide rotation and rational resistance management approaches. Abbreviations LLINs Long-Lasting Insecticide-treated mosquito Net IRS Indoor Residual Spraying NMCP National Malaria Control Program An. gambiae Anopheles gambiae CI Confidence Interval WHO World Health Organization Declarations Ethics approval and consent to participate Not applicable. Competing interests The authors declare no confict of interes Funding This study received no external funding. Author Contribution The original study was conducted by CDK, ASS, MNF, OD and SZH who also supplied the data. The idea for the study was conceptualized and generated by CDK, ASS, OD and ODC. Data were collected by CDK, ASS, SZH and MNF. RO and AD drafted the manuscript. Statistical data analysis by ASS and AS. PFL, RO and AS provided intellectual criticism on the content of the manuscript. All authors have read and approved the final submitted manuscript. Acknowledgement We would like to thank the staff of the Centre de Recherche Entomologique de Cotonou (CREC) for their dedication and commitment during the course of this work. Data Availability The data is available on reasonable request from the corresponding author. References Bhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, et al. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature . 2015;526:207–211. Ranson H, Lissenden N. Insecticide resistance in Anopheles gambiae : data, mechanisms, and consequences. Trends Parasitol . 2011;27:91–98. Hemingway J, Ranson H, Magill A, Kolaczinski J, Fornadel C, Gimnig J, et al. Averting a malaria disaster: will insecticide resistance derail malaria control? Lancet . 2016;387:1785–1788. Akogbeto M, Padonou G, Tchicaya E. Resistance of malaria vectors to pyrethroids in Benin. West Afr J Med . 1999;18:151–155. Yadouleton A, Padonou GG, Asidi A, Moiroux N, Agossa F, Djogbénou L, et al. Development of vegetable farming: a cause of the emergence of insecticide resistance in malaria vectors in Benin. Malar J . 2010;9:103. Hougbe SZ, Ossé RA, Azondékon R, Kpanou C, Ahouandjinou MJ, Affolabi Z‑K, et al. Two decades of insecticide resistance in Benin: a retrospective analysis of evolution and drivers. Malar J . 2025;24:156 Padonou GG, Agossa FR, Gnanguenon V, Yovogan B, Azondekon R, Akinro B, et al. Impact of indoor residual spraying with bendiocarb on malaria transmission in Benin. Parasites Vectors . 2012;5:147. Aïkpon R, Padonou GG, Agossa FR, Badirou K, Gnanguenon V, Akogbeto MC. Bendiocarb resistance in Anopheles gambiae populations from Benin. Parasites Vectors . 2014;7:100. Salako A, Ahogni I, Aïkpon R, Sidick A, Dagnon F, Sovi A, et al . Insecticide resistance status, frequency of L1014F kdr and G119S ace-1 mutations and expression of detoxification enzymesin Anopheles gambiae (s.l.) in two regions of northern Benin in preparation for indoor residual spraying Parasites & Vectors (2018) 11:618 World Health Organization (WHO). Test procedures for insecticide resistance monitoring in malaria vector mosquitoes. Geneva: WHO; 2016. Gnanguenon V, Agossa FR, Azondekon R, Badirou K, Aïkpon R, Baba-Moussa L, et al. Experimental hut evaluation of pyrethroid-treated nets in areas of high pyrethroid resistance in Benin. Malar J . 2015;14:315. Toe KH, Yameogo B, Dabiré RK, Diabate A, N’Fale S, Soma DD, et al. Increased pyrethroid resistance intensity in malaria vectors in Burkina Faso. Parasites Vectors . 2018;11:504. Coetzee M. Key to the females of Afrotropical Anopheles mosquitoes (Diptera: Culicidae). Malar J. 2020;19:70 Zoh MG, Koudou BG, Djenontin A, Adja AM, Ahoua Alou LP, Yeo L, et al. Pyrethroid resistance intensity in Anopheles gambiae populations from Côte d’Ivoire. Malar J . 2018;17:155. Churcher TS, Lissenden N, Griffin JT, Worrall E, Ranson H. Measuring the path toward malaria elimination. Science . 2016;344:1230–1232. World Health Organization (WHO). Global plan for insecticide resistance management in malaria vectors (GPIRM). Geneva: WHO; 2017 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8918876","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":597743114,"identity":"5f849072-7c93-45e0-b24f-8ea418a9c7ad","order_by":0,"name":"Casimir Dossou Kpanou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYJCCAwwGQAgGFQwwFtFazhCpBQQgChnbiNDC33784aEbBfeMzdnPHnzMO++wvDl78wGGHxXbcGqROJNjcDjHoNjMsicv2Zh322HDnT3HEhh7ztzG46AcBqCWBBuDAzlm0kAtjBtu5BgwM7bh0cL//AFEy/k3QC1zDtsT1iKRAHJYgpnBDZAtDYcTCWqRuPEGrMXYcsYbY8M5x9KTN5w5lnAQn1/4+9Mff875k2C4nT/H8MGbGmvbDcebDz74UYFbC1I4gEEzmDxAWD1CSx1RikfBKBgFo2BkAQCpkVsfvzswCgAAAABJRU5ErkJggg==","orcid":"","institution":"Institut Supérieur des Sciences et de Médecine Vétérinaire","correspondingAuthor":true,"prefix":"","firstName":"Casimir","middleName":"Dossou","lastName":"Kpanou","suffix":""},{"id":597743118,"identity":"17aeba8c-5333-4f84-afa7-4ff58d2e9fd6","order_by":1,"name":"Albert Sourou Salako","email":"","orcid":"","institution":"Institut Supérieur des Sciences et de Médecine Vétérinaire","correspondingAuthor":false,"prefix":"","firstName":"Albert","middleName":"Sourou","lastName":"Salako","suffix":""},{"id":597743119,"identity":"956eaea7-ba76-40ba-bb85-974181e76f0f","order_by":2,"name":"Mohammed N. 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07:42:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101013,"visible":true,"origin":"","legend":"\u003cp\u003eMortality rates of thirteen \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. populations after exposure to multiple diagnostic concentrations of deltamethrin using the CDC bottle bioassay.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8918876/v1/55f43cad900a5e195e520c8c.jpg"},{"id":103568934,"identity":"6bc479f7-bc68-497a-915f-114f632d0b12","added_by":"auto","created_at":"2026-02-27 07:41:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":119456,"visible":true,"origin":"","legend":"\u003cp\u003eMortality rates of thirteen \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. populations after exposure to multiple diagnostic concentrations of permethrin using the WHO tube assay\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8918876/v1/7ab6bbc63cd27f890141e3ed.jpg"},{"id":103569007,"identity":"fcbddaa3-ea4e-4c9d-87f5-7b116b121b75","added_by":"auto","created_at":"2026-02-27 07:42:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":96099,"visible":true,"origin":"","legend":"\u003cp\u003eMortality rates of thirteen \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. populations after exposure to multiple diagnostic concentrations of permethrin using the CDC bottle bioassay.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8918876/v1/4ea8da07a3ad8aa29951b47e.jpg"},{"id":103569005,"identity":"b715fa29-d69d-47ec-bb8e-12a462e7fe97","added_by":"auto","created_at":"2026-02-27 07:42:08","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":112827,"visible":true,"origin":"","legend":"\u003cp\u003eMortality rates of thirteen \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. populations after exposure to multiple diagnostic concentrations of bendiocarb using the WHO tube assay\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8918876/v1/29b91c608f8631f2998d8341.jpg"},{"id":103568944,"identity":"8258b5d5-0577-46f7-bbe8-d508a6bc47ad","added_by":"auto","created_at":"2026-02-27 07:42:01","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":105355,"visible":true,"origin":"","legend":"\u003cp\u003eMortality rates of thirteen \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. populations after exposure to multiple diagnostic concentrations of bendiocarb using the CDC bottle bioassay.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8918876/v1/46527b91c789837e8fa28c0c.jpg"},{"id":103568972,"identity":"ecc006a6-3a19-4b06-95ec-6140d5f700d3","added_by":"auto","created_at":"2026-02-27 07:42:07","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":132680,"visible":true,"origin":"","legend":"\u003cp\u003eMapping of insecticide resistance intensity in malaria vectors\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8918876/v1/fefec156a34681f3c884b4c7.jpg"},{"id":107860025,"identity":"0646bd83-796e-46db-84b9-d53a5bf1ff2d","added_by":"auto","created_at":"2026-04-27 05:10:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1108421,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8918876/v1/400b4517-a5f9-4b18-a4db-d8a0a801b525.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phenotypic intensity of insecticide resistance in Anopheles gambiae sensu lato along the south–north transect in Benin","fulltext":[{"header":"Background","content":"\u003cp\u003eMalaria control in sub-Saharan Africa relies primarily on insecticide-based interventions, particularly long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). These strategies have contributed substantially to reductions in malaria morbidity and mortality over the past two decades [1]. However, their effectiveness is increasingly threatened by the rapid spread and intensification of insecticide resistance in \u003cem\u003eAnopheles\u003c/em\u003e mosquito populations[2\u0026ndash;3].\u003c/p\u003e \u003cp\u003eIn Benin, resistance to pyrethroids, the only insecticide class currently approved for LLIN impregnation was first reported in the late 1990s and has since expanded across all ecological zones of the country [4\u0026ndash;6]. Continuous selection pressure exerted by large-scale LLIN deployment and IRS campaigns has likely contributed to the maintenance and intensification of resistance phenotypes [7\u0026ndash;9].\u003c/p\u003e \u003cp\u003eTraditionally, resistance surveillance has relied on mortality assessment at diagnostic doses. However, this binary approach does not fully capture the operational significance of resistance. In response, the World Health Organization (WHO) introduced resistance intensity bioassays in 2016, recommending the use of multiple concentrations (5\u0026times; and 10\u0026times; the diagnostic dose) to better characterize resistance levels and their potential impact on vector control interventions [10].\u003c/p\u003e \u003cp\u003eSeveral studies conducted in West Africa have reported high-intensity pyrethroid resistance in \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l., often associated with reduced effectiveness of insecticide-treated nets under experimental and operational conditions [11\u0026ndash;12]. Nevertheless, data on the spatial distribution and national-scale patterns of resistance intensity in Benin remain limited, particularly along ecological gradients.\u003c/p\u003e \u003cp\u003eThis study aimed to assess and map the phenotypic intensity of insecticide resistance in \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. along a south\u0026ndash;north transect in Benin using WHO tube tests and CDC bottle bioassays, in order to support resistance management strategies and evidence-based decision-making in vector control.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy sites\u003c/h2\u003e \u003cp\u003eThe study was conducted from September 2017 to September 2019 in 13 municipalities selected along a north\u0026ndash;south transect in Benin in order to capture the full range of the country\u0026rsquo;s geo-climatic, ecological, and epidemiological settings (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e):\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCotton-growing zone (Kandi, N’Dali, and Parakou):\u003c/h3\u003e\n\u003cp\u003eThis zone is characterized by intensive cotton cultivation and heavy use of pesticides for cotton pest control. Multiple classes of insecticides are routinely applied, resulting in strong and sustained selection pressure on mosquito populations.\u003c/p\u003e\n\u003ch3\u003eRice-growing zone (Malanville):\u003c/h3\u003e\n\u003cp\u003eThe Malanville area includes a 70-hectare irrigated rice perimeter where two rice cropping cycles are practiced annually, including one during the dry season. This corresponds to off-season rice cultivation with prolonged availability of aquatic breeding habitats.\u003c/p\u003e\n\u003ch3\u003eUrban market-gardening zone:\u003c/h3\u003e\n\u003cp\u003eThis zone includes the cities of Cotonou and Porto-Novo. Although no agricultural insecticide treatments are applied, vector control relies heavily on long-lasting insecticidal nets, aerosol insecticide sprays, and mosquito coils used by households.\u003c/p\u003e\n\u003ch3\u003eCereal-growing zone:\u003c/h3\u003e\n\u003cp\u003eThis zone comprises the localities of Miss\u0026eacute;r\u0026eacute;t\u0026eacute;, Bant\u0026egrave;, Ouidah, and Allada, located in the Ou\u0026eacute;m\u0026eacute;, Collines, and Atlantique departments. Agricultural activities are dominated by cereal production, with moderate use of agrochemicals.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eForest zone:\u003c/h2\u003e \u003cp\u003eThis zone is represented by Bohicon, a municipality located in the Zou department. Agricultural activities mainly include millet and maize cultivation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHilly zone:\u003c/h3\u003e\n\u003cp\u003eLocated in central Benin, this zone is characterized by rocky outcrops and heterogeneous landscapes. It is represented by the municipalities of Dassa and Sav\u0026egrave;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCollection and rearing of immature mosquitoes\u003c/h3\u003e\n\u003cp\u003eDuring the rainy seasons, mosquito larvae and pupae were collected from breeding sites using a larval dipper. The larvae and pupae were then transported to the insectary and reared to adulthood. Adult mosquitoes were morphologically identified under a binocular microscope using the identification key of [13]. Unfed female \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. aged 2\u0026ndash;5 days were used for clothianidin susceptibility tests in WHO tubes.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eResistance intensity testing\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eWHO susceptibility tube tests\u003c/h2\u003e \u003cp\u003eWHO susceptibility tube tests were conducted in accordance with WHO protocols [10] using 2\u0026ndash;5-day-old female \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. Mosquitoes were exposed to WHO test papers impregnated with permethrin, deltamethrin, and bendiocarb at 1\u0026times; (diagnostic dose), 5\u0026times;, and 10\u0026times; concentrations, respectively. Approximately 20\u0026ndash;25 mosquitoes were introduced into each of four tubes lined with insecticide-treated papers for a 1-hour exposure period. In parallel, the same number of mosquitoes was introduced into two tubes lined with untreated papers serving as controls.\u003c/p\u003e \u003cp\u003eDuring exposure, the number of knocked-down mosquitoes was recorded at regular time intervals (0, 10, 15, 20, 30, 45, and 60 min). After exposure, mosquitoes were transferred to holding tubes and maintained at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 75\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity, with free access to a 10% sugar solution. Mortality was recorded 24 h post-exposure and interpreted according to WHO criteria [10]. Following bioassays, a subset of mosquitoes was preserved for molecular analyses.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCDC bottle bioassay\u003c/h2\u003e \u003cp\u003eInsecticide resistance intensity was also assessed using the CDC bottle bioassay method. Female \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. aged 2\u0026ndash;5 days were exposed for 30 min to 250-ml Wheaton glass bottles coated with pyrethroid insecticides (permethrin and deltamethrin) and a carbamate (bendiocarb) at 1\u0026times;, 5\u0026times;, and 10\u0026times; diagnostic doses. For each insecticide dose, 20\u0026ndash;25 mosquitoes were introduced into each of four coated bottles. A bottle coated with acetone only was used as a control. Mortality was recorded at the diagnostic time of 30 min in accordance with CDC guidelines [10].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eTo determine the level of resistance intensity to pyrethroids, mortality rates recorded 24 h after WHO tube tests and at the 30-min diagnostic time for CDC bottle bioassays were interpreted according to WHO criteria [10]:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eMortality\u0026thinsp;\u0026ge;\u0026thinsp;98%: the mosquito population was considered susceptible.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eMortality between 90 and 97%: suspected resistance.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eMortality\u0026thinsp;\u0026lt;\u0026thinsp;90%: confirmed resistance.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eFor resistance intensity assays, the following criteria were applied:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e5\u0026times; diagnostic dose: mortality\u0026thinsp;\u0026ge;\u0026thinsp;98% indicates low-intensity resistance.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e10\u0026times; diagnostic dose: mortality\u0026thinsp;\u0026ge;\u0026thinsp;98% indicates moderate-intensity resistance.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eResponse of\u003c/b\u003e \u003cb\u003eAnopheles gambiae\u003c/b\u003e \u003cb\u003es.l. populations to increasing doses of deltamethrin\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing WHO tube assays, \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. populations from all thirteen localities exhibited very low mortality rates (\u0026lt;\u0026thinsp;50%) 24 h after exposure to the diagnostic dose of deltamethrin (0.05%), indicating widespread resistance across all study sites. Increasing the deltamethrin concentration to 5\u0026times; resulted in higher mortality rates; however, mortality remained low, ranging from 40% to 85%, suggesting sustained resistance. At the 10\u0026times; concentration, mortality increased further but resistance was not fully overcome, with mortality rates ranging from 66% to 98%. According to WHO criteria (WHO, 2016), high-intensity resistance was observed in all localities except Malanville, Kandi, and N\u0026rsquo;Dali, where moderate resistance intensity was recorded (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Supplementary Files, Table S1).\u003c/p\u003e \u003cp\u003eCDC bottle bioassays similarly revealed low mortality rates (50\u0026ndash;88%) at the diagnostic dose of deltamethrin (12.5 \u0026micro;g per bottle) at the 30-min diagnostic time, confirming generalized resistance across all study sites. Exposure to 5\u0026times; and 10\u0026times; doses resulted in increased mortality levels, which were generally higher than those observed using WHO tube assays; however, complete knockdown was not achieved within the diagnostic time. Consequently, mosquito populations from Bohicon, Porto-Novo, Cotonou, Kandi, Sav\u0026egrave;, Bant\u0026egrave;, Ouidah, Miss\u0026eacute;r\u0026eacute;t\u0026eacute;, Allada, and Dassa exhibited high-intensity resistance, whereas populations from Parakou, Malanville, and N\u0026rsquo;Dali showed moderate resistance intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Supplementary Files, Table S2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eResponse of\u003c/b\u003e \u003cb\u003eAnopheles gambiae\u003c/b\u003e \u003cb\u003es.l. populations to increasing doses of permethrin\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing the WHO tube assay, \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. populations from the thirteen study localities exhibited very low mortality rates (14\u0026ndash;52%) 24 h after exposure to the diagnostic dose of permethrin (0.75%), indicating generalized resistance across all sites. Increasing the permethrin concentration to 5\u0026times; resulted in higher mortality rates; however, mortality remained relatively low, ranging from 46% to 91% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Supplementary Files, Table S1), suggesting sustained resistance. At the 10\u0026times; concentration, mortality increased further but resistance was not fully overcome, with mortality rates ranging from 77% to 99% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Supplementary Files, Table S1). According to WHO criteria (WHO, 2016), high-intensity resistance was observed in all localities except Parakou and Kandi, where moderate resistance intensity was recorded.\u003c/p\u003e \u003cp\u003eUsing CDC bottle bioassays, \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. populations from all thirteen localities also showed low mortality rates (34\u0026ndash;71%) at the diagnostic time of 30 min following exposure to the diagnostic dose of permethrin (21.5 \u0026micro;g per bottle), indicating widespread resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Supplementary Files, Table S2). Exposure to 5\u0026times; and 10\u0026times; concentrations resulted in increased mortality; however, complete knockdown was not achieved within the diagnostic time. Mortality rates ranged from 76% to 97% at the 5\u0026times; dose and from 91% to 100% at the 10\u0026times; dose.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResponse of\u003c/b\u003e \u003cb\u003eAnopheles gambiae\u003c/b\u003e \u003cb\u003es.l. populations to increasing doses of bendiocarb\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing the WHO tube assay, only \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. populations from Dassa and N\u0026rsquo;Dali exhibited mortality rates below 90% (83% and 87%, respectively) 24 h after exposure to the diagnostic dose of bendiocarb (0.1%), indicating resistance in these localities. In contrast, populations from Cotonou, Porto-Novo, Bohicon, Sav\u0026egrave;, Parakou, Malanville, Bant\u0026egrave;, and Ouidah showed mortality rates ranging from 90% to 97%, suggesting suspected resistance. However, populations from Miss\u0026eacute;r\u0026eacute;t\u0026eacute; and Allada exhibited mortality rates between 98% and 100%, indicating full susceptibility to bendiocarb at 0.1% (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Supplementary Files, Table S1).\u003c/p\u003e \u003cp\u003eUsing the CDC bottle bioassay, \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. populations from Dassa, Sav\u0026egrave;, Parakou, Kandi, Bant\u0026egrave;, Malanville, and N\u0026rsquo;Dali showed mortality rates below 98% (ranging from 93% to 97.5%), indicating reduced susceptibility. In contrast, all other populations (Cotonou, Porto-Novo, Bohicon, Allada, Miss\u0026eacute;r\u0026eacute;t\u0026eacute;, and Ouidah) exhibited mortality rates between 98% and 100%, suggesting susceptibility of vectors from these localities to bendiocarb (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Supplementary Files, Table S2).\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMapping of insecticide resistance intensity in malaria vectors\u003c/h2\u003e \u003cp\u003eThe mapping of resistance intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) revealed a moderate to high distribution of resistance to pyrethroids (deltamethrin and permethrin) from southern to northern Benin. In contrast, resistance intensity to bendiocarb was low to moderate along the south\u0026ndash;north transect, with complete susceptibility observed in the municipalities of Miss\u0026eacute;r\u0026eacute;t\u0026eacute;, Ouidah, and Allada.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates widespread and high-intensity phenotypic resistance to insecticides in \u003cem\u003eAnopheles gambiae\u003c/em\u003e sensu lato along the south\u0026ndash;north transect in Benin, as evidenced by the concordant results obtained using both WHO tube assays and CDC bottle bioassays. At diagnostic doses, mortality rates associated with pyrethroids were consistently low, ranging from approximately 5\u0026ndash;30% for permethrin and 10\u0026ndash;45% for deltamethrin, confirming pronounced resistance phenotypes across all ecological zones.\u003c/p\u003e \u003cp\u003eEven at elevated concentrations (5\u0026times; and 10\u0026times;), pyrethroid-induced mortality frequently remained below 90%, with some populations exhibiting mortality rates as low as 60\u0026ndash;75%, reflecting high-intensity resistance. Resistance levels were consistently higher for permethrin than for deltamethrin in both WHO and CDC assays, suggesting differential expression of resistance within the pyrethroid class. This pattern has been previously reported in Benin and other West African settings [5;11\u0026ndash;12;14]\u003c/p\u003e \u003cp\u003eIn contrast, bendiocarb induced substantially higher mortality rates in both bioassays, generally exceeding 90\u0026ndash;98% in most study sites. Only a limited number of localities showed reduced susceptibility, with mortality rates ranging from 80\u0026ndash;89%, indicating low to moderate resistance intensity. These findings are consistent with previous studies reporting sustained efficacy of bendiocarb in areas with limited historical use of carbamates [7\u0026ndash;8;15].\u003c/p\u003e \u003cp\u003eThe strong concordance observed between WHO tube tests and CDC bottle bioassays reinforces the reliability of the resistance intensity profiles reported in this study. While WHO assays provide standardized thresholds for resistance classification, CDC assays offer complementary confirmation of resistant phenotypes under flexible exposure conditions [10;16].\u003c/p\u003e \u003cp\u003eNevertheless, several limitations should be acknowledged. The study was conducted at discrete time points and did not account for temporal or seasonal variation in mortality rates. In addition, resistance mechanisms were not investigated. Although the transect encompassed the main ecological zones of the country, fine-scale spatial heterogeneity may not have been fully captured. Finally, the study focused on a limited number of insecticides and did not include newer compounds recently introduced into vector control programs. Despite these limitations, the consistency of mortality patterns observed across methods and insecticides provides robust evidence of widespread and high-intensity resistance in Benin.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study highlights widespread and high-intensity phenotypic resistance to pyrethroids in \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. in Benin, with marked spatial variation along the south\u0026ndash;north transect. The persistence of resistance even at elevated insecticide concentrations represents a major threat to the effectiveness of pyrethroid-based interventions. Conversely, the relatively preserved susceptibility to bendiocarb supports its continued relevance for indoor residual spraying. These findings underscore the importance of integrating resistance intensity data into national vector control strategies and promoting insecticide rotation and rational resistance management approaches.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLLINs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLong-Lasting Insecticide-treated mosquito Net\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIRS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIndoor Residual Spraying\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNMCP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNational Malaria Control Program\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAn. gambiae\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnopheles gambiae\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eConfidence Interval\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWHO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWorld Health Organization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no confict of interes\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study received no external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe original study was conducted by CDK, ASS, MNF, OD and SZH who also supplied the data. The idea for the study was conceptualized and generated by CDK, ASS, OD and ODC. Data were collected by CDK, ASS, SZH and MNF. RO and AD drafted the manuscript. Statistical data analysis by ASS and AS. PFL, RO and AS provided intellectual criticism on the content of the manuscript. All authors have read and approved the final submitted manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank the staff of the Centre de Recherche Entomologique de Cotonou (CREC) for their dedication and commitment during the course of this work.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data is available on reasonable request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, et al. The effect of malaria control on \u003cem\u003ePlasmodium falciparum\u003c/em\u003e in Africa between 2000 and 2015. \u003cem\u003eNature\u003c/em\u003e. 2015;526:207\u0026ndash;211.\u003c/li\u003e\n\u003cli\u003eRanson H, Lissenden N. Insecticide resistance in \u003cem\u003eAnopheles gambiae\u003c/em\u003e: data, mechanisms, and consequences. \u003cem\u003eTrends Parasitol\u003c/em\u003e. 2011;27:91\u0026ndash;98.\u003c/li\u003e\n\u003cli\u003eHemingway J, Ranson H, Magill A, Kolaczinski J, Fornadel C, Gimnig J, et al. Averting a malaria disaster: will insecticide resistance derail malaria control? \u003cem\u003eLancet\u003c/em\u003e. 2016;387:1785\u0026ndash;1788.\u003c/li\u003e\n\u003cli\u003eAkogbeto M, Padonou G, Tchicaya E. Resistance of malaria vectors to pyrethroids in Benin. \u003cem\u003eWest Afr J Med\u003c/em\u003e. 1999;18:151\u0026ndash;155.\u003c/li\u003e\n\u003cli\u003eYadouleton A, Padonou GG, Asidi A, Moiroux N, Agossa F, Djogb\u0026eacute;nou L, et al. Development of vegetable farming: a cause of the emergence of insecticide resistance in malaria vectors in Benin. \u003cem\u003eMalar J\u003c/em\u003e. 2010;9:103.\u003c/li\u003e\n\u003cli\u003eHougbe SZ, Oss\u0026eacute; RA, Azond\u0026eacute;kon R, Kpanou C, Ahouandjinou MJ, Affolabi Z‑K, et al. Two decades of insecticide resistance in Benin: a retrospective analysis of evolution and drivers. \u003cem\u003eMalar J\u003c/em\u003e. 2025;24:156\u003c/li\u003e\n\u003cli\u003ePadonou GG, Agossa FR, Gnanguenon V, Yovogan B, Azondekon R, Akinro B, et al. Impact of indoor residual spraying with bendiocarb on malaria transmission in Benin. \u003cem\u003eParasites Vectors\u003c/em\u003e. 2012;5:147.\u003c/li\u003e\n\u003cli\u003eA\u0026iuml;kpon R, Padonou GG, Agossa FR, Badirou K, Gnanguenon V, Akogbeto MC. Bendiocarb resistance in \u003cem\u003eAnopheles gambiae\u003c/em\u003e populations from Benin. \u003cem\u003eParasites Vectors\u003c/em\u003e. 2014;7:100.\u003c/li\u003e\n\u003cli\u003eSalako A, Ahogni I, A\u0026iuml;kpon R, Sidick A, Dagnon F, Sovi A, et\u003cem\u003e al\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e Insecticide resistance status, frequency of L1014F \u003cem\u003ekdr\u003c/em\u003e and G119S \u003cem\u003eace-1\u003c/em\u003e mutations and expression of detoxification enzymesin \u003cem\u003eAnopheles gambiae (s.l.)\u003c/em\u003e in two regions of northern Benin in preparation for indoor residual spraying Parasites \u0026amp; Vectors (2018) 11:618 \u003c/li\u003e\n\u003cli\u003eWorld Health Organization (WHO). Test procedures for insecticide resistance monitoring in malaria vector mosquitoes. Geneva: WHO; 2016.\u003c/li\u003e\n\u003cli\u003eGnanguenon V, Agossa FR, Azondekon R, Badirou K, A\u0026iuml;kpon R, Baba-Moussa L, et al. Experimental hut evaluation of pyrethroid-treated nets in areas of high pyrethroid resistance in Benin. \u003cem\u003eMalar J\u003c/em\u003e. 2015;14:315.\u003c/li\u003e\n\u003cli\u003eToe KH, Yameogo B, Dabir\u0026eacute; RK, Diabate A, N\u0026rsquo;Fale S, Soma DD, et al. Increased pyrethroid resistance intensity in malaria vectors in Burkina Faso. \u003cem\u003eParasites Vectors\u003c/em\u003e. 2018;11:504.\u003c/li\u003e\n\u003cli\u003eCoetzee M. Key to the females of Afrotropical \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes (Diptera: Culicidae). Malar J. 2020;19:70\u003c/li\u003e\n\u003cli\u003eZoh MG, Koudou BG, Djenontin A, Adja AM, Ahoua Alou LP, Yeo L, et al. Pyrethroid resistance intensity in \u003cem\u003eAnopheles gambiae\u003c/em\u003e populations from C\u0026ocirc;te d\u0026rsquo;Ivoire. \u003cem\u003eMalar J\u003c/em\u003e. 2018;17:155.\u003c/li\u003e\n\u003cli\u003eChurcher TS, Lissenden N, Griffin JT, Worrall E, Ranson H. Measuring the path toward malaria elimination. \u003cem\u003eScience\u003c/em\u003e. 2016;344:1230\u0026ndash;1232.\u003c/li\u003e\n\u003cli\u003eWorld Health Organization (WHO). Global plan for insecticide resistance management in malaria vectors (GPIRM). Geneva: WHO; 2017\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anopheles gambiae s.l., resistance intensity, pyrethroids, bendiocarb, Benin","lastPublishedDoi":"10.21203/rs.3.rs-8918876/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8918876/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe emergence and widespread distribution of insecticide resistance in \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. raise serious concerns about the long-term effectiveness of vector control tools. Assessing resistance intensity provides more operationally relevant information than diagnostic-dose bioassays alone. Phenotypic resistance intensity to pyrethroids (permethrin and deltamethrin) and bendiocarb in \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. was assessed along a south\u0026ndash;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\u0026ndash;30% and 10\u0026ndash;45%, respectively, depending on the study site. Even at 5\u0026times; and 10\u0026times; concentrations, pyrethroid-induced mortality frequently remained below 90%, indicating high-intensity resistance. Resistance levels were consistently higher for permethrin than for deltamethrin in both WHO and CDC assays. In contrast, bendiocarb induced high mortality rates, generally exceeding 90\u0026ndash;98% in most localities, with low to moderate resistance detected at only a few sites. Results obtained using both bioassay methods were concordant. The intense and widespread resistance to pyrethroids observed in Benin represents a major threat to interventions relying on this insecticide class. The relatively preserved susceptibility to bendiocarb supports its potential use within insecticide rotation strategies. Integrating resistance intensity data into national surveillance systems is essential to inform evidence-based vector control decision making.\u003c/p\u003e","manuscriptTitle":"Phenotypic intensity of insecticide resistance in Anopheles gambiae sensu lato along the south–north transect in Benin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-27 07:39:27","doi":"10.21203/rs.3.rs-8918876/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f2b70393-86a0-4c1a-aa73-0465e896d156","owner":[],"postedDate":"February 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63611644,"name":"Health sciences/Diseases"},{"id":63611645,"name":"Biological sciences/Ecology"},{"id":63611646,"name":"Earth and environmental sciences/Ecology"},{"id":63611647,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-04-27T05:08:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-27 07:39:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8918876","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8918876","identity":"rs-8918876","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}