Investigating multi-resistance mechanism of Anopheles gambiae s.l to insecticides in Osun State, Nigeria

Investigating multi-resistance mechanism of Anopheles gambiae s.l to insecticides in Osun State, Nigeria | 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 Investigating multi-resistance mechanism of Anopheles gambiae s.l to insecticides in Osun State, Nigeria Zarat Oyindamola Iwalewa, Yusirat Olarinre Yusuff, Quadri Olusegun Adeshina, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9187188/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 increasing resistance to pyrethroids and other insecticide classes by Anopheles mosquitoes continues to threaten the effectiveness of core vector-control interventions and risks reversing hard-won gains. This study investigated resistance mechanisms in Anopheles gambiae s.l. populations in two urban areas (Olorunsogo and Freedom Park) in Osogbo, Osun State. Larvae were collected from the field and reared to adults for WHO bioassay insecticide susceptibility testing using different doses of pyrethroids and an organophosphate. Synergist assays following WHO procedures assessed the involvement of monooxygenases in resistance. Sibling species were identified by Polymerase Chain Reaction (PCR) - Intentional Mismatch Primer- and allelespecific PCR was used to detect the L1014F Knockdown resistance (Kdr) mutation. Metabolic enzyme activities were measured by biochemical assays. High levels of pyrethroid resistance were observed at both locations, with variation between insecticides. Preexposure to PBO produced differing recoveries in mortality: PBO+permethrin yielded 96% mortality in Olorunsogo and 75% in Freedom Park, while PBO+deltamethrin produced 99% and 87%, respectively. Kdr allele frequencies were low, at 0.84 and 0.86 in Olorunsogo and Freedom Park. Monooxygenase activity was very low in resistant populations (P = 0.000), whereas glutathione Stransferases were significantly overexpressed (59.52 ± 1.44; P = 0.001). βesterase activity was also elevated (18.86; P = 0.000). The findings reveal multiple resistance mechanisms in urban Osun State populations of An. coluzzii, including metabolic detoxification and targetsite mutation. Therefore, proactive, evidencebased resistance management strategies are urgently needed to restore and sustain the efficacy of current vectorcontrol tools and strategies in the state. Health sciences/Diseases Biological sciences/Ecology Earth and environmental sciences/Ecology Biological sciences/Molecular biology Biological sciences/Zoology Anopheles gambiae s.l. insecticide synergist resistance metabolic assay vector control Osun. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Mosquito-borne infections continue to represent a significant vector-borne health issue worldwide (WHO, 2024). Anopheles, Aedes, and Culex mosquitoes comprise species that may serve as vectors for several mosquito-borne diseases affecting people (Busari et al., 2025). Consequently, vector control is a crucial component in the eradication of malaria. In the twenty-first century, indoor residual spraying (IRS), pyrethroid-treated bed nets (ITNs) with residual insecticides, and other insecticide-based approaches were widely used as front-line tools against malaria vectors and other vector-borne diseases (Karunamoorthi and Sabesan, 2013), within an Integrated Vector Management framework (WHO, 2013). Anopheles mosquitoes are resistant to deltamethrin, permethrin, and lambda-cyhalothrin, and show varying levels of resistance to bendiocarb and pirimiphos-methyl, which is concerning as it could significantly affect the effectiveness of current vector control methods. During knockdown resistance, sensitivity declines due to mutations in the target site and metabolic pathways of the mosquito Adeogun et al ., (2025). Anopheles mosquitoes have developed two-point mutations in the voltage-gated sodium channel that confer resistance to pyrethroids and DDT: one in Burkina Faso (Soma et al ., 2021) and Côte d’Ivoire, where the leucine-phenylalanine substitution occurs at residue 1014 (L1014F) Ranson et al ., (2011). The second mutation was observed in Kenya, where leucine is replaced by serine at the same codon position (L1014S) Okoko et al., (2023). In Nigeria, both mechanisms have been shown to confer resistance in An. gambiae s.l. Haruna et al., (2025). Target site resistance involves mutations in the voltage-gated sodium channel (Vgsc) gene, mediating resistance to DDT and pyrethroids Adesoye et al ., (2024), as well as mutations in acetylcholinesterase (ACE), which is linked to resistance to carbamates and organophosphates Haruna et al., (2025). Increased activity of detoxifying enzyme families—including non-specific esterases, glutathione-S-transferases (GSTs), and monooxygenases (cytochrome P450s)—are associated with resistance to various malaria control insecticides Omotayo et al., (2021). In Nigeria, the efficacy of chemical-based strategies through the use of pyrethroid insecticides has drastically reduced across the states (Adeogun et al., 2025; Busari et al., 2025; Adeleke et al. , 2018; Awolola et al., 2003), demanding urgent need for proactive and effective control strategies. This could be due to multiple resistance of mosquito, leading to phenotypic and genotypic variations. as well as increasing the disease burden transmitted by the vector globally. In Osun State, vector control is significantly challenged by agricultural practices that involve the frequent and intensive application of pesticides (Adeleke et al., 2018; Busari et al., 2025). While some studies have indicated resistance in Anopheles gambiae s.l ., which may impede the effectiveness of insecticides used for vector control and contribute to the knockdown resistance observed in adult mosquitoes, there is a dearth of baseline data regarding both targeted and mutational resistance in An. gambiae s.l. Hence, Constant monitoring is necessary to comprehend the levels and resistance mechanisms of vectors in combating mosquito-borne diseases. This study aims to examine the multi-resistance mechanisms of An. gambiae s.l. in selected metropolitan districts of Osogbo, Osun State, Nigeria. Materials and Methods The Study Area This study was carried out in Osogbo (Lat. 7◦ 33′ N) and Olorunda Long. (4◦ 31′ E) Local Government Areas (LGA), Osun State, Nigeria (Fig 1). These were selected based on a previous report by Adeleke et al . (2025) on the widespread suitability of larval habitats for Anopheles mosquito breeding. Three communities were further purposively selected from each of the two LGAs. The communities from Osogbo LG are Olorunsogo, Owode-Ilesha garage and Ijetu while in Olohunda LG are Freedom park, Oke-fia, and Ajegunle. Osun State is a southwestern state sharing boarders with Kwara and Oyo. It is a lowland forest zone comprising of both wet and dry seasons. Residents are predominantly preoccupied by trading and farming. In addition, is the rich cultural heritage and natural resources present. Ethical approval Ethical approval was sought and obtained from the Health Planning, Research and Statistics Department of the Ministry of Health, Osogbo, Osun State, with reference number: OSHREC/PRS/569T/1264. Community entry and mobilization Before the commencement of the study, mobilization visits were made to the study communities. These was led by community health workers and heads. This was to sensitize residents on the rationale and benefits of the study. Collection Of Mosquito Larvae A thorough larval prospection was conducted throughout the study communities to identify productive and potential Anopheles larval breeding habitats. Larval collection was conducted between May and August 2024. All types of breeding habitat were surveyed, comprising of gutters, puddles and pools of water in the selected communities of prospection. The four-instar larva stage were collected via scoop and sieve with 0.55mm mesh into a well-labelled transparent container. The larvae sample was transported to the Department of Animal and Environmental Biology Laboratory, Osun State University, Osogbo, Nigeria, reared till emergence into adult. Emerged adult mosquitoes were then transferred with the aid of an aspirator into a clean and transparent container. The mosquitoes were fed with 10% sugar solution, maintained under laboratory condition at 72±1% relative humidity and 25- 27 0 C Temperature. Morphological Identification of Adult Mosquitoes The adult mosquitoes were observed and identified under Stereo microscope morphologically. All female Anopheles gambiae s.l used for insecticide bioassay were separated from the male due to their epidemiological irrelevance, using the dichotomous key described by Gilles and Coetzeez (1987). Insecticide Susceptibility Bioassay The insecticide susceptibility test was carried out following the protocol provided by WHO, with the use of impregnated papers (WHO,2016) in the Molecular Epidemiology and Vector Biology unit (MOVEB), Department of Animal and Environmental Biology, Osun State University. The recommended impregnated paper used were 0.05% deltamethrin, 0.75% permethrin and 0.05% of alphacypermethrin. Twenty-five newly emerged mosquitoes were introduced into the WHO kits with untreated paper in duplicates, followed by the ones with impregnated paper in four replicates. The bioassay was done for 60mins at 27±2 0C and 72±1% relative humidity, the knockdown was then recorded at 10min interval. After an hour of exposure, the adult mosquitoes were then transferred into a holding bottle for 24hrs with 10% sugar solution. Susceptibility status was determined after the holding hours. The susceptible were sorted from the resistance and individual sample were preserved in an eppendorf tubes containing desiccant (silica beads) for further analysis. mortality = Total number of dead mosquitoes after 24 h X 100%. Total number of exposed mosquitoes Synergy Bioassay This procedure was carried out following the WHO susceptibility bioassay (WHO, 2016). Adult mosquitoes were introduced to piperonyl butoxide (PBO) for an hour and then transferred to WHO bottle with 0.05% deltamethrin, 0.75% permethrin paper, readings and mortality were recorded Biochemical Assay This test was conducted to evaluate the mechanism of action of the enzymes and some cellular antioxidants (monooxygenases, beta-esterase, Acetylcholinesterase, Glutathione-S-Transferases) implicated for metabolic resistance in mosquitoes. An adult female mosquito, 2-5 days old, was exposed to the insecticide prior to the assay. The determination of these biochemical activities was performed according to the protocol provided by Omedes et al., (2023). P450 monooxygenase was assessed by the total content of the heme at 620nm in the end point mode. The mixture reaction contained 20µl of homogenate, 60µl of 90mM phosphate buffer (pH= 7.2), 200µl of working solution 0.2%. TMBZ with 250mM of sodium acetate buffer (pH= 5.0), and 25 µl of 3% hydrogen peroxide. For beta-esterase, β- napthyl acetate was dissolved in acetone to create a 10mM stock solution. The stock solution was then diluted to 0.5-1mM in phosphate buffer (pH =7.4). the reaction was added up with 200µl of phosphate buffer (pH =7.4), 20µl of the enzyme source and 20µl of the substrate solution (β- napthyl acetate). The absorbance was measured at 600nm. In determining AChE, 10µl of homogenate was used, then 90µl of 50mM potassium phosphate buffer (pH =7.0) and 100µl of Eliman reagent (2Mm of acetythiocholine iodide and 0.23 mM of DTNB mixed before the measurement). The optical density was measured at 405nmin the kinetic mode for 30min at 30℃. GST reaction contained 15µl of homogenate and 195µl of 100mM potassium phosphate buffer (pH= 6.5) with 9mM of GSH in 1mM of CDNB (1-chloro- 2,4-dinitrobenzene). The GST was accessed towards CDNB at 340nm in the kinetic mode for 20min at 25℃. Molecular identification of sibling species and knock-down resistance gene The DNA of hundred adult Anopheles mosquito samples was extracted using DNA extraction kit by Nigerian Institute for Medical Research (NIMR) in the MOVEB laboratory, Osun State University. Further analysis was carried out using the polymerase chain Reaction to identifying the sub-species of the complex using protocol by Wilkin et al., (2006). IMP-PCR was used for the detection of mutational genes associated with pyrethroids and organophosphate according to the method described by Huynh et al., (2007); Weil et al., (2004). Data Analysis Correlation between the insecticide mortalities were analyzed using pearson correlation. The kdr (KDT50 and KDT95) was analyzed using chi-square while a one-sample t-test was employed to compare mean activities of enzymes used in the enzymatic resistance mechanism of the mosquitoes. Insecticide susceptibility assay was analyzed using WHO standard formula (WHO, 2022). All data were significant at p ≤ 0.05 (2-tailed). Data from the Insecticide susceptibility test was analyzed and calculated using a standardized formula by WHO data was analyzed Percentage. The knock down rate (KDT50 and KDT95) was determined using SPSS software, version 21.0 (probit model). Correlation between mortality to deltamethrin, permethrin, alphacypermethrin and synergy was analysed using pearson correlation. Mutational frequency were calculated using F(R) = (2 x RR + RS)/2 N, where RR = Total number of homozygote resistant, RS = Total number. The KDR frequency was analyzed using chi-square (χ2). One sampleT-test was employed to compare mean activities of enzymes of the exposed mosquitoes (both resistance and susceptible). Result Molecular identification of Anopheles gambiae s.l sibling species A total number of six hundred mosquitoes were identified morphologically belonging to the complex of Anopheles gambiae s.l . DNA extraction was performed by randomly selecting of one hundred and fifty mosquitoes for molecular identification of the sibling species of An. gambiae s.l encountered during the study. Molecular analysis revealed only 1% of An. gambiae s.s. were present while the remaining 99% were An. coluzzii from the sample collected. Insecticide Bioassay Pyrethroids and Organophosphates All Anopheles mosquitoes collected during the study showed strong resistance to permethrin and alphacypermethrin, followed by deltamethrin, except in the case of pirimiphosmethyl. Resistance levels varied across discriminating doses of permethrin, deltamethrin, alphacypermethrin, and pirimiphosmethyl. Mortality rates at Olorunsogo were 39%, 58.6%, 82%, and 100%, while at Freedompark they were 52.78%, 49.11%, 59%, and 100% respectively (Table 1). A high level of permethrin resistance was observed in Olorunsogo, indicated by knockdown times (KDT₅₀ and KDT₉₅). Pirimiphos-methyl was the most effective insecticide tested, with no resistance detected at either site. Both locations achieved 100% mortality within 24 hours. KDT₅₀ and KDT₉₅ were 39.72 and 79.14 minutes in Olorunsogo and 45.45 and 88.98 minutes in Freedompark, respectively (Fig 2). The mortality rates across sites were significantly correlated (R2= 0.030, p = 0.01). Table 1: Knockdown time of different doses of insecticides and the percentage mortalities in the study areas. Insecticide Parameters Study site Olorunsogo Freedompark Permethrin % Mortality (24 h) KDT50(min) KDT95(min) Susceptibility status 39 >120 >120 Resistant 52.78 >120 >120 Resistant Deltamethrin % Mortality (24 h) KDT50(min) KDT95(min) Susceptibility status 82 108.05 >120 Resistance suspected 49.11 97.00 >120 Resistance suspected Alphacypermethrin % Mortality (24 h) KDT50(min) KDT95(min) Susceptibility status 58.6 >120 >120 Resistant 59 87.76 >120 Resistance suspected Pirimiphos % Mortality (24 h) KDT50(min) KDT95(min) Susceptibility status 100 39.72 79.14 Susceptible 100 45.45 88.98 Susceptible All Knockdown time values greater than 120 min is reported as >120 Synergy Bioassay The results from the synergy assay for both sites, involving pre-exposure to PBO, revealed mortality in the An. coluzzii subjected to the bioassay. However, PBO+Permethrin improved efficacy but did not fully restore susceptibility (Table 2). In Olorunsogo, the mortality rate increased significantly to 96%, with a KDT₅₀ of 70.83 minutes and a KDT₉₅ of 110.50 minutes. In Freedompark, the mortality rate was 75%, with a KDT₅₀ of 95.96 minutes and a KDT₉₅ still exceeding 120 minutes (Fig 4). PBO+ Deltamethrin showed a mortality rate reached 99%, with KDT₅₀ of 49.28 minutes and a KDT₉₅ of 106.20 minutes, indicating a high level of susceptibility, in Olorunsogo while resistance was suspected in Freedompark, with a mortality rate of 82.85%, and a KDT₅₀ of 60.20 minutes and a KDT₉₅ of 102.86 minutes (Fig 5). Table 2: Knockdown time of synergy and the percentage mortalities from the both sites. Insecticide Parameters Study site Olorunsogo Freedompark PBO+Permethrin % Mortality (24 h) KDT50(min) KDT95(min) Susceptibility status 96 70.83 110.50 Resistance suspected 75 95.96 >120 Resistance suspected PBO+Deltamethrin % Mortality (24 h) KDT50(min) KDT95(min) Susceptibility status 99 49.28 106.20 Susceptible 82.85 60.20 102.86 Resistance suspected Mutant alleles frequency The kdr genotyping mutation (L1014F) was high in both study areas. In Olorunsogo, 84% were homozygous for the resistant allele (RR), 4 individuals (16%) were homozygous for the susceptible allele (rr), and no heterozygotes (Rr) were detected (Table 3). In Freedom Park, 88% were homozygous resistant (RR), 3 individuals (12%) were homozygous susceptible (rr), and no heterozygotes were observed. Using Chi-square, P value for Hardy-Weinberg expectations for kdr gene (x2 =25, df= 1 P < 0.05). Table 3: KDR Frequency and Alleles Distribution of An. coluzzii from the two sites Locations No screened Kdr Mutation Kdr alleles Frequency (%) rr Rr RR Olorunsogo 25 4 0 21 0.84 Freedom park 25 3 0 22 0.88 Metabolic Enzymes Assay Metabolic activities based on the insecticide susceptibility of mosquitoes are presented as mean values (Table 4). The results indicated that monooxygenase activity was lower in resistant mosquitoes compared to susceptible and control groups. Conversely, GST activity was significantly higher in the resistant population (P ≤ 0.05; 0.001). β-esterase activity showed a gradual increase from the control to the susceptible and then to the resistant group, with the highest mean observed in resistant mosquitoes. A one-sample T-test revealed significant differences in enzyme activities among the susceptibility groups (P ≤ 0.05; 0.00). Acetylcholinesterase activity was slightly reduced in the susceptible group relative to the control (Table 5) (P ≤ 0.05; 0.03). Table 4: Mean Activities of Metabolic Enzymes control and exposed An. colluzi to pyrethroids Susceptibility Status (N=11) Enzymes Monooxygenase (ng/L) GST (U/L) β-esterase (nmole/mg protein) Control 55.70± 0.33 53.43± 2.32 18.16± 0.72 Susceptible 55.35± 0.51 55.71± 1.39 18.54± 0.72 Resistance 53.60± 1.02 59.52± 1.44 18.86± 0.93 Table 5: Mean Activities of Metabolic Enzymes in control and exposed An. coluzzii to pyrethroids Susceptibility Status Acetylcholinesterase Control 17.21± 1.07 Susceptible 15.65± 0.52 Discussion This study found that, among 100 An. gambiae s.l . analysed, 99% were identified as An. coluzzii and 1% as An. gambiae s.s. The predominance of An. coluzzii over An. gambiae s.s. corroborates earlier reports from Osun State (Iwalewa et al., 2025; Busari et al., 2024). Afolabi et a l. (2019) similarly reported a higher prevalence of An. coluzzii than An. gambiae in Ondo, Southwestern Nigeria. Studies from Nigeria (Oduola et al., 2012), Ghana (De Souza et al., 2010), and Benin (Yaouleton et al., 2010) also document dominance of the M form over the S form. This pattern may be related to greater larval tolerance of physico‑chemical variation in An. coluzzii , which can restrict gene flow and promote shifts in species composition. The field populations examined were highly resistant to pyrethroids, with no mortality observed in response to the organophosphate tested. Resistance to permethrin was particularly pronounced in Olorunsogo compared with Freedom Park, although mortality rates varied by site. These observations align with numerous reports of widespread pyrethroid resistance in Nigeria, including studies by Obembe et al., 2025 (Kwara State), Obembe et al., 2024 (national), Muhammad et al., 2021, Omotayo et al., 2022, Awolola et al., 2009 (Lagos), and Adeleke et al., 2018 (Osun State). Mosquito dispersal facilitates spread of resistance haplotypes, including the kdr‑W mutation that confers knockdown resistance to pyrethroids. Analysis of kdr genotypes indicated homozygous resistant (RR) frequencies of 0.84 and 0.88 at the study sites. Homozygous susceptible (rr) frequencies were 0.16 at Olorunsogo and 0.12 at Freedom Park. No heterozygotes were detected at either site. Consistent with previous reports (Djouka et al., 2008; Cukwuekezie et al., 2020; Omotayo et al., 2021; Ekedo et al., 2023), heterozygote frequencies were low in the sampled populations. The observed kdr allele distribution deviated from Hardy–Weinberg expectations, suggesting selection, nonrandom mating, or population structure affecting the L1014 locus. The prominence of kdr mutations in West Africa has been driven by prolonged use of pyrethroid‑based control measures, reducing pyrethroid efficacy in many settings. Piperonyl butoxide (PBO) is a synthetic synergist with minimal intrinsic insecticidal activity. Pre‑exposure to PBO increased mortality at both sites, although mortality in Freedom Park was slightly lower than in Olorunsogo. This contrasts with some studies reporting near‑complete restoration of susceptibility by PBO (Awolola et al., 2018; Martins et al., 2021) and likely reflects local differences in resistance profiles. PBO does not affect target‑site mutations (e.g., kdr L1014F) and may not fully restore susceptibility when multiple mechanisms such as elevated GSTs, esterases, or alternative pathways contribute to resistance. Biochemical assays showed lower monooxygenase activity in the resistant groups; monooxygenase activity was significantly higher in the susceptible and control (P = 0.000). This observation aligns with Hamid‑Adiamoh et al., 2020, but contradicts studies reporting elevated monooxygenases in pyrethroid‑resistant populations (Cuamba et al., 2010; Awolola et al., 2018). Reduced monooxygenase activity in resistant specimens could reflect the involvement of cuticular resistance or antioxidant defenses that limit insecticide uptake or mitigate oxidative damage. In contrast, GST activity was significantly overexpressed in the resistant group (P = 0.001). Earlier studies document GST involvement in Anopheles resistance (Ifeoluwa et al., 2020; Koumo et al., 2025; Adeogun et al., 2025). GST‑mediated detoxification likely contributes to the high pyrethroid resistance observed at both sites. Additionally, β‑esterase activity was significantly elevated in resistant populations (P = 0.000), consistent with reports linking increased esterases to pyrethroid resistance (Hemingway et al., 2004; Zhong et al., 2024; Olaniran et al., 2020). Some studies, however, have found no significant difference in β‑esterase activity (Omotayo et al., 2021), indicating geographic and temporal variation in resistance mechanisms. Overall, biochemical results indicate limited involvement of monooxygenases in the resistant An. coluzzii examined, while GSTs and β‑esterases are implicated as major contributors to resistance at the study sites. The significant GST overexpression and the variable effect of PBO at one site highlight a serious challenge for malaria control. Local differences in PBO efficacy imply that reliance on PBO‑based interventions may not uniformly restore pyrethroid susceptibility and could pose a risk to effective vector control. Pyrethroid resistance in Anopheles has become a major public‑health challenge in Nigeria because malaria control depends heavily on pyrethroid‑based interventions. Growing resistance demands urgent attention because it undermines control efforts and risks increasing the burden of malaria and other vector‑borne diseases. Conclusion Anopheles coluzzii remains the dominant vector in Osun State and exhibits strong resistance to pyrethroids. Although kdr mutations are present, their low frequency indicates they are not the primary resistance mechanism. Instead, metabolic enzymes, particularly GSTs and β-esterases, play a major role, with evidence of additional complex mechanisms. The limited effectiveness of PBO highlights the crucial need for locally tailored, evidence-based vector control strategies. Therefore, there is an urgent need to manage resistance and prevent the continuous transmission of malaria through proactive vector surveillance and environmental management. List of Abbreviations PCR -Polymerase Chain Reaction KDR -Knock-down Resistance IRS- Indoor Residual Spray ITN- Insecticide Treated Net DDT- Dichlorodiphenyltrichloroethane PBO -Piperonyl butoxide RR -Homozygous Resistant RS- Heterozygous Susceptible LGA -Local Government Area MOVEB -Molecular Epidemiology and Vector Biology WHO- World Health Organization IMP - Intentional Mismatch Primer pH- Potential Hydrogen TMBZ - 3,3’,5, 5’ Tetramethylbenzidine GSH- Reduced Glutathione OSMoH–REC - Osun State Ministry of Health Research and Ethics Committee Declarations Acknowledgement The authors express their sincere gratitude to the residents of the study areas for their patience and cooperation. Authors’ contributions Conceptualization–Z. O, M.A Data curation–Z.O., Y.O., Q.O., M.M., D.I., L.O., H.K., G.O., G.B., C.T., T.A., I.O. Supervision–A.M., K.A., A.O., M.A. Data analysis–Z.O. Manuscript draft—Z.O. Manuscript editing– M. A., L.O. Funding This study received no specific funding. 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Allelic variation in a cluster of epsilon glutathione S-transferase genes contributes to DDT and pyrethroid resistance in the major African malaria vector Anopheles funestus . BMC Genomics 26 , 452 (2025). https://doi.org/10.1186/s12864-025-11637-3. Martins, J. L., Mosha, F. W., Lukole, E., Rowland, M., Todd, J., Charlwood, J. D., Mosha, J. F., & Protopopoff, N. (2021). Personal protection with PBO‑pyrethroid synergist‑treated nets after two years of household use against pyrethroid‑resistant Anopheles in Tanzania. Parasites & Vectors, 14, 150. Matowo, J., Kulkarni, M.A., Mosha, F.W. et al. Biochemical basis of permethrin resistance in Anopheles arabiensis from Lower Moshi, north-eastern Tanzania. Malar J 9 , 193 (2010). https://doi.org/10.1186/1475-2875-9-193 Ministry of Health of Brazil. Quantification Methodology for Enzyme Activity Related to Insecticide Resistance in Aedes aegypti ; Ministry of Health of Brazil, Fundação Oswaldo Cruz: Brasília, Brazil, 2006. Muhammad, A., Ibrahim, S. S., Mukhtar, M. M., Irving, H., Abajue, M. C., Edith, N. M. A., Da'u, S. S., Paine, M. J. I., & Wondji, C. S. (2021). High pyrethroid/DDT resistance in major malaria vector Anopheles coluzzii from Niger-Delta of Nigeria is probably driven by metabolic resistance mechanisms. PLOS ONE, 16(3), e0247944. https://doi.org/10.1371/journal.pone.0247944 Obembe, A., Oyeniyi, T., Oduola, A. O. O., Asekun, F., Adeogun, A., & Awolola, S. (2025). First report of widespread kdr‑L995F pyrethroid‑resistant Anopheles arabiensis and temporal trends of pyrethroid resistance in urban Ilorin, Kwara State, Nigeria. BMC Infectious Diseases, 25, Article 1331. https://bmcinfectdis.biomedcentral.com/articles/10.1186/s12879-025-11789-3 Obembe, A., Oyeniyi, T., Oduola, A. O., Asekun, F., Adeogun, A., & Awolola, S. T. (2024). Multiple pyrethroid resistance in urban male and female Anopheles gambiae s.l. populations in Ilorin, Nigeria: Implications for swarm spraying and toxic sugar bait malaria vector control [Unpublished manuscript]. ResearchGate. Oduola, A. O., Idowu, E. T., Oyebola, M. K., Adeogun, A. O., Olojede, J. B., Otubanjo, O. A., & Awolola, T. S. (2012). Evidence of carbamate resistance in urban populations of Anopheles gambiae ss mosquitoes resistant to DDT and deltamethrin insecticides in Lagos, South-Western Nigeria. Parasites & vectors , 5 (1), 116. Olaniran, O., Awolola, T. S., Amajoh, C., et al. (2020). First report of AChE1 (G119S) mutation and multiple resistance mechanisms in Anopheles gambiae from Lagos State, Nigeria. Scientific Reports, 10, Article 12345. https://doi.org/10.1038/s41598-020-64412-7 Omotayo, A. I., Ande, A. T., Oduola, A. O., Adelaja, O. J., Adesalu, O., Jimoh, T. R., Ghazali, A. I., & Awolola, S. T. (2021). Multiple insecticide resistance mechanisms in urban population of Anopheles coluzzii (Diptera: Culicidae) from Lagos, South‑West Nigeria. Retrieved from https://europepmc.org/article/MED/34958768 Pauwels, (2007). "In vitro determination of beta-esterase activity in insecticides metabolism: An optimized colorimetric assay." Journal of Enzyme Inhibition and Medicinal Chemistry , 22(3), 321-329. Simma, E. A., Dermauw, W., Balabanidou, V., Snoeck, S., Bryon, A., Clark, R. M., ... & Van Leeuwen, T. (2019). Genome‐wide gene expression profiling reveals that cuticle alterations and Ranson, H., N’guessan, R., Lines, J., Moiroux, N., Nkuni, Z., & Corbel, V. (2011). Pyrethroid resistance in African anopheline mosquitoes: what are the implications for malaria control?. Trends in parasitology , 27 (2), 91-98. Soma, D. D., Poda, S. B., Hien, A. S., Namountougou, M., Sangaré, I., Sawadogo, J. M. E., ... & Dabiré, R. K. (2021). Malaria vectors diversity, insecticide resistance and transmission during the rainy season in peri-urban villages of south-western Burkina Faso. Malaria Journal , 20 (1), 63. Thiaw, O., Doucouré, S., Sougoufara, S., Bouganali, C., Konaté, L., Diagne, N., ... & Sokhna, C. (2018). Investigating insecticide resistance and knock-down resistance (kdr) mutation in Dielmo, Senegal, an area under long lasting insecticidal-treated nets universal coverage for 10 years. Malaria journal , 17 (1), 123. Weill, M., Malcolm, C., Chandre, F., Mogensen, K., Berthomieu, A., Marquine, M., & Raymond, M. (2004). The unique mutation in ace-1 giving high insecticide resistance is easily detectable in mosquito vectors. Insect Molecular Biology, 13(1), 1–7. https://doi.org/10.1111/j.1365-2583.2004.00452. Wilkins, E. E., Howell, P. I., & Benedict, M. Q. (2006). IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis. Malaria Journal, 5, Article 125 World Health Organization. (2013). World malaria report 2013. World Health Organization. https://www.who.int/publications/i/item/9789241564694. World Health Organization. (2016). World malaria report 2016. World Health Organization. https://www.who.int/publications/i/item/9789241511711SI World Health Organization. (2024). Twentieth meeting of the WHO Vector Control Advisory Group: meeting report, 25-28 March 2024. Yadouleton, A. W., Padonou, G., Asidi, A., Moiroux, N., Bio‑Banganna, S., Corbel, V., N'Guessan, R., Gbenou, D., Yacoubou, I., Gazard, K., & Akogbeto, M. C. (2010). Insecticide resistance status in Anopheles gambiae in southern Benin. Malaria Journal, 9, Article 83. https://doi.org/10.1186/1475-2875-9-83 Zhong, D., Degefa, T., Zhou, G., Lee, M.-C., Wang, C., Chen, J., Yewhalaw, D., & Yan, G. (2024). Esterase-Mediated Pyrethroid Resistance in Populations of an Invasive Malaria Vector Anopheles stephensi from Ethiopia. Genes , 15 (12). National Malaria Elimination Programme (NMEP). (2019). National Malaria Strategic Plan 2014–2020 (Updated 2019). Federal Ministry of Health, Abuja, Nigeria. https://nmcp.gov.ng/ Okoko, M., Karisa, J., Gona, R., Odongo, T., Otieno, B., Yaa, F., ... & Maia, M. F. (2025). Phenotypic and genotypic insecticide resistance profiles of main malaria vectors in Kwale county, coastal Kenya. Malaria Journal , 24 (1), 191. Additional Declarations No competing interests reported. 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sites\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9187188/v1/0af04b83b8a78cb8e070cfd1.jpg"},{"id":105728750,"identity":"eaac136a-f220-438b-bfdf-ec806e34e0ed","added_by":"auto","created_at":"2026-03-30 11:12:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":63007,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage Knockdown of \u003cem\u003eAn. coluzzii\u003c/em\u003e exposed to the Permethrin, Deltamethrin, Alphacypermethrin and Pirimiphosmethyl from Olorunsogo\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9187188/v1/9a1c4fada9854d47da636ea8.jpg"},{"id":105729068,"identity":"ed2b0412-b52e-4750-b799-31e003998cb3","added_by":"auto","created_at":"2026-03-30 11:13:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":62974,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage Knockdown of \u003cem\u003eAn. coluzzii\u003c/em\u003e exposed to the Permethrin, Deltamethrin, Alphacypermethrin and Pirimiphosmethyl from Freedompark\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9187188/v1/c4ef059ade40e39c32af9d3b.jpg"},{"id":105706023,"identity":"d2cad8e0-562a-4561-bbb9-35e75be1bf85","added_by":"auto","created_at":"2026-03-30 07:03:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53900,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage Knockdown (60mins) of \u003cem\u003eAn. coluzzii\u003c/em\u003eexposed to Permethrin and PBO+ Permethrin from Freedompark\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9187188/v1/bb97019aa924a4fa42547e83.jpg"},{"id":105706025,"identity":"a9c73172-05bc-415d-a159-65e4dc3f3ec2","added_by":"auto","created_at":"2026-03-30 07:03:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":52579,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage Knockdown (60mins) of \u003cem\u003eAn. coluzzii\u003c/em\u003e exposed to Permethrin and PBO+ Deltamethrin from the two study sites\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9187188/v1/e52f3a7c2d2046b533590ec7.jpg"},{"id":107707501,"identity":"412169a2-be12-4723-9d43-95c0ea7f26e5","added_by":"auto","created_at":"2026-04-24 09:20:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":743737,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9187188/v1/98e00a1a-a4e8-4b5b-b029-4e0811cb579a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eInvestigating multi-resistance mechanism of Anopheles gambiae s.l to insecticides in Osun State, Nigeria\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMosquito-borne infections continue to represent a significant vector-borne health issue worldwide (WHO, 2024). \u003cem\u003eAnopheles, Aedes, and Culex\u0026nbsp;\u003c/em\u003emosquitoes comprise species that may serve as vectors for several mosquito-borne diseases affecting people (Busari \u003cem\u003eet al.,\u003c/em\u003e 2025). Consequently, vector control is a crucial component in the eradication of malaria.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In the twenty-first century, indoor residual spraying (IRS), pyrethroid-treated bed nets (ITNs) with residual insecticides, and other insecticide-based approaches were widely used as front-line tools against malaria vectors and other vector-borne diseases (Karunamoorthi and Sabesan, 2013), within an Integrated Vector Management framework (WHO, 2013). \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes are resistant to deltamethrin, permethrin, and lambda-cyhalothrin, and show varying levels of resistance to bendiocarb and pirimiphos-methyl, which is concerning as it could significantly affect the effectiveness of current vector control methods. During knockdown resistance, sensitivity declines due to mutations in the target site and metabolic pathways of the mosquito Adeogun \u003cem\u003eet al\u003c/em\u003e., (2025). \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes have developed two-point mutations in the voltage-gated sodium channel that confer resistance to pyrethroids and DDT: one in Burkina Faso (Soma \u003cem\u003eet al\u003c/em\u003e., 2021) and C\u0026ocirc;te d\u0026rsquo;Ivoire, where the leucine-phenylalanine substitution occurs at residue 1014 (L1014F) Ranson \u003cem\u003eet al\u003c/em\u003e., (2011). The second mutation was observed in Kenya, where leucine is replaced by serine at the same codon position (L1014S) Okoko \u003cem\u003eet al.,\u003c/em\u003e (2023). In Nigeria, both mechanisms have been shown to confer resistance in \u003cem\u003eAn. gambiae s.l.\u0026nbsp;\u003c/em\u003eHaruna \u003cem\u003eet al.,\u003c/em\u003e (2025). Target site resistance involves mutations in the voltage-gated sodium channel (Vgsc) gene, mediating resistance to DDT and pyrethroids Adesoye \u003cem\u003eet al\u003c/em\u003e., (2024), as well as mutations in acetylcholinesterase (ACE), which is linked to resistance to carbamates and organophosphates Haruna \u003cem\u003eet al.,\u003c/em\u003e (2025). Increased activity of detoxifying enzyme families\u0026mdash;including non-specific esterases, glutathione-S-transferases (GSTs), and monooxygenases (cytochrome P450s)\u0026mdash;are associated with resistance to various malaria control insecticides Omotayo \u003cem\u003eet al.,\u003c/em\u003e (2021).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In Nigeria, the efficacy of chemical-based strategies through the use of pyrethroid insecticides has drastically reduced across the states (Adeogun \u003cem\u003eet al.,\u003c/em\u003e 2025; Busari \u003cem\u003eet al.,\u003c/em\u003e 2025; Adeleke\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, 2018; Awolola \u003cem\u003eet al.,\u003c/em\u003e 2003), demanding urgent need for proactive and effective control strategies. This could be due to multiple resistance of mosquito, leading to phenotypic and genotypic variations. \u0026nbsp; as well as increasing the disease burden transmitted by the vector globally.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn Osun State, vector control is significantly challenged by agricultural practices that involve the frequent and intensive application of pesticides (Adeleke \u003cem\u003eet al.,\u003c/em\u003e 2018; Busari \u003cem\u003eet al.,\u003c/em\u003e 2025). While some studies have indicated resistance in \u003cem\u003eAnopheles gambiae s.l\u003c/em\u003e., which may impede the effectiveness of insecticides used for vector control and contribute to the knockdown resistance observed in adult mosquitoes, there is a dearth of baseline data regarding both targeted and mutational resistance in \u003cem\u003eAn. gambiae s.l.\u003c/em\u003e Hence, Constant monitoring is necessary to comprehend the levels and resistance mechanisms of vectors in combating mosquito-borne diseases. This study aims to examine the multi-resistance mechanisms of \u003cem\u003eAn. gambiae s.l.\u003c/em\u003e in selected metropolitan districts of Osogbo, Osun State, Nigeria.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eThe Study Area\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was carried out in Osogbo (Lat. 7◦ 33′ N) and Olorunda Long. (4◦ 31′ E) Local Government Areas (LGA), Osun State, Nigeria (Fig 1). These were selected based on a previous report by Adeleke \u003cem\u003eet al\u003c/em\u003e. (2025) on the widespread suitability of larval habitats for Anopheles mosquito breeding. Three communities were further purposively selected from each of the two LGAs. The communities from Osogbo LG are Olorunsogo, Owode-Ilesha garage and Ijetu while in Olohunda LG are Freedom park, Oke-fia, and Ajegunle.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOsun State is a southwestern state sharing boarders with Kwara and Oyo. It is a lowland forest zone comprising of both wet and dry seasons. Residents are predominantly preoccupied by trading and farming. In addition, is the rich cultural heritage and natural resources present.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval was sought and obtained from the Health Planning, Research and Statistics Department of the Ministry of Health, Osogbo, Osun State, with reference number: OSHREC/PRS/569T/1264.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCommunity entry and mobilization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore the commencement of the study, mobilization visits were made to the study communities. These was led by community health workers and heads. This was to sensitize residents on the rationale and benefits of the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection Of Mosquito Larvae\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA thorough larval prospection was conducted throughout the study communities to identify productive and potential \u003cem\u003eAnopheles\u003c/em\u003e larval breeding habitats. Larval collection was conducted between\u0026nbsp;May and August 2024. \u0026nbsp;All types of breeding habitat were surveyed, comprising of gutters, puddles and pools of water in the selected communities of prospection. The four-instar larva stage were collected via scoop and sieve with 0.55mm mesh into a well-labelled transparent container. The larvae sample was transported to the Department of Animal and Environmental Biology Laboratory, Osun State University, Osogbo, Nigeria, reared till emergence into adult. Emerged adult mosquitoes were then transferred with the aid of an aspirator into a clean and transparent container. The mosquitoes were fed with\u0026nbsp;10% sugar solution,\u0026nbsp;maintained under laboratory condition at 72±1% relative humidity and 25- 27 \u003csup\u003e0\u003c/sup\u003eC Temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological Identification of Adult Mosquitoes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe adult mosquitoes were observed\u0026nbsp;and identified under Stereo microscope morphologically. All female Anopheles gambiae s.l used for insecticide bioassay were separated from the male\u0026nbsp;due to their epidemiological irrelevance,\u0026nbsp;using the dichotomous key described by Gilles and Coetzeez (1987).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInsecticide Susceptibility Bioassay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe insecticide susceptibility test was carried out following the protocol provided by WHO, with the use of impregnated papers (WHO,2016) in the Molecular Epidemiology and Vector Biology unit (MOVEB), Department of Animal and Environmental Biology, Osun State University. The recommended impregnated paper used were 0.05% deltamethrin, 0.75% permethrin and 0.05% of alphacypermethrin. Twenty-five newly emerged mosquitoes were introduced into the WHO kits with untreated paper in duplicates, followed by the ones with impregnated paper in four replicates. The bioassay was done for 60mins at\u0026nbsp;27±2 0C and 72±1% relative humidity, the knockdown was then recorded at 10min interval. After an hour of exposure, the adult mosquitoes were then transferred into a holding bottle for 24hrs with\u0026nbsp;10% sugar solution. Susceptibility status was determined after the holding hours. The susceptible were sorted from the resistance and individual sample were preserved in an\u0026nbsp;eppendorf\u0026nbsp;tubes containing desiccant (silica beads) for further analysis.\u003c/p\u003e\n\u003cp\u003emortality = \u003cu\u003eTotal number of dead mosquitoes after 24 h\u0026nbsp;\u003c/u\u003eX 100%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Total number of exposed mosquitoes\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynergy Bioassay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis procedure was carried out following the WHO susceptibility bioassay (WHO, 2016). Adult mosquitoes were introduced to\u0026nbsp;piperonyl butoxide (PBO) for an hour and then transferred to WHO bottle with 0.05% deltamethrin, 0.75% permethrin paper, readings and mortality were recorded\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis test was conducted to evaluate the mechanism of action of the enzymes and some cellular antioxidants (monooxygenases, beta-esterase, Acetylcholinesterase, Glutathione-S-Transferases) implicated for metabolic resistance in mosquitoes. An adult female mosquito, 2-5 days old, was exposed to the\u0026nbsp;insecticide prior to the assay. The determination of these biochemical activities was performed according to the protocol provided by Omedes et al., (2023). P450 monooxygenase was assessed by the total content of the heme at 620nm in the end point mode. The mixture reaction contained 20µl of homogenate, 60µl of 90mM phosphate buffer (pH= 7.2), 200µl of working solution 0.2%. TMBZ with 250mM of sodium acetate buffer (pH= 5.0), and 25 µl of 3% hydrogen peroxide. For beta-esterase, β- napthyl acetate was dissolved in acetone to create a 10mM stock solution. The stock solution was then diluted to 0.5-1mM in phosphate buffer (pH =7.4). the reaction was added up with 200µl of phosphate buffer (pH =7.4), 20µl of the enzyme source and 20µl of the substrate solution (β- napthyl acetate). The absorbance was measured at 600nm. In determining AChE, 10µl of homogenate was used, then 90µl of 50mM potassium phosphate buffer (pH =7.0) and 100µl of Eliman reagent (2Mm of acetythiocholine iodide and 0.23 mM of DTNB mixed before the measurement). The optical density was measured at 405nmin the kinetic mode for 30min at 30℃. GST reaction contained 15µl of homogenate and 195µl of 100mM potassium phosphate buffer (pH= 6.5) with 9mM of GSH in 1mM of CDNB (1-chloro- 2,4-dinitrobenzene). The GST was accessed towards CDNB at 340nm in the kinetic mode for 20min at 25℃.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular identification of sibling species and knock-down resistance gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DNA of\u0026nbsp;hundred adult Anopheles mosquito samples was extracted using DNA extraction kit by Nigerian Institute for Medical Research (NIMR) in the MOVEB laboratory, Osun State University. Further analysis was carried out using the polymerase chain Reaction to identifying the sub-species of the complex using protocol by Wilkin \u003cem\u003eet al.,\u003c/em\u003e (2006). IMP-PCR was used for the detection of mutational genes associated with pyrethroids and organophosphate according to the method described by Huynh \u003cem\u003eet al.,\u003c/em\u003e (2007); Weil \u003cem\u003eet al.,\u003c/em\u003e (2004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrelation between the insecticide mortalities were analyzed using pearson correlation. The kdr (KDT50 and KDT95) was analyzed using chi-square while a one-sample t-test was employed to compare mean activities of enzymes used in the enzymatic resistance mechanism of the mosquitoes. Insecticide susceptibility assay was analyzed using WHO standard formula (WHO, 2022). All data were significant at \u003cem\u003ep\u0026nbsp;\u003c/em\u003e≤ 0.05 (2-tailed).\u0026nbsp;Data from the Insecticide susceptibility test was analyzed and calculated using a standardized formula by WHO data was analyzed Percentage.\u003c/p\u003e\n\u003cp\u003eThe knock down rate (KDT50 and KDT95) was determined using SPSS software, version 21.0 (probit model). Correlation between mortality to deltamethrin, permethrin, alphacypermethrin and synergy was analysed using pearson correlation. Mutational frequency were calculated using F(R) = (2 x RR + RS)/2 N, where RR = Total number of homozygote resistant, RS = Total number. The KDR frequency was analyzed using chi-square (χ2). One sampleT-test was employed to compare mean activities of enzymes of the exposed mosquitoes (both resistance and susceptible).\u0026nbsp;\u003c/p\u003e"},{"header":"Result","content":"\u003cp\u003e\u003cstrong\u003eMolecular identification of \u003cem\u003eAnopheles gambiae s.l\u003c/em\u003e sibling species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total number of six hundred mosquitoes were identified morphologically belonging to the complex of \u003cem\u003eAnopheles gambiae s.l\u003c/em\u003e. \u0026nbsp;DNA extraction was performed by randomly selecting of one hundred and fifty mosquitoes for molecular identification of the sibling species of \u003cem\u003eAn. gambiae s.l\u003c/em\u003e encountered during the study. \u0026nbsp;Molecular analysis revealed only 1% of \u003cem\u003eAn. gambiae s.s.\u003c/em\u003e were present while the remaining 99% were \u003cem\u003eAn. coluzzii\u003c/em\u003e from the sample collected.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInsecticide Bioassay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePyrethroids and Organophosphates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes collected during the study showed strong resistance to permethrin and alphacypermethrin, followed by deltamethrin, except in the case of pirimiphosmethyl. Resistance levels varied across discriminating doses of permethrin, deltamethrin, alphacypermethrin, and pirimiphosmethyl. Mortality rates at Olorunsogo were 39%, 58.6%, 82%, and 100%, while at Freedompark they were 52.78%, 49.11%, 59%, and 100% respectively (Table 1). A high level of permethrin resistance was observed in Olorunsogo, indicated by knockdown times (KDT₅₀ and KDT₉₅). Pirimiphos-methyl was the most effective insecticide tested, with no resistance detected at either site. Both locations achieved 100% mortality within 24 hours. KDT₅₀ and KDT₉₅ were 39.72 and 79.14 minutes in Olorunsogo and 45.45 and 88.98 minutes in Freedompark, respectively (Fig 2). The mortality rates across sites were significantly correlated (R2= 0.030, p = 0.01).\u003c/p\u003e\n\u003cp\u003eTable 1: Knockdown time of different doses of insecticides and the percentage mortalities in the study areas.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInsecticide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eParameters \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eStudy site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOlorunsogo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFreedompark\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePermethrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% Mortality\u003c/p\u003e\n \u003cp\u003e(24 h)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eKDT50(min)\u003c/p\u003e\n \u003cp\u003eKDT95(min)\u003c/p\u003e\n \u003cp\u003eSusceptibility status\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003cp\u003e\u0026gt;120\u003c/p\u003e\n \u003cp\u003e\u0026gt;120\u003c/p\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e52.78\u003c/p\u003e\n \u003cp\u003e\u0026gt;120\u003c/p\u003e\n \u003cp\u003e\u0026gt;120\u003c/p\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDeltamethrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% Mortality\u003c/p\u003e\n \u003cp\u003e(24 h)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eKDT50(min)\u003c/p\u003e\n \u003cp\u003eKDT95(min)\u003c/p\u003e\n \u003cp\u003eSusceptibility status\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003cp\u003e108.05\u003c/p\u003e\n \u003cp\u003e\u0026gt;120\u003c/p\u003e\n \u003cp\u003eResistance suspected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e49.11\u003c/p\u003e\n \u003cp\u003e97.00\u003c/p\u003e\n \u003cp\u003e\u0026gt;120\u003c/p\u003e\n \u003cp\u003eResistance suspected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAlphacypermethrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% Mortality\u003c/p\u003e\n \u003cp\u003e(24 h)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eKDT50(min)\u003c/p\u003e\n \u003cp\u003eKDT95(min)\u003c/p\u003e\n \u003cp\u003eSusceptibility status\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e58.6\u003c/p\u003e\n \u003cp\u003e\u0026gt;120\u003c/p\u003e\n \u003cp\u003e\u0026gt;120\u003c/p\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003cp\u003e87.76\u003c/p\u003e\n \u003cp\u003e\u0026gt;120\u003c/p\u003e\n \u003cp\u003eResistance suspected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePirimiphos\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% Mortality\u003c/p\u003e\n \u003cp\u003e(24 h)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eKDT50(min)\u003c/p\u003e\n \u003cp\u003eKDT95(min)\u003c/p\u003e\n \u003cp\u003eSusceptibility status\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003cp\u003e39.72\u003c/p\u003e\n \u003cp\u003e79.14\u003c/p\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003cp\u003e45.45\u003c/p\u003e\n \u003cp\u003e88.98\u003c/p\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAll Knockdown time values greater than 120 min is reported as \u0026gt;120\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSynergy\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBioassay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results from the synergy assay for both sites, involving pre-exposure to PBO, revealed mortality in the \u003cem\u003eAn. coluzzii\u0026nbsp;\u003c/em\u003esubjected to the bioassay. However, PBO+Permethrin improved efficacy but did not fully restore susceptibility (Table 2). In Olorunsogo, the mortality rate increased significantly to 96%, with a KDT₅₀ of 70.83 minutes and a KDT₉₅ of 110.50 minutes. In Freedompark, the mortality rate was 75%, with a KDT₅₀ of 95.96 minutes and a KDT₉₅ still exceeding 120 minutes (Fig 4). PBO+ Deltamethrin showed a mortality rate reached 99%, with KDT₅₀ of 49.28 minutes and a KDT₉₅ of 106.20 minutes, indicating a high level of susceptibility, in Olorunsogo while resistance was suspected in Freedompark, with a mortality rate of 82.85%, and a KDT₅₀ of 60.20 minutes and a KDT₉₅ of 102.86 minutes (Fig 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2: Knockdown time of synergy and the percentage mortalities from the both sites.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInsecticide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eStudy site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOlorunsogo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFreedompark\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePBO+Permethrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% Mortality\u003c/p\u003e\n \u003cp\u003e(24 h)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eKDT50(min)\u003c/p\u003e\n \u003cp\u003eKDT95(min)\u003c/p\u003e\n \u003cp\u003eSusceptibility status\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003cp\u003e70.83\u003c/p\u003e\n \u003cp\u003e110.50\u003c/p\u003e\n \u003cp\u003eResistance suspected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003cp\u003e95.96\u003c/p\u003e\n \u003cp\u003e\u0026gt;120\u003c/p\u003e\n \u003cp\u003eResistance suspected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePBO+Deltamethrin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e% Mortality\u003c/p\u003e\n \u003cp\u003e(24 h)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eKDT50(min)\u003c/p\u003e\n \u003cp\u003eKDT95(min)\u003c/p\u003e\n \u003cp\u003eSusceptibility status\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e99\u003c/p\u003e\n \u003cp\u003e49.28\u003c/p\u003e\n \u003cp\u003e106.20\u003c/p\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e82.85\u003c/p\u003e\n \u003cp\u003e60.20\u003c/p\u003e\n \u003cp\u003e102.86\u003c/p\u003e\n \u003cp\u003eResistance suspected\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eMutant alleles frequency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe kdr genotyping mutation (L1014F) was high in both study areas. In Olorunsogo, 84% were homozygous for the resistant allele (RR), 4 individuals (16%) were homozygous for the susceptible allele (rr), and no heterozygotes (Rr) were detected (Table 3). In Freedom Park, 88% were homozygous resistant (RR), 3 individuals (12%) were homozygous susceptible (rr), and no heterozygotes were observed. Using Chi-square, P value for Hardy-Weinberg expectations for kdr gene (x2 =25, df= 1 P \u0026lt; 0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 3: KDR Frequency and Alleles Distribution of \u003cem\u003eAn. coluzzii\u003c/em\u003e from the two sites\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocations\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo screened\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKdr Mutation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKdr alleles Frequency (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003err\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eOlorunsogo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFreedom park\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolic Enzymes Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMetabolic activities based on the insecticide susceptibility of mosquitoes are presented as mean values (Table 4). The results indicated that monooxygenase activity was lower in resistant mosquitoes compared to susceptible and control groups. Conversely, GST activity was significantly higher in the resistant population (P \u0026le; 0.05; 0.001). \u0026beta;-esterase activity showed a gradual increase from the control to the susceptible and then to the resistant group, with the highest mean observed in resistant mosquitoes. A one-sample T-test revealed significant differences in enzyme activities among the susceptibility groups (P \u0026le; 0.05; 0.00). Acetylcholinesterase activity was slightly reduced in the susceptible group relative to the control (Table 5) (P \u0026le; 0.05; 0.03).\u003c/p\u003e\n\u003cp\u003eTable 4: Mean Activities of Metabolic Enzymes control and exposed An. colluzi to pyrethroids\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSusceptibility Status (N=11)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnzymes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMonooxygenase (ng/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGST (U/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026beta;-esterase (nmole/mg protein)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e55.70\u0026plusmn; 0.33 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e53.43\u0026plusmn; 2.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18.16\u0026plusmn; 0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSusceptible\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e55.35\u0026plusmn; 0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e55.71\u0026plusmn; 1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18.54\u0026plusmn; 0.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eResistance\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e53.60\u0026plusmn; 1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e59.52\u0026plusmn; 1.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18.86\u0026plusmn; 0.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 5: Mean Activities of Metabolic Enzymes in control and exposed \u003cem\u003eAn. coluzzii\u003c/em\u003e to pyrethroids\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSusceptibility Status\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAcetylcholinesterase\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e17.21\u0026plusmn; 1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSusceptible\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e15.65\u0026plusmn; 0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study found that, among 100 \u003cem\u003eAn. gambiae s.l\u003c/em\u003e. \u0026nbsp;analysed, 99% were identified as \u003cem\u003eAn. coluzzii\u003c/em\u003e and 1% as An. gambiae s.s. The predominance of \u003cem\u003eAn. coluzzii\u003c/em\u003e over An. gambiae s.s. corroborates earlier reports from Osun State (Iwalewa \u003cem\u003eet al.,\u003c/em\u003e 2025; Busari \u003cem\u003eet al.,\u003c/em\u003e 2024). Afolabi \u003cem\u003eet a\u003c/em\u003el. (2019) similarly reported a higher prevalence of \u003cem\u003eAn. coluzzii\u003c/em\u003e than An. gambiae in Ondo, Southwestern Nigeria. Studies from Nigeria (Oduola \u003cem\u003eet al.,\u003c/em\u003e 2012), Ghana (De Souza \u003cem\u003eet al.,\u003c/em\u003e 2010), and Benin (Yaouleton \u003cem\u003eet al.,\u003c/em\u003e 2010) also document dominance of the M form over the S form. This pattern may be related to greater larval tolerance of physico‑chemical variation in \u003cem\u003eAn. coluzzii\u003c/em\u003e, which can restrict gene flow and promote shifts in species composition.\u003c/p\u003e\n\u003cp\u003eThe field populations examined were highly resistant to pyrethroids, with no mortality observed in response to the organophosphate tested. Resistance to permethrin was particularly pronounced in Olorunsogo compared with Freedom Park, although mortality rates varied by site. These observations align with numerous reports of widespread pyrethroid resistance in Nigeria, including studies by Obembe \u003cem\u003eet al.,\u003c/em\u003e 2025 (Kwara State), Obembe \u003cem\u003eet al.,\u003c/em\u003e 2024 (national), Muhammad \u003cem\u003eet al.,\u003c/em\u003e 2021, Omotayo \u003cem\u003eet al.,\u003c/em\u003e 2022, Awolola \u003cem\u003eet al.,\u003c/em\u003e 2009 (Lagos), and Adeleke \u003cem\u003eet al.,\u003c/em\u003e 2018 (Osun State). Mosquito dispersal facilitates spread of resistance haplotypes, including the kdr‑W mutation that confers knockdown resistance to pyrethroids.\u003c/p\u003e\n\u003cp\u003eAnalysis of kdr genotypes indicated homozygous resistant (RR) frequencies of 0.84 and 0.88 at the study sites. Homozygous susceptible (rr) frequencies were 0.16 at Olorunsogo and 0.12 at Freedom Park. No heterozygotes were detected at either site. Consistent with previous reports (Djouka \u003cem\u003eet al.,\u003c/em\u003e 2008; Cukwuekezie \u003cem\u003eet al.,\u003c/em\u003e 2020; Omotayo \u003cem\u003eet al.,\u003c/em\u003e 2021; Ekedo \u003cem\u003eet al.,\u003c/em\u003e 2023), heterozygote frequencies were low in the sampled populations. The observed kdr allele distribution deviated from Hardy\u0026ndash;Weinberg expectations, suggesting selection, nonrandom mating, or population structure affecting the L1014 locus. The prominence of kdr mutations in West Africa has been driven by prolonged use of pyrethroid‑based control measures, reducing pyrethroid efficacy in many settings.\u003c/p\u003e\n\u003cp\u003ePiperonyl butoxide (PBO) is a synthetic synergist with minimal intrinsic insecticidal activity. Pre‑exposure to PBO increased mortality at both sites, although mortality in Freedom Park was slightly lower than in Olorunsogo. This contrasts with some studies reporting near‑complete restoration of susceptibility by PBO (Awolola \u003cem\u003eet al.,\u003c/em\u003e 2018; Martins \u003cem\u003eet al.,\u003c/em\u003e 2021) and likely reflects local differences in resistance profiles. PBO does not affect target‑site mutations (e.g., kdr L1014F) and may not fully restore susceptibility when multiple mechanisms such as elevated GSTs, esterases, or alternative pathways contribute to resistance.\u003c/p\u003e\n\u003cp\u003eBiochemical assays showed lower monooxygenase activity in the resistant groups; monooxygenase activity was significantly higher in the susceptible and control (P = 0.000). This observation aligns with Hamid‑Adiamoh \u003cem\u003eet al.,\u003c/em\u003e 2020, but contradicts studies reporting elevated monooxygenases in pyrethroid‑resistant populations (Cuamba \u003cem\u003eet al.,\u003c/em\u003e 2010; Awolola \u003cem\u003eet al.,\u003c/em\u003e 2018). Reduced monooxygenase activity in resistant specimens could reflect the involvement of cuticular resistance or antioxidant defenses that limit insecticide uptake or mitigate oxidative damage.\u003c/p\u003e\n\u003cp\u003eIn contrast, GST activity was significantly overexpressed in the resistant group (P = 0.001). Earlier studies document GST involvement in Anopheles resistance (Ifeoluwa \u003cem\u003eet al.,\u003c/em\u003e 2020; Koumo \u003cem\u003eet al.,\u003c/em\u003e 2025; Adeogun \u003cem\u003eet al.,\u003c/em\u003e 2025). GST‑mediated detoxification likely contributes to the high pyrethroid resistance observed at both sites. Additionally, \u0026beta;‑esterase activity was significantly elevated in resistant populations (P = 0.000), consistent with reports linking increased esterases to pyrethroid resistance (Hemingway \u003cem\u003eet al.,\u003c/em\u003e 2004; Zhong \u003cem\u003eet al.,\u003c/em\u003e 2024; Olaniran \u003cem\u003eet al.,\u003c/em\u003e 2020). Some studies, however, have found no significant difference in \u0026beta;‑esterase activity (Omotayo \u003cem\u003eet al.,\u003c/em\u003e 2021), indicating geographic and temporal variation in resistance mechanisms.\u003c/p\u003e\n\u003cp\u003eOverall, biochemical results indicate limited involvement of monooxygenases in the resistant \u003cem\u003eAn. coluzzii\u003c/em\u003e examined, while GSTs and \u0026beta;‑esterases are implicated as major contributors to resistance at the study sites. The significant GST overexpression and the variable effect of PBO at one site highlight a serious challenge for malaria control. Local differences in PBO efficacy imply that reliance on PBO‑based interventions may not uniformly restore pyrethroid susceptibility and could pose a risk to effective vector control.\u003c/p\u003e\n\u003cp\u003ePyrethroid resistance in Anopheles has become a major public‑health challenge in Nigeria because malaria control depends heavily on pyrethroid‑based interventions. Growing resistance demands urgent attention because it undermines control efforts and risks increasing the burden of malaria and other vector‑borne diseases.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e\u003cem\u003eAnopheles coluzzii\u003c/em\u003e remains the dominant vector in Osun State and exhibits strong resistance to pyrethroids. Although \u003cem\u003ekdr\u003c/em\u003e mutations are present, their low frequency indicates they are not the primary resistance mechanism. Instead, metabolic enzymes, particularly GSTs and \u0026beta;-esterases, play a major role, with evidence of additional complex mechanisms. The limited effectiveness of PBO highlights the crucial need for locally tailored, evidence-based vector control strategies. Therefore, there is an urgent need to manage resistance and prevent the continuous transmission of malaria through proactive vector surveillance and environmental management.\u003c/p\u003e"},{"header":"List of Abbreviations","content":"\u003cp\u003ePCR -Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eKDR -Knock-down Resistance\u003c/p\u003e\n\u003cp\u003eIRS- Indoor Residual Spray\u003c/p\u003e\n\u003cp\u003eITN- Insecticide Treated Net\u003c/p\u003e\n\u003cp\u003eDDT- Dichlorodiphenyltrichloroethane\u003c/p\u003e\n\u003cp\u003ePBO -Piperonyl butoxide\u003c/p\u003e\n\u003cp\u003eRR -Homozygous Resistant\u003c/p\u003e\n\u003cp\u003eRS- Heterozygous Susceptible\u003c/p\u003e\n\u003cp\u003eLGA -Local Government Area\u003c/p\u003e\n\u003cp\u003eMOVEB -Molecular Epidemiology and Vector Biology\u003c/p\u003e\n\u003cp\u003eWHO- World Health Organization\u003c/p\u003e\n\u003cp\u003eIMP\u0026nbsp;- Intentional Mismatch Primer\u003c/p\u003e\n\u003cp\u003epH- Potential Hydrogen\u003c/p\u003e\n\u003cp\u003eTMBZ - 3,3\u0026rsquo;,5, 5\u0026rsquo; Tetramethylbenzidine\u003c/p\u003e\n\u003cp\u003eGSH- Reduced Glutathione\u003c/p\u003e\n\u003cp\u003eOSMoH\u0026ndash;REC - Osun State Ministry of Health Research and Ethics Committee\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their sincere gratitude to the residents of the study areas for their patience and cooperation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization\u0026ndash;Z. O, M.A\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData curation\u0026ndash;Z.O., Y.O., Q.O., M.M., D.I., L.O., \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eH.K., G.O., G.B., C.T., T.A., I.O.\u003c/p\u003e\n\u003cp\u003eSupervision\u0026ndash;A.M., K.A., A.O., M.A.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData analysis\u0026ndash;Z.O.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eManuscript draft\u0026mdash;Z.O.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eManuscript editing\u0026ndash; M. A., L.O.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received no specific funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were generated and analyzed during the study are present in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVerbal consent of resident was obtained durig the community mobilization visit\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest regarding the article\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdeleke, M. A., Adeyemi, J. A., Fasasi, K. A., Oforka, L. 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Esterase-Mediated Pyrethroid Resistance in Populations of an Invasive Malaria Vector \u003cem\u003eAnopheles stephensi\u003c/em\u003e from Ethiopia. \u003cem\u003eGenes\u003c/em\u003e, \u003cem\u003e15\u003c/em\u003e(12).\u003c/li\u003e\n\u003cli\u003eNational Malaria Elimination Programme (NMEP). (2019). National Malaria Strategic Plan 2014\u0026ndash;2020 (Updated 2019). Federal Ministry of Health, Abuja, Nigeria. https://nmcp.gov.ng/\u003c/li\u003e\n\u003cli\u003eOkoko, M., Karisa, J., Gona, R., Odongo, T., Otieno, B., Yaa, F., ... \u0026amp; Maia, M. F. (2025). Phenotypic and genotypic insecticide resistance profiles of main malaria vectors in Kwale county, coastal Kenya. \u003cem\u003eMalaria Journal\u003c/em\u003e, \u003cem\u003e24\u003c/em\u003e(1), 191.\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. insecticide, synergist, resistance, metabolic assay, vector control, Osun.","lastPublishedDoi":"10.21203/rs.3.rs-9187188/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9187188/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The increasing resistance to pyrethroids and other insecticide classes by Anopheles mosquitoes continues to threaten the effectiveness of core vector-control interventions and risks reversing hard-won gains. This study investigated resistance mechanisms in Anopheles gambiae s.l. populations in two urban areas (Olorunsogo and Freedom Park) in Osogbo, Osun State. Larvae were collected from the field and reared to adults for WHO bioassay insecticide susceptibility testing using different doses of pyrethroids and an organophosphate. Synergist assays following WHO procedures assessed the involvement of monooxygenases in resistance. Sibling species were identified by Polymerase Chain Reaction (PCR) - Intentional Mismatch Primer- and allelespecific PCR was used to detect the L1014F Knockdown resistance (Kdr) mutation. Metabolic enzyme activities were measured by biochemical assays. High levels of pyrethroid resistance were observed at both locations, with variation between insecticides. Preexposure to PBO produced differing recoveries in mortality: PBO+permethrin yielded 96% mortality in Olorunsogo and 75% in Freedom Park, while PBO+deltamethrin produced 99% and 87%, respectively. Kdr allele frequencies were low, at 0.84 and 0.86 in Olorunsogo and Freedom Park. Monooxygenase activity was very low in resistant populations (P = 0.000), whereas glutathione Stransferases were significantly overexpressed (59.52 ± 1.44; P = 0.001). βesterase activity was also elevated (18.86; P = 0.000). The findings reveal multiple resistance mechanisms in urban Osun State populations of An. coluzzii, including metabolic detoxification and targetsite mutation. Therefore, proactive, evidencebased resistance management strategies are urgently needed to restore and sustain the efficacy of current vectorcontrol tools and strategies in the state.","manuscriptTitle":"Investigating multi-resistance mechanism of Anopheles gambiae s.l to insecticides in Osun State, Nigeria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-30 07:03:18","doi":"10.21203/rs.3.rs-9187188/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":"e536c686-01c4-4642-b7e0-4f3a3499ee5e","owner":[],"postedDate":"March 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65367733,"name":"Health sciences/Diseases"},{"id":65367734,"name":"Biological sciences/Ecology"},{"id":65367735,"name":"Earth and environmental sciences/Ecology"},{"id":65367736,"name":"Biological sciences/Molecular biology"},{"id":65367737,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-04-24T08:11:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-30 07:03:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9187188","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9187188","identity":"rs-9187188","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}