Comparative transcriptomic analysis of pyrethroid-resistant Anopheles gambiae s.l. from Ghana, reveals concentration-dependent and site-specific patterns of gene expression

Comparative transcriptomic analysis of pyrethroid-resistant Anopheles gambiae s.l. from Ghana, reveals concentration-dependent and site-specific patterns of gene expression | 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 Comparative transcriptomic analysis of pyrethroid-resistant Anopheles gambiae s.l. from Ghana, reveals concentration-dependent and site-specific patterns of gene expression Christopher Mfum Owusu-Asenso, Anisa Abdulai, Isaac Kwame Sraku, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7760720/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract The role of detoxification enzymes in pyrethroid resistance intensity among malaria vectors remains a critical area of research. This study evaluated the role of detoxifying enzymes in driving resistance intensity in Anopheles mosquitoes from high insecticide resistance sites in Ghana. Larvae were collected from Tema, Abossey Okai, and Dansoman, and bioassays were performed on 3–5 days old adult females by exposing them to deltamethrin at discriminating concentrations (1× = 0.05%, 5× = 0.25%, and 10× = 0.5%) to assess resistance intensity. A piperonyl butoxide (PBO) synergist assay was used to test the involvement of cytochrome P450s , while qRT-PCR quantified expression of detoxification genes ( CYP6P1 , CYP9K1 , CYP6M2 , CYP6P3 , CYP4G16 , GSTE2 , and CYP6Z1 ). kdr mutations ( L995F , L995S ) were genotyped. High-intensity resistance was observed across all sites [deltamethrin 10× MR = 75–91%]. Pre-exposure to PBO significantly increased mortality (Tema: 13–56%; Abossey Okai: 20–91%; Dansoman: 34–88%, P < 0.001), however, complete susceptibility was not restored. The L995F mutation was present at similar frequencies in resistant and susceptible mosquitoes. Transcriptomic profiling revealed concentration-dependent and site-specific expression: Tema; CYP9K1 , CYP6M2 , CYP6P1 , and CYP6P3 were significantly overexpressed (FC = 43.71–1222.98, P < 0.05), while CYP4G16 expression increased with insecticide concentration. In Abossey Okai, CYP9K1 , CYP6P1 , CYP6M2 , and CYP6P3 were overexpressed (FC = 5.54–162.84). Mosquitoes from Dansoman showed generally low expression, however, CYP6M2 and CYP6P3 were overexpressed (FC = 120.80–292.68). These findings may suggest the dominant role of metabolic resistance, particularly P450 -mediated detoxification in driving high pyrethroid resistance intensity in Ghana. Health sciences/Diseases Biological sciences/Genetics Biological sciences/Molecular biology Biological sciences/Zoology Pyrethroid resistance intensity metabolic gene expression concentration-dependent gene expression site-specific gene expression Anopheles gambiae s.l. kdr mutations Figures Figure 1 Figure 2 Figure 3 Figure 4 Background The widespread emergence of pyrethroid resistance in malaria vectors poses a critical challenge to global malaria control initiatives, particularly in Ghana, where insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are heavily reliant on this class of insecticides 1 . The upsurge of pyrethroid resistance among Anopheles mosquito populations has severely undermined the effectiveness of these interventions 2 , contributing to ongoing malaria transmission even in areas with high coverage of these vector control tools. The observed resistance pattern is likely influenced not only by widespread kdr mutations which alter the voltage-gated sodium channels targeted by pyrethroids, but could also be due to the overexpression of detoxification enzymes such as cytochrome P450 monooxygenases , glutathione S transferases ( GSTs ), and carboxylesterases 3 . These enzymes play a pivotal role in enhancing the mosquitoes' ability to metabolize and neutralize insecticides, thereby increasing their survival rate despite insecticide exposure 4 . The kdr mutations are prevalent across malaria-endemic areas, and in some regions, have reached fixation in mosquito populations 5 . The coexistence of kdr mutations and elevated detoxification enzyme activity may create a polygenic resistance profile threatening malaria elimination efforts 6 , 7 . While kdr mutations are well-documented and widely studied, the contribution of detoxification enzymes to pyrethroid resistance intensity, particularly in areas with already high kdr frequencies, remains less understood. This knowledge gap is critical, as detoxification enzymes may enhance resistance and influence the effectiveness of insecticides that are increasingly used for vector control. Given the critical role that detoxification enzymes may play in sustaining and intensifying pyrethroid resistance, it is imperative to investigate their contribution to driving resistance intensity in areas where kdr mutations are prevalent and possibly fixed. This study aimed to investigate how detoxifying enzymes drive pyrethroid resistance intensity in mosquito populations in areas with high insecticide resistance. Understanding these molecular pathways is crucial, providing insights into how detoxifying genes may heighten resistance intensity in malaria vectors, and also help develop potential interventions that could disrupt these resistance mechanisms and restore the efficacy of current control strategies. Results Insecticide Resistance Intensity Bioassays and Synergist Bioassays on An. gambiae s.l. High-intensity resistance was observed in Anopheles mosquitoes across all sites; Tema [1x Mortality rate (MR) = 13%, 5x = 81%, 10x = 90%], Abossey Okai [1x MR = 20%, 5x = 34%, 10x = 75%] and Dansoman [1x MR = 34%, 5x = 66%, 10x = 91%]. Exposure of the Kisumu susceptible strain to a standard baseline insecticide concentration [deltamethrin (1x MR = 0.05%)] resulted in full mortality (100%), confirming the susceptibility of this population to the insecticide and effectiveness of the insecticide-impregnated papers, Fig. 2 a. Pre-exposure to PBO (4%) for 1 hour before deltamethrin (0.05%) significantly increased mortality in An. gambiae s.l. populations from Tema (13% to 56%), Abossey Okai (20% to 91%), and Dansoman (34% to 88%) (χ² = 40, df = 1, P < 0.001), although full susceptibility was not restored (Fig. 2 b). Figure 2 : WHO susceptibility bioassays, a: intensity Bioassays b: PBO bioassays on Anopheles Mosquitoes from Tema, Abossey Okai, Dansoman and Kisumu Susceptible Strain . Concentration-Dependent Gene Expression Dynamics in Anopheles Mosquitoes Transcriptomic analysis revealed concentration-dependent metabolic gene expression in deltamethrin-resistant mosquitoes at 1x, 5x, and 10x discrimination doses, with variable gene responses across different insecticide concentrations, Table 1 . In Tema, CYP9K1 , CYP6M2 , CYP6P1 and CYP6P3 were significantly overexpressed in 1x and 10x insecticide concentrations [fold change (FC) = 43.71–1222.98] in deltamethrin-resistant mosquitoes in comparison to the Kisumu susceptible laboratory strain (P < 0.05). Expression levels of CYP4G16 increased with insecticide concentration [FC: 1x = 11.5, 5x = 15.9, 10x = 26.3], whilst GSTe2 and CYP6Z1 were moderately expressed 1x and 10x insecticide concentrations (FC = 1.85–4.20). In Abossey Okai, CYP9K1 , CYP6P1 were overexpressed in 1x and 10x concentration [FC = 5.54–162.84] in deltamethrin-resistant mosquitoes, while GSTe2 [FC = 0.01–2.52], CYP6M2 [FC = 0.07–9.71], CYP6P3 [FC = 5.54–41.38] and CYP4G16 [FC = 0.01–0.22] expression remained consistently low across all insecticide concentrations. In Dansoman, the control site; CYP6M2 and CYP6P3 were overexpressed in 1x and 10x deltamethrin-resistant mosquitoes (FC = 120.80–292.68), whilst low expression of GSTe2 , CYP9K1 , CYP6P1 , CYP6Z1 and CYP4G16 (FC = 0.00 to 1.43) were observed across all insecticide concentrations, Table 1 . Table 1 Association Between Resistance Intensity and Mean Relative Gene Expression Levels Across Insecticide Treatment Study site Metabolic genes Kisumu RI/Relative mean expression Fold change (FC = 2^-ΔΔCt) P- value (Bonferroni) ANOVA 1x 5x 10x 1x/5x 1x/10x 5x/10x P-value CYP4G16 1.16 11.5 15.9 26.3 ns 0.036 ns 0.00 CYP6M2 0.73 832.25 6.05 152.41 < 0.001 0.001 ns 0.00 Tema GSTE2 1.20 1.85 0.24 4.20 ns ns 0.020 0.02 CYP9K1 1.25 583.25 13.11 1222.98 0.005 0.002 < 0.001 0.00 CYP6P1 1.11 43.71 10.75 108.20 ns 0.029 0.002 0.00 CYP6P3 1.17 937.41 32.51 916.52 0.011 ns 0.013 0.00 Abossey Okai CYP9K1 1.25 138.97 0.23 162.84 0.049 0.021 ns 0.01 CYP6P1 1.11 5.64 0.97 43.94 ns 0.001 < 0.001 0.00 Dansoman CYP6M2 0.73 70.78 25.75 292.68 ns 0.016 0.005 0.00 CYP6P3 1.17 118.79 22.47 120.80 0.001 ns 0.001 0.00 RI = resistance intensity, ns = not significant, 1x, 5x, 10x = discriminating concentration of deltamethrin insecticide, Kisumu = Kisumu susceptible strain Site-specific Variations in Gene Expression Profiles in Anopheles mosquitoes Gene expression profiles varied significantly across the three study sites; Tema, Abossey Okai, and Dansoman, indicating site-specific differences in metabolic resistance mechanisms. In Tema, metabolic genes such as CYP9K1 [FC = 13.11–1222.98] and CYP6M2 [FC = 6.05–832.25] were highly overexpressed in deltamethrin-resistant An. gambiae s.l. compared to that of Abossey Okai and Dansoman, Fig. 3 . Comparatively, CYP6P1 [FC = 10.75–108.20], CYP6P3 [FC = 32.51–937.41] and GSTe2 [FC = 0.24–4.20] were also relatively highly expressed in Tema compared to the other study sites, Fig. 3 . Gene expression in An. gambiae s.l. from Abossey Okai, was moderate to relatively high, with CYP9K1 overexpressed [FC = 0.23–162.84] as compared to lower expression levels observed in Dansoman. GSTe2 [FC = 0.01–2.52], CYP6M2 [FC = 0.07–9.71] and CYP6P3 [FC = 5.54–41.38] expression remained consistently low across all insecticide concentrations, Fig. 3 . Anopheles gambiae s.l. from Dansoman exhibited the lowest overall gene expression levels, with minimal expression of GSTe2 [FC = 0.03–0.09], CYP6Z1 [FC = 0.04–0.16] and CYP9K1 [FC = 1.07–1.43]. However, CYP6M2 [FC = 25.75–292.68] was overexpressed, Fig. 3 . Figure 3 : Relative gene expression ( CYP4G16 , CYP6M2 , GSTe2 and CYP6Z1 and CYP9K1 , CYP6P1 , CYP6P3 ) for deltamethrin-resistant An. gambiae s.l. populations from Tema, Abossey Okai, Dansoman and Kisumu susceptible strain. Gene Expression in Deltamethrin-Resistant and Susceptible An . gambiae s.l. To understand the molecular basis of resistance intensity, gene expression between two biologically distinct groups: mosquitoes that died at the diagnostic dose (1×, representing the susceptible phenotype), and those that survived the highest dose (10×, representing the resistant phenotype) were analysed. Comparative gene expression levels between 10x deltamethrin-resistant An. gambiae s.l. in comparison to 1x deltamethrin-susceptible An. gambiae s.l. indicated substantial overexpression of specific detoxification genes. CYP6P1 [FC = 43.94] was overexpressed in 10x deltamethrin-resistant An. gambiae s.l.in comparison to the 1x deltamethrin-susceptible An. gambiae s.l. [FC = 1.98]. Moreover, CYP9K1 was significantly overexpressed in 10x deltamethrin-resistant An. gambiae s.l. [ FC = 162.84, P < 0.001]. Other genes, including CYP6P3 [resistant: FC = 35.92 vs susceptible: FC = 2.59; P = 0.002] and CYP4G16 [resistant: FC = 26.33 vs susceptible: FC = 0.13; P = 0.002] were significantly overexpressed in 10x deltamethrin-resistant An. gambiae s.l. in comparison to 1x deltamethrin-susceptible An. gambiae s.l., Table 2 . Table 2 Association Between Mean Expression Levels of Resistant and Susceptible Anopheles Mosquitoes Genes Mean relative expression level Fold change (FC = 2^-ΔΔCt) Kruskal Wallis P-value Kisumu DELTA 1x S DELTA 10x R CYP6P1 1.11 1.98 43.94 0.001 CYP4G16 1.13 0.13 26.33 0.002 CYP6Z1 1.09 0.41 0.65 ns GSTE2 1.20 0.09 0.73 ns CYP9K1 1.25 0.85 162.84 < 0.001 CYP6P3 1.17 2.59 35.92 0.002 CYP6M2 0.73 0.53 9.79 ns Kisumu: Kisumu susceptible strain, DELTA 1x S: deltamethrin-susceptible An. gambiae s.l. exposed to 1x concentration of deltamethrin, DELTA 10x R: deltamethrin-resistant An. gambiae s.l. exposed to 10x concentration of deltamethrin kdr Mutation in deltamethrin-resistant and susceptible Anopheles gambiae s.l. A total of 177 An. gambiae s.l. were genotyped for the presence of kdr mutations. A high allele frequency (0.78) of L995F was observed in Tema. In contrast, the L995S mutation was present at a much lower frequency in Tema (0.12) (𝜒2 = 57, 𝑃 < 0.001). In Abossey Okai, significantly high allele frequency (0.72) of L995F were observed (𝜒 2 = 60, 𝑃 < 0.001). However, low allele frequency of 0.03 was recorded for the L995S mutation in Abossey (𝜒 2 = 60, 𝑃 < 0.001). The L995F mutation had a relatively low frequency (0.41) in Dansoman compared to Tema and Abossey Okai. Similarly, the L995S mutation frequency in Dansoman was low (0.19), Table 3 . Table 3 kdr Allele Frequency Distribution in An. gambiae s.l. Stratified by Study Site, Phenotype and Species. Study site Phenotype N L995F L995S RR RS SS F P -value RR RS SS F P -value Tema Resistant 27 15 11 1 0.78 0.18 3 0 24 0.12 0.00 Susceptible 30 18 12 0 4 0 26 Total 57 33 23 1 7 0 50 Abossey Okai Resistant 30 23 0 7 0.72 0.00 2 0 28 0.03 0.00 Susceptible 30 20 0 10 0 0 30 Total 60 43 0 17 2 0 58 Dansoman Resistant 30 3 20 7 0 11 19 Susceptible 30 0 23 7 0.41 0.00 0 12 18 0.19 0.07 Total 60 3 43 14 0 23 37 Species An. gambiae s.s. Resistant 7 4 2 1 0.71 0.43 1 1 5 0.25 0.46 Susceptible 8 4 1 3 0.56 0.03 0 0 8 0.00 - An. coluzzii Resistant 53 35 11 7 0.76 0.00 4 9 65 0.08 0.00 Susceptible 78 34 30 14 0.63 0.12 4 11 63 0.07 0.00 Hybrid Resistant 2 0 2 0 0.50 0.16 0 1 1 0.25 0.64 Susceptible 4 0 4 0 0.50 0.05 0 1 3 0.13 0.78 N = samples tested, F = Allele frequency , L995F = kdr west , L995S = kdr east, (if P-value < 0.05, not consistent with HWE) Species Discrimination in Anopheles gambiae s.l. A total of 177 An. gambiae s.l. specimens were analysed to differentiate between the sibling species. Overall, An. coluzzii was the dominant species 88.1% (156), followed by An. gambiae s.s. 8.5% (15) and then hybrids 3.4% (6). Site-specific data revealed that, An. coluzzii was the most dominant species in all study sites. Hybrids were only present in Dansoman, Fig. 4 . Figure 4 : Species Discrimination of Anopheles gambiae s.l. from Tema Abossey Okai and Dansoman. Discussion Understanding insecticide resistance mechanisms in Anopheles mosquito populations is essential for effectively combating malaria transmission in areas with high insecticide resistance. This study highlights the significant role of detoxification enzymes in insecticide resistance intensity. The overexpression of key metabolic genes may signify an adaptive response of these malaria vectors under increased insecticide pressure and environmental contaminants, indicating the significant role of metabolic resistance mechanisms in driving insecticide resistance intensity in Anopheles mosquitoes. The observed high-intensity resistance to deltamethrin indicates the possibility of strong selection pressure within these sites. A similar trend was observed in studies that reported significant pyrethroid resistance in An. gambiae populations from Ghana 8 , Nigeria 9 and Burkina Faso 10 . These findings may suggest a challenge in controlling these vectors using pyrethroid-based interventions. Pre-exposure to PBO bioassays significantly increased deltamethrin-induced mortality in malaria vectors from all study sites, suggesting that monooxygenases may play a major role in the observed pyrethroid resistance in this study 11 . However, full susceptibility was not restored. This observation may indicate that other resistance mechanisms or genes, beyond monooxygenases such as cuticular resistance genes, may be contributing to resistance in the mosquitoes. This is particular concerning, indicating that the recently introduced PBO-bednets distributed by the National Malaria Elimination Program may have no operational effect on the control of An. gambiae s.l.. The concentration-dependent overexpression of CYP4G16 in Tema, with fold-change values significantly increasing systematically from 1x to 10x, could suggest that this gene actively contributes to detoxification under increasing pyrethroid insecticide. CYP4G16 is associated with cuticular resistance mechanisms in Anopheles species, where its elevated expression improves cuticle impermeability to insecticides 12 . This systematic increase in CYP4G16 expression aligns gene expression patterns from other studies where increasing deltamethrin concentrations resulted in overexpression of detoxifying genes 11 , 13 . However, CYP4G16 expression was negligible in Abossey Okai and Dansoman, which may indicate its site-specific role, potentially due to the heightened xenobiotics in Tema 14 . These site-specific variations may reflect the effect of localized selection pressures influencing detoxification gene regulation in An. gambiae s.l. populations. The CYP6M2 and CYP9K1 genes showed significant overexpression in the Anopheles population from Tema indicating their possible role in pyrethroid detoxification. This elevated expression contrasts with much lower levels observed in Abossey Okai, indicating a possible variation in selective pressures between the sites. Other studies have reported similar variations in gene expression patterns from sites with different environmental pressures 14 , 15 . High gene expression levels in deltamethrin-resistant An. gambiae s.l. exposed to 1x and 10x insecticide concentration followed by a sharp decrease in gene expression levels in Anopheles mosquito samples exposed at 5x insecticide concentration may suggest a complex regulatory mechanism that may depend on insecticide concentration 13 . This expression pattern could imply a possible saturation of detoxification at 5x, followed by adaptive upregulation at 10x to cope with higher insecticide exposure 13 . This finding may suggest that reducing insecticide use could help manage resistance by lowering selective pressure. Interestingly, 1x deltamethrin-susceptible An. gambiae s.l., despite harbouring kdr mutations relatively similar to the 10x deltamethrin-resistant An. gambiae s.l., showed lower expression of these detoxifying genes. The low expression of detoxifying genes in the 1x deltamethrin-susceptible An. gambiae s.l., may have contributed to their higher mortality despite the presence of these kdr mutations. This finding suggests that detoxifying enzymes may play a more significant role in high-pyrethroid resistance intensity than kdr mutations, as metabolic resistance mechanisms may be the primary factor driving survival under pyrethroid exposure. These results are consistent with previous studies that reported on the importance of detoxification pathways, such as cytochrome P450s , in insecticide resistance 11 , 13 . However, GSTE2 and CYP6Z1 showed minimal variation in expression (DELTA 1x S vs DELTA 10x R), suggesting that they may play a lesser role in pyrethroid resistance, supporting previous studies that reported variable contributions of different detoxification enzymes to insecticide resistance 10 15 . The L995F mutation was observed at high frequencies across all study sites. These findings are consistent with other studies documenting the widespread presence of the L995F mutation in Anopheles populations exposed to intense pyrethroid use 16 , 17 , 18 . Furthermore, the polygenic nature of insecticide resistance, as indicated by the co-occurrence of kdr mutations and elevated expression of detoxification genes could pose a significant challenge to malaria control efforts in these localities. Species composition analysis revealed that An. coluzzii was the predominant species of the sampled Anopheles population. This aligns with previous findings that have reported that An. coluzzii has adapted well to urbanized and polluted environments, likely due to its ecological flexibility 19 , 20 . Conclusion This study revealed critical knowledge on the insecticide resistance mechanisms in An. gambiae s.l. population in Ghana, indicating the concentration-specific and site-specific roles of detoxification enzymes in high pyrethroid-resistance intensity. Overexpression of detoxifying genes was significantly associated with high pyrethroid-resistance intensity in Anopheles gambiae mosquitoes. Allele frequency of kdr mutation was relatively similar among resistant and susceptible Anopheles mosquitoes. These findings may suggest the dominant role of metabolic resistance in driving high-pyrethroid resistance intensity in An. gambiae s.l. in Ghana. Materials and Methods Study Design This study was conducted in three sites within Ghana's coastal savannah ecozone: Tema (5.6698° N, 0.0200° E), Abossey Okai (09°24.886 N, 000°50.939 E), and Dansoman (5° 33′ 0″ N, 0° 16′ 0″ W), Fig. 1 . Tema Community 1 (5.6698° N, 0.0200° W), located in the coastal savannah of southern Ghana, is an industrial and port city. The dense concentration of manufacturing facilities and port operations generates significant industrial effluents, many of which are discharged into open drains and stagnant pools that double as mosquito breeding grounds. The Tema port experiences a constant influx of goods, vehicles, and people from other countries, potentially facilitating the introduction of mosquito populations with diverse genetic backgrounds, including those carrying resistance traits. Abossey Okai (5.5480° N, 0.2424° W), located in the city of Accra, in the coastal savannah zone of southern Ghana. Oil spills from engine oil changes and vehicle repairs, and the leaching of metal compounds into mosquito breeding habitats, may trigger an adaptive response and increase resistance in the vectors. These contaminants may activate detoxification enzyme pathways such as cytochrome P450s in mosquitoes, helping them metabolize toxins and insecticides, and contributing to enhanced resistance in mosquito populations. Dansoman, a suburb of Accra, was selected as a control site due to its limited exposure to industrial activities and automobile contamination by oil-spills. This site provides a baseline for comparison with the high-resistance sites in Tema and Abossey Okai. Figure 1 : A map of Ghana showing the study sites Larval Collection and Raising in Insectary Anopheles larvae sampling was carried out from January 2023 to July 2024. To avoid the collection of sibling species, larvae were sampled randomly from different breeding habitats in each study site. The collected larvae were carefully transferred into sterile plastic containers and promptly transported to the insectary at the Department of Medical Microbiology, University of Ghana Medical School. In the insectary, larvae were fed with Tetramin Baby Fish meal and reared under controlled, standardized temperature (26 ± 2°C) and relative humidity (80% ± 10%) with 12 h: 12 h light/dark cycle. Upon pupation, pupae were collected, transferred to cages, and allowed to emerge as adults. From the day of emergence, adults were provided with a wad of cotton wool soaked with 10% sugar solution until ready to be used for bioassay tests. WHO Intensity Bioassay on Adult Anopheles mosquitoes To determine the intensity of resistance in the An. gambiae s.l. population, batches of 25 non-blood-fed female mosquitoes aged 3–5 days were subjected to the WHO susceptibility test bioassay. For each insecticide concentration, four replicates and two control tubes were used. Mosquitoes were exposed to papers impregnated with deltamethrin at 1× (0.05%), 5× (0.25%), and 10× (0.5%) concentrations, alongside oil-impregnated papers as controls, following the standard WHO tube assay procedure. 21 . Mosquitoes were exposed for 1 h and the knockdown was recorded every 10 min during the 60-min exposure period. Mortality was recorded after a 24-h recovery period. Post-bioassay, alive (resistant), dead, and moribund mosquitoes from each insecticide treatment (1x, 5x, 10x) were processed separately. For DNA-based analyses, including molecular species identification and genotyping of kdr mutations, the head and thorax of resistant (alive) and dead mosquitoes were individually tweezed using sterile forceps and placed in 1.5 ml Eppendorf tubes containing silica gel and cotton. For RNA-based analyses, both resistant and moribund (susceptible) mosquitoes (mosquitoes unable to stand or fly properly, showing signs of severe incapacitation, such as twitching or lying on their backs) were used. The abdomen, legs, and wings of each moribund and resistant mosquito were submerged in separate 1.5 ml Eppendorf tubes containing RNA later (Ambion), treated according to the manufacturer's instructions, and stored overnight at 4°C to allow the solution to penetrate the mosquito tissue before transfer to a − 20°C freezer. Piperonyl Butoxide (PBO) Synergist Bioassays To understand the role of metabolic detoxification in pyrethroid resistance, piperonyl butoxide (PBO), a synergist that inhibits the specific activity of P450 monooxygenases in insects was used in the resistance bioassay. Each test had four replicates of 25 unfed female Anopheles mosquitoes aged 3–5 days were pre-exposed to 4% PBO-impregnated test papers for 1 hr, and then immediately exposed to 0.05% deltamethrin for another hour. One batch of 25 females were exposed to 4% PBO without insecticide and another batch of 25 females were also exposed to deltamethrin (0.05%) only, these served as controls. The number of mosquitoes knocked down after one hour of exposition to the insecticides were recorded. Mosquitoes were then transferred into holding tubes and supplied with a 10% sugar solution soaked in a wad of cotton. Mortality was scored after the 24-hr recovery period. RNA extraction and cDNA synthesis for metabolic resistance determination To investigate the role of detoxifying genes in resistance, deltamethrin-resistant and susceptible Anopheles mosquitoes (1x, 5x, 10x) from each site and Kisumu susceptible strain stored in RNA later at − 20°C were grouped in pools of 10 in 1.5-ml Eppendorf tubes. Total RNA was extracted from each pool (deltamethrin-resistant, deltamethrin-susceptible mosquitoes, as well as the Kisumu susceptible strain) using the ZYMO Quick-RNA™ Miniprep Kit following the manufacturer’s protocol. The cDNA was synthesized from 1 µg of total RNA of three biological replicates each [1x, 5x, 10x deltamethrin-resistant and 1x, 5x, 10x susceptible mosquitoes and the Kisumu susceptible strain using Protoscript III (Invitrogen) cDNA synthesis kit with oligo-dT20 and RNase H, (Invitrogen, New England Biolabs - United Kingdom) according to the manufacturer’s protocol. The total RNA and synthesized cDNA were stored at − 80ºC. Expression profile of detoxifying genes in Deltamethrin-resistant An . gambiae s.l. The level of expression of seven resistance-associated genes was validated by qRT-PCR. These include the GSTe2 , CYP6P3 , CYP6M2 , CYP9K1 , CYP9Z1 , CYP64G16 , CYP6P1 . Reactions were carried out in a final volume of 10µl consisting of 5µl SYBR Green Master Mix (Roche, Indianapolis, IN), 10 µM of each primer and 2.0µl of cDNA. The qRT-PCR assay was performed on the Bio-Rad Opus 96 PCR System (Bio-Rad) with an initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 10s, 60°C for 10s). Standard curves for all primer sets were carried out using Kisumu cDNA as a reference. kdr Genotyping and Species Discrimination of Anopheles Mosquitoes kdr genotyping was done by extracting DNA using the alcohol precipitation method 22 from the head and thorax of deltamethrin-resistant and susceptible individual mosquitoes preserved on silica gel. The L995F and L995S kdr mutations were identified using AS-PCR 23 . Mosquitoes collected were morphologically identified using identification keys by Gillies and Coetzee 24 . Members of the An. gambiae s.l. were further identified to distinguish sibling species, with a leg of each mosquito as DNA template using protocols of rDNA PCR by Scott et al. 25 and PCR-RFLP by Fanello et al. 26 . Data Management and Analysis Descriptive analyses were performed to visualize WHO susceptibility data, resistant allele frequencies, and mosquito species composition in graphs and tables. WHO insecticide susceptibility levels were classified according to the WHO criteria 21 . The chi-square test was utilised to determine differences in resistant alleles among mosquito populations. The allele frequency of resistance gene markers in the vector population from each site was calculated using the Hardy-Weinberg equilibrium (HWE) formula. The oneway ANOVA and Kruskal-Wallis test was employed to compare relative gene expression levels in An. gambiae s.l. population. The cycling threshold (CT) values obtained were used in determining the expression levels of the selected genes using the delta CT method 13 . The housekeeping gene Ribosomal Protein S7 (RPS7) (VectorBase: AGAP010592) was used as an internal control. The fold expression of the genes was calculated using the formula, Fold change (FC) = 2 −∆∆CT with normalization against the ribosomal protein S7. In all analyses, a P-value ≤ 0.05 was considered statistically significant. Declarations Competing Interests The authors declared no conflict of interest. Ethics declaration This study was approved by the Ethics and Protocol Review Committee of the College of Health Sciences, University of Ghana (protocol identification number: CHS-Et/M.8-P4.6/2023–2024). Meetings were conducted at each study site with the chiefs, community leaders, and residents to introduce the research. Permission to conduct the study at the various sites was obtained from the community leaders. All methods were carried out in accordance with relevant guidelines and regulations, including the ethical principles outlined in the Declaration of Helsinki for medical research. Meetings were held at each study site with chiefs, community leaders, and residents to introduce the research. Permission to conduct the study at the various sites was obtained from community leaders. Verbal informed consent was obtained residents for mosquito sampling activities. Consent to publish Not applicable Funding This study was supported by grants from the National Institute of Health (R01 A1 123074, R03 AI 186018 and D43 TW 011513). The funders had no role or influence on the design of this study, the collection, analyses, and interpretation of the data collected, as well as in writing this manuscript. Author Contribution CMO-A, AA, SKA, FA-A and YAA were responsible for the study design, supervised the data collection, and contributed to the writing of the manuscript. CMO-A carried out the sample collection. CMO-A and IKS carried out the laboratory work. CMO-A performed the data visualization and analysis. CMO-A drafted and revised the manuscript. All authors read and approved the final manuscript. Acknowledgement Special appreciation to the entire inhabitants within our study sites. The base map for the study site depiction was sourced from https://ghana-mission.co.in/mapofghana/ and modified using Adobe Photoshop (Version 7.0.1). Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Cook, J. et al. Implications of insecticide resistance for malaria vector control with long-lasting insecticidal nets: trends in pyrethroid resistance during a WHO-coordinated multi-country prospective study. Parasites Vectors . 11 , 550 (2018). Nguiffo-Nguete, D. et al. Evidence of intensification of pyrethroid resistance in the major malaria vectors in Kinshasa, Democratic Republic of Congo. Sci. Rep. 13 , 14711 (2023). Silva, A. P. B., Santos, J. M. M. & Martins, A. J. Mutations in the voltage-gated sodium channel gene of anophelines and their association with resistance to pyrethroids–a review. Parasites vectors . 7 , 450 (2014). Adedeji, E. O. et al. Anopheles metabolic proteins in malaria transmission, prevention and control: a review. Parasites Vectors . 13 , 465 (2020). Wondji, C. S. et al. Impact of pyrethroid resistance on operational malaria control in Malawi. Proc. Natl. Acad. Sci. U S A . 109 , 19063–19070 (2012). Edi, C. V. et al. CYP6 P450 Enzymes and ACE-1 Duplication Produce Extreme and Multiple Insecticide Resistance in the Malaria Mosquito Anopheles gambiae. PLoS Genet. 10 , e1004236 (2014). Riveron, J. M. et al. Insecticide Resistance in Malaria Vectors: An Update at a Global Scale (InTech, 2018). Owusu-Asenso, C. M. I.K.S., Nana Aba Setorwu Eyeson, Anisa Abdulai, Abdul Rahim Mohammed Sabtiu, Simon K Attah, Fred Aboagye-Antwi, Yaw Asare Afrane. Environmental contaminants drive Insecticide resistance in Anopheles Mosquitoes in Ghana. Scientific Reports (2025). Awolola, T. S. et al. Pyrethroids resistance intensity and resistance mechanisms in Anopheles gambiae from malaria vector surveillance sites in Nigeria. PLOS ONE . 13 , e0205230 (2018). Ibrahim, S. S., Ndula, M., Riveron, J. M., Irving, H. & Wondji, C. S. The P450 CYP6Z1confers carbamate/pyrethroid cross-resistance in a major African malaria vector beside a novel carbamate-insensitive N485I acetylcholinesterase-1mutation. Mol. Ecol. 25 , 3436–3452 (2016). Riveron, J. M. et al. Multiple insecticide resistance in the major malaria vector Anopheles funestus in southern Ghana: implications for malaria control. Parasites & Vectors 9 (2016). Balabanidou, V. et al. Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae. Proc. Natl. Acad. Sci. U S A . 113 , 9268–9273 (2016). Stica, C. et al. Characterizing the molecular and metabolic mechanisms of insecticide resistance in Anopheles gambiae in Faranah, Guinea. Malaria Journal 18 (2019). Kouadio, F. P. A. et al. Relationship between insecticide resistance profiles in Anopheles gambiae sensu lato and agricultural practices in Côte d’Ivoire. Parasites Vectors . 16 , 270 (2023). Mitchell, S. N. et al. Metabolic and target-site mechanisms combine to confer strong DDT resistance in Anopheles gambiae. PLoS One . 9 , e92662 (2014). Dabiré, R. K. et al. Distribution and Frequency of kdr Mutations within Anopheles gambiae s.l. Populations and First Report of the Ace.1G119S Mutation in Anopheles arabiensis from Burkina Faso (West Africa). PLOS ONE . 9 , e101484 (2014). Akuoko, O. K. et al. Biting behaviour, spatio-temporal dynamics, and the insecticide resistance status of malaria vectors in different ecological zones in Ghana. Parasites Vectors . 17 , 16 (2024). Djègbè, I. et al. Dynamics of insecticide resistance in malaria vectors in Benin: first evidence of the presence of L1014S kdr mutation in Anopheles gambiae from West Africa. Malar. J. 10 , 261 (2011). Coetzee, M. et al. Anopheles coluzzii and Anopheles amharicus, new members of the Anopheles gambiae complex. Zootaxa 3619 , 246–274 (2013). Mwangangi, J. M. et al. Shifts in malaria vector species composition and transmission dynamics along the Kenyan coast over the past 20 years. Malar. J. 12 , 13 (2013). WHO. Standard operating procedure for testing insecticide susceptibility of adult mosquitoes in WHO tube tests. World Health Organization , 16 (2022a). Green, M. R. & Sambrook, J. Precipitation of DNA with ethanol. Cold Spring Harbor Protocols pdb. prot093377 (2016). (2016). Jones, C. M. et al. Footprints of positive selection associated with a mutation (N1575Y) in the voltage-gated sodium channel of Anopheles gambiae. Proc. Natl. Acad. Sci. U S A . 109 , 6614–6619 (2012). Coetzee, M. Key to the females of Afrotropical Anopheles mosquitoes (Diptera: Culicidae). Malar. J. 19 , 70 (2020). Scott, J. A., Brogdon, W. G. & Collins, F. H. Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction. Am. J. Trop. Med. Hyg. 49 , 520–529 (1993). Fanello, C. & Santolamazza, F. della Torre, A. Simultaneous identification of species and molecular forms of the Anopheles gambiae complex by PCR-RFLP. Med. Vet. Entomol. 16 , 461–464 (2002). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 20 Nov, 2025 Reviews received at journal 06 Nov, 2025 Reviews received at journal 30 Oct, 2025 Reviewers agreed at journal 25 Oct, 2025 Reviewers agreed at journal 24 Oct, 2025 Reviewers agreed at journal 24 Oct, 2025 Reviewers agreed at journal 24 Oct, 2025 Reviewers agreed at journal 18 Oct, 2025 Reviewers invited by journal 17 Oct, 2025 Editor invited by journal 08 Oct, 2025 Editor assigned by journal 06 Oct, 2025 Submission checks completed at journal 04 Oct, 2025 First submitted to journal 01 Oct, 2025 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. 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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-7760720","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":548265723,"identity":"8a327ffd-fe8f-4bc2-a9b0-7d1ff54dd8db","order_by":0,"name":"Christopher Mfum Owusu-Asenso","email":"","orcid":"","institution":"University of Ghana Medical School","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"Mfum","lastName":"Owusu-Asenso","suffix":""},{"id":548265724,"identity":"15cdc308-8bec-46da-906f-ce0482ae88e6","order_by":1,"name":"Anisa Abdulai","email":"","orcid":"","institution":"University of Ghana Medical 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1","display":"","copyAsset":false,"role":"figure","size":43302,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA map of Ghana showing the study sites\u003c/strong\u003e. \u003cstrong\u003e(\u003c/strong\u003eThe base map for the study site depiction was sourced from \u003cem\u003ehttps://ghana-mission.co.in/mapofghana/\u003c/em\u003eand modified using Adobe Photoshop (Version 7.0.1).)\u003c/p\u003e","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7760720/v1/5743d8544b2825a9425a9205.png"},{"id":98437702,"identity":"1dc83efb-d491-4ca4-8c0f-48ae576ea33a","added_by":"auto","created_at":"2025-12-17 16:57:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWHO susceptibility bioassays, a: intensity Bioassays b: PBO bioassays on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAnopheles\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eMosquitoes from Tema, Abossey Okai, Dansoman and Kisumu Susceptible Strain\u003c/strong\u003e. Error bars indicate 95% confidence intervals, (R): resistant or alive mosquitoes.\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7760720/v1/8a3cb1a55418f49ecbb1534c.png"},{"id":98316196,"identity":"3cbf5b79-c238-48e9-8be3-dc6f66c1d2eb","added_by":"auto","created_at":"2025-12-16 13:21:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":29945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative gene expression (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCYP4G16\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCYP6M2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGSTe2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCYP6Z1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCYP9K1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCYP6P1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCYP6P3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) for deltamethrin-resistant \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAn. gambiae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e s.l. populations from Tema, Abossey Okai, Dansoman and Kisumu susceptible strain\u003c/strong\u003e. Error bars indicate 95% confidence intervals, (R): resistant or alive mosquitoes.\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7760720/v1/29e6b9756c5c92658c57e3f6.png"},{"id":98316193,"identity":"15c34e07-c169-4290-aaf8-e97b2eb807b5","added_by":"auto","created_at":"2025-12-16 13:21:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecies Discrimination of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAn. gambiae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e s.l. from Tema Abossey Okai and Dansoman.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7760720/v1/2ed0559d22dabfe3d9a0642b.png"},{"id":100069048,"identity":"bf94b2f5-e293-4578-8ea7-a2843f7f68ca","added_by":"auto","created_at":"2026-01-12 16:07:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2105007,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7760720/v1/05ddb305-f3dd-4585-bde5-1589355217d2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative transcriptomic analysis of pyrethroid-resistant Anopheles gambiae s.l. from Ghana, reveals concentration-dependent and site-specific patterns of gene expression","fulltext":[{"header":"Background","content":"\u003cp\u003eThe widespread emergence of pyrethroid resistance in malaria vectors poses a critical challenge to global malaria control initiatives, particularly in Ghana, where insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are heavily reliant on this class of insecticides \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The upsurge of pyrethroid resistance among \u003cem\u003eAnopheles\u003c/em\u003e mosquito populations has severely undermined the effectiveness of these interventions \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, contributing to ongoing malaria transmission even in areas with high coverage of these vector control tools.\u003c/p\u003e\u003cp\u003eThe observed resistance pattern is likely influenced not only by widespread \u003cem\u003ekdr\u003c/em\u003e mutations which alter the voltage-gated sodium channels targeted by pyrethroids, but could also be due to the overexpression of detoxification enzymes such as \u003cem\u003ecytochrome P450 monooxygenases\u003c/em\u003e, \u003cem\u003eglutathione S transferases\u003c/em\u003e (\u003cem\u003eGSTs\u003c/em\u003e), and \u003cem\u003ecarboxylesterases\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. These enzymes play a pivotal role in enhancing the mosquitoes' ability to metabolize and neutralize insecticides, thereby increasing their survival rate despite insecticide exposure \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003ekdr\u003c/em\u003e mutations are prevalent across malaria-endemic areas, and in some regions, have reached fixation in mosquito populations \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The coexistence of \u003cem\u003ekdr\u003c/em\u003e mutations and elevated detoxification enzyme activity may create a polygenic resistance profile threatening malaria elimination efforts \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWhile \u003cem\u003ekdr\u003c/em\u003e mutations are well-documented and widely studied, the contribution of detoxification enzymes to pyrethroid resistance intensity, particularly in areas with already high \u003cem\u003ekdr\u003c/em\u003e frequencies, remains less understood. This knowledge gap is critical, as detoxification enzymes may enhance resistance and influence the effectiveness of insecticides that are increasingly used for vector control. Given the critical role that detoxification enzymes may play in sustaining and intensifying pyrethroid resistance, it is imperative to investigate their contribution to driving resistance intensity in areas where \u003cem\u003ekdr\u003c/em\u003e mutations are prevalent and possibly fixed. This study aimed to investigate how detoxifying enzymes drive pyrethroid resistance intensity in mosquito populations in areas with high insecticide resistance. Understanding these molecular pathways is crucial, providing insights into how detoxifying genes may heighten resistance intensity in malaria vectors, and also help develop potential interventions that could disrupt these resistance mechanisms and restore the efficacy of current control strategies.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eInsecticide Resistance Intensity Bioassays and Synergist Bioassays on\u003c/b\u003e \u003cb\u003eAn. gambiae\u003c/b\u003e \u003cb\u003es.l.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHigh-intensity resistance was observed in \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes across all sites; Tema [1x Mortality rate (MR)\u0026thinsp;=\u0026thinsp;13%, 5x\u0026thinsp;=\u0026thinsp;81%, 10x\u0026thinsp;=\u0026thinsp;90%], Abossey Okai [1x MR\u0026thinsp;=\u0026thinsp;20%, 5x\u0026thinsp;=\u0026thinsp;34%, 10x\u0026thinsp;=\u0026thinsp;75%] and Dansoman [1x MR\u0026thinsp;=\u0026thinsp;34%, 5x\u0026thinsp;=\u0026thinsp;66%, 10x\u0026thinsp;=\u0026thinsp;91%]. Exposure of the Kisumu susceptible strain to a standard baseline insecticide concentration [deltamethrin (1x MR\u0026thinsp;=\u0026thinsp;0.05%)] resulted in full mortality (100%), confirming the susceptibility of this population to the insecticide and effectiveness of the insecticide-impregnated papers, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePre-exposure to PBO (4%) for 1 hour before deltamethrin (0.05%) significantly increased mortality in An. gambiae s.l. populations from Tema (13% to 56%), Abossey Okai (20% to 91%), and Dansoman (34% to 88%) (χ\u0026sup2; = 40, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), although full susceptibility was not restored (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e: \u003cb\u003eWHO susceptibility bioassays, a: intensity Bioassays b: PBO bioassays on\u003c/b\u003e \u003cb\u003eAnopheles\u003c/b\u003e \u003cb\u003eMosquitoes from Tema, Abossey Okai, Dansoman and Kisumu Susceptible Strain\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConcentration-Dependent Gene Expression Dynamics in\u003c/b\u003e \u003cb\u003eAnopheles\u003c/b\u003e \u003cb\u003eMosquitoes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTranscriptomic analysis revealed concentration-dependent metabolic gene expression in deltamethrin-resistant mosquitoes at 1x, 5x, and 10x discrimination doses, with variable gene responses across different insecticide concentrations, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eIn Tema, \u003cem\u003eCYP9K1\u003c/em\u003e, \u003cem\u003eCYP6M2\u003c/em\u003e, \u003cem\u003eCYP6P1\u003c/em\u003e and \u003cem\u003eCYP6P3\u003c/em\u003e were significantly overexpressed in 1x and 10x insecticide concentrations [fold change (FC)\u0026thinsp;=\u0026thinsp;43.71\u0026ndash;1222.98] in deltamethrin-resistant mosquitoes in comparison to the Kisumu susceptible laboratory strain (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Expression levels of \u003cem\u003eCYP4G16\u003c/em\u003e increased with insecticide concentration [FC: 1x\u0026thinsp;=\u0026thinsp;11.5, 5x\u0026thinsp;=\u0026thinsp;15.9, 10x\u0026thinsp;=\u0026thinsp;26.3], whilst \u003cem\u003eGSTe2\u003c/em\u003e and \u003cem\u003eCYP6Z1\u003c/em\u003e were moderately expressed 1x and 10x insecticide concentrations (FC\u0026thinsp;=\u0026thinsp;1.85\u0026ndash;4.20). In Abossey Okai, \u003cem\u003eCYP9K1\u003c/em\u003e, \u003cem\u003eCYP6P1\u003c/em\u003e were overexpressed in 1x and 10x concentration [FC\u0026thinsp;=\u0026thinsp;5.54\u0026ndash;162.84] in deltamethrin-resistant mosquitoes, while \u003cem\u003eGSTe2\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;0.01\u0026ndash;2.52], \u003cem\u003eCYP6M2\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;0.07\u0026ndash;9.71], \u003cem\u003eCYP6P3\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;5.54\u0026ndash;41.38] and \u003cem\u003eCYP4G16\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;0.01\u0026ndash;0.22] expression remained consistently low across all insecticide concentrations. In Dansoman, the control site; \u003cem\u003eCYP6M2\u003c/em\u003e and \u003cem\u003eCYP6P3\u003c/em\u003e were overexpressed in 1x and 10x deltamethrin-resistant mosquitoes (FC\u0026thinsp;=\u0026thinsp;120.80\u0026ndash;292.68), whilst low expression of \u003cem\u003eGSTe2\u003c/em\u003e, \u003cem\u003eCYP9K1\u003c/em\u003e, \u003cem\u003eCYP6P1\u003c/em\u003e, \u003cem\u003eCYP6Z1\u003c/em\u003e and \u003cem\u003eCYP4G16\u003c/em\u003e (FC\u0026thinsp;=\u0026thinsp;0.00 to 1.43) were observed across all insecticide concentrations, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAssociation Between Resistance Intensity and Mean Relative Gene Expression Levels Across Insecticide Treatment\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"12\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStudy site\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMetabolic genes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eKisumu\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eRI/Relative mean expression\u003c/p\u003e\u003cp\u003eFold change (FC\u0026thinsp;=\u0026thinsp;2^-ΔΔCt)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u003cp\u003eP- value (Bonferroni)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003eANOVA\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e1x\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e5x\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e10x\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e1x/5x\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e1x/10x\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003e\u003cb\u003e5x/10x\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003eP-value\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCYP4G16\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e26.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCYP6M2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e832.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e152.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTema\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eGSTE2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003e0.020\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCYP9K1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e583.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e13.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1222.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCYP6P1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e43.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e10.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e108.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.029\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCYP6P3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e937.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e32.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e916.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.011\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003e0.013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbossey Okai\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCYP9K1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e138.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e162.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.049\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCYP6P1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e43.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDansoman\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCYP6M2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e70.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e25.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e292.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003e0.005\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eCYP6P3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e118.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e22.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e120.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"12\"\u003e\u003cb\u003eRI\u0026thinsp;=\u0026thinsp;resistance intensity, ns\u0026thinsp;=\u0026thinsp;not significant, 1x, 5x, 10x\u0026thinsp;=\u0026thinsp;discriminating concentration of deltamethrin insecticide, Kisumu\u0026thinsp;=\u0026thinsp;Kisumu susceptible strain\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSite-specific Variations in Gene Expression Profiles in\u003c/b\u003e \u003cb\u003eAnopheles\u003c/b\u003e \u003cb\u003emosquitoes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGene expression profiles varied significantly across the three study sites; Tema, Abossey Okai, and Dansoman, indicating site-specific differences in metabolic resistance mechanisms. In Tema, metabolic genes such as \u003cem\u003eCYP9K1\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;13.11\u0026ndash;1222.98] and \u003cem\u003eCYP6M2\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;6.05\u0026ndash;832.25] were highly overexpressed in deltamethrin-resistant \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. compared to that of Abossey Okai and Dansoman, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Comparatively, \u003cem\u003eCYP6P1\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;10.75\u0026ndash;108.20], \u003cem\u003eCYP6P3\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;32.51\u0026ndash;937.41] and \u003cem\u003eGSTe2\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;0.24\u0026ndash;4.20] were also relatively highly expressed in Tema compared to the other study sites, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGene expression in \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. from Abossey Okai, was moderate to relatively high, with \u003cem\u003eCYP9K1\u003c/em\u003e overexpressed [FC\u0026thinsp;=\u0026thinsp;0.23\u0026ndash;162.84] as compared to lower expression levels observed in Dansoman. \u003cem\u003eGSTe2\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;0.01\u0026ndash;2.52], \u003cem\u003eCYP6M2\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;0.07\u0026ndash;9.71] and \u003cem\u003eCYP6P3\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;5.54\u0026ndash;41.38] expression remained consistently low across all insecticide concentrations, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e. \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. from Dansoman exhibited the lowest overall gene expression levels, with minimal expression of \u003cem\u003eGSTe2\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;0.03\u0026ndash;0.09], \u003cem\u003eCYP6Z1\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;0.04\u0026ndash;0.16] and \u003cem\u003eCYP9K1\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;1.07\u0026ndash;1.43]. However, \u003cem\u003eCYP6M2\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;25.75\u0026ndash;292.68] was overexpressed, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e: \u003cb\u003eRelative gene expression (\u003c/b\u003e\u003cb\u003eCYP4G16\u003c/b\u003e, \u003cb\u003eCYP6M2\u003c/b\u003e, \u003cb\u003eGSTe2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eCYP6Z1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eCYP9K1\u003c/b\u003e, \u003cb\u003eCYP6P1\u003c/b\u003e, \u003cb\u003eCYP6P3\u003c/b\u003e\u003cb\u003e) for deltamethrin-resistant\u003c/b\u003e \u003cb\u003eAn. gambiae\u003c/b\u003e \u003cb\u003es.l. populations from Tema, Abossey Okai, Dansoman and Kisumu susceptible strain.\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene Expression in Deltamethrin-Resistant and Susceptible\u003c/b\u003e \u003cb\u003eAn\u003c/b\u003e. \u003cb\u003egambiae\u003c/b\u003e \u003cb\u003es.l.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo understand the molecular basis of resistance intensity, gene expression between two biologically distinct groups: mosquitoes that died at the diagnostic dose (1\u0026times;, representing the susceptible phenotype), and those that survived the highest dose (10\u0026times;, representing the resistant phenotype) were analysed. Comparative gene expression levels between 10x deltamethrin-resistant \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. in comparison to 1x deltamethrin-susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. indicated substantial overexpression of specific detoxification genes. \u003cem\u003eCYP6P1\u003c/em\u003e [FC\u0026thinsp;=\u0026thinsp;43.94] was overexpressed in 10x deltamethrin-resistant \u003cem\u003eAn. gambiae\u003c/em\u003e s.l.in comparison to the 1x deltamethrin-susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. [FC\u0026thinsp;=\u0026thinsp;1.98]. Moreover, \u003cem\u003eCYP9K1\u003c/em\u003e was significantly overexpressed in 10x deltamethrin-resistant \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. [ FC\u0026thinsp;=\u0026thinsp;162.84, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001]. Other genes, including \u003cem\u003eCYP6P3\u003c/em\u003e [resistant: FC\u0026thinsp;=\u0026thinsp;35.92 vs susceptible: FC\u0026thinsp;=\u0026thinsp;2.59; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002] and \u003cem\u003eCYP4G16\u003c/em\u003e [resistant: FC\u0026thinsp;=\u0026thinsp;26.33 vs susceptible: FC\u0026thinsp;=\u0026thinsp;0.13; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002] were significantly overexpressed in 10x deltamethrin-resistant \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. in comparison to 1x deltamethrin-susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e s.l., Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAssociation Between Mean Expression Levels of Resistant and Susceptible \u003cem\u003eAnopheles\u003c/em\u003e Mosquitoes\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGenes\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eMean relative expression level\u003c/p\u003e\u003cp\u003eFold change (FC\u0026thinsp;=\u0026thinsp;2^-ΔΔCt)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eKruskal Wallis\u003c/p\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eKisumu\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eDELTA 1x S\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eDELTA 10x R\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCYP6P1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e43.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCYP4G16\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCYP6Z1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eGSTE2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCYP9K1\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e162.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCYP6P3\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35.92\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eCYP6M2\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003eKisumu: Kisumu susceptible strain, DELTA 1x S: deltamethrin-susceptible\u003c/b\u003e \u003cb\u003eAn. gambiae\u003c/b\u003e \u003cb\u003es.l. exposed to 1x concentration of deltamethrin, DELTA 10x R: deltamethrin-resistant\u003c/b\u003e \u003cb\u003eAn. gambiae\u003c/b\u003e \u003cb\u003es.l. exposed to 10x concentration of deltamethrin\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ekdr\u003c/b\u003e \u003cb\u003eMutation in deltamethrin-resistant and susceptible\u003c/b\u003e \u003cb\u003eAnopheles gambiae\u003c/b\u003e \u003cb\u003es.l.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 177 \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. were genotyped for the presence of \u003cem\u003ekdr\u003c/em\u003e mutations. A high allele frequency (0.78) of \u003cem\u003eL995F\u003c/em\u003e was observed in Tema. In contrast, the \u003cem\u003eL995S\u003c/em\u003e mutation was present at a much lower frequency in Tema (0.12) (\u0026#120594;2\u0026thinsp;=\u0026thinsp;57, \u0026#119875; \u0026lt; 0.001). In Abossey Okai, significantly high allele frequency (0.72) of \u003cem\u003eL995F\u003c/em\u003e were observed (\u0026#120594;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;60, \u0026#119875; \u0026lt; 0.001). However, low allele frequency of 0.03 was recorded for the \u003cem\u003eL995S\u003c/em\u003e mutation in Abossey (\u0026#120594;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;60, \u0026#119875; \u0026lt; 0.001).\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eL995F\u003c/em\u003e mutation had a relatively low frequency (0.41) in Dansoman compared to Tema and Abossey Okai. Similarly, the \u003cem\u003eL995S\u003c/em\u003e mutation frequency in Dansoman was low (0.19), Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003ekdr\u003c/em\u003e Allele Frequency Distribution in \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. Stratified by Study Site, Phenotype and Species.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"15\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eStudy site\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePhenotype\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eN\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c9\" namest=\"c4\"\u003e\u003cp\u003e\u003cem\u003eL995F\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c15\" namest=\"c10\"\u003e\u003cp\u003e\u003cem\u003eL995S\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eRR\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eRS\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eSS\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eF\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003cb\u003e-value\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003eRR\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cb\u003eRS\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003eSS\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u003cb\u003eF\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c15\" namest=\"c14\"\u003e\u003cp\u003e\u003cb\u003eP\u003c/b\u003e\u003cb\u003e-value\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTema\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResistant\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c8\" namest=\"c7\" rowspan=\"2\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSusceptible\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAbossey Okai\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResistant\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c8\" namest=\"c7\" rowspan=\"2\"\u003e\u003cp\u003e0.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSusceptible\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDansoman\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResistant\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSusceptible\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSpecies\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e s.s.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResistant\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSusceptible\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eAn. coluzzii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResistant\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSusceptible\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHybrid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eResistant\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e0.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSusceptible\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003e0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"1\" nameend=\"c15\" namest=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eN\u0026thinsp;=\u0026thinsp;samples tested, F\u0026thinsp;=\u0026thinsp;Allele frequency\u003c/b\u003e, \u003cb\u003eL995F\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u003c/b\u003e\u0026thinsp;\u003cb\u003ekdr\u003c/b\u003e \u003cb\u003ewest\u003c/b\u003e, \u003cb\u003eL995S\u003c/b\u003e\u0026thinsp;\u003cb\u003e=\u003c/b\u003e\u0026thinsp;\u003cb\u003ekdr\u003c/b\u003e \u003cb\u003eeast, (if P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, not consistent with HWE)\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpecies Discrimination in\u003c/b\u003e \u003cb\u003eAnopheles gambiae\u003c/b\u003e \u003cb\u003es.l.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 177 \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. specimens were analysed to differentiate between the sibling species. Overall, \u003cem\u003eAn. coluzzii\u003c/em\u003e was the dominant species 88.1% (156), followed by \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. 8.5% (15) and then hybrids 3.4% (6). Site-specific data revealed that, \u003cem\u003eAn. coluzzii\u003c/em\u003e was the most dominant species in all study sites. Hybrids were only present in Dansoman, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e: \u003cb\u003eSpecies Discrimination of\u003c/b\u003e \u003cb\u003eAnopheles gambiae\u003c/b\u003e \u003cb\u003es.l. from Tema Abossey Okai and Dansoman.\u003c/b\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUnderstanding insecticide resistance mechanisms in \u003cem\u003eAnopheles\u003c/em\u003e mosquito populations is essential for effectively combating malaria transmission in areas with high insecticide resistance. This study highlights the significant role of detoxification enzymes in insecticide resistance intensity. The overexpression of key metabolic genes may signify an adaptive response of these malaria vectors under increased insecticide pressure and environmental contaminants, indicating the significant role of metabolic resistance mechanisms in driving insecticide resistance intensity in \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes.\u003c/p\u003e\u003cp\u003eThe observed high-intensity resistance to deltamethrin indicates the possibility of strong selection pressure within these sites. A similar trend was observed in studies that reported significant pyrethroid resistance in \u003cem\u003eAn. gambiae\u003c/em\u003e populations from Ghana\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, Nigeria \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and Burkina Faso \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These findings may suggest a challenge in controlling these vectors using pyrethroid-based interventions. Pre-exposure to PBO bioassays significantly increased deltamethrin-induced mortality in malaria vectors from all study sites, suggesting that monooxygenases may play a major role in the observed pyrethroid resistance in this study \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, full susceptibility was not restored. This observation may indicate that other resistance mechanisms or genes, beyond \u003cem\u003emonooxygenases\u003c/em\u003e such as cuticular resistance genes, may be contributing to resistance in the mosquitoes. This is particular concerning, indicating that the recently introduced PBO-bednets distributed by the National Malaria Elimination Program may have no operational effect on the control of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l..\u003c/p\u003e\u003cp\u003eThe concentration-dependent overexpression of \u003cem\u003eCYP4G16\u003c/em\u003e in Tema, with fold-change values significantly increasing systematically from 1x to 10x, could suggest that this gene actively contributes to detoxification under increasing pyrethroid insecticide. \u003cem\u003eCYP4G16\u003c/em\u003e is associated with cuticular resistance mechanisms in \u003cem\u003eAnopheles\u003c/em\u003e species, where its elevated expression improves cuticle impermeability to insecticides \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This systematic increase in \u003cem\u003eCYP4G16\u003c/em\u003e expression aligns gene expression patterns from other studies where increasing deltamethrin concentrations resulted in overexpression of detoxifying genes \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, \u003cem\u003eCYP4G16\u003c/em\u003e expression was negligible in Abossey Okai and Dansoman, which may indicate its site-specific role, potentially due to the heightened xenobiotics in Tema \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. These site-specific variations may reflect the effect of localized selection pressures influencing detoxification gene regulation in \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. populations.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eCYP6M2\u003c/em\u003e and \u003cem\u003eCYP9K1\u003c/em\u003e genes showed significant overexpression in the \u003cem\u003eAnopheles\u003c/em\u003e population from Tema indicating their possible role in pyrethroid detoxification. This elevated expression contrasts with much lower levels observed in Abossey Okai, indicating a possible variation in selective pressures between the sites. Other studies have reported similar variations in gene expression patterns from sites with different environmental pressures \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHigh gene expression levels in deltamethrin-resistant \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. exposed to 1x and 10x insecticide concentration followed by a sharp decrease in gene expression levels in \u003cem\u003eAnopheles\u003c/em\u003e mosquito samples exposed at 5x insecticide concentration may suggest a complex regulatory mechanism that may depend on insecticide concentration \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This expression pattern could imply a possible saturation of detoxification at 5x, followed by adaptive upregulation at 10x to cope with higher insecticide exposure \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. This finding may suggest that reducing insecticide use could help manage resistance by lowering selective pressure.\u003c/p\u003e\u003cp\u003eInterestingly, 1x deltamethrin-susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e s.l., despite harbouring \u003cem\u003ekdr\u003c/em\u003e mutations relatively similar to the 10x deltamethrin-resistant \u003cem\u003eAn. gambiae\u003c/em\u003e s.l., showed lower expression of these detoxifying genes. The low expression of detoxifying genes in the 1x deltamethrin-susceptible \u003cem\u003eAn. gambiae\u003c/em\u003e s.l., may have contributed to their higher mortality despite the presence of these \u003cem\u003ekdr\u003c/em\u003e mutations. This finding suggests that detoxifying enzymes may play a more significant role in high-pyrethroid resistance intensity than \u003cem\u003ekdr\u003c/em\u003e mutations, as metabolic resistance mechanisms may be the primary factor driving survival under pyrethroid exposure. These results are consistent with previous studies that reported on the importance of detoxification pathways, such as \u003cem\u003ecytochrome P450s\u003c/em\u003e, in insecticide resistance \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. However, \u003cem\u003eGSTE2\u003c/em\u003e and \u003cem\u003eCYP6Z1\u003c/em\u003e showed minimal variation in expression (DELTA 1x S vs DELTA 10x R), suggesting that they may play a lesser role in pyrethroid resistance, supporting previous studies that reported variable contributions of different detoxification enzymes to insecticide resistance \u003csup\u003e10 15\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eL995F\u003c/em\u003e mutation was observed at high frequencies across all study sites. These findings are consistent with other studies documenting the widespread presence of the \u003cem\u003eL995F\u003c/em\u003e mutation in \u003cem\u003eAnopheles\u003c/em\u003e populations exposed to intense pyrethroid use \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Furthermore, the polygenic nature of insecticide resistance, as indicated by the co-occurrence of \u003cem\u003ekdr\u003c/em\u003e mutations and elevated expression of detoxification genes could pose a significant challenge to malaria control efforts in these localities.\u003c/p\u003e\u003cp\u003eSpecies composition analysis revealed that \u003cem\u003eAn. coluzzii\u003c/em\u003e was the predominant species of the sampled \u003cem\u003eAnopheles\u003c/em\u003e population. This aligns with previous findings that have reported that \u003cem\u003eAn. coluzzii\u003c/em\u003e has adapted well to urbanized and polluted environments, likely due to its ecological flexibility \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study revealed critical knowledge on the insecticide resistance mechanisms in \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. population in Ghana, indicating the concentration-specific and site-specific roles of detoxification enzymes in high pyrethroid-resistance intensity. Overexpression of detoxifying genes was significantly associated with high pyrethroid-resistance intensity in \u003cem\u003eAnopheles gambiae\u003c/em\u003e mosquitoes. Allele frequency of \u003cem\u003ekdr\u003c/em\u003e mutation was relatively similar among resistant and susceptible \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes. These findings may suggest the dominant role of metabolic resistance in driving high-pyrethroid resistance intensity in \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. in Ghana.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStudy Design\u003c/h2\u003e\u003cp\u003eThis study was conducted in three sites within Ghana's coastal savannah ecozone: Tema (5.6698\u0026deg; N, 0.0200\u0026deg; E), Abossey Okai (09\u0026deg;24.886 N, 000\u0026deg;50.939 E), and Dansoman (5\u0026deg; 33\u0026prime; 0\u0026Prime; N, 0\u0026deg; 16\u0026prime; 0\u0026Prime; W), Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTema Community 1 (5.6698\u0026deg; N, 0.0200\u0026deg; W), located in the coastal savannah of southern Ghana, is an industrial and port city. The dense concentration of manufacturing facilities and port operations generates significant industrial effluents, many of which are discharged into open drains and stagnant pools that double as mosquito breeding grounds. The Tema port experiences a constant influx of goods, vehicles, and people from other countries, potentially facilitating the introduction of mosquito populations with diverse genetic backgrounds, including those carrying resistance traits.\u003c/p\u003e\u003cp\u003eAbossey Okai (5.5480\u0026deg; N, 0.2424\u0026deg; W), located in the city of Accra, in the coastal savannah zone of southern Ghana. Oil spills from engine oil changes and vehicle repairs, and the leaching of metal compounds into mosquito breeding habitats, may trigger an adaptive response and increase resistance in the vectors. These contaminants may activate detoxification enzyme pathways such as cytochrome P450s in mosquitoes, helping them metabolize toxins and insecticides, and contributing to enhanced resistance in mosquito populations.\u003c/p\u003e\u003cp\u003eDansoman, a suburb of Accra, was selected as a control site due to its limited exposure to industrial activities and automobile contamination by oil-spills. This site provides a baseline for comparison with the high-resistance sites in Tema and Abossey Okai.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e: \u003cb\u003eA map of Ghana showing the study sites\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLarval Collection and Raising in Insectary\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eAnopheles\u003c/em\u003e larvae sampling was carried out from January 2023 to July 2024. To avoid the collection of sibling species, larvae were sampled randomly from different breeding habitats in each study site. The collected larvae were carefully transferred into sterile plastic containers and promptly transported to the insectary at the Department of Medical Microbiology, University of Ghana Medical School. In the insectary, larvae were fed with Tetramin Baby Fish meal and reared under controlled, standardized temperature (26\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) and relative humidity (80% \u0026plusmn; 10%) with 12 h: 12 h light/dark cycle. Upon pupation, pupae were collected, transferred to cages, and allowed to emerge as adults. From the day of emergence, adults were provided with a wad of cotton wool soaked with 10% sugar solution until ready to be used for bioassay tests.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWHO Intensity Bioassay on Adult\u003c/b\u003e \u003cb\u003eAnopheles\u003c/b\u003e \u003cb\u003emosquitoes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the intensity of resistance in the \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. population, batches of 25 non-blood-fed female mosquitoes aged 3\u0026ndash;5 days were subjected to the WHO susceptibility test bioassay. For each insecticide concentration, four replicates and two control tubes were used. Mosquitoes were exposed to papers impregnated with deltamethrin at 1\u0026times; (0.05%), 5\u0026times; (0.25%), and 10\u0026times; (0.5%) concentrations, alongside oil-impregnated papers as controls, following the standard WHO tube assay procedure. \u003csup\u003e21\u003c/sup\u003e. Mosquitoes were exposed for 1 h and the knockdown was recorded every 10 min during the 60-min exposure period. Mortality was recorded after a 24-h recovery period.\u003c/p\u003e\u003cp\u003ePost-bioassay, alive (resistant), dead, and moribund mosquitoes from each insecticide treatment (1x, 5x, 10x) were processed separately. For DNA-based analyses, including molecular species identification and genotyping of \u003cem\u003ekdr\u003c/em\u003e mutations, the head and thorax of resistant (alive) and dead mosquitoes were individually tweezed using sterile forceps and placed in 1.5 ml Eppendorf tubes containing silica gel and cotton. For RNA-based analyses, both resistant and moribund (susceptible) mosquitoes (mosquitoes unable to stand or fly properly, showing signs of severe incapacitation, such as twitching or lying on their backs) were used. The abdomen, legs, and wings of each moribund and resistant mosquito were submerged in separate 1.5 ml Eppendorf tubes containing RNA later (Ambion), treated according to the manufacturer's instructions, and stored overnight at 4\u0026deg;C to allow the solution to penetrate the mosquito tissue before transfer to a \u0026minus;\u0026thinsp;20\u0026deg;C freezer.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePiperonyl Butoxide (PBO) Synergist Bioassays\u003c/h2\u003e\u003cp\u003eTo understand the role of metabolic detoxification in pyrethroid resistance, piperonyl butoxide (PBO), a synergist that inhibits the specific activity of P450 monooxygenases in insects was used in the resistance bioassay. Each test had four replicates of 25 unfed female \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes aged 3\u0026ndash;5 days were pre-exposed to 4% PBO-impregnated test papers for 1 hr, and then immediately exposed to 0.05% deltamethrin for another hour. One batch of 25 females were exposed to 4% PBO without insecticide and another batch of 25 females were also exposed to deltamethrin (0.05%) only, these served as controls. The number of mosquitoes knocked down after one hour of exposition to the insecticides were recorded. Mosquitoes were then transferred into holding tubes and supplied with a 10% sugar solution soaked in a wad of cotton. Mortality was scored after the 24-hr recovery period.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRNA extraction and cDNA synthesis for metabolic resistance determination\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of detoxifying genes in resistance, deltamethrin-resistant and susceptible \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes (1x, 5x, 10x) from each site and Kisumu susceptible strain stored in RNA later at \u0026minus;\u0026thinsp;20\u0026deg;C were grouped in pools of 10 in 1.5-ml Eppendorf tubes. Total RNA was extracted from each pool (deltamethrin-resistant, deltamethrin-susceptible mosquitoes, as well as the Kisumu susceptible strain) using the ZYMO \u003cem\u003eQuick-RNA\u0026trade; Miniprep Kit\u003c/em\u003e following the manufacturer\u0026rsquo;s protocol. The cDNA was synthesized from 1 \u0026micro;g of total RNA of three biological replicates each [1x, 5x, 10x deltamethrin-resistant and 1x, 5x, 10x susceptible mosquitoes and the Kisumu susceptible strain using Protoscript III (Invitrogen) cDNA synthesis kit with oligo-dT20 and RNase H, (Invitrogen, New England Biolabs - United Kingdom) according to the manufacturer\u0026rsquo;s protocol. The total RNA and synthesized cDNA were stored at \u0026minus;\u0026thinsp;80\u0026ordm;C.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression profile of detoxifying genes in Deltamethrin-resistant\u003c/b\u003e \u003cb\u003eAn\u003c/b\u003e. \u003cb\u003egambiae\u003c/b\u003e \u003cb\u003es.l.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe level of expression of seven resistance-associated genes was validated by qRT-PCR. These include the \u003cem\u003eGSTe2\u003c/em\u003e, \u003cem\u003eCYP6P3\u003c/em\u003e, \u003cem\u003eCYP6M2\u003c/em\u003e, \u003cem\u003eCYP9K1\u003c/em\u003e, \u003cem\u003eCYP9Z1\u003c/em\u003e, \u003cem\u003eCYP64G16\u003c/em\u003e, \u003cem\u003eCYP6P1\u003c/em\u003e. Reactions were carried out in a final volume of 10\u0026micro;l consisting of 5\u0026micro;l SYBR Green Master Mix (Roche, Indianapolis, IN), 10 \u0026micro;M of each primer and 2.0\u0026micro;l of cDNA. The qRT-PCR assay was performed on the Bio-Rad Opus 96 PCR System (Bio-Rad) with an initial denaturation at 95\u0026deg;C for 10 min, followed by 40 cycles of 95\u0026deg;C for 10s, 60\u0026deg;C for 10s). Standard curves for all primer sets were carried out using Kisumu cDNA as a reference.\u003c/p\u003e\u003cp\u003e\u003cb\u003ekdr\u003c/b\u003e \u003cb\u003eGenotyping and Species Discrimination of\u003c/b\u003e \u003cb\u003eAnopheles\u003c/b\u003e \u003cb\u003eMosquitoes\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003ekdr\u003c/em\u003e genotyping was done by extracting DNA using the alcohol precipitation method \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e from the head and thorax of deltamethrin-resistant and susceptible individual mosquitoes preserved on silica gel. The \u003cem\u003eL995F\u003c/em\u003e and \u003cem\u003eL995S kdr\u003c/em\u003e mutations were identified using AS-PCR \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Mosquitoes collected were morphologically identified using identification keys by Gillies and Coetzee \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Members of the \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. were further identified to distinguish sibling species, with a leg of each mosquito as DNA template using protocols of rDNA PCR by Scott \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e25\u003c/sup\u003e and PCR-RFLP by Fanello \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e26\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eData Management and Analysis\u003c/h3\u003e\n\u003cp\u003eDescriptive analyses were performed to visualize WHO susceptibility data, resistant allele frequencies, and mosquito species composition in graphs and tables. WHO insecticide susceptibility levels were classified according to the WHO criteria \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The chi-square test was utilised to determine differences in resistant alleles among mosquito populations. The allele frequency of resistance gene markers in the vector population from each site was calculated using the Hardy-Weinberg equilibrium (HWE) formula. The oneway ANOVA and Kruskal-Wallis test was employed to compare relative gene expression levels in \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. population. The cycling threshold (CT) values obtained were used in determining the expression levels of the selected genes using the delta CT method \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The housekeeping gene Ribosomal Protein S7 (RPS7) (VectorBase: AGAP010592) was used as an internal control. The fold expression of the genes was calculated using the formula, Fold change (FC)\u0026thinsp;=\u0026thinsp;2 \u0026minus;∆∆CT with normalization against the ribosomal protein S7. In all analyses, a P-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors declared no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthics declaration\u003c/h2\u003e\u003cp\u003eThis study was approved by the Ethics and Protocol Review Committee of the College of Health Sciences, University of Ghana (protocol identification number: CHS-Et/M.8-P4.6/2023\u0026ndash;2024). Meetings were conducted at each study site with the chiefs, community leaders, and residents to introduce the research. Permission to conduct the study at the various sites was obtained from the community leaders. All methods were carried out in accordance with relevant guidelines and regulations, including the ethical principles outlined in the Declaration of Helsinki for medical research. Meetings were held at each study site with chiefs, community leaders, and residents to introduce the research. Permission to conduct the study at the various sites was obtained from community leaders. Verbal informed consent was obtained residents for mosquito sampling activities.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConsent to publish\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was supported by grants from the National Institute of Health (R01 A1 123074, R03 AI 186018 and D43 TW 011513). The funders had no role or influence on the design of this study, the collection, analyses, and interpretation of the data collected, as well as in writing this manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCMO-A, AA, SKA, FA-A and YAA were responsible for the study design, supervised the data collection, and contributed to the writing of the manuscript. CMO-A carried out the sample collection. CMO-A and IKS carried out the laboratory work. CMO-A performed the data visualization and analysis. CMO-A drafted and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eSpecial appreciation to the entire inhabitants within our study sites. The base map for the study site depiction was sourced from https://ghana-mission.co.in/mapofghana/ and modified using Adobe Photoshop (Version 7.0.1).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCook, J. et al. Implications of insecticide resistance for malaria vector control with long-lasting insecticidal nets: trends in pyrethroid resistance during a WHO-coordinated multi-country prospective study. \u003cem\u003eParasites Vectors\u003c/em\u003e. \u003cb\u003e11\u003c/b\u003e, 550 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNguiffo-Nguete, D. et al. Evidence of intensification of pyrethroid resistance in the major malaria vectors in Kinshasa, Democratic Republic of Congo. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 14711 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilva, A. P. B., Santos, J. M. M. \u0026amp; Martins, A. J. Mutations in the voltage-gated sodium channel gene of anophelines and their association with resistance to pyrethroids\u0026ndash;a review. \u003cem\u003eParasites vectors\u003c/em\u003e. \u003cb\u003e7\u003c/b\u003e, 450 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdedeji, E. O. et al. Anopheles metabolic proteins in malaria transmission, prevention and control: a review. \u003cem\u003eParasites Vectors\u003c/em\u003e. \u003cb\u003e13\u003c/b\u003e, 465 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWondji, C. S. et al. Impact of pyrethroid resistance on operational malaria control in Malawi. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e. \u003cb\u003e109\u003c/b\u003e, 19063\u0026ndash;19070 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEdi, C. V. et al. CYP6 P450 Enzymes and ACE-1 Duplication Produce Extreme and Multiple Insecticide Resistance in the Malaria Mosquito Anopheles gambiae. \u003cem\u003ePLoS Genet.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, e1004236 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRiveron, J. M. et al. \u003cem\u003eInsecticide Resistance in Malaria Vectors: An Update at a Global Scale\u003c/em\u003e (InTech, 2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOwusu-Asenso, C. M. I.K.S., Nana Aba Setorwu Eyeson, Anisa Abdulai, Abdul Rahim Mohammed Sabtiu, Simon K Attah, Fred Aboagye-Antwi, Yaw Asare Afrane. Environmental contaminants drive Insecticide resistance in Anopheles Mosquitoes in Ghana. \u003cem\u003eScientific Reports\u003c/em\u003e (2025).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAwolola, T. S. et al. Pyrethroids resistance intensity and resistance mechanisms in Anopheles gambiae from malaria vector surveillance sites in Nigeria. \u003cem\u003ePLOS ONE\u003c/em\u003e. \u003cb\u003e13\u003c/b\u003e, e0205230 (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIbrahim, S. S., Ndula, M., Riveron, J. M., Irving, H. \u0026amp; Wondji, C. S. The P450 CYP6Z1confers carbamate/pyrethroid cross-resistance in a major African malaria vector beside a novel carbamate-insensitive N485I acetylcholinesterase-1mutation. \u003cem\u003eMol. Ecol.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e, 3436\u0026ndash;3452 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRiveron, J. M. et al. Multiple insecticide resistance in the major malaria vector Anopheles funestus in southern Ghana: implications for malaria control. \u003cem\u003eParasites \u0026amp; Vectors\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBalabanidou, V. et al. Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e. \u003cb\u003e113\u003c/b\u003e, 9268\u0026ndash;9273 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStica, C. et al. Characterizing the molecular and metabolic mechanisms of insecticide resistance in Anopheles gambiae in Faranah, Guinea. \u003cem\u003eMalaria Journal\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKouadio, F. P. A. et al. Relationship between insecticide resistance profiles in Anopheles gambiae sensu lato and agricultural practices in C\u0026ocirc;te d\u0026rsquo;Ivoire. \u003cem\u003eParasites Vectors\u003c/em\u003e. \u003cb\u003e16\u003c/b\u003e, 270 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMitchell, S. N. et al. Metabolic and target-site mechanisms combine to confer strong DDT resistance in Anopheles gambiae. \u003cem\u003ePLoS One\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e, e92662 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDabir\u0026eacute;, R. K. et al. Distribution and Frequency of kdr Mutations within Anopheles gambiae s.l. Populations and First Report of the Ace.1G119S Mutation in Anopheles arabiensis from Burkina Faso (West Africa). \u003cem\u003ePLOS ONE\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e, e101484 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAkuoko, O. K. et al. Biting behaviour, spatio-temporal dynamics, and the insecticide resistance status of malaria vectors in different ecological zones in Ghana. \u003cem\u003eParasites Vectors\u003c/em\u003e. \u003cb\u003e17\u003c/b\u003e, 16 (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDj\u0026egrave;gb\u0026egrave;, I. et al. Dynamics of insecticide resistance in malaria vectors in Benin: first evidence of the presence of L1014S kdr mutation in Anopheles gambiae from West Africa. \u003cem\u003eMalar. J.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 261 (2011).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCoetzee, M. et al. Anopheles coluzzii and Anopheles amharicus, new members of the Anopheles gambiae complex. \u003cem\u003eZootaxa\u003c/em\u003e \u003cb\u003e3619\u003c/b\u003e, 246\u0026ndash;274 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMwangangi, J. M. et al. Shifts in malaria vector species composition and transmission dynamics along the Kenyan coast over the past 20 years. \u003cem\u003eMalar. J.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 13 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWHO. Standard operating procedure for testing insecticide susceptibility of adult mosquitoes in WHO tube tests. \u003cem\u003eWorld Health Organization\u003c/em\u003e, \u003cb\u003e16\u003c/b\u003e (2022a).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGreen, M. R. \u0026amp; Sambrook, J. Precipitation of DNA with ethanol. \u003cem\u003eCold Spring Harbor Protocols\u003c/em\u003e pdb. prot093377 (2016). (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJones, C. M. et al. Footprints of positive selection associated with a mutation (N1575Y) in the voltage-gated sodium channel of Anopheles gambiae. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e. \u003cb\u003e109\u003c/b\u003e, 6614\u0026ndash;6619 (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCoetzee, M. Key to the females of Afrotropical Anopheles mosquitoes (Diptera: Culicidae). \u003cem\u003eMalar. J.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 70 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eScott, J. A., Brogdon, W. G. \u0026amp; Collins, F. H. Identification of single specimens of the Anopheles gambiae complex by the polymerase chain reaction. \u003cem\u003eAm. J. Trop. Med. Hyg.\u003c/em\u003e \u003cb\u003e49\u003c/b\u003e, 520\u0026ndash;529 (1993).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFanello, C. \u0026amp; Santolamazza, F. della Torre, A. Simultaneous identification of species and molecular forms of the Anopheles gambiae complex by PCR-RFLP. \u003cem\u003eMed. Vet. Entomol.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 461\u0026ndash;464 (2002).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pyrethroid resistance intensity, metabolic gene expression, concentration-dependent gene expression, site-specific gene expression, Anopheles gambiae s.l., kdr mutations","lastPublishedDoi":"10.21203/rs.3.rs-7760720/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7760720/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe role of detoxification enzymes in pyrethroid resistance intensity among malaria vectors remains a critical area of research. This study evaluated the role of detoxifying enzymes in driving resistance intensity in \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes from high insecticide resistance sites in Ghana. Larvae were collected from Tema, Abossey Okai, and Dansoman, and bioassays were performed on 3\u0026ndash;5 days old adult females by exposing them to deltamethrin at discriminating concentrations (1\u0026times; = 0.05%, 5\u0026times; = 0.25%, and 10\u0026times; = 0.5%) to assess resistance intensity. A piperonyl butoxide (PBO) synergist assay was used to test the involvement of \u003cem\u003ecytochrome P450s\u003c/em\u003e, while qRT-PCR quantified expression of detoxification genes (\u003cem\u003eCYP6P1\u003c/em\u003e, \u003cem\u003eCYP9K1\u003c/em\u003e, \u003cem\u003eCYP6M2\u003c/em\u003e, \u003cem\u003eCYP6P3\u003c/em\u003e, \u003cem\u003eCYP4G16\u003c/em\u003e, \u003cem\u003eGSTE2\u003c/em\u003e, and \u003cem\u003eCYP6Z1\u003c/em\u003e). \u003cem\u003ekdr\u003c/em\u003e mutations (\u003cem\u003eL995F\u003c/em\u003e, \u003cem\u003eL995S\u003c/em\u003e) were genotyped. High-intensity resistance was observed across all sites [deltamethrin 10\u0026times; MR\u0026thinsp;=\u0026thinsp;75\u0026ndash;91%]. Pre-exposure to PBO significantly increased mortality (Tema: 13\u0026ndash;56%; Abossey Okai: 20\u0026ndash;91%; Dansoman: 34\u0026ndash;88%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), however, complete susceptibility was not restored. The \u003cem\u003eL995F\u003c/em\u003e mutation was present at similar frequencies in resistant and susceptible mosquitoes. Transcriptomic profiling revealed concentration-dependent and site-specific expression: Tema; \u003cem\u003eCYP9K1\u003c/em\u003e, \u003cem\u003eCYP6M2\u003c/em\u003e, \u003cem\u003eCYP6P1\u003c/em\u003e, and \u003cem\u003eCYP6P3\u003c/em\u003e were significantly overexpressed (FC\u0026thinsp;=\u0026thinsp;43.71\u0026ndash;1222.98, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while \u003cem\u003eCYP4G16\u003c/em\u003e expression increased with insecticide concentration. In Abossey Okai, \u003cem\u003eCYP9K1\u003c/em\u003e, \u003cem\u003eCYP6P1\u003c/em\u003e, \u003cem\u003eCYP6M2\u003c/em\u003e, and \u003cem\u003eCYP6P3\u003c/em\u003e were overexpressed (FC\u0026thinsp;=\u0026thinsp;5.54\u0026ndash;162.84). Mosquitoes from Dansoman showed generally low expression, however, \u003cem\u003eCYP6M2\u003c/em\u003e and \u003cem\u003eCYP6P3\u003c/em\u003e were overexpressed (FC\u0026thinsp;=\u0026thinsp;120.80\u0026ndash;292.68). These findings may suggest the dominant role of metabolic resistance, particularly \u003cem\u003eP450\u003c/em\u003e-mediated detoxification in driving high pyrethroid resistance intensity in Ghana.\u003c/p\u003e","manuscriptTitle":"Comparative transcriptomic analysis of pyrethroid-resistant Anopheles gambiae s.l. from Ghana, reveals concentration-dependent and site-specific patterns of gene expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-16 13:21:23","doi":"10.21203/rs.3.rs-7760720/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-20T16:13:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-06T09:52:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-30T22:27:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"256052543847445802093969164970564946713","date":"2025-10-25T19:51:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180202010288309353539100998061415172676","date":"2025-10-24T14:22:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"294999236704915685646402684382795815276","date":"2025-10-24T10:10:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230076631050107530438414388892494206529","date":"2025-10-24T06:57:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271472393074611964833188067673940206940","date":"2025-10-18T05:08:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-17T12:00:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-08T06:49:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-06T10:44:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-04T04:45:17+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-01T14:56:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"61a6d570-a45c-48c5-9036-bfede3b530cf","owner":[],"postedDate":"December 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58339417,"name":"Health sciences/Diseases"},{"id":58339418,"name":"Biological sciences/Genetics"},{"id":58339419,"name":"Biological sciences/Molecular biology"},{"id":58339420,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-01-12T16:00:28+00:00","versionOfRecord":{"articleIdentity":"rs-7760720","link":"https://doi.org/10.1038/s41598-025-33720-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-09 15:57:14","publishedOnDateReadable":"January 9th, 2026"},"versionCreatedAt":"2025-12-16 13:21:23","video":"","vorDoi":"10.1038/s41598-025-33720-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-33720-1","workflowStages":[]},"version":"v1","identity":"rs-7760720","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7760720","identity":"rs-7760720","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}