The Susceptibility Status of Aedes aegypti (Diptera: Culicidae) Mosquitoes in Malaysia on Pyrethroid and Organophosphate Insecticides with First Detection of T1520I Mutation

The Susceptibility Status of Aedes aegypti (Diptera: Culicidae) Mosquitoes in Malaysia on Pyrethroid and Organophosphate Insecticides with First Detection of T1520I Mutation | 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 The Susceptibility Status of Aedes aegypti (Diptera: Culicidae) Mosquitoes in Malaysia on Pyrethroid and Organophosphate Insecticides with First Detection of T1520I Mutation Teng Ma, Wan Fatma Zuharah This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7708119/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Background Aedes aegypti , the primary vector of dengue, has developed widespread resistance to insecticides, posing a threat to the efficacy of vector control programs. This study assessed the susceptibility status of Ae. aegypti across Malaysia from seven dengue hotspot localities and characterized knockdown resistance ( kdr ) mutations, including the first detection of T1520I. Methods Susceptibility was assessed using World Health Organization (WHO, 2022) bioassays with four insecticides: deltamethrin (0.03%), permethrin (0.4%), pirimiphos-methyl (60 mg/m²), and malathion (5%). Knockdown times (KT₅₀ and KT₉₅) were determined using probit regression, and resistance ratios were calculated relative to a laboratory-susceptible strain. Genomic DNA was extracted from survivors, and sequencing of the voltage-gated sodium channel (VGSC) gene was conducted to detect knockdown resistance (kdr) mutations. Results All field populations remained susceptible to malathion, with mortality ≥ 95%, though one Johor strain (ABS) showed suspected resistance (95%). Pirimiphos-methyl resistance was widespread, with mortality as low as 6% (ABS). Whereas resistance to pyrethroids was pronounced, with deltamethrin mortality ranging from 22% (ABS) to 95% (AA), and permethrin from 0% (TMB, ABS) to 95% (AA). Knockdown assays revealed high resistance ratios, particularly for permethrin (TMB: RR₅₀=9.98, RR₉₅=14.98; ABS: RR₉₅=41.46). Sequencing identified multiple kdr mutations, including S989P, V1016G, F1534C, A1007G, and the novel detection of T1520I in Malaysian populations. F1534C was strongly associated with permethrin resistance, while V1016G and S989P predominated under deltamethrin exposure. Conclusions Aedes aegypti populations in Malaysia exhibit widespread pyrethroid resistance and emerging organophosphate resistance, underpinned by multiple kdr mutations. The first detection of T1520I underscores the evolving genetic basis of resistance. These findings highlight the urgent need for integrated resistance management, including molecular surveillance and insecticide rotation, to sustain effective dengue control. Health sciences/Diseases Biological sciences/Genetics Biological sciences/Microbiology Biological sciences/Molecular biology Biological sciences/Zoology Aedes aegypti Insecticide Resistance kdr mutation mosquito pyrethroid organophosphate Figures Figure 1 Figure 2 Figure 3 Introduction Aedes aegypti is one of the most widespread and medically important mosquito species, inhabiting tropical and subtropical regions worldwide. It serves as the primary vector of several arboviruses, including dengue virus (DENV), chikungunya virus (CHIKV), yellow fever virus, and Zika virus 1 , 2 , 3 . Dengue has emerged as the most significant arboviral disease affecting humans, with nearly 390 million infections estimated annually, of which approximately 96 million are clinically apparent 4 , 5 . In Malaysia, dengue poses an escalating public health burden, characterized by the hyperendemic circulation of all four DENV serotypes and recurrent outbreaks, resulting in substantial morbidity and economic costs 6 , 7 , 8 . Over the long term, dengue incidence in Malaysia has shown a marked increase, rising from approximately 44 cases per 100,000 population in 1999 to about 399 cases per 100,000 in 2015, as reported in a decade-scale study on dengue morbidity and mortality 9 . Despite advances in dengue vaccines, including a tetravalent formulation that has demonstrated partial efficacy, large-scale implementation remains limited due to variable performance across serostatus groups and regulatory concerns 10 , 11 . As a result, vector control continues to serve as the primary method of dengue prevention in Malaysia. Vector control strategies largely rely on environmental management to reduce larval habitats and the extensive use of chemical insecticides to suppress adult mosquito populations 12 , 13 . Pyrethroids (e.g., deltamethrin, permethrin) and organophosphates (e.g., malathion, pirimiphos-methyl) are the most widely deployed insecticides in Malaysia’s dengue control programs 14 . However, this sustained reliance on chemical interventions has exerted strong selective pressure on mosquito populations, leading to the emergence and spread of insecticide resistance. Insecticide resistance in Ae. aegypti has been increasingly documented across Malaysia. A nationwide study by Asgarian et al. (2023) 15 revealed heterogeneous but widespread resistance to pyrethroids in major dengue hotspots, including Penang, Johor, and Selangor. While Ae. aegypti populations remain largely susceptible to malathion, localized resistance has been detected in Kuala Lumpur and Selangor 16 . At the molecular level, resistance is frequently associated with knockdown resistance ( kdr ) mutations in the voltage-gated sodium channel (VGSC) gene. Several key mutations—S989P, V1016G, F1534C, and more recently A1007G—have been reported in Malaysian Ae. aegypti populations 17 , 18 , 19 , 20 , reflecting the cumulative effects of insecticide selection pressure and highlighting complex, region-specific resistance dynamics. While insecticides remain indispensable in Malaysia’s vector control programs, their effectiveness is increasingly undermined by resistance. The over-reliance on chemical interventions as a primary control strategy has inadvertently accelerated the selection of resistant phenotypes, threatening the long-term sustainability of dengue control. Understanding the prevalence and geographic distribution of kdr mutations provides valuable insights into resistance dynamics, enabling vector control programs to adapt their interventions and maintain the efficacy of chemical-based measures. Therefore, the study aims to assess the susceptibility status of Ae. aegypti populations from diverse Malaysian locations and to characterize the frequency of key kdr mutations associated with pyrethroid resistance. Methods Mosquito Collections Aedes aegypti populations were collected from seven dengue hotspot localities distributed across five states in Malaysia (Fig. 1 ). The sampling sites were selected based on dengue hotspot designations provided by the Ministry of Health Malaysia (MOH), following an analysis of national dengue case distributions. The selected sites represented geographically distinct regions of Malaysia, including, (1) Northern Malaysia (Penang): Apartment Asoka (AA), Penang Island (5°21′N, 100°18′E); Taman Machang Bubuk (TMB), Penang Mainland (5°30′02″N, 100°25′70″E); and Flat Sri Pauh (FSP), Penang Mainland (5°33′92″N, 100°50′46″E), (2) East Coast Malaysia (Kelantan): Taman Desa Rahmat (TDR), Kelantan (6°11′49″N, 102°28′09″E), (3) Central Malaysia: Taman Dahlia (TD), Negeri Sembilan (2°84′37″N, 101°82′65″E); and PPR Raya Permai (PRP), Kuala Lumpur (3°06′17″N, 101°70′56″E), and (4) Southern Malaysia (Johor): Apartment Bukit Saujana (ABS), Johor (1°47′44″N, 103°74′87″E). At each locality, sixty ovitraps were deployed randomly for five consecutive days. Hardwood paddles served as oviposition substrates and were subsequently collected and transported to the Medical Entomology Laboratory, Universiti Sains Malaysia. Eggs were reared to adults under controlled insectary conditions (27 ± 2°C; 70–80% relative humidity). Only the dominant Aedes species obtained from each locality were subjected to subsequent insecticide susceptibility assays. Adult Mosquito Bioassay Adult susceptibility bioassays were performed following the World Health Organization (WHO) standard protocol 21 . Four replicates of 25 non-blood-fed female Ae. aegypti (2–5 days old) from each field population were exposed to insecticide-impregnated filter papers prepared by the Vector Control Research Unit (VCRU), Universiti Sains Malaysia. The insecticides tested were: (1) 0.03% deltamethrin, (2) 0.4% permethrin, (3) pirimiphos-methyl at 60 mg/m², and (4) 5% malathion. Mosquitoes were initially introduced into holding tubes and acclimatized for one hour. Any damaged, injured, or dead individuals were replaced prior to testing. A total of 25 females were then transferred into exposure tubes lined with insecticide-impregnated papers. Knockdown data was recorded at 5-minute intervals until one hour. Following exposure, mosquitoes were transferred into clean paper cups supplied with 10% sucrose solution and maintained at 27 ± 2°C and 60–80% relative humidity. Mortality was assessed 24 hours post-exposure, and four replicates were performed for each insecticide. Two control replicates were prepared with silicone oil for pyrethroids, olive oil for malathion, and acetone for pirimiphos-methyl. Laboratory-susceptible Ae. aegypti strain maintained at VCRU (Penang, Malaysia; 5°21′N, 100°18′E) which were reared for over 300 generations since the 1960s was used as the reference baseline. Genomic DNA Extraction Genomic DNA was extracted from individual adult Ae. aegypti mosquitoes that survived the susceptibility bioassays using the PrimeWay Genomic II DNA Extraction Kit (Apical Scientific Sdn Bhd, Malaysia), following the manufacturer’s protocol with slight modifications to optimize yield. Prior to extraction, the elution buffer was preheated to 60°C to improve recovery efficiency. Each mosquito was homogenized in 100 µl of GL1 buffer using a sterile pestle, which was subsequently rinsed with an additional 100 µl of GL1 buffer to maximize tissue recovery. Protein digestion was carried out by adding 20 µl of Proteinase K solution to the homogenate, followed by incubation at 60°C for 3 h with occasional inversion to ensure complete lysis of cellular and nuclear membranes. After incubation, the lysate was centrifuged at 12,000 × g for 2.5 min at room temperature, and the supernatant was carefully transferred to a new 1.5 mL microcentrifuge tube to prevent disruption of the pellet. The supernatant was mixed with 200 µl of GL2 buffer and vortexed thoroughly. Afterward, 4 µL of RNase A solution was added. The mixture was vortexed again and incubated at room temperature for 5 min to degrade RNA contaminants. DNA precipitation was induced by adding 200 µl of absolute ethanol, and the lysate–ethanol mixture was vortexed until homogeneous. A total of 750 µL of the resulting mixture was applied to a PrimeWay silica spin column inserted into a clean collection tube and centrifuged at 12,000 x g for 1.2 min. Afterward, the flow-through was discarded. To remove residual impurities, 400 µl of Wash Buffer G1 was added to the column, followed by centrifugation at 12,000 x g for 35 s; the flow-through was then discarded. This was followed by a second wash step with 600 µl of Wash Buffer G2 and centrifugation at 12,000 × g for 35 s. To ensure complete removal of residual ethanol and drying of the membrane, the column was subjected to an additional centrifugation step at 12,000 x g for 3.5 min. For DNA elution, the spin column was transferred into a new 1.5 ml microcentrifuge tube, and 50 µl of preheated (60°C) elution buffer was added directly to the center of the membrane to maximize contact. After incubating at room temperature for at least 3 min, the column was centrifuged at 12,000 x g for 35 s to obtain the final DNA yield. The eluted DNA was quantified and assessed for purity using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) at an absorbance of 260 nm. The extracted DNA was subsequently stored at − 20°C until further molecular analysis. Detection of knockdown resistance mutation ( kdr ) in domains II and III Two fragments of the voltage-gated sodium channel (VGSC) coding region from domains II and III, encompassing positions 989, 1007, 1011, 1016, 1520, and 1534, were amplified and sequenced to detect potential kdr mutations. Each 30 µl polymerase chain reaction (PCR) consisted of 15 µl of OneTaq Quick-Load 2× Master Mix (for domain II) or 15 µl of EconoTaq Plus Green 2× Master Mix (for domain III), combined with 3 µl of forward primer, 3 µl of reverse primer, and 3 µl of genomic DNA template. Prior to thermal cycling, the mixtures were briefly centrifuged (10 s) in a mini centrifuge to ensure homogeneity. Amplification of domain II was performed using primers AaSCF1 (AGACAATGTGGATCGCTTCC) and AaSCR4 (GGACGCAATCTGGCTTGTTA), while domain III was amplified using primers AaSCF7 (GAGAACTCGGCCGATGAACTT) and AaSCR7 (GACGACGAAATCGAACAGGT) (Kawada et al., 2016a). Thermal cycling conditions comprised an initial denaturation at 94°C for 5 min, followed by 36 cycles of 94°C for 30 s (denaturation), 50°C for 30 s (annealing, domain II) or 60.4°C for 30 s (annealing, domain III), and 72°C for 1 min (extension), with a final elongation at 72°C for 10 min, and a subsequent hold at 4°C. PCR amplicons were resolved by electrophoresis on 1.7% agarose gels prepared with FluoroSafe nucleic acid stain in 1× TAE buffer. Electrophoresis was conducted at 140 V for 50 min, and DNA fragments were visualized under UV illumination. PCR products were subsequently purified and submitted to MyTACG DNA Sequencing Services (Malaysia) for sequencing. Sequencing was carried out using primers AaSCR8 (CGACTTGATCCAGTTTGGAGA) for domain II and AaSCR9 (TAGCTTTCAGCGGCTTCTTC) for domain III 21 (Kawada et al., 2016). Data analysis The susceptibility status of Ae. aegypti mosquitoes were determined according to World Health Organization (WHO) criteria, based on 24-hour post-exposure mortality. Populations exhibiting 98–100% mortality were classified as susceptible, those with 90–97% mortality as possibly resistant, and those with < 90% mortality as resistant 20 (WHO, 2022). Statistical comparisons of insecticide susceptibility among mosquito populations from different geographical localities were conducted using one-way analysis of variance (ANOVA). Knockdown times for 50% (KT₅₀) and 95% (KT₉₅) of exposed mosquitoes were estimated using probit regression analysis in SPSS (IBM Corp., USA) version 30.0 after log-transformation of time data to satisfy model assumptions. Resistance ratios (RR) were calculated as the ratio of KT₅₀ and KT₉₅ values for field-collected populations in comparison to the laboratory-susceptible VCRU strain. Resistance was interpreted as follows: susceptible (RR < 1), low resistance (1 ≤ RR < 10), moderate resistance (10 ≤ RR ≤ 30), high resistance (30 < RR < 100), and very high resistance (RR ≥ 100) 22,23 (Khan et al., 2011; Ramdan et al., 2022). DNA sequence data generated by MyTAGC DNA Sequencing Services were aligned using ClustalW, and the nucleotide sequences were translated into amino acid sequences with the protein translation function implemented in MEGA X software 24 (Kumar et al., 2018). Results Bioassay of insecticides The susceptibility status of Ae. aegypti populations varied markedly across insecticides and collection sites (Fig. 2 ). For pirimiphos-methyl (60 mg/m²), only the laboratory reference strain (VCRU) remained fully susceptible (100% mortality), whereas all field populations demonstrated resistance, with mortality ranging from 6% (ABS) to 88% (TD). TMB strains showed a significantly lower mosquito mortality compared to other strains (p < 0.05). In contrast, malahion exposure resulted in consistently high mortality across all populations. One-way ANOVA revealed the significant differences between strains in the mortality of Ae. aegypti mosquitoes (F = 0.000, df = 7, p < 0.001; Table 1 ). The VCRU strain and six field populations (AA, TMB, FSP, TDR, TD, and PRP) were fully susceptible (100% mortality), while only one site (ABS) exhibited suspected resistance, with slightly reduced mortality (95%) but showed significant differences between strains (p < 0.001). Table 1 The results of a one-way ANOVA represent the mean differences of 24 hours of Aedes aegypti mortality between each locality against pyrethroid and organophosphate insecticides. Class Insecticides df MS F Sign. Organophosphate 5% malathion Between group 7 0.000 25.550 < 0.001 Within group 24 0.000 Total 31 60mg/m 2 Between group 7 4139.960 8.876 < 0.001 pirimiphos- Within group 24 466.406 methyl Total 31 0.4% Between group 7 0.178 6.998 < 0.01 Pyrethroid permethrin Within group 24 0.025 Total 31 0.03% Between group 7 0.441 29.323 < 0.001 deltamethrin Within group 24 0.015 Total 31 For deltamethrin, susceptibility was limited to the laboratory reference strain (VCRU), which achieved complete mortality. Among field-collected populations, only AA strain (95%) showed signs of possible resistance (95%), whereas other sites demonstrated resistance, with mortality as low as 22% (ABS). All of the strains were found to have significantly lower mortality than the VCRU strain (P < 0.01). Whereas, permethrin also displayed the same pattern as deltamethrin. The Negeri Sembilan population (TD strain) showed significantly higher resistance with lower mortality compared to AA (p = 0.011), FSP (p = 0.005), and VCRU (p < 0.001) strains. Thus indicates, the pyrethroid group is more resistant than organophosphate group in almost all of the localities. Knockdown Time Knockdown time (KT) assays revealed clear differences in susceptibility patterns of Ae. aegypti across insecticides and localities, with resistance ratios (RR) providing insight into the relative speed of knockdown compared to the laboratory reference strain (VCRU), as in Table 2 . For malathion (5%), VCRU recorded KT50​ and KT95​ values of 64.40 and 177.75 minutes, respectively, while most field populations displayed shorter knockdown times, reflected in RR50​ and RR95 values below 1.0 (e.g., ABS: RR50 = 0.26, RR9 = 0.22; TDR: RR50 = 0.34, RR95 = 0.26), indicating that malathion was more effective in field populations than in the susceptible reference strain. Table 2 Knockdown time 50 (KT50)​ and knockdown time 95 (KT95) values estimated using Probit analysis of Aedes aegypti populations from different localities following insecticide exposure. Insecticide Strain/ Locality KT 50 (min)(95%Cl) KT 95 (min)(95%Cl) RR 50 /RR 95 R 2 5% malathion VCRU 64.396 (44.045–100.540) 177.753 (87.129-1582.547) 1.00/1.00 1 AA 63.113 (42.774-221.944) 173.231 (83.255-2002.683) 0.98/0.97 1 TMB 47.129 (34.668-116.714) 120.002 (65.176-811.281) 0.73/0.68 1 FSP 38.369 (31.156–58.678) 92.918 (60.199-239.387) 0.60/0.52 0.945 TDR 21.661 (17.816–35.539) 46.936 (30.687-165.791) 0.34/0.26 0.993 TD 29.024 (25.924–34.191) 66.689 (52.072–97.045) 0.45/0.38 0.991 PRP 47.181 (36.510-67.479) 100.619 (77.200-146.646) 0.73/0.57 0.674 ABS 16.786 (12.958–23.276) 39.465 (30.839–55.254) 0.26/0.22 0.732 0.03% Deltamethrin VCRU 9.837 (8.646–12.842) 18.797 (13.930-39.835) 1.00/1.00 0.848 AA 16.099 (14.009–20.638) 33.275 (24.552–61.387) 1.64/1.77 0.859 TMB NA NA NA NA FSP 34.25 (31.413–38.250) 79.827 (66.082-103.255) 3.48/4.25 0.981 TDR 34.656 (31.261–39.513) 100.343 (79.824-136.492) 3.52/5.34 0.980 TD 30.326 (27.519–34.499) 68.914 (55.745–93.949) 3.08/3.67 0.941 PRP 6.093 (4.748–8.471) 224.505 (93.010-983.667) 0.62/11.94 0.916 ABS 11.359 (8.226–18.176) 178.485 (75.567-884.836) 1.15/9.50 0.892 VCRU 9.313 (8.251–11.873) 17.717 (13.305–34.535) 1.00/1.00 0.951 0.4% Permethrin AA 20.773 (19.379–22.660) 44.329 (37.826–54.987) 2.23/2.50 0.958 TMB 92.905 (76.824-126.023) 265.474 (179.482-510.348) 9.98/14.98 0.990 FSP NA NA NA NA TDR NA NA NA NA TD NA NA NA NA PRP NA NA NA NA ABS 26.300 (16.064–66.377) 734.612 (195.569-14841.980) 2.82/41.46 0.812 * KT50 = Knockdown Time when 50% of the mosquito population died; KT95 = Knockdown time when 95% of the mosquito population died; 95% CI = 95% Confidence Interval; RR-resistance ratio; R2 value = Regression coefficient value, NA = Not available because no knockdown was recorded. * pirimiphos-methyl: pirimiphos-methyl does not cause rapid knockdown, and therefore KT cannot be calculated as no data was obtained within one hour. * AA: Apartment Asoka; TMB: Taman Machang Bubuk; FSP: Flat Sri Pauh; TDR: Taman Desa Rahmat; TD: Taman Dahlia. In contrast, exposure to 0.03% deltamethrin revealed delayed knockdown among several populations, with FSP and TDR exhibiting markedly elevated values (KT50 = 34–35 min, KT95 = 80–100 min; RR50​>3.4, RR95 > 4.2), consistent with strong resistance. Interestingly, PRP and ABS demonstrated discordant patterns, where KT50 values were close to or below the reference (RR50 = 0.62–1.15), but KT95​ values were substantially prolonged (RR95 = 9.50–11.94), suggesting heterogeneous responses within these populations. For 0.4% permethrin, pronounced resistance was evident in TMB, which exhibited extremely high knockdown times (KT50 = 92.91 min, KT95 = 265.47 min) with RR50​ and RR95​ values of 9.98 and 14.98, respectively, indicating nearly ten- to fifteen-fold slower knockdown relative to VCRU. The ABS strain also displayed variable responses, with a moderate KT50 (26.30 min; RR50 = 2.82) but an exceptionally prolonged KT95 (734.61 min; RR95 = 41.46), pointing to substantial intra-population variability and delayed knockdown in a subset of individuals. In contrast, the AA strain showed more moderate resistance to permethrin (RR50 = 2.23; RR95 = 2.50). The results highlight a consistent trend of susceptibility to malathion across populations, but widespread and variable resistance to pyrethroids, with resistance ratios quantifying both moderate (2–5 fold slower knockdown) and extreme (> 10-fold slower knockdown) resistance phenotypes among field-collected Ae. aegypti . The results for pirimiphos-methyl could not be computed due to no knockdown on Ae. aegypti mosquitoes during the one-hour observation. Detection of kdr mutations The distribution of kdr genotypes differed between populations exposed to permethrin and deltamethrin, with the resistant alleles present at multiple loci, but their frequency and fixation levels varied by insecticide (Fig. 3 ). For the S989P mutation, resistant alleles were detected under both insecticide exposures, with all of Ae. aegypti mosquitoes TDR strain was detected with homozygous resistance (PP) for both pyrethroid exposure (frequency allele = 1.0). At the same time, the AA strain showed a high frequency of heterozygous resistance (SP) to both pyrethroid exposures (frequency allele = 1.0). At the V1016G locus, resistance alleles (VG and GG) were widespread under both insecticides. At the V1016G locus, resistant allele frequencies (V/G) were high under both insecticides, but consistently greater with deltamethrin (0.67–1.0) compared to permethrin (0.34–0.8). The frequency of homozygous resistant individuals (GG) was higher in deltamethrin-exposed groups (e.g., Asoka Apartment, AA; TMB; PRP) compared to permethrin, where heterozygotes were more common. The TD strain exhibited homozygous resistance to all tested mosquitoes when exposed to both permethrin and deltamethrin (frequency allele = 1.0). No detection of I1011M mutation was found for insecticide exposure (Fig. 3 ). In the case of A1007G, homozygous resistance alleles were found in the TDR strain only when exposed to permethrin. While, for T1520I, resistance was nearly fixed in deltamethrin-exposed populations, with all individuals carrying heterozygous resistance genotypes (TI). The F1534C locus showed the most variation between insecticides. For permethrin, resistant heterozygotes and homozygotes (FC and CC) were frequently observed, with resistance allele frequencies ranging from 0.17 (TD) to 0.83 (TMB). Interestingly, the kdr mutation for the FSP strain was only detected in domain II and not in domain III for T1520I and F1534C. Under deltamethrin exposure, however, several populations (e.g., AA, TDR) retained higher proportions of the wild-type (FF) allele, and resistance allele frequencies were generally lower (0.17–0.67). This suggests that F1534C contributes more to permethrin resistance than to deltamethrin resistance. Discussion This study revealed Ae. aegypti in Malaysia are responding differently to the insecticides used in control programs, being more impacted by pyrethroids, which have caused resistance. Malathion still works well in most sites, but resistance to pirimiphos-methyl was already widespread for the organophosphate group. Pyrethroids such as deltamethrin and permethrin were the least effective. In many places, survival rates were far higher than the WHO limit for resistance, as in the WHO (2022) 21 protocol. These findings confirm what has been reported in Malaysia before, that the pyrethroids are losing their effectiveness in dengue-prone regions because of long and repeated use 18 . The present findings reveal notable spatial heterogeneity in insecticide resistance across Malaysia. Populations from Johor (ABS) exhibited particularly high levels of pyrethroid resistance, whereas populations from Penang (TMB and FSP) demonstrated only moderate resistance. Localized ecological and operational pressures most likely shape such spatial variation in resistance. These include differences in human population density, variation in the frequency and intensity of fogging activities, and the extent of household insecticide use. Previous studies in Malaysia have reported similar trends, where areas subjected to intensive vector control interventions showed elevated frequencies of resistance alleles compared to less-treated localities 17 , 20 . Sustained reliance on chemical interventions in densely populated dengue hotspots, such as Penang, Johor, and Selangor, has been shown to impose strong selective pressure on mosquito populations, accelerating the emergence and spread of resistance alleles 15 . Although malathion remains largely effective in most surveyed sites, the early indications of reduced susceptibility in Johor raise concerns that continued reliance on this compound may lead to the development of resistance, analogous to that observed with pyrethroids. Knockdown assays provided additional insight, revealing that mosquitoes exposed to pyrethroids exhibited markedly prolonged knockdown times compared to the laboratory-susceptible strain, in some cases requiring over tenfold longer durations to achieve comparable knockdown. This delay suggests a combination of reduced insecticide absorption and stronger metabolic defenses, particularly the activity of detoxifying enzymes such as cytochrome P450s. These mechanisms have been reported before in Malaysian mosquito populations 17 , 18 . A notable observation was the pronounced heterogeneity in knockdown responses within certain populations. For example, in Johor, individual mosquitoes displayed highly variable susceptibility, with some exhibiting rapid knockdowns. In contrast, others remained active for several hours, highlighting that resistance dynamics can be heterogeneous and complex even within a single geographic population. Molecular analysis provided insight into the resistance mechanisms underlying the phenotypic variation. Several established knockdown resistance ( kdr ) mutations, including S989P, V1016G, and F1534C, were detected, together with more recently reported substitutions such as A1007G and T1520I, within Malaysia. In certain populations, resistant alleles were found at near-fixation levels, indicating that almost all individuals carried these mutations. Notably, the F1534C mutation appeared to confer a stronger association with permethrin resistance than with deltamethrin resistance, a pattern consistent with reports from other Southeast Asian populations 5 , 26 , 27 . Plernsub et al. (2016) 27 demonstrated that the F1534C mutation is particularly important for type I pyrethroid (permethrin) resistance, while other combinations (S989P + V1016G) contribute more to type II pyrethroid (deltamethrin) resistance. These findings underscore that individual kdr mutations differ in their functional impact across pyrethroid compounds, emphasizing the importance of monitoring the distribution and frequency of specific alleles to guide the continued efficacy of vector control strategies 27 . When placed in a global context, the resistance profile observed in Malaysia reflects broader patterns that have been documented elsewhere. Multiple mutations were detected in the voltage-gated sodium channel (VGSC) gene, including S989P, V1016G, F1534C, A1007G, and the newly observed T1520I. The present study reveals a complex kdr resistance profile in Malaysian Ae. aegypti , shaped by prolonged pyrethroid use. The widespread occurrence of S989P and V1016G, often in combination, is consistent with their established role in conferring resistance to type II pyrethroids such as deltamethrin, whereas F1534C was more strongly associated with resistance to type I compounds such as permethrin, supporting earlier reports from Southeast Asia that different alleles exhibit compound-specific effects 26 , 28 . In Asia, particularly in Thailand and Vietnam, the combined presence of S989P and V1016G mutations has been linked to significantly reduced efficacy of pyrethroid-based interventions 29 , 30 . The detection of A1007G at localized sites, and the near fixation of T1520I under deltamethrin exposure, indicate that novel mutations are emerging under ongoing selection pressure, mirroring findings from India and Myanmar, where these alleles often occur in combination with F1534C 28 , 31 . In Latin America, widespread pyrethroid resistance has been strongly associated with V1016I/G and F1534C mutations, which have been directly implicated in operational control failures1 13,32 . The coexistence of multiple kdr alleles across different regions suggests that Malaysian populations are evolving heterogeneous but increasingly robust resistance phenotypes, posing a substantial challenge to vector control. The first detection of T1520I in Malaysian Ae. aegypti broadens the regional picture of vgsc evolution under pyrethroid selection. In our study, the T1520I mutation was present at a high frequency under deltamethrin exposure, with TI heterozygotes approaching fixation in several populations. Comparable occurrences of mutation have been reported initially in India, where it co-segregated with F1534C in pyrethroid-resistant populations and was associated with reduced permethrin susceptibility 5 . Myanmar documented T1520I alongside S989P, V1016G, and F1534C, including a quadruple haplotype (S989P/V1016G/T1520I/F1534C) indicative of strong multi-locus selection 31 . Mechanistic work using expressed sodium channels further shows that T1520I modifies the PyR1 binding site and, in combination with F1534C, preferentially elevates resistance to type I pyrethroids (e.g., permethrin) while retaining sensitivity to type II compounds (e.g., deltamethrin) 34 . Regionally, reviews and multi-country syntheses have noted that T1520I, typically linked with F1534C, has now been detected across parts of South and Southeast Asia, reinforcing its relevance as a surveillance marker for operational programs 22 . These findings highlight a divergence in susceptibility within the organophosphate class, where malathion continues to serve as a viable option in most settings, whereas pirimiphos-methyl efficacy is severely compromised. In India and Sri Lanka, Ae. aegypti have already developed reduced susceptibility to malathion through enzyme-based detoxification mechanisms like esterases and GSTs 5 , 34 . The overall profile emphasizes that although organophosphates remain more effective than pyrethroids, localized resistance is emerging, and continued surveillance is crucial to prevent further erosion of their utility in dengue control programs. In short, this study shows that Ae. aegypti in Malaysia are already resistant to pyrethroids and are showing the first signs of resistance to organophosphates. The observed resistance patterns have critical implications for vector control and pest management in Malaysia. The widespread resistance to pyrethroids, combined with the emergence of resistance to organophosphates such as pirimiphos-methyl, indicates that relying solely on chemical insecticides will increasingly undermine the effectiveness of dengue control programs 15 , 16 , 18 . Keeping track of the spread of resistance mutations can help predict where and when insecticides are likely to fail. In Malaysia, embedding routine monitoring of kdr mutations into dengue control programs would provide a strong early-warning system, similar to how malaria vector programs already use genetic tools to track resistance trends 26 , 34 . Importantly, molecular surveillance of kdr mutations, particularly the novel T1520I identified in this study, should be incorporated into routine monitoring frameworks to provide early warnings of resistance spread and to guide evidence-based interventions. The latter path offers the best chance to maintain effective dengue control in the long term 32 , 34 . To address this challenge, integrated resistance management strategies should be prioritized, including insecticide rotation with compounds of different modes of action 21 , the strengthening of larval source reduction and environmental management 12 , and the integration of biological control measures where feasible 35 . Additionally, community education and the regulation of domestic insecticide use are crucial in reducing household-level selection pressures that accelerate the development of resistance. Collectively, these strategies will strengthen the resilience of pest management programs and help sustain the long-term efficacy of dengue control efforts in Malaysia. Conclusions In conclusion, this study demonstrates that, Ae. aegypti populations in Malaysia exhibit a complex resistance landscape characterized by widespread pyrethroid resistance and emerging, though more localized, resistance to organophosphates. The detection of multiple kdr mutations, including well-established alleles (S989P, V1016G, F1534C) and newer variants (A1007G, T1520I), underscores the adaptive capacity of vector populations under sustained chemical pressure and highlights the compound-specific nature of resistance mechanisms. While malathion remains broadly effective, the compromised efficacy of pirimiphos-methyl signals that even organophosphates are not invulnerable to resistance development. Collectively, these findings underscore the urgent need for integrated resistance management strategies that incorporate molecular surveillance, insecticide rotation, and non-chemical interventions to sustain the long-term effectiveness of vector control programs and mitigate the risk of operational failure in dengue prevention. Data Availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Declarations Data Availability The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Acknowledgements We would like to express our sincere gratitude to the School of Biological Sciences, Universiti Sains Malaysia, for their invaluable support throughout this research. The institution's resources and facilities greatly contributed to the successful completion of this work. We want to acknowledge the collaborative efforts of the Ministry of Health Malaysia. We also extend our heartfelt appreciation to the Ministry of Higher Education Malaysia for the funding that is essential in facilitating the research and enabling the publication of this manuscript (FRGS/1/2023/STG03/USM/02/4). Funding Declaration This research/project was funded by the Ministry of Higher Education Malaysia under the Fundamental Research Grant Scheme, grant number [FRGS/1/2023/STG03/USM/02/4]. The funding agency had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript/report. Author Information Authors and Affiliations Medical Entomology Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, MALAYSIA Ma Teng & Wan Fatma Zuharah Authors’ Contributions The authors' contributions are as follows: WFZ: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Supervision, Writing—original draft; Writing—review & editing, MT: Project administration, Methodology, Data curation, Formal analysis. Corresponding author Correspondence to Wan Fatma Zuharah, [email protected] Ethics declarations Competing Interests Each author has approved the submitted version (and any substantially modified version that involves the author's contribution to the study) and has agreed both to be personally accountable for the author's own contributions. 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PLoS Negl. Trop. Dis. 11 (7), e0005625. https://doi.org/10.1371/journal.pntd.0005625 (2017). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 24 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 18 Dec, 2025 Reviews received at journal 14 Dec, 2025 Reviewers agreed at journal 10 Dec, 2025 Reviewers agreed at journal 22 Oct, 2025 Reviews received at journal 17 Oct, 2025 Reviewers agreed at journal 14 Oct, 2025 Reviewers invited by journal 14 Oct, 2025 Editor assigned by journal 14 Oct, 2025 Editor invited by journal 03 Oct, 2025 Submission checks completed at journal 01 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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18:18:55","extension":"xml","order_by":95,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139135,"visible":true,"origin":"","legend":"","description":"","filename":"0ec4bacb5bcf4a6896608fbd58c458841structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7708119/v1/dc29499f66a77fa6d43786cc.xml"},{"id":94588596,"identity":"9581f934-16d3-4505-b37f-24b1d6b36ab2","added_by":"auto","created_at":"2025-10-28 18:19:36","extension":"html","order_by":96,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":151423,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7708119/v1/ed1145194ff7853f5b902188.html"},{"id":94588425,"identity":"4193d2aa-2266-4992-95e8-bd1e38194dd6","added_by":"auto","created_at":"2025-10-28 18:19:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":165001,"visible":true,"origin":"","legend":"\u003cp\u003eThe locations of \u003cem\u003eAedes aegypti\u003c/em\u003e mosquito sampling sites in Malaysia.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7708119/v1/6c9146ba177b64a71a61ef3b.png"},{"id":94588677,"identity":"f10254ec-5c10-446d-89ea-ec5d890ed23f","added_by":"auto","created_at":"2025-10-28 18:19:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80063,"visible":true,"origin":"","legend":"\u003cp\u003eSusceptibility status of \u003cem\u003eAedes aegypti\u003c/em\u003e laboratory and field populations against four types of insecticides (pirimiphos-methyl, malathion, deltamethrin, and permethrin). The same small letter represents no significant differences between strains for each insecticide. ** S-Susceptibility, PR-Possible Resistance, R-Resistance; VCRU-Vector Control Research Unit, AA-Apartment Asoka, TMB-Taman Machang Bubuk, FSP-Flat Sri Pauh, TDR-Taman Desa Rahmat, TD-Taman Dahlia, PRP-PPR Raya Permai, ABS-Apartment Bukit Saujana.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7708119/v1/bcddc8a04a59a28703f714d0.png"},{"id":94587856,"identity":"1e7f09bc-c6d9-4884-a087-c32903e0bfaa","added_by":"auto","created_at":"2025-10-28 18:18:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":206771,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid proportions of the six principal insecticide resistance–associated mutations across all sampling locations\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7708119/v1/11afaa9ee0c4525e5a3ea440.png"},{"id":103765845,"identity":"6a63df64-7c61-4707-87ad-8915358e7cc0","added_by":"auto","created_at":"2026-03-02 16:10:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1365362,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7708119/v1/bf760707-8d14-4246-ad0a-ccc8e61f1453.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Susceptibility Status of Aedes aegypti (Diptera: Culicidae) Mosquitoes in Malaysia on Pyrethroid and Organophosphate Insecticides with First Detection of T1520I Mutation","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eAedes aegypti\u003c/em\u003e is one of the most widespread and medically important mosquito species, inhabiting tropical and subtropical regions worldwide. It serves as the primary vector of several arboviruses, including dengue virus (DENV), chikungunya virus (CHIKV), yellow fever virus, and Zika virus\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Dengue has emerged as the most significant arboviral disease affecting humans, with nearly 390\u0026nbsp;million infections estimated annually, of which approximately 96\u0026nbsp;million are clinically apparent\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn Malaysia, dengue poses an escalating public health burden, characterized by the hyperendemic circulation of all four DENV serotypes and recurrent outbreaks, resulting in substantial morbidity and economic costs\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Over the long term, dengue incidence in Malaysia has shown a marked increase, rising from approximately 44 cases per 100,000 population in 1999 to about 399 cases per 100,000 in 2015, as reported in a decade-scale study on dengue morbidity and mortality\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Despite advances in dengue vaccines, including a tetravalent formulation that has demonstrated partial efficacy, large-scale implementation remains limited due to variable performance across serostatus groups and regulatory concerns\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. As a result, vector control continues to serve as the primary method of dengue prevention in Malaysia.\u003c/p\u003e\u003cp\u003eVector control strategies largely rely on environmental management to reduce larval habitats and the extensive use of chemical insecticides to suppress adult mosquito populations\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Pyrethroids (e.g., deltamethrin, permethrin) and organophosphates (e.g., malathion, pirimiphos-methyl) are the most widely deployed insecticides in Malaysia\u0026rsquo;s dengue control programs\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, this sustained reliance on chemical interventions has exerted strong selective pressure on mosquito populations, leading to the emergence and spread of insecticide resistance.\u003c/p\u003e\u003cp\u003eInsecticide resistance in \u003cem\u003eAe. aegypti\u003c/em\u003e has been increasingly documented across Malaysia. A nationwide study by Asgarian et al. (2023)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e revealed heterogeneous but widespread resistance to pyrethroids in major dengue hotspots, including Penang, Johor, and Selangor. While \u003cem\u003eAe. aegypti\u003c/em\u003e populations remain largely susceptible to malathion, localized resistance has been detected in Kuala Lumpur and Selangor\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. At the molecular level, resistance is frequently associated with knockdown resistance (\u003cem\u003ekdr\u003c/em\u003e) mutations in the voltage-gated sodium channel (VGSC) gene. Several key mutations\u0026mdash;S989P, V1016G, F1534C, and more recently A1007G\u0026mdash;have been reported in Malaysian \u003cem\u003eAe. aegypti\u003c/em\u003e populations\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, reflecting the cumulative effects of insecticide selection pressure and highlighting complex, region-specific resistance dynamics.\u003c/p\u003e\u003cp\u003eWhile insecticides remain indispensable in Malaysia\u0026rsquo;s vector control programs, their effectiveness is increasingly undermined by resistance. The over-reliance on chemical interventions as a primary control strategy has inadvertently accelerated the selection of resistant phenotypes, threatening the long-term sustainability of dengue control. Understanding the prevalence and geographic distribution of \u003cem\u003ekdr\u003c/em\u003e mutations provides valuable insights into resistance dynamics, enabling vector control programs to adapt their interventions and maintain the efficacy of chemical-based measures. Therefore, the study aims to assess the susceptibility status of \u003cem\u003eAe. aegypti\u003c/em\u003e populations from diverse Malaysian locations and to characterize the frequency of key \u003cem\u003ekdr\u003c/em\u003e mutations associated with pyrethroid resistance.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMosquito Collections\u003c/h2\u003e\u003cp\u003e\u003cem\u003eAedes aegypti\u003c/em\u003e populations were collected from seven dengue hotspot localities distributed across five states in Malaysia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The sampling sites were selected based on dengue hotspot designations provided by the Ministry of Health Malaysia (MOH), following an analysis of national dengue case distributions. The selected sites represented geographically distinct regions of Malaysia, including, (1) Northern Malaysia (Penang): Apartment Asoka (AA), Penang Island (5\u0026deg;21\u0026prime;N, 100\u0026deg;18\u0026prime;E); Taman Machang Bubuk (TMB), Penang Mainland (5\u0026deg;30\u0026prime;02\u0026Prime;N, 100\u0026deg;25\u0026prime;70\u0026Prime;E); and Flat Sri Pauh (FSP), Penang Mainland (5\u0026deg;33\u0026prime;92\u0026Prime;N, 100\u0026deg;50\u0026prime;46\u0026Prime;E), (2) East Coast Malaysia (Kelantan): Taman Desa Rahmat (TDR), Kelantan (6\u0026deg;11\u0026prime;49\u0026Prime;N, 102\u0026deg;28\u0026prime;09\u0026Prime;E), (3) Central Malaysia: Taman Dahlia (TD), Negeri Sembilan (2\u0026deg;84\u0026prime;37\u0026Prime;N, 101\u0026deg;82\u0026prime;65\u0026Prime;E); and PPR Raya Permai (PRP), Kuala Lumpur (3\u0026deg;06\u0026prime;17\u0026Prime;N, 101\u0026deg;70\u0026prime;56\u0026Prime;E), and (4) Southern Malaysia (Johor): Apartment Bukit Saujana (ABS), Johor (1\u0026deg;47\u0026prime;44\u0026Prime;N, 103\u0026deg;74\u0026prime;87\u0026Prime;E).\u003c/p\u003e\u003cp\u003eAt each locality, sixty ovitraps were deployed randomly for five consecutive days. Hardwood paddles served as oviposition substrates and were subsequently collected and transported to the Medical Entomology Laboratory, Universiti Sains Malaysia. Eggs were reared to adults under controlled insectary conditions (27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C; 70\u0026ndash;80% relative humidity). Only the dominant \u003cem\u003eAedes\u003c/em\u003e species obtained from each locality were subjected to subsequent insecticide susceptibility assays.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAdult Mosquito Bioassay\u003c/h3\u003e\n\u003cp\u003eAdult susceptibility bioassays were performed following the World Health Organization (WHO) standard protocol\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Four replicates of 25 non-blood-fed female \u003cem\u003eAe. aegypti\u003c/em\u003e (2\u0026ndash;5 days old) from each field population were exposed to insecticide-impregnated filter papers prepared by the Vector Control Research Unit (VCRU), Universiti Sains Malaysia. The insecticides tested were: (1) 0.03% deltamethrin, (2) 0.4% permethrin, (3) pirimiphos-methyl at 60 mg/m\u0026sup2;, and (4) 5% malathion.\u003c/p\u003e\u003cp\u003eMosquitoes were initially introduced into holding tubes and acclimatized for one hour. Any damaged, injured, or dead individuals were replaced prior to testing. A total of 25 females were then transferred into exposure tubes lined with insecticide-impregnated papers. Knockdown data was recorded at 5-minute intervals until one hour. Following exposure, mosquitoes were transferred into clean paper cups supplied with 10% sucrose solution and maintained at 27\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 60\u0026ndash;80% relative humidity. Mortality was assessed 24 hours post-exposure, and four replicates were performed for each insecticide. Two control replicates were prepared with silicone oil for pyrethroids, olive oil for malathion, and acetone for pirimiphos-methyl. Laboratory-susceptible \u003cem\u003eAe. aegypti\u003c/em\u003e strain maintained at VCRU (Penang, Malaysia; 5\u0026deg;21\u0026prime;N, 100\u0026deg;18\u0026prime;E) which were reared for over 300 generations since the 1960s was used as the reference baseline.\u003c/p\u003e\n\u003ch3\u003eGenomic DNA Extraction\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from individual adult \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes that survived the susceptibility bioassays using the PrimeWay Genomic II DNA Extraction Kit (Apical Scientific Sdn Bhd, Malaysia), following the manufacturer\u0026rsquo;s protocol with slight modifications to optimize yield. Prior to extraction, the elution buffer was preheated to 60\u0026deg;C to improve recovery efficiency. Each mosquito was homogenized in 100 \u0026micro;l of GL1 buffer using a sterile pestle, which was subsequently rinsed with an additional 100 \u0026micro;l of GL1 buffer to maximize tissue recovery. Protein digestion was carried out by adding 20 \u0026micro;l of Proteinase K solution to the homogenate, followed by incubation at 60\u0026deg;C for 3 h with occasional inversion to ensure complete lysis of cellular and nuclear membranes. After incubation, the lysate was centrifuged at 12,000 \u0026times; g for 2.5 min at room temperature, and the supernatant was carefully transferred to a new 1.5 mL microcentrifuge tube to prevent disruption of the pellet.\u003c/p\u003e\u003cp\u003eThe supernatant was mixed with 200 \u0026micro;l of GL2 buffer and vortexed thoroughly. Afterward, 4 \u0026micro;L of RNase A solution was added. The mixture was vortexed again and incubated at room temperature for 5 min to degrade RNA contaminants. DNA precipitation was induced by adding 200 \u0026micro;l of absolute ethanol, and the lysate\u0026ndash;ethanol mixture was vortexed until homogeneous. A total of 750 \u0026micro;L of the resulting mixture was applied to a PrimeWay silica spin column inserted into a clean collection tube and centrifuged at 12,000 x g for 1.2 min. Afterward, the flow-through was discarded. To remove residual impurities, 400 \u0026micro;l of Wash Buffer G1 was added to the column, followed by centrifugation at 12,000 x g for 35 s; the flow-through was then discarded. This was followed by a second wash step with 600 \u0026micro;l of Wash Buffer G2 and centrifugation at 12,000 \u0026times; g for 35 s. To ensure complete removal of residual ethanol and drying of the membrane, the column was subjected to an additional centrifugation step at 12,000 x g for 3.5 min.\u003c/p\u003e\u003cp\u003eFor DNA elution, the spin column was transferred into a new 1.5 ml microcentrifuge tube, and 50 \u0026micro;l of preheated (60\u0026deg;C) elution buffer was added directly to the center of the membrane to maximize contact. After incubating at room temperature for at least 3 min, the column was centrifuged at 12,000 x g for 35 s to obtain the final DNA yield. The eluted DNA was quantified and assessed for purity using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) at an absorbance of 260 nm. The extracted DNA was subsequently stored at \u0026minus;\u0026thinsp;20\u0026deg;C until further molecular analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of knockdown resistance mutation (\u003c/b\u003e\u003cb\u003ekdr\u003c/b\u003e\u003cb\u003e) in domains II and III\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTwo fragments of the voltage-gated sodium channel (VGSC) coding region from domains II and III, encompassing positions 989, 1007, 1011, 1016, 1520, and 1534, were amplified and sequenced to detect potential \u003cem\u003ekdr\u003c/em\u003e mutations. Each 30 \u0026micro;l polymerase chain reaction (PCR) consisted of 15 \u0026micro;l of OneTaq Quick-Load 2\u0026times; Master Mix (for domain II) or 15 \u0026micro;l of EconoTaq Plus Green 2\u0026times; Master Mix (for domain III), combined with 3 \u0026micro;l of forward primer, 3 \u0026micro;l of reverse primer, and 3 \u0026micro;l of genomic DNA template. Prior to thermal cycling, the mixtures were briefly centrifuged (10 s) in a mini centrifuge to ensure homogeneity.\u003c/p\u003e\u003cp\u003eAmplification of domain II was performed using primers AaSCF1 (AGACAATGTGGATCGCTTCC) and AaSCR4 (GGACGCAATCTGGCTTGTTA), while domain III was amplified using primers AaSCF7 (GAGAACTCGGCCGATGAACTT) and AaSCR7 (GACGACGAAATCGAACAGGT) (Kawada et al., 2016a). Thermal cycling conditions comprised an initial denaturation at 94\u0026deg;C for 5 min, followed by 36 cycles of 94\u0026deg;C for 30 s (denaturation), 50\u0026deg;C for 30 s (annealing, domain II) or 60.4\u0026deg;C for 30 s (annealing, domain III), and 72\u0026deg;C for 1 min (extension), with a final elongation at 72\u0026deg;C for 10 min, and a subsequent hold at 4\u0026deg;C.\u003c/p\u003e\u003cp\u003ePCR amplicons were resolved by electrophoresis on 1.7% agarose gels prepared with FluoroSafe nucleic acid stain in 1\u0026times; TAE buffer. Electrophoresis was conducted at 140 V for 50 min, and DNA fragments were visualized under UV illumination. PCR products were subsequently purified and submitted to MyTACG DNA Sequencing Services (Malaysia) for sequencing. Sequencing was carried out using primers AaSCR8 (CGACTTGATCCAGTTTGGAGA) for domain II and AaSCR9 (TAGCTTTCAGCGGCTTCTTC) for domain III\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e (Kawada et al., 2016).\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eThe susceptibility status of \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes were determined according to World Health Organization (WHO) criteria, based on 24-hour post-exposure mortality. Populations exhibiting 98\u0026ndash;100% mortality were classified as susceptible, those with 90\u0026ndash;97% mortality as possibly resistant, and those with \u0026lt;\u0026thinsp;90% mortality as resistant\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e (WHO, 2022). Statistical comparisons of insecticide susceptibility among mosquito populations from different geographical localities were conducted using one-way analysis of variance (ANOVA).\u003c/p\u003e\u003cp\u003eKnockdown times for 50% (KT₅₀) and 95% (KT₉₅) of exposed mosquitoes were estimated using probit regression analysis in SPSS (IBM Corp., USA) version 30.0 after log-transformation of time data to satisfy model assumptions. Resistance ratios (RR) were calculated as the ratio of KT₅₀ and KT₉₅ values for field-collected populations in comparison to the laboratory-susceptible VCRU strain. Resistance was interpreted as follows: susceptible (RR\u0026thinsp;\u0026lt;\u0026thinsp;1), low resistance (1\u0026thinsp;\u0026le;\u0026thinsp;RR\u0026thinsp;\u0026lt;\u0026thinsp;10), moderate resistance (10\u0026thinsp;\u0026le;\u0026thinsp;RR\u0026thinsp;\u0026le;\u0026thinsp;30), high resistance (30\u0026thinsp;\u0026lt;\u0026thinsp;RR\u0026thinsp;\u0026lt;\u0026thinsp;100), and very high resistance (RR\u0026thinsp;\u0026ge;\u0026thinsp;100)\u003csup\u003e22,23\u003c/sup\u003e (Khan et al., 2011; Ramdan et al., 2022).\u003c/p\u003e\u003cp\u003eDNA sequence data generated by MyTAGC DNA Sequencing Services were aligned using ClustalW, and the nucleotide sequences were translated into amino acid sequences with the protein translation function implemented in MEGA X software\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e (Kumar et al., 2018).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eBioassay of insecticides\u003c/h2\u003e\u003cp\u003eThe susceptibility status of \u003cem\u003eAe. aegypti\u003c/em\u003e populations varied markedly across insecticides and collection sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For pirimiphos-methyl (60 mg/m\u0026sup2;), only the laboratory reference strain (VCRU) remained fully susceptible (100% mortality), whereas all field populations demonstrated resistance, with mortality ranging from 6% (ABS) to 88% (TD). TMB strains showed a significantly lower mosquito mortality compared to other strains (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eIn contrast, malahion exposure resulted in consistently high mortality across all populations. One-way ANOVA revealed the significant differences between strains in the mortality of \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes (F\u0026thinsp;=\u0026thinsp;0.000, df\u0026thinsp;=\u0026thinsp;7, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The VCRU strain and six field populations (AA, TMB, FSP, TDR, TD, and PRP) were fully susceptible (100% mortality), while only one site (ABS) exhibited suspected resistance, with slightly reduced mortality (95%) but showed significant differences between strains (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\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\u003eThe results of a one-way ANOVA represent the mean differences of 24 hours of \u003cem\u003eAedes aegypti\u003c/em\u003e mortality between each locality against pyrethroid and organophosphate insecticides.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClass\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInsecticides\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003edf\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMS\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSign.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrganophosphate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5% malathion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBetween group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e25.550\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" 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colname=\"c4\"\u003e\u003cp\u003e31\u003c/p\u003e\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\" colname=\"c7\"\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\u003e60mg/m\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBetween group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4139.960\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e8.876\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\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\u003epirimiphos-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWithin group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e466.406\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\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\u003emethyl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e31\u003c/p\u003e\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\" colname=\"c7\"\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\u003e0.4%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBetween group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.178\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e6.998\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePyrethroid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epermethrin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWithin group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\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\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e31\u003c/p\u003e\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\" colname=\"c7\"\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\u003e0.03%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBetween group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.441\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e29.323\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\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\u003edeltamethrin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWithin group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\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\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e31\u003c/p\u003e\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\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eFor deltamethrin, susceptibility was limited to the laboratory reference strain (VCRU), which achieved complete mortality. Among field-collected populations, only AA strain (95%) showed signs of possible resistance (95%), whereas other sites demonstrated resistance, with mortality as low as 22% (ABS). All of the strains were found to have significantly lower mortality than the VCRU strain (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Whereas, permethrin also displayed the same pattern as deltamethrin. The Negeri Sembilan population (TD strain) showed significantly higher resistance with lower mortality compared to AA (p\u0026thinsp;=\u0026thinsp;0.011), FSP (p\u0026thinsp;=\u0026thinsp;0.005), and VCRU (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) strains. Thus indicates, the pyrethroid group is more resistant than organophosphate group in almost all of the localities.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eKnockdown Time\u003c/h3\u003e\n\u003cp\u003eKnockdown time (KT) assays revealed clear differences in susceptibility patterns of \u003cem\u003eAe. aegypti\u003c/em\u003e across insecticides and localities, with resistance ratios (RR) providing insight into the relative speed of knockdown compared to the laboratory reference strain (VCRU), as in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. For malathion (5%), VCRU recorded KT50​ and KT95​ values of 64.40 and 177.75 minutes, respectively, while most field populations displayed shorter knockdown times, reflected in RR50​ and RR95 values below 1.0 (e.g., ABS: RR50\u0026thinsp;=\u0026thinsp;0.26, RR9\u0026thinsp;=\u0026thinsp;0.22; TDR: RR50\u0026thinsp;=\u0026thinsp;0.34, RR95\u0026thinsp;=\u0026thinsp;0.26), indicating that malathion was more effective in field populations than in the susceptible reference strain.\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\u003eKnockdown time 50 (KT50)​ and knockdown time 95 (KT95) values estimated using Probit analysis of \u003cem\u003eAedes aegypti\u003c/em\u003e populations from different localities following insecticide exposure.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInsecticide\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStrain/\u003c/p\u003e\u003cp\u003eLocality\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eKT\u003csub\u003e50\u003c/sub\u003e (min)(95%Cl)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eKT\u003csub\u003e95\u003c/sub\u003e (min)(95%Cl)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRR\u003csub\u003e50\u003c/sub\u003e/RR\u003csub\u003e95\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003e5%\u003c/p\u003e\u003cp\u003emalathion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVCRU\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e64.396 (44.045\u0026ndash;100.540)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e177.753 (87.129-1582.547)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.00/1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e63.113 (42.774-221.944)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e173.231 (83.255-2002.683)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.98/0.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTMB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47.129 (34.668-116.714)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e120.002 (65.176-811.281)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.73/0.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFSP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e38.369 (31.156\u0026ndash;58.678)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e92.918 (60.199-239.387)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.60/0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.945\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTDR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e21.661 (17.816\u0026ndash;35.539)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e46.936 (30.687-165.791)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.34/0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.993\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29.024 (25.924\u0026ndash;34.191)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e66.689 (52.072\u0026ndash;97.045)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.45/0.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.991\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePRP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47.181 (36.510-67.479)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.619 (77.200-146.646)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.73/0.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.674\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eABS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.786 (12.958\u0026ndash;23.276)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39.465 (30.839\u0026ndash;55.254)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.26/0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.732\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003e0.03%\u003c/p\u003e\u003cp\u003eDeltamethrin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVCRU\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.837 (8.646\u0026ndash;12.842)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18.797 (13.930-39.835)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.00/1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.848\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16.099 (14.009\u0026ndash;20.638)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33.275 (24.552\u0026ndash;61.387)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.64/1.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.859\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTMB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFSP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.25 (31.413\u0026ndash;38.250)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e79.827 (66.082-103.255)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.48/4.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.981\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTDR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34.656 (31.261\u0026ndash;39.513)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.343 (79.824-136.492)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.52/5.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.980\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30.326 (27.519\u0026ndash;34.499)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e68.914 (55.745\u0026ndash;93.949)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3.08/3.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.941\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePRP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.093 (4.748\u0026ndash;8.471)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e224.505 (93.010-983.667)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.62/11.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.916\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eABS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.359 (8.226\u0026ndash;18.176)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e178.485 (75.567-884.836)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.15/9.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.892\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\u003eVCRU\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.313 (8.251\u0026ndash;11.873)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.717 (13.305\u0026ndash;34.535)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.00/1.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.951\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e\u003cp\u003e0.4%\u003c/p\u003e\u003cp\u003ePermethrin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20.773 (19.379\u0026ndash;22.660)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44.329 (37.826\u0026ndash;54.987)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.23/2.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.958\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTMB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e92.905 (76.824-126.023)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e265.474 (179.482-510.348)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.98/14.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.990\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFSP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTDR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePRP\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eABS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.300 (16.064\u0026ndash;66.377)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e734.612 (195.569-14841.980)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.82/41.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.812\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e* KT50\u0026thinsp;=\u0026thinsp;Knockdown Time when 50% of the mosquito population died; KT95\u0026thinsp;=\u0026thinsp;Knockdown time when 95% of the mosquito population died; 95% CI\u0026thinsp;=\u0026thinsp;95% Confidence Interval; RR-resistance ratio; R2 value\u0026thinsp;=\u0026thinsp;Regression coefficient value, NA\u0026thinsp;=\u0026thinsp;Not available because no knockdown was recorded.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e* pirimiphos-methyl: pirimiphos-methyl does not cause rapid knockdown, and therefore KT cannot be calculated as no data was obtained within one hour.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003e* AA: Apartment Asoka; TMB: Taman Machang Bubuk; FSP: Flat Sri Pauh; TDR: Taman Desa Rahmat; TD: Taman Dahlia.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn contrast, exposure to 0.03% deltamethrin revealed delayed knockdown among several populations, with FSP and TDR exhibiting markedly elevated values (KT50\u0026thinsp;=\u0026thinsp;34\u0026ndash;35 min, KT95\u0026thinsp;=\u0026thinsp;80\u0026ndash;100 min; RR50​\u0026gt;3.4, RR95\u0026thinsp;\u0026gt;\u0026thinsp;4.2), consistent with strong resistance. Interestingly, PRP and ABS demonstrated discordant patterns, where KT50 values were close to or below the reference (RR50\u0026thinsp;=\u0026thinsp;0.62\u0026ndash;1.15), but KT95​ values were substantially prolonged (RR95\u0026thinsp;=\u0026thinsp;9.50\u0026ndash;11.94), suggesting heterogeneous responses within these populations. For 0.4% permethrin, pronounced resistance was evident in TMB, which exhibited extremely high knockdown times (KT50\u0026thinsp;=\u0026thinsp;92.91 min, KT95\u0026thinsp;=\u0026thinsp;265.47 min) with RR50​ and RR95​ values of 9.98 and 14.98, respectively, indicating nearly ten- to fifteen-fold slower knockdown relative to VCRU. The ABS strain also displayed variable responses, with a moderate KT50 (26.30 min; RR50\u0026thinsp;=\u0026thinsp;2.82) but an exceptionally prolonged KT95 (734.61 min; RR95\u0026thinsp;=\u0026thinsp;41.46), pointing to substantial intra-population variability and delayed knockdown in a subset of individuals. In contrast, the AA strain showed more moderate resistance to permethrin (RR50\u0026thinsp;=\u0026thinsp;2.23; RR95\u0026thinsp;=\u0026thinsp;2.50).\u003c/p\u003e\u003cp\u003eThe results highlight a consistent trend of susceptibility to malathion across populations, but widespread and variable resistance to pyrethroids, with resistance ratios quantifying both moderate (2\u0026ndash;5 fold slower knockdown) and extreme (\u0026gt;\u0026thinsp;10-fold slower knockdown) resistance phenotypes among field-collected \u003cem\u003eAe. aegypti\u003c/em\u003e. The results for pirimiphos-methyl could not be computed due to no knockdown on \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes during the one-hour observation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of\u003c/b\u003e \u003cb\u003ekdr\u003c/b\u003e \u003cb\u003emutations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe distribution of \u003cem\u003ekdr\u003c/em\u003e genotypes differed between populations exposed to permethrin and deltamethrin, with the resistant alleles present at multiple loci, but their frequency and fixation levels varied by insecticide (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For the S989P mutation, resistant alleles were detected under both insecticide exposures, with all of \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes TDR strain was detected with homozygous resistance (PP) for both pyrethroid exposure (frequency allele\u0026thinsp;=\u0026thinsp;1.0). At the same time, the AA strain showed a high frequency of heterozygous resistance (SP) to both pyrethroid exposures (frequency allele\u0026thinsp;=\u0026thinsp;1.0). At the V1016G locus, resistance alleles (VG and GG) were widespread under both insecticides. At the V1016G locus, resistant allele frequencies (V/G) were high under both insecticides, but consistently greater with deltamethrin (0.67\u0026ndash;1.0) compared to permethrin (0.34\u0026ndash;0.8). The frequency of homozygous resistant individuals (GG) was higher in deltamethrin-exposed groups (e.g., Asoka Apartment, AA; TMB; PRP) compared to permethrin, where heterozygotes were more common. The TD strain exhibited homozygous resistance to all tested mosquitoes when exposed to both permethrin and deltamethrin (frequency allele\u0026thinsp;=\u0026thinsp;1.0). No detection of I1011M mutation was found for insecticide exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the case of A1007G, homozygous resistance alleles were found in the TDR strain only when exposed to permethrin. While, for T1520I, resistance was nearly fixed in deltamethrin-exposed populations, with all individuals carrying heterozygous resistance genotypes (TI). The F1534C locus showed the most variation between insecticides. For permethrin, resistant heterozygotes and homozygotes (FC and CC) were frequently observed, with resistance allele frequencies ranging from 0.17 (TD) to 0.83 (TMB).\u003c/p\u003e\u003cp\u003eInterestingly, the \u003cem\u003ekdr\u003c/em\u003e mutation for the FSP strain was only detected in domain II and not in domain III for T1520I and F1534C. Under deltamethrin exposure, however, several populations (e.g., AA, TDR) retained higher proportions of the wild-type (FF) allele, and resistance allele frequencies were generally lower (0.17\u0026ndash;0.67). This suggests that F1534C contributes more to permethrin resistance than to deltamethrin resistance.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study revealed \u003cem\u003eAe. aegypti\u003c/em\u003e in Malaysia are responding differently to the insecticides used in control programs, being more impacted by pyrethroids, which have caused resistance. Malathion still works well in most sites, but resistance to pirimiphos-methyl was already widespread for the organophosphate group. Pyrethroids such as deltamethrin and permethrin were the least effective. In many places, survival rates were far higher than the WHO limit for resistance, as in the WHO (2022)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e protocol. These findings confirm what has been reported in Malaysia before, that the pyrethroids are losing their effectiveness in dengue-prone regions because of long and repeated use\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe present findings reveal notable spatial heterogeneity in insecticide resistance across Malaysia. Populations from Johor (ABS) exhibited particularly high levels of pyrethroid resistance, whereas populations from Penang (TMB and FSP) demonstrated only moderate resistance. Localized ecological and operational pressures most likely shape such spatial variation in resistance. These include differences in human population density, variation in the frequency and intensity of fogging activities, and the extent of household insecticide use. Previous studies in Malaysia have reported similar trends, where areas subjected to intensive vector control interventions showed elevated frequencies of resistance alleles compared to less-treated localities\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Sustained reliance on chemical interventions in densely populated dengue hotspots, such as Penang, Johor, and Selangor, has been shown to impose strong selective pressure on mosquito populations, accelerating the emergence and spread of resistance alleles\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Although malathion remains largely effective in most surveyed sites, the early indications of reduced susceptibility in Johor raise concerns that continued reliance on this compound may lead to the development of resistance, analogous to that observed with pyrethroids.\u003c/p\u003e\u003cp\u003eKnockdown assays provided additional insight, revealing that mosquitoes exposed to pyrethroids exhibited markedly prolonged knockdown times compared to the laboratory-susceptible strain, in some cases requiring over tenfold longer durations to achieve comparable knockdown. This delay suggests a combination of reduced insecticide absorption and stronger metabolic defenses, particularly the activity of detoxifying enzymes such as cytochrome P450s. These mechanisms have been reported before in Malaysian mosquito populations\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. A notable observation was the pronounced heterogeneity in knockdown responses within certain populations. For example, in Johor, individual mosquitoes displayed highly variable susceptibility, with some exhibiting rapid knockdowns. In contrast, others remained active for several hours, highlighting that resistance dynamics can be heterogeneous and complex even within a single geographic population.\u003c/p\u003e\u003cp\u003eMolecular analysis provided insight into the resistance mechanisms underlying the phenotypic variation. Several established knockdown resistance (\u003cem\u003ekdr\u003c/em\u003e) mutations, including S989P, V1016G, and F1534C, were detected, together with more recently reported substitutions such as A1007G and T1520I, within Malaysia. In certain populations, resistant alleles were found at near-fixation levels, indicating that almost all individuals carried these mutations. Notably, the F1534C mutation appeared to confer a stronger association with permethrin resistance than with deltamethrin resistance, a pattern consistent with reports from other Southeast Asian populations\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Plernsub et al. (2016)\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e demonstrated that the F1534C mutation is particularly important for type I pyrethroid (permethrin) resistance, while other combinations (S989P\u0026thinsp;+\u0026thinsp;V1016G) contribute more to type II pyrethroid (deltamethrin) resistance. These findings underscore that individual \u003cem\u003ekdr\u003c/em\u003e mutations differ in their functional impact across pyrethroid compounds, emphasizing the importance of monitoring the distribution and frequency of specific alleles to guide the continued efficacy of vector control strategies\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWhen placed in a global context, the resistance profile observed in Malaysia reflects broader patterns that have been documented elsewhere. Multiple mutations were detected in the voltage-gated sodium channel (VGSC) gene, including S989P, V1016G, F1534C, A1007G, and the newly observed T1520I. The present study reveals a complex \u003cem\u003ekdr\u003c/em\u003e resistance profile in Malaysian \u003cem\u003eAe. aegypti\u003c/em\u003e, shaped by prolonged pyrethroid use. The widespread occurrence of S989P \u003cem\u003eand\u003c/em\u003e V1016G, often in combination, is consistent with their established role in conferring resistance to type II pyrethroids such as deltamethrin, whereas F1534C was more strongly associated with resistance to type I compounds such as permethrin, supporting earlier reports from Southeast Asia that different alleles exhibit compound-specific effects\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In Asia, particularly in Thailand and Vietnam, the combined presence of S989P and V1016G mutations has been linked to significantly reduced efficacy of pyrethroid-based interventions\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe detection of A1007G at localized sites, and the near fixation of T1520I under deltamethrin exposure, indicate that novel mutations are emerging under ongoing selection pressure, mirroring findings from India and Myanmar, where these alleles often occur in combination with F1534C\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In Latin America, widespread pyrethroid resistance has been strongly associated with V1016I/G and F1534C mutations, which have been directly implicated in operational control failures1\u003csup\u003e13,32\u003c/sup\u003e. The coexistence of multiple \u003cem\u003ekdr\u003c/em\u003e alleles across different regions suggests that Malaysian populations are evolving heterogeneous but increasingly robust resistance phenotypes, posing a substantial challenge to vector control.\u003c/p\u003e\u003cp\u003eThe first detection of T1520I in Malaysian \u003cem\u003eAe. aegypti\u003c/em\u003e broadens the regional picture of vgsc evolution under pyrethroid selection. In our study, the T1520I mutation was present at a high frequency under deltamethrin exposure, with TI heterozygotes approaching fixation in several populations. Comparable occurrences of mutation have been reported initially in India, where it co-segregated with F1534C in pyrethroid-resistant populations and was associated with reduced permethrin susceptibility\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Myanmar documented T1520I alongside S989P, V1016G, and F1534C, including a quadruple haplotype (S989P/V1016G/T1520I/F1534C) indicative of strong multi-locus selection\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Mechanistic work using expressed sodium channels further shows that T1520I modifies the PyR1 binding site and, in combination with F1534C, preferentially elevates resistance to type I pyrethroids (e.g., permethrin) while retaining sensitivity to type II compounds (e.g., deltamethrin)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Regionally, reviews and multi-country syntheses have noted that T1520I, typically linked with F1534C, has now been detected across parts of South and Southeast Asia, reinforcing its relevance as a surveillance marker for operational programs\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThese findings highlight a divergence in susceptibility within the organophosphate class, where malathion continues to serve as a viable option in most settings, whereas pirimiphos-methyl efficacy is severely compromised. In India and Sri Lanka, \u003cem\u003eAe. aegypti\u003c/em\u003e have already developed reduced susceptibility to malathion through enzyme-based detoxification mechanisms like esterases and GSTs\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. The overall profile emphasizes that although organophosphates remain more effective than pyrethroids, localized resistance is emerging, and continued surveillance is crucial to prevent further erosion of their utility in dengue control programs.\u003c/p\u003e\u003cp\u003eIn short, this study shows that \u003cem\u003eAe. aegypti\u003c/em\u003e in Malaysia are already resistant to pyrethroids and are showing the first signs of resistance to organophosphates. The observed resistance patterns have critical implications for vector control and pest management in Malaysia. The widespread resistance to pyrethroids, combined with the emergence of resistance to organophosphates such as pirimiphos-methyl, indicates that relying solely on chemical insecticides will increasingly undermine the effectiveness of dengue control programs\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Keeping track of the spread of resistance mutations can help predict where and when insecticides are likely to fail. In Malaysia, embedding routine monitoring of \u003cem\u003ekdr\u003c/em\u003e mutations into dengue control programs would provide a strong early-warning system, similar to how malaria vector programs already use genetic tools to track resistance trends\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Importantly, molecular surveillance of \u003cem\u003ekdr\u003c/em\u003e mutations, particularly the novel T1520I identified in this study, should be incorporated into routine monitoring frameworks to provide early warnings of resistance spread and to guide evidence-based interventions. The latter path offers the best chance to maintain effective dengue control in the long term\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. To address this challenge, integrated resistance management strategies should be prioritized, including insecticide rotation with compounds of different modes of action\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, the strengthening of larval source reduction and environmental management\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and the integration of biological control measures where feasible\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Additionally, community education and the regulation of domestic insecticide use are crucial in reducing household-level selection pressures that accelerate the development of resistance. Collectively, these strategies will strengthen the resilience of pest management programs and help sustain the long-term efficacy of dengue control efforts in Malaysia.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this study demonstrates that, \u003cem\u003eAe. aegypti\u003c/em\u003e populations in Malaysia exhibit a complex resistance landscape characterized by widespread pyrethroid resistance and emerging, though more localized, resistance to organophosphates. The detection of multiple \u003cem\u003ekdr\u003c/em\u003e mutations, including well-established alleles (S989P, V1016G, F1534C) and newer variants (A1007G, T1520I), underscores the adaptive capacity of vector populations under sustained chemical pressure and highlights the compound-specific nature of resistance mechanisms. While malathion remains broadly effective, the compromised efficacy of pirimiphos-methyl signals that even organophosphates are not invulnerable to resistance development. Collectively, these findings underscore the urgent need for integrated resistance management strategies that incorporate molecular surveillance, insecticide rotation, and non-chemical interventions to sustain the long-term effectiveness of vector control programs and mitigate the risk of operational failure in dengue prevention.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our sincere gratitude to the School of Biological Sciences, Universiti Sains Malaysia, for their invaluable support throughout this research. The institution's resources and facilities greatly contributed to the successful completion of this work. We want to acknowledge the collaborative efforts of the Ministry of Health Malaysia. \u0026nbsp;We also extend our heartfelt appreciation to the Ministry of Higher Education Malaysia for the funding that is essential in facilitating the research and enabling the publication of this manuscript (FRGS/1/2023/STG03/USM/02/4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research/project was funded by the Ministry of Higher Education Malaysia under the Fundamental Research Grant Scheme, grant number [FRGS/1/2023/STG03/USM/02/4]. The funding agency had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript/report.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMedical Entomology Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800, Penang, MALAYSIA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMa Teng \u0026amp; Wan Fatma Zuharah\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors' contributions are as follows: WFZ: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Supervision, Writing—original draft; Writing—review \u0026amp; editing, MT: Project administration, Methodology, Data curation, Formal analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Wan Fatma Zuharah, [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach author has approved the submitted version (and any substantially modified version that involves the author's contribution to the study) and has agreed both to be personally accountable for the author's own contributions. The funders did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo ethical approval is needed for this project.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLeta, S. et al. Global risk mapping for major diseases transmitted by Aedes aegypti and Aedes albopictus. \u003cem\u003eInt. J. Infect. Dis.\u003c/em\u003e \u003cb\u003e67\u003c/b\u003e, 25\u0026ndash;35. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijid.2017.11.026\u003c/span\u003e\u003cspan address=\"10.1016/j.ijid.2017.11.026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSimmons, C. P., Farrar, J. J., Nguyen, V. V. 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Insecticide resistance status and mechanisms in Aedes aegypti and Aedes albopictus populations from Malaysia. \u003cem\u003eInsects\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (5), 456. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/insects13050456\u003c/span\u003e\u003cspan address=\"10.3390/insects13050456\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKumar, S., Stecher, G., Li, M., Knyaz, C. \u0026amp; Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e (6), 1547\u0026ndash;1549. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/molbev/msy096\u003c/span\u003e\u003cspan address=\"10.1093/molbev/msy096\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKawada, H. et al. Discovery of point mutations in the voltage-gated sodium channel gene of Aedes aegypti populations in Myanmar, suspected to confer pyrethroid resistance. \u003cem\u003ePLoS Negl. Trop. Dis.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e (7), e0004780. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pntd.0004780\u003c/span\u003e\u003cspan address=\"10.1371/journal.pntd.0004780\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePlernsub, S. et al. Additive effect of knockdown resistance mutations, S989P\u0026thinsp;+\u0026thinsp;V1016G\u0026thinsp;+\u0026thinsp;F1534C, in Aedes aegypti. \u003cem\u003eParasites Vectors\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e, 1\u0026ndash;12 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKushwah, R. B. S., Dykes, C. L., Kapoor, N., Adak, T. \u0026amp; Singh, O. P. Pyrethroid-resistance and presence of two knockdown resistance (kdr) mutations, F1534C and a novel mutation T1520I, in Indian Aedes aegypti. \u003cem\u003ePLoS Negl. Trop. 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Dis.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e (7), e0005625. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pntd.0005625\u003c/span\u003e\u003cspan address=\"10.1371/journal.pntd.0005625\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Aedes aegypti, Insecticide Resistance, kdr mutation, mosquito, pyrethroid, organophosphate","lastPublishedDoi":"10.21203/rs.3.rs-7708119/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7708119/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003e\u003cem\u003eAedes aegypti\u003c/em\u003e, the primary vector of dengue, has developed widespread resistance to insecticides, posing a threat to the efficacy of vector control programs. This study assessed the susceptibility status of \u003cem\u003eAe. aegypti across Malaysia\u003c/em\u003e from seven dengue hotspot localities and characterized knockdown resistance (\u003cem\u003ekdr\u003c/em\u003e) mutations, including the first detection of T1520I.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eSusceptibility was assessed using World Health Organization (WHO, 2022) bioassays with four insecticides: deltamethrin (0.03%), permethrin (0.4%), pirimiphos-methyl (60 mg/m\u0026sup2;), and malathion (5%). Knockdown times (KT₅₀ and KT₉₅) were determined using probit regression, and resistance ratios were calculated relative to a laboratory-susceptible strain. Genomic DNA was extracted from survivors, and sequencing of the voltage-gated sodium channel (VGSC) gene was conducted to detect knockdown resistance (kdr) mutations.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eAll field populations remained susceptible to malathion, with mortality\u0026thinsp;\u0026ge;\u0026thinsp;95%, though one Johor strain (ABS) showed suspected resistance (95%). Pirimiphos-methyl resistance was widespread, with mortality as low as 6% (ABS). Whereas resistance to pyrethroids was pronounced, with deltamethrin mortality ranging from 22% (ABS) to 95% (AA), and permethrin from 0% (TMB, ABS) to 95% (AA). Knockdown assays revealed high resistance ratios, particularly for permethrin (TMB: RR₅₀=9.98, RR₉₅=14.98; ABS: RR₉₅=41.46). Sequencing identified multiple \u003cem\u003ekdr\u003c/em\u003e mutations, including S989P, V1016G, F1534C, A1007G, and the novel detection of T1520I in Malaysian populations. F1534C was strongly associated with permethrin resistance, while V1016G and S989P predominated under deltamethrin exposure.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003e\u003cem\u003eAedes aegypti\u003c/em\u003e populations in Malaysia exhibit widespread pyrethroid resistance and emerging organophosphate resistance, underpinned by multiple \u003cem\u003ekdr\u003c/em\u003e mutations. The first detection of T1520I underscores the evolving genetic basis of resistance. These findings highlight the urgent need for integrated resistance management, including molecular surveillance and insecticide rotation, to sustain effective dengue control.\u003c/p\u003e","manuscriptTitle":"The Susceptibility Status of Aedes aegypti (Diptera: Culicidae) Mosquitoes in Malaysia on Pyrethroid and Organophosphate Insecticides with First Detection of T1520I Mutation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-28 16:40:15","doi":"10.21203/rs.3.rs-7708119/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-18T08:48:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-14T18:51:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"172669101243380277291291288343211292514","date":"2025-12-10T14:20:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104096454371970543744843687510609377219","date":"2025-10-22T17:39:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-18T03:07:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"109064601537066247712803317443378396053","date":"2025-10-15T01:57:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-14T15:20:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-14T14:38:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-03T12:15:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-01T09:51:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-01T09:42:48+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":"68fc5c9f-3755-4454-b425-30a227462eb9","owner":[],"postedDate":"October 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":56981448,"name":"Health sciences/Diseases"},{"id":56981449,"name":"Biological sciences/Genetics"},{"id":56981450,"name":"Biological sciences/Microbiology"},{"id":56981451,"name":"Biological sciences/Molecular biology"},{"id":56981452,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-03-02T16:07:42+00:00","versionOfRecord":{"articleIdentity":"rs-7708119","link":"https://doi.org/10.1038/s41598-026-41000-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-24 15:59:06","publishedOnDateReadable":"February 24th, 2026"},"versionCreatedAt":"2025-10-28 16:40:15","video":"","vorDoi":"10.1038/s41598-026-41000-9","vorDoiUrl":"https://doi.org/10.1038/s41598-026-41000-9","workflowStages":[]},"version":"v1","identity":"rs-7708119","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7708119","identity":"rs-7708119","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}