Insecticide resistance status and high frequency of kdr mutations in Aedes aegypti in Tegucigalpa, Honduras

Insecticide resistance status and high frequency of kdr mutations in Aedes aegypti in Tegucigalpa, Honduras | 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 Research Article Insecticide resistance status and high frequency of kdr mutations in Aedes aegypti in Tegucigalpa, Honduras Cindy Reyes-Perdomo, Denis Escobar, Luis Galo, Oscar Urrutia, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6329329/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Aug, 2025 Read the published version in Parasites & Vectors → Version 1 posted 9 You are reading this latest preprint version Abstract Background: Aedes aegypti is the primary vector of arboviruses including dengue, Zika, and chikungunya. The primary approach to control vector populations and reduce the transmission of these arboviruses during outbreaks is the use of insecticide spraying. However, the prolonged use of insecticides promotes resistance due to the selective pressure exerted on mosquito populations. This study aimed to conduct a phenotypic assessment of insecticide resistance and characterize the main knockdown resistance ( kdr ) mutations in Ae. aegypti populations collected in the Central District of Honduras. Methods: Larvae of Ae. aegypti were collected from four localities in the Central District of Honduras between May and June of 2023. Bioassays to determine susceptibility to deltamethrin, permethrin, malathion, and bendiocarb were carried out. For each location and phenotype, F1534C and V1016I kdr allele frequencies and haplotypes were calculated. Sequencing analyses were employed to genotype additional positions of interest on the vgsc gene. Results: A total of 1,592 Ae. aegypti females were phenotyped in the bioassays. Only two populations were resistant to deltamethrin. Conversely, all populations were resistant to permethrin and malathion, and all populations were susceptible to bendiocarb. The genotyping of 275 individuals revealed the presence of mutant alleles at both kdr loci (1016I and 1534C). The overall allele frequencies for 1534C and 1016I were 1.0 and 0.89, respectively. Variability of frequencies for 1016I was observed between localities, with two populations exhibiting a frequency of 1.0 for the mutant allele, while the rest ranged from 0.48 to 0.97. No additional mutations were detected on the vgsc gene. Conclusion: This study provides evidence regarding the resistance status of Ae. aegypti to insecticides used for vector control in Honduras. Additionally, the high frequency of permethrin resistance and of kdr mutations suggests that the mosquito populations have been under selective pressure from pyrethroids. This information could be integrated into vector control policies in Honduras to develop more targeted and effective strategies for combating mosquito-borne diseases. Aedes aegypti Dengue Honduras Insecticide resistance kdr Figures Figure 1 Figure 2 Figure 3 Background Aedes (Stegomyia) aegypti is a major vector that has contributed to outbreaks of multiple arboviral diseases throughout the past century ( 1 , 2 ). Its vectorial capacity for new and re-emerging diseases has been projected to increase as a result of the impact of climate change, urbanization, and human movement patterns ( 3 , 4 ). Modeling analyses have provided indications that the distribution patterns of Ae. aegypti are anticipated to undergo significant transformations over the current century, as previously hostile habitats are transformed into more favorable environments ( 5 , 6 ). In recent decades, most tropical countries of the Americas have been severely impacted by recurring dengue epidemics, as well as the spread of new arboviruses such as chikungunya and Zika ( 7 , 8 ). The first cases of dengue in Honduras were documented during the 1978 epidemic, with a total of over 130,000 cases. In the following years, there were documented reports of epidemics in 1987, impacting around 29,000 individuals, as well as in 1989 and 1991. At that time, dengue occurred in large outbreaks with a particular concentration in the two most densely inhabited cities, Tegucigalpa and San Pedro Sula ( 9 ). Between 2015 and 2016, Honduras experienced the emergence of two additional arboviruses (chikungunya and Zika) transmitted by Ae. aegypti , in addition to ongoing dengue epidemics ( 10 – 12 ). The main approach for controlling epidemic outbreaks of Aedes -borne arboviruses remains the control of the adult stage of the mosquito ( 13 ). In Honduras, the Ministry of Health carries out vector control activities with insecticide fogging, often using a combination of permethrin plus a synergist (piperonyl butoxide) ( 14 ). The recurrent use of insecticides often leads to the emergence of mosquito populations that are resistant to insecticides. Phenotypic resistance to insecticides arises by at least four previously reported biological mechanisms ( 15 ). The most commonly reported mechanisms include an increase in the activity of detoxifying enzymes (a metabolic mechanism) and the occurrence of mutations at the insecticide target site within the mosquito (a genetic mechanism), referred to as knockdown resistance ( kdr ) ( 16 ). So far, a total of eleven mutations of the kdr type, linked to resistance against pyrethroids, have been documented. These mutations have also been observed as multiple mutations at positions 989, 1016, and 1534 (V1016G/S989P, F1534C/V1016G and F1534C/V1016I) ( 17 – 20 ). Although insecticides are critical for controlling vectors, there is a lack of information regarding the status of resistance and the distribution of kdr mutations in Ae. aegypti in Central America. The available data from Latin America is primarily generated from Mexico and Brazil, with some additional reports from countries including Colombia, Costa Rica, Cuba, Peru, and some Caribbean nations ( 21 – 26 ). Nevertheless, significant knowledge gaps remain regarding the prevalence and magnitude of insecticide resistance in Ae. aegypti within the Central American region. Thus, this study aimed to conduct a phenotypic assessment of insecticide resistance in Ae. aegypti populations collected in the Central District of Honduras. Additionally, the study sought to characterize the frequency of kdr alleles in the populations. Methods Study site Aedes aegypti were collected in the municipality of the Central District of Honduras. This region was selected because it includes the capital city of Tegucigalpa and reports the highest prevalence of dengue and other arboviruses in the country ( 27 , 28 ). The Central District is located between 900 and 1000 meters above sea level and has an approximate population of 1.1 million inhabitants. There are two seasons, a dry season (from December to May) and a rainy season (from June to November). The average temperature ranges between 16 and 30°C. Four neighborhoods were selected to collect larvae: “Loarque - LO” (14.041576, -87.211082), “La Concordia - LC” (14.112068, -87.208535), “Altos de Villa Vieja - AVV” (14.059852, -87.162463) and “Río Abajo - RA” (14.173899, -87.211295) (Fig. 1 ). These neighborhoods were selected because they were reported as those with the highest routine use of insecticide spraying by the Ministry of Health of Honduras. Collection and rearing of mosquitoes The study involved visiting between five to seven locations inside each of the four chosen neighborhoods that exhibited clear evidence of water accumulation, including sinks, pans, and automobile tires. Mosquito larvae and pupae were collected at each site ( 29 ). The live larvae and pupae were transferred in plastic containers to the Medical Entomology Laboratory of the National Autonomous University of Honduras (UNAH). Larvae and pupae obtained from the field (F0 generation) were reared to adults under standard insectary settings. Each group of larvae collected per neighborhood was placed separately in trays (28 × 18 × 6 cm) with 1500 mL of distilled water. The larvae were fed daily with fish food (Biomaa, Super Flakes, Mexico) and maintained at a relative humidity of 70–80%, a temperature range of 25–27°C, and a light and dark cycle of 12 hours each, until they reached maturity ( 30 – 32 ). The emerged adult mosquitoes were identified using taxonomic keys ( 33 ), separating all individuals identified as Ae. aegypti. An F1 was generated from each population, following the previously described procedure ( 31 ), and fed with a 10% sugar solution until use in insecticide susceptibility bioassays. Phenotypic bioassays Susceptibility bioassays were performed following the procedures described for the CDC bottle bioassay method ( 34 ). Briefly, the diagnostic concentrations of four insecticides were prepared from technical grade active ingredients (Chem Services, West Chester, PA, USA): two pyrethroids (deltamethrin, 10 µg/mL and permethrin, 15 µg/mL); a carbamate (bendiocarb, 12.5 µg/mL), and an organophosphate (malathion, 50 µg/mL). The bioassays were performed using 3- to 5-day-old non-blood fed adult female Ae. aegypti . Between 20–25 mosquitoes were placed into 250 mL Wheaton bottles previously impregnated with the diagnostic concentration of insecticide and kept under observation for 30 minutes. The number of knocked down individuals was recorded at 0, 15, and 30 minutes of exposure. The diagnostic time to determine the phenotype was 30 minutes, according to what is described by the CDC method ( 34 ). Subsequently, all exposed mosquitoes were transferred to holding containers with 10% sugar solution to evaluate mortality after 24 hours (as a proxy for recovery). After recording the mortality rate at 24 hours, both susceptible and resistant individuals were separated and kept at -20°C until molecular analyses were performed. DNA extraction and genotyping of positions 1534 and 1016 of the VGSC gene For the detection of kdr mutations, a sample of 285 individuals from the F1 generation, who were phenotyped in deltamethrin and permethrin bioassays, was randomly chosen from the four study locations: 70 individuals (25%) from “AVV”, 80 (28%) from “LC”, 74 (26%) from “LO”, and 61 (21%) from “RA”. Individuals of both resistant (n = 135) and susceptible (n = 150) phenotypes were selected. DNA was extracted separately from each mosquito using the ReliaPrep™ Blood gDNA Miniprep System (Promega, Madison, Wisconsin, USA), according to the manufacturer's instructions. Genotyping of positions 1534 and 1016 was carried out using allele-specific PCR (AS-PCR) according to the primers and protocols described by Yanola et al ( 11 ) and the primers modified by Contreras-Perera et al ( 35 ) (Table 1 ). The possible genotypes for position 1534 were F1534F (homozygous wild type), F1534C (heterozygous mutant), and C1534C (homozygous mutant). For position 1016, the possible genotypes were V1016V (homozygous wild type), V1016I (heterozygous mutant), and I1016I (homozygous mutant). For both loci, the presence of heterozygotes was registered by observing the presence of double bands according to the base pair sizes previously reported. Briefly, PCR reactions to analyze position 1534 were carried out in a final volume of 20 µL, including 10 µL of Taq Master Mix 2X (Promega, Madison, Wisconsin, USA), 0.8 µL of each primer (10 µM) (Table 1 ), 1 µL of DNA and 6.6 µL of nuclease-free water (NFW). The amplification conditions were as follows: 95°C for 5 min, 35 cycles of 95°C for 1 min, 57°C for 1 min, 72°C for 1 min, with a final extension step at 72°C for 4 min. For position 1016, the PCR reaction was carried out in a volume of 25 µL, with 12.5 µL of Taq Master Mix 2X (Promega, Madison, Wisconsin, USA), 1 µL of each primer (10 µM) (Table 1 ), 4 µL of DNA and 5.5 µL of NFW. The amplification conditions were as follows: 95°C for 5 min, 29 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min, with a final extension step at 72°C for 10 min. The amplification products were separated by electrophoresis on a 3% agarose gel and visualized with ethidium bromide under UV light. Genotyping of positions 989, 1011, and 1520 of the VGSC gene In order to detect additional mutations at 989,1011 and 1520 positions at the voltage-gated sodium channel gene, a partial region of the IIS5-6 and IIIS5-6 segments was amplified using specific primers reported elsewhere (Table 1 ) ( 36 , 37 ). To identify additional kdr mutations associated with resistance to pyrethroid insecticides, 60 individuals were selected: Twenty-eight individuals resistant to the pyrethroids deltamethrin (n = 9) and permethrin (n = 19), and 32 individuals susceptible to deltamethrin (n = 17) and permethrin (n = 15).. Briefly, the IIS5-6 fragment was amplified in a final volume of 50 µL including 25 µL of Taq Master Mix 2X (Promega, Madison, Wisconsin, USA), 2 µL of each primer (10 µM) (Table 1 ), 4 µL of DNA and 17 µL of NFW. The amplification conditions were as follows: 95°C for 2 min, 40 cycles of 94°C for 30 sec, 50°C for 30 sec, 72°C for 1 min, with a final extension at 72°C for 10 min. PCR reactions for the IIIS5-6 fragment were carried out in a final volume of 50 µL including 25 µL of Taq Master Mix 2X (Promega, Madison, Wisconsin, USA), 2.5 µL of each primer (10 µM) (Table 1 ), 2 µL of DNA and 18 µL of NFW, under the following conditions: 95°C for 5 min, 35 cycles of 94°C for 30 sec, 59°C for 30 sec, 72°C for 1 min, with a final extension at 72°C for 10 min. The amplified products were separated on 1% agarose gels and visualized with ethidium bromide under UV light. The amplified products were sequenced by Psomagen® ( https://www.psomagen.com ) using internal primers: 508R: 5′- TTG TTC GTT TCG TTG TCG GC − 3´ for the IIS6 fragment and 71F: 5′- GTC CTC GAT CCT TCC AGG TG − 3´ for the IIIS6 fragment. The sequences obtained were curated and analyzed with Geneious Prime 2024.0.2 software (Dotmatics, Auckland, New Zealand). Each sequence was aligned and compared with a reference sequence of Ae. aegypti (Accession number AAEL023266) available from VectorBase ( https://vectorbase.org/vectorbase ) ( 38 ) and other sequences available in NCBI (Accession numbers KY626180.1, KY626197.1, KY046222.1, XM_021852349.1) previously reported with mutations at positions 989, 1011 and 1016. Table 1 Sequences of the primers and PCR conditions used for genotyping positions 1534 and 1016 of the VGSC gene. Target Primer Primer sequence Annealing temperature (°C) PCR product size (bp) IIS5-6 C1534fw 5′-GCG GGC AGG GCG GCG GGG GCG GGG CCT CTA CTT TGT GTT CTT CAT CAT GTG-3′ 57 93 113 F1534fw 5′-GCG GGC TCT ACT TTG TGT TCT TCA TCA TAT T-3′ F1534R 5′-TCT GCT CGT TGA AGT TGT CGA T-3′ IIIS5-6 Val1016fw 5′- GCG GGC AGG GCG GCG GGG GCG GGG CCA CAA ATT GTT TCC CAC CCG CAC CGG- 3´ 60 102 82 Ile1016f 5′-GCG GGC ACA AAT TGT TTC CCA CCC GCA CTG A- 3′ Ile1016r 5′-TGA TGA ACC SGA ATT GGA CAA AAG C- 3′ IIS5-6 IIS5-6F 5′-ATC GCT TCC CGG ACA AAG AC- 3′ 50 562 – Intron A 579 – Intron B IIS5-6R 5′-GTT GGC GAT GTT CGA CTT GA- 3′ IIIS5-6 AaNa31F 5′- GAC TCG CGG GAG GTA AGT T – 3′ 59 512 AaNa31R 5′- CCG TCT GCT TGT AGT GAT CG – 3′ Statistical analysis Mortality was defined as the percent of individuals knocked down at the diagnostic time. To categorize the susceptibility status of the populations, the ranges described by the CDC were followed: 98–100% mortality indicated susceptibility of the population, 90–97% indicated the development of resistance, and ˂90% indicated resistance. Both allele frequencies and haplotype frequencies were calculated for each population and phenotype ( 39 ). Wright's inbreeding coefficient (FIS) was calculated to evaluate the population structure, where possible, considering the loci of interest using the formula: FIS = 1- ( Ho / He ), where Ho is the number of observed heterozygotes and He was calculated as follows: He = 2 np (1- p ), where n is the sample size ( 40 , 41 ). The Chi-square test was calculated in R software (version 2024.04.1; https://www.R-project.org/ ) under the package “stats -> “ chisq.test” to determine a significant association between phenotypic susceptibility and the F1534C, V1016I and V1016V alleles, ( p < 0.05) ( 42 ). Results Bioassays A total of 1,592 Ae. aegypti females of the F1 generation were analyzed in bioassays. Four sets of bioassays were carried out per location, one per insecticide. All populations were resistant to permethrin and malathion. For permethrin, the mortality ranged between 1% and 48%, with LC showing the lowest mortality (1%) and RA showing the highest (48%) (Fig. 2 ). For malathion, mortality ranged between 24% (AVV) and 74% (LO). Mortality rates for deltamethrin ranged between 86% (LC) and 100% (RA and AVV). All populations showed 100% mortality at the diagnostic time for bendiocarb. Of the four locations evaluated, only the LC population showed changes in mortality for both pyrethroids 24 hours after exposure, with a recovery rate of 15% and 1% for deltamethrin and permethrin, respectively. (Additional file S1, Table 1 ). Kdr genotypes and haplotypes for positions 1016 and 1534 Out of the 285 individuals tested, 275 (96.5%) were successfully genotyped for at least one of the two loci, n = 275 for position 1534 and n = 273 for position 1016. No permethrin-susceptible phenotypes were detected from LC, nor deltamethrin-resistant phenotypes from AVV or RA, so these were not included in the genetic analyses. The allele frequencies by insecticide and phenotype are summarized in Table 2 . Genotyping analyses revealed the presence of individuals with the F1534C mutation in its homozygous mutant (C/C) and heterozygous (F/C) variants. In these populations, no individual was found with the wild variant (F/F). In contrast, the three genotypes were observed at position 1016 (V/V, V/I, and I/I). The double homozygous mutant haplotype (1534 C/C, 1016 I/I) was detected in 196 of 285 individuals analyzed (68.8%). The mosquitoes from LC accounted for 38% of the individuals with the double homozygous mutant haplotype, closely followed by the population from AVV. The allele frequencies for the mutant alleles were 1.0 and 0.89 for F1534C and V1016I, respectively. Among the four populations evaluated, the 1534C allele exhibited a frequency of 1.0 across all locations. Although homozygote mutant allele was highly frequent, heterozygotes were observed in three out of the four populations studied: AVV (0.06), RA (0.22) and LO (0.15); in this latter, particularly only five individuals (0.07) resistant to permethrin showed this genotype. On the contrary, the frequency of the V1016I resistant alleles showed variability between locations, ranging from 0.69 in RA to 0.99 in LC populations. Heterozygotes alleles (V/I) were observed in the four populations; of those, the highest frequency was observed in LO (0.33). Wright's inbreeding coefficient (FIS) calculations showed a low relationship between the populations analyzed at position 1016, with a slight excess of heterozygosity in the RA and AVV populations (Additional file 1-Table 2). Table 2 Frequency of 1534 and 1016 kdr alleles per site. Neighborhood n CC FC FF Freq C Freq FC n II VI VV Freq I Freq VI AVV 66 62 4 0 1.00 0.06 67 55 4 8 0.88 0.06 LC 78 78 0 0 1.00 0 78 75 2 1 0.99 0.03 LO 71 60 11 0 1.00 0.15 73 46 24 3 0.96 0.33 RA 60 47 13 0 1.00 0.22 55 30 8 17 0.69 0.15 Based on the observed genotypes, the haplotypes (1016/1534) by locality were calculated (Fig. 3 ). Six haplotypes were observed: R1R2 (II/CC), R1H2 (II/FC), H1R2 (VI/CC), H1H2 (VI/FC), S1R2 (VV/CC), S1H2 (VV/FC). Haplotype R1R2 (II/CC) was the most frequent haplotype in all localities, with the highest frequency observed in LC (0.99), followed by AVV (0.81). On the other hand, the S1H2 haplotype (VV/FC) had an overall frequency of 0.03 and was reported only in the AVV and RA localities. When combining results from phenotype and haplotype, only populations from two sites, LO and LC, were resistant to both deltamethrin and permethrin. Both populations showed a high proportion of R1R2 haplotype (˃0.6) and nearly no S1R2 (˂0.03). These results contrast with AVV, whose population was susceptible to deltamethrin and where R1R2 was present at a frequency of 0.81, but with a frequency of 0.11 of the S1R2 haplotype. As F1534C heterozygotes were observed mostly in susceptible individuals, Chi-square tests were applied to evaluate an association between the F1534C alelle and the susceptible phenotype, and the V1016I allele underwent the same analysis, an additional test were carried out to test association between V1016V allele and susceptible phenotype. This revealed a significant association between heterozygotes and the susceptible phenotype, both for F1534C (χ 2 = 5.6209; df = 1; p = 0.01775) and V1016I (χ2 = 5.9001; df = 1; p = 0.01514). Finally, V1016V showed an strong association with susceptible phenotype (χ 2 = 20.881; df = 1; p =˂ 0.00001). Genotyping of positions 989, 1011, and 1520 on the voltage-gated sodium channel (VGSC) gene Sequence analyses for the IIS6 and IIIS6 segments showed that all individuals presented the wild genotypes at positions 1520 (ACC), 989 (ATC) and 1011 (ATA). Additionally, the sequencing data confirmed the genotypes identified by AS-PCR at positions 1016 and 1534. Discussion The long-term efficacy of vector control interventions is significantly compromised by the emergence of insecticide resistance ( 43 ). The monitoring of insecticide susceptibility and characterization of resistance mechanisms provide crucial information for designing vector control strategies ( 44 , 45 ). The present study provides evidence of the state of resistance to insecticides and the high frequency of kdr alleles in Ae. aegypti in four locations around the capital of Honduras. There have been numerous reports of Ae. aegypti populations exhibiting resistance to multiple insecticides around the world. However, information on insecticide susceptibility in Honduras and the Central American region is still scarce ( 21 ). The results of the present study showed populations resistant to two pyrethroids and the organophosphate malathion, but full susceptibility to the carbamate bendiocarb. The Ministry of Health of Honduras has used commercial formulations with pyrethroids for control interventions for more than two decades, which has probably led to the selection of resistance in populations of Ae. aegypti . This phenomenon has been reported elsewhere in the Americas, such as in Mexico and Brazil, where populations highly resistant to pyrethroids have been observed after consecutive periods of insecticide application ( 16 , 40 , 46 , 47 ). The presence of two mosquito populations (RA and AVV) resistant to permethrin (a type I pyrethroid) but susceptible to deltamethrin (a type II pyrethroid) suggests that resistance to one pyrethroid doesn’t always result in cross-resistance across the pyrethroid class. In the Americas, there are reports with similar results, showing lower mortality to permethrin compared to deltamethrin ( 22 , 48 ). The LC and LO populations showed resistance to both pyrethroids. These populations came from primarily residential areas, which may be subject to additional selection pressure by insecticides for domestic use, a phenomenon also observed in some areas of Brazil and Mexico ( 49 , 50 ). In Honduras, organophosphate insecticides were replaced by pyrethroids in the late 1990s to control Ae. aegypti . However, the results reported here reveal that populations continue to show resistance to the organophosphate malathion. A study in Jamaica in 2015 observed that populations of Ae. aegypti showed heterogeneous results to malathion in five populations, with mortalities between 84 to 90%, after a rotation from malathion to permethrin in 2014 during a chikungunya outbreak ( 25 ). In addition, a study published by Rubio-Palis et al. in 2023 found that, despite reducing the use of organophosphates since 2010, four populations of Ae. aegypti continued to present resistance to malathion in Venezuela in 2020, demonstrating that despite the absence of pressure, phenotypic resistance was maintained ( 25 , 51 ). In addition to the phenotypic resistance results, this study detected high frequencies of both the F1534C and V1016I mutations. These kdr alleles in Ae. aegypti are distributed worldwide, and the F1534C mutation is the most frequently reported that has been associated with loss of susceptibility to pyrethroids ( 16 , 52 ). Although the 1534C mutation seems almost fixed in the populations analyzed, association tests revealed an association between the heterozygous individuals from two neighborhoods and the susceptible phenotype, similar to what was observed for the V1016I allele. Reports from Brazil and Mexico have demonstrated the rapid evolution and distribution of kdr mutations in Ae. aegypti , revealing that these mutations are capable of arising independently and fixing quickly in local populations, probably driven by highly local selection factors ( 46 , 47 , 53 – 55 ). It is notable that in this study F1534C and V1016I mutant individuals were detected among both susceptible and resistant individuals. A previous study in Venezuela showed that despite being fixed in the population, the F1534C mutation was found in populations susceptible to cypermethrin, evidencing the complexity of the multiple mechanisms involved in eliciting resistance ( 48 ). To further elucidate the mechanisms contributing to the resistance in the populations studied here, the next step would be the characterization of families of key enzymes, such as multiple function oxidases (MOF) and glutathione-S-transferases (GST), which help detoxify insecticide molecules and may also be contributing to resistant phenotypes ( 56 , 57 ). The combination of multiple mutant alleles on the mosquito vgsc gene can result in haplotypes that are associated with high levels of resistance to pyrethroids ( 58 , 59 ). Reports from Brazil have demonstrated the wide distribution of the 1534/1016 homozygous mutant haplotype (C/C, I/I). A recent national survey carried out in 123 municipalities in Brazil showed that this haplotype was the second most frequent nationally (24%). In Florida, USA, another investigation examined populations of Ae. aegypti in 62 locations, revealing a significant occurrence of the double mutant combination ( 60 ). This double mutant haplotype was also the most frequent in the four populations we studied from the Central District of Honduras. Although the haplotypes weren’t predictive of resistance phenotype (likely due to the contributions of additional resistance mechanisms), the variation in the frequencies of kdr haplotypes across the neighborhoods suggests a highly focal variation in selection that is occurring at the local level. Additional kdr mutations have been reported on the vgsc gene (T1520I, S989P, and I1011V/M), mainly in Asian mosquito populations ( 16 , 37 ). Although the results of this study show the absence of non-synonymous mutations in positions 1520, 989, and 1011, the constant use of pyrethroids and the potential introduction of Ae. aegypti from elsewhere could lead to the appearance of additional mutations in the future ( 17 ). Conclusion This study revealed phenotypic resistance to permethrin and malathion in Ae. aegypti populations across the study sites, and variable levels of deltamethrin susceptibility. The kdr alleles F1534C and V1016I were detected at high frequencies in Ae. aegypti , together with a predominant double-mutant haplotype (C/C, I/I). These results can inform vector control decision-making, and can be considered as a baseline for prospective monitoring of resistance trends. Abbreviations CDC Centers for Disease Control and Prevention Kdr Knockdown resistance vgsc Voltage-Gated Sodium Channel Declarations Ethics approval and consent to participate. Not applicable. Consent for publication. Not applicable. Availability of data and materials . All data generated or analysed during this study are included in this published article. Competing interests. The authors declare that they have no competing interests. Funding . Funding for this study was provided by the Genetic Research Center, CIG-UNAH. The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript. Authors' contributions . Conceptualization, D.E, O.U., A.L., and G.F.; methodology, A.L., D.E., R.L.V and G.F.; validation, R.L.V. A.L. and D.E.; experiments and analysis, C.R.P., L.G., and D.E; resources, A.L., D.E. and G.F.; data curation, G.F., and D.E.; writing—original draft preparation, G.F. and D.E.; writing—review and editing, G.F., D.E.; visualization, G.F.; supervision, D.E., and G.F.; funding acquisition, A.L., D.E., O.U., and G.F. All authors have read and agreed to the published version of the manuscript. Acknowledgements . The authors wish to express their gratitude to the staff of the Vigilancia Entomológica-Unidad de Vigilancia de la Salud, and the Region Metropolitana del Distrito Central of the Ministry of Health of Honduras for the collaboration for the development of this project. Many thanks to Mr. Hector Videa, Mr. Carlos Doblado, Mr. Ramón Martín Luque and Mr. Juan Zepeda for their support in the entomological collections. Disclaimer: The views expressed in this manuscript are those of the authors and do not necessarily reflect the official policy or position of the Centers for Disease Control and Prevention. References Kraemer MUG, Sinka ME, Duda KA, Mylne AQN, Shearer FM, Barker CM, et al. The global distribution of the arbovirus vectors Aedes aegypti and Ae. albopictus. eLife. 2015;4:e08347. Kraemer MUG, Sinka ME, Duda KA, Mylne A, Shearer FM, Brady OJ, et al. The global compendium of Aedes aegypti and Ae. albopictus occurrence. Sci Data. 2015;2:150035. 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Zambrano LI, Sierra M, Lara B, Rodríguez-Núñez I, Medina MT, Lozada-Riascos CO, et al. Estimating and mapping the incidence of dengue and chikungunya in Honduras during 2015 using Geographic Information Systems (GIS). J Infect Public Health. 2017;10(4):446–56. Roiz D, Wilson AL, Scott TW, Fonseca DM, Jourdain F, Müller P, et al. Integrated Aedes management for the control of Aedes-borne diseases. PLoS Negl Trop Dis. 2018;12(12):e0006845. En 20 municipios que acumulan el 85% de los casos de dengue se masificarán acciones - Honduras | ReliefWeb [Internet]. 2019 [cited 2024 Mar 14]. Available from: https://reliefweb.int/report/honduras/en-20-municipios-que-acumulan-el-85-de-los-casos-de-dengue-se-masificar-n-acciones Du Y, Nomura Y, Zhorov B, Dong K. Sodium Channel Mutations and Pyrethroid Resistance in Aedes aegypti. Insects. 2016;7(4):60. Moyes CL, Vontas J, Martins AJ, Ng LC, Koou SY, Dusfour I, et al. Contemporary status of insecticide resistance in the major Aedes vectors of arboviruses infecting humans. Sinnis P, editor. PLoS Negl Trop Dis. 2017;11(7):e0005625. Chen M, Du Y, Wu S, Nomura Y, Zhu G, Zhorov BS, et al. Molecular evidence of sequential evolution of DDT- and pyrethroid-resistant sodium channel in Aedes aegypti. PLoS Negl Trop Dis. 2019;13(6):e0007432. Zardkoohi A, Castañeda D, Lol JC, Castillo C, Lopez F, Marín Rodriguez R, et al. Co-occurrence of kdr Mutations V1016I and F1534C and Its Association With Phenotypic Resistance to Pyrethroids in Aedes aegypti (Diptera: Culicidae) Populations From Costa Rica. J Med Entomol. 2020;57(3):830–6. Linss JGB, Brito LP, Garcia GA, Araki AS, Bruno RV, Lima JBP, et al. Distribution and dissemination of the Val1016Ile and Phe1534Cys Kdr mutations in Aedes aegypti Brazilian natural populations. Parasit Vectors. 2014;7(1):25. Ishak IH, Jaal Z, Ranson H, Wondji CS. 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Available from: https://www.salud.gob.hn/sshome/index.php/dengue#boletin Abdulai A, Owusu-Asenso CM, Akosah-Brempong G, Mohammed AR, Sraku IK, Attah SK, et al. Insecticide resistance status of Aedes aegypti in southern and northern Ghana. Parasit Vectors. 2023;16(1):135. Consoli RAGB, Oliveira RL de. Principais mosquitos de importância sanitária no Brasil [Internet]. Editora Fiocruz; 1994 [cited 2024 May 9]. Available from: https://www.arca.fiocruz.br/handle/icict/2708 Secretaria de Salud México. Guía metodológica para la instalación y mantenimiento del insectario [Internet]. Segunda. 27 p. Available from: https://www.gob.mx/cms/uploads/attachment/file/598095/Guia_Metodologica_para_la_Instalacion_y_Mantenimiento_del__Insectario_compressed.pdf Danis-Lozano, R, Correa-Morales F. Cría de mosquitos Culicidae y evaluación de insecticidas de uso en salud pública [Internet]. Primera. Morelos, México: Instituto Nacional de Salud Pública; 2021 [cited 2024 May 9]. 120 p. 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Combined target site (kdr) mutations play a primary role in highly pyrethroid resistant phenotypes of Aedes aegypti from Saudi Arabia. Parasit Vectors. 2017;10(1):161. Alvarez-Jarreta J, Amos B, Aurrecoechea C, Bah S, Barba M, Barreto A, et al. VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center in 2023. Nucleic Acids Res. 2024;52(D1):D808–16. Deming R, Manrique-Saide P, Medina Barreiro A, Cardeña EUK, Che-Mendoza A, Jones B, et al. Spatial variation of insecticide resistance in the dengue vector Aedes aegypti presents unique vector control challenges. Parasit Vectors. 2016;9(1):67. García GP, Flores AE, Fernández-Salas I, Saavedra-Rodríguez K, Reyes-Solis G, Lozano-Fuentes S, et al. Recent Rapid Rise of a Permethrin Knock Down Resistance Allele in Aedes aegypti in México. PLoS Negl Trop Dis. 2009;3(10):e531. Wright S. Systems of Mating. II. the Effects of Inbreeding on the Genetic Composition of a Population. Genetics. 1921;6(2):124–43. R Core Team (2024). R: A Language and Environment for Statistical Computing_. R Foundation for Statistical Computing [Internet]. Vienna, Austria.; 2024. Available from: https://www.R-project.org/ World Health Organization. Global Malaria Programme. Global plan for insecticide resistance management in malaria vectors. 2012; Available from: https://apps.who.int/iris/handle/10665/44846 Pan American Health Organization. Monitoring and managing. insecticide resistance in Aedes mosquito populations Interim guidance for entomologists; 2016 - PAHO/WHO | Pan American Health Organization [Internet]. 2016 [cited 2024 Mar 11]. Available from: https://www.paho.org/en/documents/monitoring-and-managing-insecticide-resistance-aedes-mosquito-populations-interim World Health Organization. Manual for monitoring insecticide resistance in mosquito vectors and selecting appropriate interventions [Internet]. Geneva; 2022 [cited 2023 Feb 27]. Available from: https://www.who.int/publications-detail-redirect/9789240051089 Solis-Santoyo F, Rodriguez AD, Penilla-Navarro RP, Sanchez D, Castillo-Vera A, Lopez-Solis AD, et al. Insecticide resistance in Aedes aegypti from Tapachula, Mexico: Spatial variation and response to historical insecticide use. PLoS Negl Trop Dis. 2021;15(9):e0009746. Melo Costa M, Campos KB, Brito LP, Roux E, Melo Rodovalho C, Bellinato DF, et al. Kdr genotyping in Aedes aegypti from Brazil on a nation-wide scale from 2017 to 2018. Sci Rep. 2020;10(1):13267. Rubio-Palis Y, Dzuris N, Sandi C, Vizcaino-Cabarrus RL, Corredor-Medina C, González JA, et al. Insecticide resistance levels and associated mechanisms in three Aedes aegypti populations from Venezuela. Mem Inst Oswaldo Cruz. 118:e220210. Garcia G de A, David MR, Martins A de J, Maciel-de-Freitas R, Linss JGB, Araújo SC, et al. The impact of insecticide applications on the dynamics of resistance: The case of four Aedes aegypti populations from different Brazilian regions. PLoS Negl Trop Dis. 2018;12(2):e0006227. Gray L, Florez SD, Barreiro AM, Vadillo-Sánchez J, González-Olvera G, Lenhart A, et al. Experimental evaluation of the impact of household aerosolized insecticides on pyrethroid resistant Aedes aegypti. Sci Rep. 2018;8(1):12535. Alvarez LC, Ponce G, Oviedo M, Lopez B, Flores AE. Resistance to malathion and deltamethrin in Aedes aegypti (Diptera: Culicidae) from western Venezuela. J Med Entomol. 2013;50(5):1031–9. Du Y, Nomura Y, Satar G, Hu Z, Nauen R, He SY, et al. Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel. Proc Natl Acad Sci. 2013;110(29):11785–90. Saavedra-Rodriguez K, Urdaneta-Marquez L, Rajatileka S, Moulton M, Flores AE, Fernandez-Salas I, et al. A mutation in the voltage-gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti. Insect Mol Biol. 2007;16(6):785–98. Saavedra-Rodriguez K, Maloof FV, Campbell CL, Garcia-Rejon J, Lenhart A, Penilla P, et al. Parallel evolution of vgsc mutations at domains IS6, IIS6 and IIIS6 in pyrethroid resistant Aedes aegypti from Mexico. Sci Rep. 2018;8(1):6747. Cosme LV, Gloria-Soria A, Caccone A, Powell JR, Martins AJ. Evolution of kdr haplotypes in worldwide populations of Aedes aegypti: Independent origins of the F1534C kdr mutation. Pietrantonio P, editor. PLoS Negl Trop Dis. 2020;14(4):e0008219. Kasai S, Komagata O, Itokawa K, Shono T, Ng LC, Kobayashi M, et al. Mechanisms of Pyrethroid Resistance in the Dengue Mosquito Vector, Aedes aegypti: Target Site Insensitivity, Penetration, and Metabolism. PLoS Negl Trop Dis. 2014;8(6):e2948. Schluep SM, Buckner EA. Metabolic Resistance in Permethrin-Resistant Florida Aedes aegypti (Diptera: Culicidae). Insects. 2021;12(10):866. Xu Q, Zhang L, Li T, Zhang L, He L, Dong K, et al. Evolutionary adaptation of the amino acid and codon usage of the mosquito sodium channel following insecticide selection in the field mosquitoes. PloS One. 2012;7(10):e47609. Carvajal TM, Ogishi K, Yaegeshi S, Hernandez LFT, Viacrusis KM, Ho HT, et al. Fine-scale population genetic structure of dengue mosquito vector, Aedes aegypti, in Metropolitan Manila, Philippines. PLoS Negl Trop Dis. 2020;14(5):e0008279. Estep AS, Sanscrainte ND, Waits CM, Bernard SJ, Lloyd AM, Lucas KJ, et al. Quantification of permethrin resistance and kdr alleles in Florida strains of Aedes aegypti (L.) and Aedes albopictus (Skuse). PLoS Negl Trop Dis. 2018;12(10):e0006544. Additional Declarations No competing interests reported. Supplementary Files AdditionalfilesPV2025Clean.docx Cite Share Download PDF Status: Published Journal Publication published 01 Aug, 2025 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 24 Jun, 2025 Reviews received at journal 24 Jun, 2025 Reviewers agreed at journal 30 May, 2025 Reviews received at journal 22 May, 2025 Reviewers agreed at journal 02 May, 2025 Reviewers invited by journal 29 Apr, 2025 Editor assigned by journal 02 Apr, 2025 Submission checks completed at journal 01 Apr, 2025 First submitted to journal 28 Mar, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Tegucigalpa","correspondingAuthor":false,"prefix":"","firstName":"Cindy","middleName":"","lastName":"Reyes-Perdomo","suffix":""},{"id":451150311,"identity":"fd05f659-9f28-4bab-b665-89232499f15e","order_by":1,"name":"Denis Escobar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYDCCA2AEAjxAXAHEEhB2ApFazhCphQGuhbGNCC18x3sPHvzBYGPP39578HPlvMN5/LMbGB+8bWPIM8ehRfLMuYTDPAxpiTPOnEuWPLvtcLHEnQPMhnPbGIotG7BrMbiRY3CYgeFwgoFEjoFk47bDiQ03EtikedsYEjccwKHl/hsDoMP+2wO1GP9snHM4cf6NBPbfeLXc4DE4wMNwgHGDRI6ZZGPD4cQNQFuY8WmRPAN0GI9BMsgvaZYNx9ITN95IbJacc04Cpxa+42eMP/6osAOF2OGbDTXWifNuJB/88KbMBqcWqPNQeIwNDJDYGQWjYBSMglFALgAAox1lUNCPGbsAAAAASUVORK5CYII=","orcid":"","institution":"Universidad Nacional Autónoma de Honduras. Tegucigalpa","correspondingAuthor":true,"prefix":"","firstName":"Denis","middleName":"","lastName":"Escobar","suffix":""},{"id":451150312,"identity":"4f5b6a38-ede0-42af-8dcd-46e2b5d4529d","order_by":2,"name":"Luis Galo","email":"","orcid":"","institution":"Universidad Nacional Autónoma de Honduras. Tegucigalpa","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"","lastName":"Galo","suffix":""},{"id":451150313,"identity":"da4d78b1-959b-48a4-81ed-69a207a62e13","order_by":3,"name":"Oscar Urrutia","email":"","orcid":"","institution":"Unidad de Vigilancia de la Salud, Secretaría de Salud de Honduras. Tegucigalpa","correspondingAuthor":false,"prefix":"","firstName":"Oscar","middleName":"","lastName":"Urrutia","suffix":""},{"id":451150314,"identity":"545b0bb5-4553-45ee-89fb-0244bdd7e36f","order_by":4,"name":"Rita Lucrecia Vizcaíno","email":"","orcid":"","institution":"U.S. Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Rita","middleName":"Lucrecia","lastName":"Vizcaíno","suffix":""},{"id":451150315,"identity":"d3cc26ab-9072-4685-be3f-c8aedf3979a3","order_by":5,"name":"Audrey Lenhart","email":"","orcid":"","institution":"U.S. Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Audrey","middleName":"","lastName":"Lenhart","suffix":""},{"id":451150316,"identity":"6c41ac05-1fef-4639-8d86-d0825e09e20c","order_by":6,"name":"Gustavo Fontecha","email":"","orcid":"","institution":"Universidad Nacional Autónoma de Honduras. Tegucigalpa","correspondingAuthor":false,"prefix":"","firstName":"Gustavo","middleName":"","lastName":"Fontecha","suffix":""}],"badges":[],"createdAt":"2025-03-28 15:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6329329/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6329329/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-025-06953-2","type":"published","date":"2025-08-01T16:38:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":81965663,"identity":"f580f408-6363-4291-907d-a07c15fa2bdb","added_by":"auto","created_at":"2025-05-05 11:30:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2306311,"visible":true,"origin":"","legend":"\u003cp\u003eMap of the Central District of Honduras showing the four collection sites. Green dot: “Loarque”; Blue dot: “Altos de Villa Vieja”; Yellow dot: “La Concordia”; Orange dot: “Río Abajo”.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6329329/v1/5f59f9447db32c618f8b53f0.png"},{"id":81963687,"identity":"405cc0f2-0fea-4a55-81c6-118e5140ebf6","added_by":"auto","created_at":"2025-05-05 11:22:52","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":267250,"visible":true,"origin":"","legend":"\u003cp\u003eBioassay mortality for each population at the diagnostic time of 30 minutes. A: Deltamethrin; B: Permethrin; C: Malathion.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6329329/v1/2bd27aeabe5acc2f9f91c849.jpeg"},{"id":81963693,"identity":"25424298-3ec6-485e-a2ca-a726bbd8c881","added_by":"auto","created_at":"2025-05-05 11:22:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1796053,"visible":true,"origin":"","legend":"\u003cp\u003eHaplotype distribution within each locality. LO: “Loarque”; AVV: “Altos de Villa Vieja”; LC: “La Concordia”; RA: “Río Abajo”.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6329329/v1/4d1b44fedc4a3c98d8791e09.png"},{"id":88268758,"identity":"5737928d-d874-4dda-b776-51eea9f7d289","added_by":"auto","created_at":"2025-08-04 16:52:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4821163,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6329329/v1/0630c1a4-7150-4ad7-ada9-5685f3576589.pdf"},{"id":81963685,"identity":"0d90df58-9ae5-48fe-8c59-20bfceac1382","added_by":"auto","created_at":"2025-05-05 11:22:52","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19319,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalfilesPV2025Clean.docx","url":"https://assets-eu.researchsquare.com/files/rs-6329329/v1/c99246768de74e99f83739f4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Insecticide resistance status and high frequency of kdr mutations in Aedes aegypti in Tegucigalpa, Honduras","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eAedes (Stegomyia) aegypti\u003c/em\u003e is a major vector that has contributed to outbreaks of multiple arboviral diseases throughout the past century (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Its vectorial capacity for new and re-emerging diseases has been projected to increase as a result of the impact of climate change, urbanization, and human movement patterns (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Modeling analyses have provided indications that the distribution patterns of \u003cem\u003eAe. aegypti\u003c/em\u003e are anticipated to undergo significant transformations over the current century, as previously hostile habitats are transformed into more favorable environments (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn recent decades, most tropical countries of the Americas have been severely impacted by recurring dengue epidemics, as well as the spread of new arboviruses such as chikungunya and Zika (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The first cases of dengue in Honduras were documented during the 1978 epidemic, with a total of over 130,000 cases. In the following years, there were documented reports of epidemics in 1987, impacting around 29,000 individuals, as well as in 1989 and 1991. At that time, dengue occurred in large outbreaks with a particular concentration in the two most densely inhabited cities, Tegucigalpa and San Pedro Sula (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Between 2015 and 2016, Honduras experienced the emergence of two additional arboviruses (chikungunya and Zika) transmitted by \u003cem\u003eAe. aegypti\u003c/em\u003e, in addition to ongoing dengue epidemics (\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe main approach for controlling epidemic outbreaks of \u003cem\u003eAedes\u003c/em\u003e-borne arboviruses remains the control of the adult stage of the mosquito (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In Honduras, the Ministry of Health carries out vector control activities with insecticide fogging, often using a combination of permethrin plus a synergist (piperonyl butoxide) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The recurrent use of insecticides often leads to the emergence of mosquito populations that are resistant to insecticides.\u003c/p\u003e \u003cp\u003ePhenotypic resistance to insecticides arises by at least four previously reported biological mechanisms (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The most commonly reported mechanisms include an increase in the activity of detoxifying enzymes (a metabolic mechanism) and the occurrence of mutations at the insecticide target site within the mosquito (a genetic mechanism), referred to as knockdown resistance (\u003cem\u003ekdr\u003c/em\u003e) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). So far, a total of eleven mutations of the \u003cem\u003ekdr\u003c/em\u003e type, linked to resistance against pyrethroids, have been documented. These mutations have also been observed as multiple mutations at positions 989, 1016, and 1534 (V1016G/S989P, F1534C/V1016G and F1534C/V1016I) (\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough insecticides are critical for controlling vectors, there is a lack of information regarding the status of resistance and the distribution of \u003cem\u003ekdr\u003c/em\u003e mutations in \u003cem\u003eAe. aegypti\u003c/em\u003e in Central America. The available data from Latin America is primarily generated from Mexico and Brazil, with some additional reports from countries including Colombia, Costa Rica, Cuba, Peru, and some Caribbean nations (\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Nevertheless, significant knowledge gaps remain regarding the prevalence and magnitude of insecticide resistance in \u003cem\u003eAe. aegypti\u003c/em\u003e within the Central American region.\u003c/p\u003e \u003cp\u003eThus, this study aimed to conduct a phenotypic assessment of insecticide resistance in \u003cem\u003eAe. aegypti\u003c/em\u003e populations collected in the Central District of Honduras. Additionally, the study sought to characterize the frequency of \u003cem\u003ekdr\u003c/em\u003e alleles in the populations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy site\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAedes aegypti\u003c/em\u003e were collected in the municipality of the Central District of Honduras. This region was selected because it includes the capital city of Tegucigalpa and reports the highest prevalence of dengue and other arboviruses in the country (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). The Central District is located between 900 and 1000 meters above sea level and has an approximate population of 1.1\u0026nbsp;million inhabitants. There are two seasons, a dry season (from December to May) and a rainy season (from June to November). The average temperature ranges between 16 and 30\u0026deg;C.\u003c/p\u003e \u003cp\u003eFour neighborhoods were selected to collect larvae: \u0026ldquo;Loarque - LO\u0026rdquo; (14.041576, -87.211082), \u0026ldquo;La Concordia - LC\u0026rdquo; (14.112068, -87.208535), \u0026ldquo;Altos de Villa Vieja - AVV\u0026rdquo; (14.059852, -87.162463) and \u0026ldquo;R\u0026iacute;o Abajo - RA\u0026rdquo; (14.173899, -87.211295) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These neighborhoods were selected because they were reported as those with the highest routine use of insecticide spraying by the Ministry of Health of Honduras.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCollection and rearing of mosquitoes\u003c/h3\u003e\n\u003cp\u003eThe study involved visiting between five to seven locations inside each of the four chosen neighborhoods that exhibited clear evidence of water accumulation, including sinks, pans, and automobile tires. Mosquito larvae and pupae were collected at each site (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The live larvae and pupae were transferred in plastic containers to the Medical Entomology Laboratory of the National Autonomous University of Honduras (UNAH).\u003c/p\u003e \u003cp\u003eLarvae and pupae obtained from the field (F0 generation) were reared to adults under standard insectary settings. Each group of larvae collected per neighborhood was placed separately in trays (28 \u0026times; 18 \u0026times; 6 cm) with 1500 mL of distilled water. The larvae were fed daily with fish food (Biomaa, Super Flakes, Mexico) and maintained at a relative humidity of 70\u0026ndash;80%, a temperature range of 25\u0026ndash;27\u0026deg;C, and a light and dark cycle of 12 hours each, until they reached maturity (\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). The emerged adult mosquitoes were identified using taxonomic keys (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), separating all individuals identified as \u003cem\u003eAe. aegypti.\u003c/em\u003e An F1 was generated from each population, following the previously described procedure (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), and fed with a 10% sugar solution until use in insecticide susceptibility bioassays.\u003c/p\u003e\n\u003ch3\u003ePhenotypic bioassays\u003c/h3\u003e\n\u003cp\u003eSusceptibility bioassays were performed following the procedures described for the CDC bottle bioassay method (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Briefly, the diagnostic concentrations of four insecticides were prepared from technical grade active ingredients (Chem Services, West Chester, PA, USA): two pyrethroids (deltamethrin, 10 \u0026micro;g/mL and permethrin, 15 \u0026micro;g/mL); a carbamate (bendiocarb, 12.5 \u0026micro;g/mL), and an organophosphate (malathion, 50 \u0026micro;g/mL). The bioassays were performed using 3- to 5-day-old non-blood fed adult female \u003cem\u003eAe. aegypti\u003c/em\u003e. Between 20\u0026ndash;25 mosquitoes were placed into 250 mL Wheaton bottles previously impregnated with the diagnostic concentration of insecticide and kept under observation for 30 minutes. The number of knocked down individuals was recorded at 0, 15, and 30 minutes of exposure. The diagnostic time to determine the phenotype was 30 minutes, according to what is described by the CDC method (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Subsequently, all exposed mosquitoes were transferred to holding containers with 10% sugar solution to evaluate mortality after 24 hours (as a proxy for recovery). After recording the mortality rate at 24 hours, both susceptible and resistant individuals were separated and kept at -20\u0026deg;C until molecular analyses were performed.\u003c/p\u003e\n\u003ch3\u003eDNA extraction and genotyping of positions 1534 and 1016 of the VGSC gene\u003c/h3\u003e\n\u003cp\u003eFor the detection of \u003cem\u003ekdr\u003c/em\u003e mutations, a sample of 285 individuals from the F1 generation, who were phenotyped in deltamethrin and permethrin bioassays, was randomly chosen from the four study locations: 70 individuals (25%) from \u0026ldquo;AVV\u0026rdquo;, 80 (28%) from \u0026ldquo;LC\u0026rdquo;, 74 (26%) from \u0026ldquo;LO\u0026rdquo;, and 61 (21%) from \u0026ldquo;RA\u0026rdquo;. Individuals of both resistant (n\u0026thinsp;=\u0026thinsp;135) and susceptible (n\u0026thinsp;=\u0026thinsp;150) phenotypes were selected. DNA was extracted separately from each mosquito using the ReliaPrep\u0026trade; Blood gDNA Miniprep System (Promega, Madison, Wisconsin, USA), according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003eGenotyping of positions 1534 and 1016 was carried out using allele-specific PCR (AS-PCR) according to the primers and protocols described by Yanola et al (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) and the primers modified by Contreras-Perera et al (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The possible genotypes for position 1534 were F1534F (homozygous wild type), F1534C (heterozygous mutant), and C1534C (homozygous mutant). For position 1016, the possible genotypes were V1016V (homozygous wild type), V1016I (heterozygous mutant), and I1016I (homozygous mutant). For both loci, the presence of heterozygotes was registered by observing the presence of double bands according to the base pair sizes previously reported. Briefly, PCR reactions to analyze position 1534 were carried out in a final volume of 20 \u0026micro;L, including 10 \u0026micro;L of Taq Master Mix 2X (Promega, Madison, Wisconsin, USA), 0.8 \u0026micro;L of each primer (10 \u0026micro;M) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), 1 \u0026micro;L of DNA and 6.6 \u0026micro;L of nuclease-free water (NFW). The amplification conditions were as follows: 95\u0026deg;C for 5 min, 35 cycles of 95\u0026deg;C for 1 min, 57\u0026deg;C for 1 min, 72\u0026deg;C for 1 min, with a final extension step at 72\u0026deg;C for 4 min.\u003c/p\u003e \u003cp\u003eFor position 1016, the PCR reaction was carried out in a volume of 25 \u0026micro;L, with 12.5 \u0026micro;L of Taq Master Mix 2X (Promega, Madison, Wisconsin, USA), 1 \u0026micro;L of each primer (10 \u0026micro;M) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), 4 \u0026micro;L of DNA and 5.5 \u0026micro;L of NFW. The amplification conditions were as follows: 95\u0026deg;C for 5 min, 29 cycles of 95\u0026deg;C for 1 min, 60\u0026deg;C for 1 min, and 72\u0026deg;C for 1 min, with a final extension step at 72\u0026deg;C for 10 min. The amplification products were separated by electrophoresis on a 3% agarose gel and visualized with ethidium bromide under UV light.\u003c/p\u003e\n\u003ch3\u003eGenotyping of positions 989, 1011, and 1520 of the VGSC gene\u003c/h3\u003e\n\u003cp\u003eIn order to detect additional mutations at 989,1011 and 1520 positions at the voltage-gated sodium channel gene, a partial region of the IIS5-6 and IIIS5-6 segments was amplified using specific primers reported elsewhere (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). To identify additional \u003cem\u003ekdr\u003c/em\u003e mutations associated with resistance to pyrethroid insecticides, 60 individuals were selected: Twenty-eight individuals resistant to the pyrethroids deltamethrin (n\u0026thinsp;=\u0026thinsp;9) and permethrin (n\u0026thinsp;=\u0026thinsp;19), and 32 individuals susceptible to deltamethrin (n\u0026thinsp;=\u0026thinsp;17) and permethrin (n\u0026thinsp;=\u0026thinsp;15).. Briefly, the IIS5-6 fragment was amplified in a final volume of 50 \u0026micro;L including 25 \u0026micro;L of Taq Master Mix 2X (Promega, Madison, Wisconsin, USA), 2 \u0026micro;L of each primer (10 \u0026micro;M) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), 4 \u0026micro;L of DNA and 17 \u0026micro;L of NFW. The amplification conditions were as follows: 95\u0026deg;C for 2 min, 40 cycles of 94\u0026deg;C for 30 sec, 50\u0026deg;C for 30 sec, 72\u0026deg;C for 1 min, with a final extension at 72\u0026deg;C for 10 min.\u003c/p\u003e \u003cp\u003ePCR reactions for the IIIS5-6 fragment were carried out in a final volume of 50 \u0026micro;L including 25 \u0026micro;L of Taq Master Mix 2X (Promega, Madison, Wisconsin, USA), 2.5 \u0026micro;L of each primer (10 \u0026micro;M) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), 2 \u0026micro;L of DNA and 18 \u0026micro;L of NFW, under the following conditions: 95\u0026deg;C for 5 min, 35 cycles of 94\u0026deg;C for 30 sec, 59\u0026deg;C for 30 sec, 72\u0026deg;C for 1 min, with a final extension at 72\u0026deg;C for 10 min. The amplified products were separated on 1% agarose gels and visualized with ethidium bromide under UV light. The amplified products were sequenced by Psomagen\u0026reg; (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.psomagen.com\u003c/span\u003e\u003cspan address=\"https://www.psomagen.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using internal primers: 508R: 5\u0026prime;- TTG TTC GTT TCG TTG TCG GC \u0026minus;\u0026thinsp;3\u0026acute; for the IIS6 fragment and 71F: 5\u0026prime;- GTC CTC GAT CCT TCC AGG TG \u0026minus;\u0026thinsp;3\u0026acute; for the IIIS6 fragment. The sequences obtained were curated and analyzed with Geneious Prime 2024.0.2 software (Dotmatics, Auckland, New Zealand). Each sequence was aligned and compared with a reference sequence of \u003cem\u003eAe. aegypti\u003c/em\u003e (Accession number AAEL023266) available from VectorBase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://vectorbase.org/vectorbase\u003c/span\u003e\u003cspan address=\"https://vectorbase.org/vectorbase\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) and other sequences available in NCBI (Accession numbers KY626180.1, KY626197.1, KY046222.1, XM_021852349.1) previously reported with mutations at positions 989, 1011 and 1016.\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\u003eSequences of the primers and PCR conditions used for genotyping positions 1534 and 1016 of the VGSC gene.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrimer sequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnnealing temperature (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePCR product size (bp)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIIS5-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC1534fw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-GCG GGC AGG GCG GCG GGG GCG GGG CCT CTA CTT TGT GTT CTT CAT CAT GTG-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e93\u003c/p\u003e \u003cp\u003e113\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF1534fw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-GCG GGC TCT ACT TTG TGT TCT TCA TCA TAT T-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF1534R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-TCT GCT CGT TGA AGT TGT CGA T-3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIIIS5-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVal1016fw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;- GCG GGC AGG GCG GCG GGG GCG GGG CCA CAA ATT GTT TCC CAC CCG CAC CGG- 3\u0026acute;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e102\u003c/p\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIle1016f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-GCG GGC ACA AAT TGT TTC CCA CCC GCA CTG A- 3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIle1016r\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-TGA TGA ACC SGA ATT GGA CAA AAG C- 3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIIS5-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIIS5-6F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-ATC GCT TCC CGG ACA AAG AC- 3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e562 \u0026ndash; Intron A\u003c/p\u003e \u003cp\u003e579 \u0026ndash; Intron B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIIS5-6R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;-GTT GGC GAT GTT CGA CTT GA- 3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIIIS5-6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAaNa31F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;- GAC TCG CGG GAG GTA AGT T \u0026ndash; 3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e512\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAaNa31R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u0026prime;- CCG TCT GCT TGT AGT GAT CG \u0026ndash; 3\u0026prime;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eMortality was defined as the percent of individuals knocked down at the diagnostic time. To categorize the susceptibility status of the populations, the ranges described by the CDC were followed: 98\u0026ndash;100% mortality indicated susceptibility of the population, 90\u0026ndash;97% indicated the development of resistance, and ˂90% indicated resistance. Both allele frequencies and haplotype frequencies were calculated for each population and phenotype (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Wright's inbreeding coefficient (FIS) was calculated to evaluate the population structure, where possible, considering the loci of interest using the formula: FIS\u0026thinsp;=\u0026thinsp;1- (\u003cem\u003eHo\u003c/em\u003e / \u003cem\u003eHe\u003c/em\u003e), where \u003cem\u003eHo\u003c/em\u003e is the number of observed heterozygotes and \u003cem\u003eHe\u003c/em\u003e was calculated as follows: \u003cem\u003eHe\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2\u003cem\u003enp\u003c/em\u003e(1-\u003cem\u003ep\u003c/em\u003e), where \u003cem\u003en\u003c/em\u003e is the sample size (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The Chi-square test was calculated in R software (version 2024.04.1; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.R-project.org/\u003c/span\u003e\u003cspan address=\"https://www.R-project.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) under the package \u0026ldquo;stats -\u0026gt; \u0026ldquo;\u003cem\u003echisq.test\u0026rdquo;\u003c/em\u003e to determine a significant association between phenotypic susceptibility and the F1534C, V1016I and V1016V alleles, (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBioassays\u003c/h2\u003e \u003cp\u003eA total of 1,592 \u003cem\u003eAe. aegypti\u003c/em\u003e females of the F1 generation were analyzed in bioassays. Four sets of bioassays were carried out per location, one per insecticide. All populations were resistant to permethrin and malathion. For permethrin, the mortality ranged between 1% and 48%, with LC showing the lowest mortality (1%) and RA showing the highest (48%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). For malathion, mortality ranged between 24% (AVV) and 74% (LO). Mortality rates for deltamethrin ranged between 86% (LC) and 100% (RA and AVV). All populations showed 100% mortality at the diagnostic time for bendiocarb.\u003c/p\u003e \u003cp\u003eOf the four locations evaluated, only the LC population showed changes in mortality for both pyrethroids 24 hours after exposure, with a recovery rate of 15% and 1% for deltamethrin and permethrin, respectively. (Additional file S1, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eKdr \u003cem\u003egenotypes and haplotypes for positions 1016 and 1534\u003c/em\u003e\u003c/p\u003e \u003cp\u003eOut of the 285 individuals tested, 275 (96.5%) were successfully genotyped for at least one of the two loci, n\u0026thinsp;=\u0026thinsp;275 for position 1534 and n\u0026thinsp;=\u0026thinsp;273 for position 1016. No permethrin-susceptible phenotypes were detected from LC, nor deltamethrin-resistant phenotypes from AVV or RA, so these were not included in the genetic analyses. The allele frequencies by insecticide and phenotype are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eGenotyping analyses revealed the presence of individuals with the F1534C mutation in its homozygous mutant (C/C) and heterozygous (F/C) variants. In these populations, no individual was found with the wild variant (F/F). In contrast, the three genotypes were observed at position 1016 (V/V, V/I, and I/I). The double homozygous mutant haplotype (1534 C/C, 1016 I/I) was detected in 196 of 285 individuals analyzed (68.8%). The mosquitoes from LC accounted for 38% of the individuals with the double homozygous mutant haplotype, closely followed by the population from AVV. The allele frequencies for the mutant alleles were 1.0 and 0.89 for F1534C and V1016I, respectively.\u003c/p\u003e \u003cp\u003eAmong the four populations evaluated, the 1534C allele exhibited a frequency of 1.0 across all locations. Although homozygote mutant allele was highly frequent, heterozygotes were observed in three out of the four populations studied: AVV (0.06), RA (0.22) and LO (0.15); in this latter, particularly only five individuals (0.07) resistant to permethrin showed this genotype. On the contrary, the frequency of the V1016I resistant alleles showed variability between locations, ranging from 0.69 in RA to 0.99 in LC populations. Heterozygotes alleles (V/I) were observed in the four populations; of those, the highest frequency was observed in LO (0.33). Wright's inbreeding coefficient (FIS) calculations showed a low relationship between the populations analyzed at position 1016, with a slight excess of heterozygosity in the RA and AVV populations (Additional file 1-Table\u0026nbsp;2).\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\u003eFrequency of 1534 and 1016 \u003cem\u003ekdr\u003c/em\u003e alleles per site.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" 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=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeighborhood\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFF\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFreq C\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFreq FC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eVI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eVV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eFreq I\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eFreq VI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAVV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBased on the observed genotypes, the haplotypes (1016/1534) by locality were calculated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Six haplotypes were observed: R1R2 (II/CC), R1H2 (II/FC), H1R2 (VI/CC), H1H2 (VI/FC), S1R2 (VV/CC), S1H2 (VV/FC). Haplotype R1R2 (II/CC) was the most frequent haplotype in all localities, with the highest frequency observed in LC (0.99), followed by AVV (0.81). On the other hand, the S1H2 haplotype (VV/FC) had an overall frequency of 0.03 and was reported only in the AVV and RA localities.\u003c/p\u003e \u003cp\u003eWhen combining results from phenotype and haplotype, only populations from two sites, LO and LC, were resistant to both deltamethrin and permethrin. Both populations showed a high proportion of R1R2 haplotype (˃0.6) and nearly no S1R2 (˂0.03). These results contrast with AVV, whose population was susceptible to deltamethrin and where R1R2 was present at a frequency of 0.81, but with a frequency of 0.11 of the S1R2 haplotype. As F1534C heterozygotes were observed mostly in susceptible individuals, Chi-square tests were applied to evaluate an association between the F1534C alelle and the susceptible phenotype, and the V1016I allele underwent the same analysis, an additional test were carried out to test association between V1016V allele and susceptible phenotype. This revealed a significant association between heterozygotes and the susceptible phenotype, both for F1534C (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;5.6209; df\u0026thinsp;=\u0026thinsp;1; \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.01775) and V1016I (χ2\u0026thinsp;=\u0026thinsp;5.9001; df\u0026thinsp;=\u0026thinsp;1; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01514). Finally, V1016V showed an strong association with susceptible phenotype (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;20.881; df\u0026thinsp;=\u0026thinsp;1; \u003cem\u003ep =˂\u003c/em\u003e0.00001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGenotyping of positions 989, 1011, and 1520 on the voltage-gated sodium channel (VGSC) gene\u003c/h2\u003e \u003cp\u003eSequence analyses for the IIS6 and IIIS6 segments showed that all individuals presented the wild genotypes at positions 1520 (ACC), 989 (ATC) and 1011 (ATA). Additionally, the sequencing data confirmed the genotypes identified by AS-PCR at positions 1016 and 1534.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe long-term efficacy of vector control interventions is significantly compromised by the emergence of insecticide resistance (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). The monitoring of insecticide susceptibility and characterization of resistance mechanisms provide crucial information for designing vector control strategies (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). The present study provides evidence of the state of resistance to insecticides and the high frequency of \u003cem\u003ekdr\u003c/em\u003e alleles in \u003cem\u003eAe. aegypti\u003c/em\u003e in four locations around the capital of Honduras.\u003c/p\u003e \u003cp\u003eThere have been numerous reports of \u003cem\u003eAe. aegypti\u003c/em\u003e populations exhibiting resistance to multiple insecticides around the world. However, information on insecticide susceptibility in Honduras and the Central American region is still scarce (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The results of the present study showed populations resistant to two pyrethroids and the organophosphate malathion, but full susceptibility to the carbamate bendiocarb. The Ministry of Health of Honduras has used commercial formulations with pyrethroids for control interventions for more than two decades, which has probably led to the selection of resistance in populations of \u003cem\u003eAe. aegypti\u003c/em\u003e. This phenomenon has been reported elsewhere in the Americas, such as in Mexico and Brazil, where populations highly resistant to pyrethroids have been observed after consecutive periods of insecticide application (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe presence of two mosquito populations (RA and AVV) resistant to permethrin (a type I pyrethroid) but susceptible to deltamethrin (a type II pyrethroid) suggests that resistance to one pyrethroid doesn\u0026rsquo;t always result in cross-resistance across the pyrethroid class. In the Americas, there are reports with similar results, showing lower mortality to permethrin compared to deltamethrin (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). The LC and LO populations showed resistance to both pyrethroids. These populations came from primarily residential areas, which may be subject to additional selection pressure by insecticides for domestic use, a phenomenon also observed in some areas of Brazil and Mexico (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Honduras, organophosphate insecticides were replaced by pyrethroids in the late 1990s to control \u003cem\u003eAe. aegypti\u003c/em\u003e. However, the results reported here reveal that populations continue to show resistance to the organophosphate malathion. A study in Jamaica in 2015 observed that populations of \u003cem\u003eAe. aegypti\u003c/em\u003e showed heterogeneous results to malathion in five populations, with mortalities between 84 to 90%, after a rotation from malathion to permethrin in 2014 during a chikungunya outbreak (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In addition, a study published by Rubio-Palis \u003cem\u003eet al.\u003c/em\u003e in 2023 found that, despite reducing the use of organophosphates since 2010, four populations of \u003cem\u003eAe. aegypti\u003c/em\u003e continued to present resistance to malathion in Venezuela in 2020, demonstrating that despite the absence of pressure, phenotypic resistance was maintained (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to the phenotypic resistance results, this study detected high frequencies of both the F1534C and V1016I mutations. These \u003cem\u003ekdr\u003c/em\u003e alleles in \u003cem\u003eAe. aegypti\u003c/em\u003e are distributed worldwide, and the F1534C mutation is the most frequently reported that has been associated with loss of susceptibility to pyrethroids (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Although the 1534C mutation seems almost fixed in the populations analyzed, association tests revealed an association between the heterozygous individuals from two neighborhoods and the susceptible phenotype, similar to what was observed for the V1016I allele. Reports from Brazil and Mexico have demonstrated the rapid evolution and distribution of \u003cem\u003ekdr\u003c/em\u003e mutations in \u003cem\u003eAe. aegypti\u003c/em\u003e, revealing that these mutations are capable of arising independently and fixing quickly in local populations, probably driven by highly local selection factors (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is notable that in this study F1534C and V1016I mutant individuals were detected among both susceptible and resistant individuals. A previous study in Venezuela showed that despite being fixed in the population, the F1534C mutation was found in populations susceptible to cypermethrin, evidencing the complexity of the multiple mechanisms involved in eliciting resistance (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). To further elucidate the mechanisms contributing to the resistance in the populations studied here, the next step would be the characterization of families of key enzymes, such as multiple function oxidases (MOF) and glutathione-S-transferases (GST), which help detoxify insecticide molecules and may also be contributing to resistant phenotypes (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe combination of multiple mutant alleles on the mosquito \u003cem\u003evgsc\u003c/em\u003e gene can result in haplotypes that are associated with high levels of resistance to pyrethroids (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Reports from Brazil have demonstrated the wide distribution of the 1534/1016 homozygous mutant haplotype (C/C, I/I). A recent national survey carried out in 123 municipalities in Brazil showed that this haplotype was the second most frequent nationally (24%). In Florida, USA, another investigation examined populations of \u003cem\u003eAe. aegypti\u003c/em\u003e in 62 locations, revealing a significant occurrence of the double mutant combination (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). This double mutant haplotype was also the most frequent in the four populations we studied from the Central District of Honduras. Although the haplotypes weren\u0026rsquo;t predictive of resistance phenotype (likely due to the contributions of additional resistance mechanisms), the variation in the frequencies of \u003cem\u003ekdr\u003c/em\u003e haplotypes across the neighborhoods suggests a highly focal variation in selection that is occurring at the local level.\u003c/p\u003e \u003cp\u003eAdditional \u003cem\u003ekdr\u003c/em\u003e mutations have been reported on the \u003cem\u003evgsc\u003c/em\u003e gene (T1520I, S989P, and I1011V/M), mainly in Asian mosquito populations (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Although the results of this study show the absence of non-synonymous mutations in positions 1520, 989, and 1011, the constant use of pyrethroids and the potential introduction of \u003cem\u003eAe. aegypti\u003c/em\u003e from elsewhere could lead to the appearance of additional mutations in the future (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study revealed phenotypic resistance to permethrin and malathion in \u003cem\u003eAe. aegypti\u003c/em\u003e populations across the study sites, and variable levels of deltamethrin susceptibility. The \u003cem\u003ekdr\u003c/em\u003e alleles F1534C and V1016I were detected at high frequencies in \u003cem\u003eAe. aegypti\u003c/em\u003e, together with a predominant double-mutant haplotype (C/C, I/I). These results can inform vector control decision-making, and can be considered as a baseline for prospective monitoring of resistance trends.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCDC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCenters for Disease Control and Prevention\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003eKdr\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKnockdown resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cem\u003evgsc\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eVoltage-Gated Sodium Channel\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u003cbr\u003e\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e.\u0026nbsp;\u003cbr\u003e\u0026nbsp;Funding for this study was provided by the Genetic Research Center, CIG-UNAH. The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization, D.E, O.U., A.L., and G.F.; methodology, A.L., D.E., R.L.V and G.F.; validation, R.L.V. A.L. and D.E.; experiments and analysis, C.R.P., L.G., and D.E; resources, A.L., D.E. and G.F.; data curation, G.F., and D.E.; writing—original draft preparation, G.F. and D.E.; writing—review and editing, G.F., D.E.; visualization, G.F.; supervision, D.E., and G.F.; funding acquisition, A.L., D.E., O.U., and G.F. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e. The authors wish to express their gratitude to the staff of the Vigilancia Entomológica-Unidad de Vigilancia de la Salud, and the Region Metropolitana del Distrito Central of the Ministry of Health of Honduras for the collaboration for the development of this project. Many thanks to Mr. Hector Videa, Mr. Carlos Doblado, Mr. Ramón Martín Luque and Mr. Juan Zepeda for their support in the entomological collections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclaimer:\u003c/strong\u003e The views expressed in this manuscript are those of the authors and do not necessarily reflect the official policy or position of the Centers for Disease Control and Prevention.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKraemer MUG, Sinka ME, Duda KA, Mylne AQN, Shearer FM, Barker CM, et al. 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Quantification of permethrin resistance and kdr alleles in Florida strains of Aedes aegypti (L.) and Aedes albopictus (Skuse). PLoS Negl Trop Dis. 2018;12(10):e0006544.\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":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Aedes aegypti, Dengue, Honduras, Insecticide resistance, kdr","lastPublishedDoi":"10.21203/rs.3.rs-6329329/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6329329/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e \u003cem\u003eAedes aegypti\u003c/em\u003eis the primary vector of arboviruses including dengue, Zika, and chikungunya. The primary approach to control vector populations and reduce the transmission of these arboviruses during outbreaks is the use of insecticide spraying. However, the prolonged use of insecticides promotes resistance due to the selective pressure exerted on mosquito populations. This study aimed to conduct a phenotypic assessment of insecticide resistance and characterize the main knockdown resistance (\u003cem\u003ekdr\u003c/em\u003e) mutations in \u003cem\u003eAe. aegypti\u003c/em\u003e populations collected in the Central District of Honduras.\u003cstrong\u003e\u003cbr\u003e\nMethods: \u003c/strong\u003eLarvae of \u003cem\u003eAe. aegypti\u003c/em\u003e were collected from four localities in the Central District of Honduras between May and June of 2023. Bioassays to determine susceptibility to deltamethrin, permethrin, malathion, and bendiocarb were carried out. For each location and phenotype, F1534C and V1016I\u003cem\u003e kdr \u003c/em\u003eallele frequencies and haplotypes were calculated. Sequencing analyses were employed to genotype additional positions of interest on the \u003cem\u003evgsc\u003c/em\u003e gene.\u003cstrong\u003e\u003cbr\u003e\nResults: \u003c/strong\u003eA total of 1,592 \u003cem\u003eAe. aegypti\u003c/em\u003e females were phenotyped in the bioassays. Only two populations were resistant to deltamethrin. Conversely, all populations were resistant to permethrin and malathion, and all populations were susceptible to bendiocarb. The genotyping of 275 individuals revealed the presence of mutant alleles at both \u003cem\u003ekdr\u003c/em\u003e loci (1016I and 1534C). The overall allele frequencies for 1534C and 1016I were 1.0 and 0.89, respectively. Variability of frequencies for 1016I was observed between localities, with two populations exhibiting a frequency of 1.0 for the mutant allele, while the rest ranged from 0.48 to 0.97. No additional mutations were detected on the \u003cem\u003evgsc\u003c/em\u003e gene.\u003cbr\u003e\n \u003cstrong\u003eConclusion:\u003c/strong\u003e This study provides evidence regarding the resistance status of \u003cem\u003eAe. aegypti\u003c/em\u003e to insecticides used for vector control in Honduras. Additionally, the high frequency of permethrin resistance and of \u003cem\u003ekdr \u003c/em\u003emutations suggests that the mosquito populations have been under selective pressure from pyrethroids. This information could be integrated into vector control policies in Honduras to develop more targeted and effective strategies for combating mosquito-borne diseases.\u003c/p\u003e","manuscriptTitle":"Insecticide resistance status and high frequency of kdr mutations in Aedes aegypti in Tegucigalpa, Honduras","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 11:22:47","doi":"10.21203/rs.3.rs-6329329/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-24T18:40:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-24T16:30:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"245708746857694902781558827740828696074","date":"2025-05-30T11:26:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-22T19:24:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26885065612176838093609744025515769475","date":"2025-05-02T14:10:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-29T17:55:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-02T07:31:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-02T02:17:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-03-28T15:08:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"06846fc7-b7ca-46e4-b9d7-666a0d71bb93","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-04T16:47:46+00:00","versionOfRecord":{"articleIdentity":"rs-6329329","link":"https://doi.org/10.1186/s13071-025-06953-2","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-08-01 16:38:23","publishedOnDateReadable":"August 1st, 2025"},"versionCreatedAt":"2025-05-05 11:22:47","video":"","vorDoi":"10.1186/s13071-025-06953-2","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06953-2","workflowStages":[]},"version":"v1","identity":"rs-6329329","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6329329","identity":"rs-6329329","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}