First Detection of the L1014F kdr Mutation in Triatoma dimidiata, a Vector of Chagas Disease in Mexico.

Triatoma species from thephyllosoma subcomplex are sympatrically distributed and include some of the main vectors of Chagas disease in Mexico. Species within this subcomplex, including Triatoma pallidipennis, T. mazzottii, T. picturata, and T. longipennis, have shown resistance to pyrethroid insecticides, associated with mutations in the para gene of the voltage gate sodium channel (VGSC) and the activity of detoxifying enzymes such as β-esterases and glutathione s-transferases (GST). In this study, we evaluated resistance to deltamethrin in hybrids of T. pallidipennis × T. mazzottii (T.pal × T.maz) and T. pallidipennis × T. picturata (T.pal × T.pic) under laboratory conditions, and the inheritance was determined based on the degree of dominance (DO). Additionally, associated resistance mechanisms were analyzed, including detoxifying enzymes and knockdown resistance (kdr) mutations. High levels of resistance to deltamethrin were found in the hybrids of T.pal × T.maz when compared with the susceptible strain of T. mazzottii (RR50 = 17.50). Dominance levels calculated for each hybrid showed values < - 1, confirming that resistance to deltamethrin was recessive. Hybrids exhibited reduced α-, β-esterases, and cytochrome P450 mixed-function oxidases (MFO) activity. However, both hybrids showed significantly increased GST activity, particularly in T.pal × T.pic, suggesting enhanced detoxification through this pathway. The kdr mutation A943V, present in T. mazzottii, was found in T.pal × T.maz hybrids. These results emphasize the importance of considering hybridization in resistance management programs and its potential impact on the success of insecticide-based control measures.

Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) gene are a key mechanism of insecticide resistance in mosquitoes. In Asian Aedes aegypti populations two main VGSC haplogroups with kdr mutations have been identified: one carrying the F1534C mutation and another with V1016G and/or S989P mutations. Previous functional studies have demonstrated that these three mutations on a single haplotype confer up to a 1100-fold increase in pyrethroid resistance, underscoring the importance of monitoring these triple mutations in distinct populations. This study investigates the prevalence of kdr mutations in Indian populations and explores the linkage association between these mutations and two distinct conserved types of introns located between exons 20 and 21. Ae. aegypti specimens collected from eight different locations were genotyped for kdr alleles and intron (between exons 20 and 21) haplotypes using PCR-based assays. Representative samples underwent DNA sequencing of VGSC regions. Five kdr mutations namely S989P, V1016G, T1520I, F1534C, and F1534L were identified, each exhibiting varying distribution and frequencies across different geographical regions. Two distinct and stably-diverged intron haplotypes, designated as intron-A and intron-B, were identified between exons 20 and 21. Seven haplotypes, including two wild-type variants, were observed among Indian populations. The kdr-bearing haplotypes can be classified into three distinct haplogroups: haplogroup G (V1016G with/or without S989P and with intron-A), haplogroup L (F1534L and intron-A), and haplogroup C (F1534C with/or without T1520I and with intron-B). Importantly, no evidence of recombination within Indian populations was detected among these three haplogroups. Five kdr mutations were identified in the VGSC of Indian Ae. aegypti populations, each showing a definitive linkage with one of the two types of intron haplotypes. The lack of recombination among haplogroups bearing 1016G with 989P, 1534C and 1534L mutations suggests that the most potent insecticide resistance haplotype, bearing the triple kdr mutation, is currently absent. This finding has significant operational implications, as it may indicate that current vector control measures remain effective against these populations, potentially delaying the emergence of highly resistant phenotypes.

BackgroundThe Aedes aegypti mosquito is the primary vector for dengue, chikungunya, yellow fever and Zika viruses worldwide. The first record of Ae. aegypti in southwestern Saudi Arabia was in 1956. However, the first outbreak and cases of dengue fever were reported in 1994, and cases have increased in recent years. Vector control for Ae. aegypti mainly uses pyrethroid insecticides in outdoor and indoor space spraying. The constant use of pyrethroids has exerted intense selection pressure for developing target-site mutations in the voltage-gated sodium channel (vgsc) gene in Ae. Aegypti against pyrethroids—mutations that have led to knockdown resistance (kdr).MethodsAedes aegypti field populations from five regions (Jazan, Sahil, Makkah, Jeddah and Madinah) of southwestern Saudi Arabia were genotyped for known kdr mutations in domains IIS6 and IIIS6 of the vgsc gene using polymerase chain reaction (PCR) amplification and sequencing. We estimated the frequency of kdr mutations and genotypes from Saudi Arabia as well as from other countries, Thailand, Myanmar (Southeast Asia) and Uganda (East Africa). We constructed haplotype networks to infer the evolutionary relationships of these gene regions.ResultsThe three known kdr mutations, S989P, V1016G (IIS6) and F1534C (IIIS6), were detected in all five regions of Saudi Arabia. Interestingly, the triple homozygous wild genotype was reported for the first time in two individuals from the highlands of the Jazan region and one from the Al-Quoz, Sahil region. Overall, nine genotypes comprising four haplotypes were observed in southwestern Saudi Arabia. The median-joining haplotype networks of eight populations from Saudi Arabia, Southeast Asia and East Africa for both the IIS6 and IIIS6 domains revealed that haplotype diversity was highest in Uganda and in the Jazan and Sahil regions of Saudi Arabia, whereas haplotype diversity was low in the Jeddah, Makkah and Madinah regions. Median-joining haplotype networks of both domains indicated selection acting on the kdr-mutation containing haplotypes in Saudi Arabia.ConclusionsThe presence of wild type haplotypes without any of the three kdr mutations, i.e. that are fully susceptible, in Saudi Arabia indicates that further consideration should be given to insecticide resistance management strategies that could restore pyrethroid sensitivity to the populations of Ae. aegypti in Saudi Arabia as part of an integrative vector control strategy.Graphical

Detection and population genetic analysis of Aedes albopictus (Diptera: Culicidae) based on knockdown resistance (kdr) mutations in the Yangtze River basin of China

The ongoing fight against mosquito-borne diseases such as dengue, Zika, and chikungunya is increasingly challenged by Aedes aegypti resistance to pyrethroid insecticides. This resistance is primarily driven by knockdown resistance (kdr) mutations in the voltage-gated sodium channel (Vgsc) gene, which have been identified in different regions with significant variability in their prevalence and impact. Managing Ae. aegypti populations in the face of this resistance require in-depth understanding of the global distribution of kdr mutations and their relationship to insecticide use patterns. This narrative review identifies a diverse landscape of kdr mutations, with common mutations such as V1016I, F1534C, and L982W, the latter of which has recently gained attention as an important mutation contributing to resistance in various regions, often co-occurring with other mutations and leading to enhanced resistance phenotypes. Additional mutations including V410L, G923V, S989P, I1011M/V, and D1763Y further complicate the resistance profile, indicating a dynamic evolution of resistance in response to pyrethroid use. This review also highlights significant gaps in current research, particularly in understanding the interaction between kdr mutations and other resistance mechanisms, such as metabolic detoxification by enzymes like cytochrome P450s, glutathione S-transferases (GST), and esterases, and the need for more standardized methodologies for data collection and reporting. The widespread use of pyrethroids has significantly contributed to the global spread of kdr mutations in Ae. aegypti, thereby challenging the efficacy of the current chemical vector control strategies. Despite these challenges, pyrethroids remain essential for mosquito control owing to their proven efficacy, rapid action, and versatility. However, the rising levels of resistance highlight the need for an integrated approach to vector management that combines chemical, biological, and community-based strategies. Future research should address these gaps through longitudinal studies and the development of new insecticides and resistance management strategies to address the growing threat of insecticide resistance in Ae. aegypti and protect public health.Graphical

Simple SummaryKnockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) of mosquitoes confer resistance to pyrethroid insecticides. Analysis of kdr mutations in Aedes aegypti mosquitoes collected from five different townships in the Mandalay area, Myanmar, revealed high levels of validated kdr mutations in domains II and III of vgsc. Moreover, high frequencies of concurrent kdr mutations were also detected. The results of this study suggest that kdr mutations associated with pyrethroid resistance are widespread in the Ae. aegypti population of the study area. Our results provide a valuable molecular basis to understand the pyrethroid resistance status of the Ae. aegypti population in the area and underscore the need for an effective vector control program in Myanmar.Aedes aegypti is an important mosquito vector transmitting diverse arboviral diseases in Myanmar. Pyrethroid insecticides have been widely used in Myanmar as the key mosquito control measure, but the efforts are constrained by increasing resistance. Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) are related to pyrethroid resistance in Ae. aegypti. We analyzed the patterns and distributions of the kdr mutations in Ae. aegypti in the Mandalay area of Myanmar. The segment 6 regions of domains II and III of vgsc were separately amplified from individual Ae. aegypti genomic DNA via polymerase chain reaction. The amplified gene fragments were sequenced. High proportions of three major kdr mutations, including S989P (54.8%), V1016G (73.6%), and F1534C (69.5%), were detected in the vgsc of Ae. aegypti from all studied areas. Other kdr mutations, T1520I and F1534L, were also found. These kdr mutations represent 11 distinct haplotypes of the vgsc population. The S989P/V1016G/F1534C was the most prevalent, followed by S989P/V1016V and V1016G/F1534C. A quadruple mutation, S989P/V1016G/T1520I/F1534C, was also identified. High frequencies of concurrent kdr mutations were observed in vgsc of Myanmar Ae. aegypti, suggesting a high level of pyrethroid resistance in the population. These findings underscore the need for an effective vector control program in Myanmar.

BackgroundAedes aegypti is a primary vector of dengue, chikungunya and Zika infections in India. In the absence of specific drugs or safe and effective vaccines for these infections, their control relies mainly on vector control measures. The emergence of insecticide resistance in vectors, especially against pyrethroids, is a serious threat to the insecticide-based vector control programme. This study reports the presence of multiple knockdown resistance (kdr) mutations present in an Ae. aegypti population from Bengaluru (India), including a new mutation F1534L.MethodsAedes aegypti collected from Bengaluru were subjected to insecticide susceptibility tests with DDT, deltamethrin and permethrin. The DNA sequencing of partial domain II, III and IV of the voltage-gated sodium channel (VGSC) was performed to screen kdr mutations present in the population and PCR-based assays were developed for their detection. Genotyping of kdr mutations was done using PCR-based assays, allelic frequencies were determined, and tests of genetic association of kdr mutations with the insecticide resistance phenotype were performed.ResultsThe Ae. aegypti population was resistant to DDT, deltamethrin and permethrin. The DNA sequencing of the VGSC revealed the presence of four kdr mutations, i.e. S989P and V1016G in domain II and two alternative kdr mutations F1534C and F1534L in domain III. Allele-specific PCR assays (ASPCR) were developed for the detection of kdr mutations S989P and V1016G and an existing PCR-RFLP based strategy was modified for the genotyping of all three known kdr mutations in domain III (F1534L, F1534C and T1520I). Genotyping of Ae. aegypti samples revealed a moderate frequency of S989P/V1016G (18.27%) and F1534L (17.48%), a relatively high frequency of F1534C (50.61%) and absence of T1520I in the population. Mutations S989P and V1016G were in complete linkage disequilibrium in this population while they were in linkage equilibrium with kdr mutations F1534C and F1534L. The alleles F1534C and F1534L are genetically associated with permethrin resistance.ConclusionsA new kdr mutation, F1534L, was found in an Ae. aegypti population from Bengaluru (India), co-occurring with the other three mutations S989P, V1016G and F1534C. The findings of a new mutation have implications for insecticide resistance management.

Triatoma dimidiata is a widely distributed vector of Trypanosoma cruzi in Mexico and Central America, found across a range of habitats from sylvatic to domestic. Vector control has relied heavily on indoor residual spraying with pyrethroids; however, reinfestation and emerging resistance have limited its long-term effectiveness. In this study, we analyzed the genetic diversity and population structure of T. dimidiata from Veracruz, Oaxaca, and Yucatan using mitochondrial markers (cyt b and ND4) and screened for knockdown resistance (kdr)-type mutations in the voltage-gated sodium channel (VGSC) gene. High haplotype diversity and regional differentiation were observed, with most genetic variation occurring between populations. The ND4 marker provided greater resolution than cyt b, revealing ten haplotypes and supporting evidence of recent population expansion. Haplotype networks showed clear geographic segregation, particularly between populations east and west of the Isthmus of Tehuantepec. The L925I mutation, highly associated with pyrethroid resistance, was detected for the first time in Mexican populations of T. dimidiata, albeit at low frequencies. These findings highlight the importance of integrating population genetic data and resistance surveillance into regionally adapted vector control strategies for Chagas disease.

Aedes aegypti Linnaeus (Diptera: Culicidae) is an important vector of mosquito-borne viruses, including dengue, Zika and chikungunya. In the case of dengue, approximately half of the world’s population is at risk, and an estimated 100–400 million new infections are reported each year. To control Ae. aegypti populations, mosquito management programmes rely heavily on insecticides, which have been the mainstay of mosquito management programmes for almost 50 years. The most common class of insecticide used to control mosquito vectors of human disease are the pyrethroids. These target the voltage-gated sodium channel, an essential protein that facilitates electrical signalling within the nervous system. Due to the intense selection pressures applied by public health programmes and the incidental exposure of mosquitoes to agricultural and domestic insecticides, pyrethroid-resistant Ae. aegypti populations are now common.Target site resistance, one of the common mechanisms associated with resistance to pyrethroids, occurs most frequently in mosquitoes through non-synonymous mutations in the voltage-gated sodium channel gene. These point mutations, commonly known as knockdown resistance (kdr) mutations, can cause a change in the shape of the voltage-gated sodium channel, which decreases the efficacy with which pyrethroids bind to them. Knockdown resistance mutations in Ae. aegypti populations have been reported from many parts of the world and reflect the increasingly common reports of phenotypic resistance to pyrethroids on a global scale. The geographically widespread distribution of pyrethroid-resistant Ae. aegypti has emphasised the requirement for insecticide resistance management strategies that conserve susceptibility and ensure the availability of effective insecticidal chemistries to control mosquito-borne disease. Insecticide Resistance Management (IRM) strategies rely on reducing selection pressure, which can result in the immigration of susceptible genotypes and, where they exist, allow fitness costs to reduce the frequency of resistance mutations. With regard to the latter, our understanding of the consequences of kdr mutations on the fitness and behaviour of Ae. aegypti, in the absence of pyrethroids, is limited. Furthermore, we do not have a clear understanding of how Ae. aegypti with or without these mutations are affected by the sublethal doses of insecticide that are common in urban and peri-urban habitats.I backcrossed a pyrethroid-resistant strain of Ae. aegypti from Timor-Leste, harbouring the V1016G and S989P kdr mutations, with an insecticide susceptible strain from Queensland, Australia that does not carry kdr mutations. This produced a strain of Ae. aegypti that was homozygous for the V1016G and S989P kdr mutations in an otherwise susceptible genetic background. Using double-digest restriction associated sequencing, I confirmed that >99.9% of the backcrossed strain’s genome originated from the susceptible parental strain. The creation of the backcrossed strain allowed me to compare differences in life-history traits and behaviour between the backcrossed-resistant and susceptible parent strain, in the absence or presence of permethrin, and to directly attribute those distinctions to the effects of the homozygous V1016G/S989P genotype.The results presented within this thesis demonstrate that, in comparison to the susceptible strain, mosquitoes with the V1016G/S989P genotype exhibited longer larval development times, had smaller wing lengths and females had a shorter average lifespan (Chapter II). Investigations into behavioural changes showed a significant reduction in mean wing beat frequency in males and a significant reduction in estimated male mating success, associated with the introgressed kdr genotype (Chapter III). These results suggest specific and significant fitness costs and behavioural changes associated with the double homozygous V1016G/S989P genotype, in the absence of insecticides. These likely consequences of resistance evolution may encourage reversion to susceptibility in the absence of insecticide selection pressures.I also examined the impact of sublethal permethrin exposure on the mosquito fitness parameters of survival, fecundity, fertility, blood-feeding, host location and mating success. For the first time, I demonstrated some expected, negative impacts of sublethal exposure on susceptible Ae. aegypti mosquitoes but also showed that detrimental effects on longevity and host location can occur even in highly resistant mosquitoes (Chapter IV). Overall, I demonstrated that significant differences in fitness exists between mosquitoes with susceptible and resistant genotypes, in the presence of sublethal doses of insecticides. The competitive advantage conferred to resistant mosquito strains in the presence of sublethal doses of permethrin from various sources, questions our ability to create and exploit untreated refugia or mosaics and therefore remove selection pressures and permit resistance-associated fitness costs to manifest.My results focus on the pleiotropic effects of a common kdr genotype on the biology and behaviour of Ae. aegypti in the absence and presence of insecticides. By cataloguing these effects, this thesis provides evidence to inform the introduction of specific IRM measures into vector control programmes and predict their potential impact by cataloguing these effects. My results are discussed in relation to reducing pyrethroid selection pressures and the subsequent impact on the frequency of resistant genotypes by temporarily removing pyrethroid selection pressures as part of IRM and encouraging the consequent recovery or conservation of pyrethroid susceptibility.

Aedes albopictus is the only vector that can transmit the dengue virus in Zhejiang Province, central China, and it can develop insecticide resistance due to long-term exposure to pyrethroids. The presence of knockdown resistance (kdr) mutations is one of the mechanisms responsible for pyrethroid resistance, and has been reported in some Ae. albopictus populations in southern China. However, little is known about the DNA diversity of the voltage-gated sodium channel (VGSC) gene in Ae. albopictus populations in central China. Four Ae. albopictus field populations were collected, in Yiwu (YW), Quzhou (QZ), Wenzhou (WZ), and Jiaxing (JX) from Zhejiang Province, central China. The susceptibility of Ae. albopictus adults to three pyrethroids (beta-cypermethrin, deltamethrin, and permethrin) was tested using the WHO tube assay, and Kdr mutations were identified via PCR and sequencing. The relationship between kdr mutations and pyrethroid phenotypes was also analyzed. Of the four populations, none was sensitive to any pyrethroid tested, and the YW population showed the strongest pyrethroid resistance. Non-synonymous kdr mutations were detected in codons 1532 and 1534, domain III. At codon 1534, one mutant allele, TCC(S), was detected in the four populations with a frequency of 42.08%, while at codon 1532, one mutant allele, ACC(T), was detected in the JX and QZ populations, with frequencies of 4.22 and 3.03%, respectively. The F1534S mutant allele was positively correlated with both beta-cypermethrin and deltamethrin resistance phenotypes (OR > 1, P < 0.05), whereas the I1532T mutant allele was possibly negatively correlated with beta-cypermethrin, deltamethrin, and permethrin resistance phenotypes (OR < 1, P > 0.05). In conclusion, resistance and resistance mutations regarding to three pyrethroids are already present in the Ae. Albopictus populations from Zhejiang, central China, which prompts the need to use non-insecticide-based methods of insect control.

Females of Aedes aegypti transmit emerging arboviruses including Zika, dengue, yellow fever, and chikungunya. Control of these adult mosquitoes heavily relies on synthetic insecticides, including pyrethroids. However, insecticide resistance development in populations poses a significant challenge to vector control, particularly from knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC), the target of pyrethroids. This study investigated the field efficacy of Permanone, a pyrethroid-based insecticide, against Ae. aegypti by assessing the impact of three common kdr mutations (V410L, V1016I, F1534C) on mosquito survival under a real operational mosquito control scenario, by quantifying the pesticide delivered in the field. Field cage tests (FCTs) were conducted while conducting a realistic mosquito control application. Female mosquitoes from six operational areas from Harris County, TX, USA were exposed to Permanone delivered with a handheld sprayer. Permanone deposited near the cages was estimated from aluminum boats placed in the field during FCTs using gas chromatography-mass spectrometry (GC-MS). Mortality rates were recorded, and individual mosquitoes were genotyped for kdr mutations. A probit regression model was used to analyze the factors influencing mosquito survivorship. As the distance from the application source route increased, the amount of Permanone deposited decreased, resulting in higher survivorship frequency of Ae. aegypti females with the triple-resistant kdr genotype (LL/II/CC). The L allele at the 410-site significantly contributed to an increased resistance level when co-occurring with other kdr mutations. This study linked the survival probabilities of mosquitoes with different kdr genotypes, and the amount of pesticide they received in the field. Pesticide quantification, control efficacy results and genotyping allowed us to empirically determine the impact of genotypic resistance on vector control in the field. © 2024 Society of Chemical Industry.

Despite ongoing vector control programs, dengue incidence in Malaysia continues to rise. A significant contributing factor is the emergence of insecticide resistance in Aedes mosquitoes, particularly against pyrethroids such as permethrin. This study aimed to evaluate permethrin resistance in Aedes aegypti and Aedes albopictus from Kuala Lumpur and Selangor and to investigate the prevalence of knockdown resistance (kdr) mutations in the voltage-gated sodium channel (Vgsc) gene. Due to limited sample quantity and quality, molecular assessment of kdr mutations was performed only for Ae. aegypti. Ovitraps were deployed in three dengue hotspot areas to collect Aedes eggs. Eggs were reared to adulthood under controlled laboratory conditions. Adult female mosquitoes were subjected to the WHO tube bioassay using permethrin to assess phenotypic resistance. Genomic DNA was extracted from individual Ae. aegypti, and polymerase chain reaction (PCR) was conducted to amplify target regions of the Vgsc gene using published kdr-associated primers. Direct sequencing of PCR products was performed to identify point mutations associated with pyrethroid resistance. Both Ae. aegypti and Ae. albopictus populations showed high levels of permethrin resistance, with mortality rates of <5% and <50%, respectively, indicating confirmed resistance. Sequencing of Ae. aegypti revealed the presence of pyrethroid resistance-associated kdr mutations, including S989P, A1007G, V1016G, and F1534C. These findings suggest strong selective pressure on local Ae. aegypti populations, reflecting ongoing adaptation to insecticidal interventions. The identification of these mutations emphasizes the challenges faced by current vector control strategies relying predominantly on chemical control.The detection of both phenotypic permethrin resistance and kdr mutations in Ae. aegypti populations from Kuala Lumpur and Selangor highlights the urgent need to revise and diversify vector control strategies. Improved sampling and molecular surveillance of Ae. albopictus are recommended to provide a more comprehensive understanding of insecticide resistance dynamics in Malaysian dengue vectors.

BackgroundMosquitoes are important vectors for a range of diseases, contributing to high rates of morbidity and mortality in the human population. Culex pipiens pallens is dominant species of Culex mosquito in northern China and a major vector for both West Nile virus and Bancroftian filariasis. Insecticide application were largely applied to control the mosquito-mediated spread of these diseases, contributing to increasing rates of resistance in the mosquito population. The voltage-gated sodium channel (Vgsc) gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). While these kdr mutations are known to be critical to pyrethroid resistance, their evolutionary origins remain poorly understood. Clarifying the origins of these mutations is potential to guide further vector control and disease prevention efforts. Accordingly, the present study was designed to study the evolutionary genetics of kdr mutations and their association with the population structure of Cx. p. pallens in Shandong province, China.MethodsAdult Culex females were collected from Shandong province and subjected to morphological identification under a dissection microscope. Genomic DNA were extracted from the collected mosquitoes, the Vgsc gene were amplified via PCR and sequenced to assess kdr allele frequencies, intron polymorphisms, and kdr codon evolution. In addition, population genetic diversity and related population characteristics were assessed by amplifying and sequencing the mitochondrial cytochrome C oxidase I (COI) gene.ResultsTotally, 263 Cx. p. pallens specimens were used for DNA barcoding and sequencing analyses to assess kdr allele frequencies in nine Culex populations. The kdr codon L1014 in the Vgsc gene identified two non-synonymous mutations (L1014F and L1014S) in the analyzed population. These mutations were present in the eastern hilly area and west plain region of Shandong Province. However, only L1014F mutation was detected in the southern mountainous area and Dongying city of Shandong Province, where the mutation frequency was low. Compared to other cities, population in Qingdao revealed significant genetic differentiation. Spatial kdr mutation patterns are likely attributable to some combination of prolonged insecticide-mediated selection coupled with the genetic isolation of these mosquito populations.ConclusionsThese data suggest that multiple kdr alleles associated with insecticide resistance are present within the Cx. p. pallens populations of Shandong Province, China. The geographical distributions of kdr mutations in this province are likely that the result of prolonged and extensive insecticide application in agricultural contexts together with frequent mosquito population migrations. In contrast, the low-frequency kdr mutation detected in central Shandong Province populations may originate from the limited selection pressure in this area and the relative genetic isolation. Overall, the study compares the genetic patterns revealed by a functional gene with a neutral marker and demonstrates the combined impact of demographic and selection factors on population structure.

BackgroundIn the final phase of China’s national programme to eliminate malaria by 2020, it is vitally important to monitor the resistance of malaria vectors for developing effective vector control strategies. In 2017 Shanghai declared that it had eliminated malaria; however, the insecticide resistance status of the primary malaria vector Anopheles sinensis remains unknown.MethodsWe examined the pyrethroid and organophosphate resistance of An. sinensis via a bioassay of two populations from the Chongming District of Shanghai. The voltage-gated sodium channel (VGSC) and acetylcholinesterase 1 (ace-1) genes were partially sequenced to examine the association between resistance phenotype and target site genotype. In addition, the geographical distribution, polymorphism and genotype frequencies of insecticide resistance genes were examined using samples collected during routine mosquito surveillance in 2016 and 2017 from Chongming, Songjiang, Jiading and Qingpu Districts.ResultsIn Chongming District, the An. sinensis population near Dongtan National Nature Reserve showed resistance to pyrethroids, sensitivity to organophosphate, no knockdown resistance (kdr) mutations in the VGSC gene, and a low frequency (1.71%) of the ace-1 119S allele. An An. sinensis population near the Chongming central area (CM-Xinhe population) showed high resistance to pyrethroids and organophosphates and high frequencies of kdr 1014F and 1014C (80.73%) and ace-1 119S (85.98%) alleles. A significant association was detected between the homozygous kdr mutation 1014F/1014F and pyrethroid resistance in the CM-Xinhe population, indicating that the kdr mutation is probably recessive. Eight kdr genotypes with 1014F and 1014C substitutions were detected in the four surveyed districts of Shanghai. TTT and GGC/AGC were the dominant kdr allele and ace-1 genotype, respectively, and were prevalent in most Shanghai An. sinensis populations.ConclusionsOn the basis of our assessment of insecticide resistance gene mutations in Shanghai, we identified a kdr mutation-free population in Chongming Dongtan. However, high frequencies of target-site mutations of insecticide resistance genes were observed in most areas of Shanghai. Bioassays of An. sinensis populations in the central Chongming District indicated the high insecticide resistance status of An. sinensis populations in Shanghai. We accordingly recommend a restriction on insecticide usage and development of effective integrated pest/vector management interventions to support disease control efforts.

First report of two kdr mutations L1014F/S in natural populations of Triatoma pallidipennis Stal and Triatoma picturata Usinger vectors of Chagas disease in Mexico.