Insecticide Resistance Alleles Research Articles

Dominance reversal protects large-effect resistance polymorphisms in temporally varying environments.

A central challenge in evolutionary biology is to uncover mechanisms maintaining functional genetic variation in heterogeneous environments 1,2 . Population genetics theory suggests that beneficial reversal of dominance, where alleles are dominant when beneficial and recessive when deleterious, can help maintain such variation in temporally varying environments 3,4 . However, empirical examples are scarce 5 due to difficulties in measuring dominance in fitness in field experiments 6 . Here we quantify the selective effects and dominance of large-effect insecticide-resistant alleles at the Ace enzymatic locus in Drosophila melanogaster 7 . In laboratory assays, we identify a beneficial reversal of dominance of the Ace alleles for fitness-associated phenotypes under selection in the presence and absence of malathion, a widely used organophosphate insecticide. Using highly replicated field mesocosms, we show that resistant Ace alleles fluctuate rapidly in response to a pulse of malathion, but are maintained in its absence. We show that this pattern is only consistent with beneficial reversal of dominance, where the resistant Ace alleles are dominant and beneficial for resistance and recessive and deleterious for enzymatic function. Moreover, we find that seasonally fluctuating selection due to insecticide resistance can generate fluctuating chromosome-scale genomic perturbations of allele frequencies. We propose an extension to Wright's physiological theory of dominance 8 , arguing that under fluctuating selection the currently deleterious allele should often behave as loss-of-function and thus recessive to the currently beneficial allele. Overall, our results suggest that beneficial reversals of dominance can be common and can help both maintain genetic variation and allow for rapid evolutionary responses to environmental shifts.

MGSurvE: A framework to optimize trap placement for genetic surveillance of mosquito populations.

Genetic surveillance of mosquito populations is becoming increasingly relevant as genetics-based mosquito control strategies advance from laboratory to field testing. Especially applicable are mosquito gene drive projects, the potential scale of which leads monitoring to be a significant cost driver. For these projects, monitoring will be required to detect unintended spread of gene drive mosquitoes beyond field sites, and the emergence of alternative alleles, such as drive-resistant alleles or non-functional effector genes, within intervention sites. This entails the need to distribute mosquito traps efficiently such that an allele of interest is detected as quickly as possible-ideally when remediation is still viable. Additionally, insecticide-based tools such as bednets are compromised by insecticide-resistance alleles for which there is also a need to detect as quickly as possible. To this end, we present MGSurvE (Mosquito Gene SurveillancE): a computational framework that optimizes trap placement for genetic surveillance of mosquito populations such that the time to detection of an allele of interest is minimized. A key strength of MGSurvE is that it allows important biological features of mosquitoes and the landscapes they inhabit to be accounted for, namely: i) resources required by mosquitoes (e.g., food sources and aquatic breeding sites) can be explicitly distributed through a landscape, ii) movement of mosquitoes may depend on their sex, the current state of their gonotrophic cycle (if female) and resource attractiveness, and iii) traps may differ in their attractiveness profile. Example MGSurvE analyses are presented to demonstrate optimal trap placement for: i) an Aedes aegypti population in a suburban landscape in Queensland, Australia, and ii) an Anopheles gambiae population on the island of São Tomé, São Tomé and Príncipe. Further documentation and use examples are provided in project's documentation. MGSurvE is intended as a resource for both field and computational researchers interested in mosquito gene surveillance.

Impact of a spatial repellent intervention on Anopheles kdr insecticide resistance allele in Sumba, Indonesia

BackgroundThe emergence of insecticide resistance and outdoor transmission in malaria-endemic areas underlines the urgent need to develop innovative tools, such as spatial repellents (SR), that may circumvent this residual transmission. With limited options for effective insecticides, regular resistance monitoring is warranted for selecting and using appropriate tools. This study evaluates the pyrethroid knockdown resistance (kdr) allele before and after implementing a transfluthrin-based spatial repellent (SR) intervention in placebo-treated clusters.MethodsThis study looks at the frequency distribution of the kdr allele in Sumba Island from June 2015 to August 2018. Insecticide susceptibility tests were carried out on female Anopheles sp. aged 3–5 days against permethrin 21.5 μg/ml, deltamethrin 12.5 μg/ml, and transfluthrin 10 μg/ml using CDC bottle assay. PCR sequencing of representative samples from adult mosquito collections and insecticide tests revealed the presence of kdr mutations (L1014F and L1014S) in the VGSC gene.ResultsA total of 12 Anopheles species, Anopheles tesselatus, Anopheles. aconitus, Anopheles barbirostris, Anopheles kochi, Anopheles annularis, Anopheles maculatus, Anopheles sundaicus, Anopheles flavirostris, Anopheles balabacensis, Anopheles indefinitus, Anopheles subpictus, and Anopheles vagus were analysed. Anopheles vagus and An. sundaicus predominated in the larval populations. Susceptibility assays for all insecticides identified fully susceptible phenotypes in all species examined. Anopheles increasing frequency of kdr mutant alleles during the 3 year SR deployment was observed in both SR-treated and placebo areas, a statistically significant increase occurred in each arm. However, it is unclear how significant SR is in causing the increase in mutant alleles. The L1014S, knockdown resistance east type (kdr-e) allele was detected for the first time among the mosquito samples in this study. The L1014F, knockdown resistance west type (kdr-w) allele and heteroduplex form (wild-type—mutant) were found in almost all Anopheles species examined, including An. vagus, An. aconitus, An. subpictus, An. tesselatus, An. annularis, An. flavirostris and An. sundaicus.ConclusionThe presence of fully susceptible phenotypes over time, along with an increase in the frequency distribution of the L1014F/S mutations post-intervention, suggest drivers of resistance external to the study, including pyrethroid use in agriculture and long-lasting insecticidal nets (LLINs). However, this does not negate possible SR impacts that support resistance. More studies that enable the comprehension of possible SR-based drivers of resistance in mosquitoes need to be conducted.

Species composition, infection rate and detection of resistant alleles in Anopheles funestus (Diptera: Culicidae) from Lare, a malaria hotspot district of Ethiopia

BackgroundAnopheles funestus, which is considered as secondary vector of malaria in Ethiopia, is known to have several morphologically indistinguishable (sibling) species. Accurate identification of sibling species is crucial to understand their biology, behaviour and vector competence. In this study, molecular identification was conducted on the Ethiopian An. funestus populations. Moreover, insecticide resistance mechanism markers were detected, including ace N485I, kdr L1014F, L1014S, and CYP6P9a TaqMan qPCR was used to detect the infective stage of the parasite from field collected adult female An. funestus populations.MethodsAdult female mosquito collection was conducted from Lare, Gambella Regional State of Ethiopia between June 2018 to July 2020 using CDC light traps and HLC. Sub-samples of the morphologically identified An. funestus mosquitoes were molecularly identified using species-specific PCR, and the possible presence of insecticide resistance alleles was investigated using TaqMan qPCR (N485I-Ace-1), PCR-Sanger sequencing (L1014F-kdr), and PCR–RFLP (CYP6P9a resistance allele). Following head/thorax dissection, the TaqMan qPCR assay was used to investigate the presence of the infective stage Plasmodium parasite species.ResultsA total of 1086 adult female An. funestus mosquitoes were collected during the study period. All sub-samples (N = 20) that were morphologically identified as An. funestus sensu lato (s.l.) were identified as An. funestus sensu stricto (s.s.) using species- specific PCR assay. The PCR–RFLP assay that detects the CYP6P9a resistance allele that confers pyrethroid resistance in An. funestus was applied in N = 30 randomly selected An. funestus s.l. specimens. None of the specimens showed a digestion pattern consistent with the presence of the CYP6P9a resistance allele in contrast to what was observed in the positive control. Consequently, all samples were characterized as wild type. The qPCR TaqMan assay that detects the N485I acetylcholinesterase-1 mutation conferring resistance to organophosphates/carbamates in An. funestus was used in (N = 144) samples. All samples were characterized as wild type. The kdr L1014F and L1014S mutations in the VGSC gene that confer resistance to pyrethroids and DDT were analysed with direct Sanger sequencing after PCR and clean-up of the PCR products were also characterized as wild type. None of the samples (N = 169) were found positive for Plasmodium (P. falciparum/ovale/malariae/vivax) detection.ConclusionAll An. funestus s.l. samples from Lare were molecularly identified as An. funestus s.s. No CYP6P9, N485I acetylcholinesterase 1, kdr L1014F or L1014S mutations were detected in the An. funestus samples. None of the An. funestus samples were positive for Plasmodium. Although the current study did not detect any insecticide resistant mechanism, it provides a reference for future vector monitoring programmes. Regular monitoring of resistance mechanisms covering wider geographical areas of Ethiopia where this vector is distributed is important for improving the efficacy of vector control programs.

Frequencies and distribution of kdr and Ace alleles that cause insecticide resistance in house flies in the United States

House flies (Musca domestica L) are nuisances and vectors of pathogens between and among humans and livestock. Population suppression has been accomplished for decades with pyrethroids and acetylcholinesterase (AChE) inhibitors, but recurrent selection has led to increased frequency of alleles conferring resistance to those two classes of active ingredients (Geden et al., 2021). A common mechanism of resistance to both classes involves an altered target site (mutations in Voltage gated sodium channel (Vgsc) for pyrethroids or in Ace for AChE inhibitors). As part of ongoing efforts to understand the origin, spread and evolution of insecticide resistance alleles in house fly populations, we sampled flies in 11 different US states, sequenced, and then estimated frequencies of the Vgsc and Ace alleles.There was substantial variation in frequencies of the four common knockdown resistance alleles (kdr (L1014F), kdr-his (L1014H), super-kdr (M918T + L10414F) and 1B (T929I + L1014F) across the sampled states. The kdr allele was found in all 11 states and was the most common allele in four of them. The super-kdr allele was detected in only six collections, with the highest frequencies found in the north, northeast and central United States. The kdr-his allele was the most common allele in PA, NC, TN and TX. In addition, a novel super-kdr-like mutation in mutually exclusive exon 17a was found.The overall frequencies of the different Ace alleles, which we name based on the amino acid present at the mutation sites (V260L, A316S, G342A/V and F407Y), varied considerably between states. Five Ace alleles were identified: VAGF, VAVY, VAGY, VAAY and VSAY. Generally, the VSAY allele was the most common in the populations sampled. The susceptible allele (VAGF) was found in all populations, ranging in frequency from 3% (KS) to 41% (GA).Comparisons of these resistance allele frequencies with those previously found suggests a dynamic interaction between the different alleles, in terms of levels of resistance they confer and likely fitness costs they impose in the absence of insecticides.

Lack of Known Target-Site Mutations in Field Populations of Ostrinia furnacalis in China from 2019 to 2021.

The Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera; Pyralidae), is one of the most destructive insect pests of corn, for which chemical insecticides have been the primary method of control, especially during outbreaks. Little information is currently available on the status of insecticide resistance and associated mechanisms in O. furnacalis field populations. Invasions and outbreaks of Spodoptera frugiperda in China in recent years have increased chemical application in corn fields, which adds to the selection pressure on O. furnacalis. This study was conducted to estimate the risk of insecticide resistance by investigating the frequency of insecticide resistant alleles associated with target site insensitivity in field populations of O. furnacalis. Using the individual-PCR genotype sequencing analysis, none of the six target-site insecticide resistant mutations were detected in O. furnacalis field populations collected from 2019 to 2021 in China. These investigated insecticide resistance alleles are common in resistant Lepidoptra pests and are responsible for resistance to pyrethroids, organophosphorus, carbamates, diamide, and Cry1Ab. Our results support the low insecticide resistance status in field O. furnacalis populations and betokens the unlikely development of high resistance mediated by the common target-site resistance alleles. Additionally, the findings would serve as references for further efforts toward the sustainable management of O. furnacalis.

A Globally Distributed Insecticide Resistance Allele Confers a Fitness Cost in the Absence of Insecticide in Aedes aegypti (Diptera: Culicidae), the Yellow Fever Mosquito.

The cosmopolitan mosquito Aedes aegypti is a vector of harmful arboviruses. Pyrethroid insecticides are used to reduce adult populations and prevent the spread of disease. Pyrethroids target the insect voltage-gated sodium channel (VGSC). Collectively, mutations in Vgsc that confer resistance are referred to as knock-down resistance or kdr. There are numerous kdr mutations found in A. aegypti Vgsc, and there is co-occurrence of some mutations. Full-length cDNA sequences have identified nine known kdr (e.g., 1534C) alleles. The 1534C allele is among the most common kdr alleles, but allele frequencies between populations vary considerably. We used the 1534C:RK strain, which has the 1534C (kdr) allele in the genetic background of the insecticide susceptible Rockefeller (ROCK) strain, and conducted population cage experiments to assess the potential intrinsic fitness cost of the 1534C allele relative to the susceptible allele (F1534) in the ROCK strain. Individuals were genotyped across generations using allele specific PCR. A fitness cost of the 1534C allele was detected across seven generations of mosquitos reared in the absence of insecticide selection pressure. The decrease in allele frequency was not due to drift. Comparison of our results to previous studies suggests that the magnitude of the fitness cost of kdr alleles in the absence of insecticide is disconnected from the level of resistance they confer, and that the fitness costs of different kdr alleles can be variable.

Acetylcholinesterase and Knockdown Resistant Mutations in Anopheles Gambiae S.L. (Culicidae: Diptera) In Three Vegetations of the Niger Delta Region

Comprehending the genetic arrangement of resistant mosquito populations is essential for addressing biological and public health concerns as, spread of insecticide resistant alleles. Anopheles gambiae sensu lato, which spread malaria parasites comprises genetically different but morphologically indistinguishable species. This study: determine presence of resistant genes, in the lowland forest, mangrove and fresh water swamp in Rivers state, Niger delta. Sample collection was done from May to December. Laboratory-reared larvae collected from their breeding sites, were used for the molecular bioassay. Resistant genes were determined from DNAs extracted from the tissues of the samples and subjected to polymerase chain reaction (PCR), to identify knockdown and acetylcholinesterase resistance mechanisms. Acetylcholinesterase resistance mutation with accession number OQ802845 was found in all the three vegetations while knockdown resistance mutation was scantily found in the lowland forest vegetation only. The study makes available reference base-line data of various insecticide resistant genes in the Niger delta.

Spodoptera frugiperda: Ecology, Evolution, and Management Options of an Invasive Species.

The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera, Noctuidae), is a well-known agricultural pest in its native range, North and South America, and has become a major invasive pest around the globe in the past decade. In this review, we provide an overview to update what is known about S. frugiperda in its native geographic ranges. This is followed by discussion of studies from the invaded areas to gain insights into S. frugiperda's ecology, specifically its reproductive biology, host plant use, status of insecticide resistance alleles, and biocontrol methods in native and invasive regions. We show that reference to host strains is uninformative in the invasive populations because multidirectional introduction events likely underpinned its recent rapid spread. Given that recent genomic analyses show that FAW is much more diverse than was previously assumed, and natural selection forces likely differ geographically, region-specific approaches will be needed to control this global pest.

Highly sensitive droplet digital PCR-based diagnostics for the surveillance of malaria vector populations in low transmission and incipient resistance settings

ABSTRACT Background Sensitive monitoring of Plasmodium infective mosquitoes in low malaria transmission settings is of high priority for disease control. Early detection of insecticide resistance at low frequencies is also key for vector monitoring nowadays, when new insecticides are launched to control vector populations. Research design and methods An. gambiae mosquitoes with predetermined infection and resistance status were used to produce populations with various malaria infection rates and mutant allelic frequencies (MAFs) of target site insecticide resistance traits. Total RNA and gDNA were isolated and used in droplet Digital PCR (ddPCR) and Reverse Transcription (RT) ddPCR performed in the QX200 ddPCR System. Results We developed a novel ddPCR for detecting P. falciparum DNA in pooled mosquito head-thoraces with infective rate as low as 1.0%. A dissection-free RT-ddPCR assay for specific infective-stage detection was additionally developed and validated (accuracy = 100%) in mosquito pools with infective rates down to 1.0%. A novel ddPCR assay for insecticide resistant alleles, which was able to reliably quantify MAFs as low as 0.050% in pooled mosquito specimens, is also reported. Conclusions We developed highly sensitive and efficient (RT-) ddPCR assays for contemporary operational needs that require monitoring of low malaria transmission and emerging insecticide resistance.

Phenotypic, genotypic and biochemical changes during pyrethroid resistance selection in Anopheles gambiae mosquitoes

The directional selection for insecticide resistance due to indiscriminate use of insecticides in public health and agricultural system favors an increase in the frequency of insecticide-resistant alleles in the natural populations. Similarly, removal of selection pressure generally leads to decay in resistance. Past investigations on the emergence of insecticide resistance in mosquitoes mostly relied on field survey of resistance in vector populations that typically had a complex history of exposure to various public health and agricultural pest control insecticides in nature, and thus the effect of specific insecticides on rate of resistance emergency or resistance decay rate is not known. This study examined the phenotypic, genotypic, and biochemical changes that had occurred during the process of selection for pyrethroid resistance in Anopheles gambiae, the most important malaria vector in Africa. In parallel, we also examined these changes in resistant populations when there is no selection pressure applied. Through repeated deltamethrin selection in adult mosquitoes from a field population collected in western Kenya for 12 generations, we obtained three independent and highly pyrethroid-resistant An. gambiae populations. Three susceptible populations from the same parental population were generated by removing selection pressure. These two lines of mosquito populations differed significantly in monooxygenase and beta-esterase activities, but not in Vgsc gene mutation frequency, suggesting metabolic detoxification mechanism plays a major role in generating moderate-intensity resistance or high-intensity resistance. Pre-exposure to the synergist piperonyl butoxide restored the susceptibility to insecticide among the highly resistant mosquitoes, confirming the role of monooxygenases in pyrethroid resistance. The rate of resistance decay to become fully susceptible from moderate-intensity resistance took 15 generations, supporting at least 2-years interval is needed when the rotational use of insecticides with different modes of action is considered for resistance management.

Origin, selection, and spread of diamide insecticide resistance allele in field populations of diamondback moth in east and southeast Asia.

The investigation of molecular mechanisms and evolution of resistance to insecticides is an ongoing challenge, as researchers must provide guidance to manage the resistance to achieve sustainable production in agriculture. Predicting, monitoring, and managing insecticide resistance requires information on the origins, selection, and spread of resistance genes. The resistance of Plutella xylostella (L.) against diamide insecticides is becoming an increasingly severe problem in east and southeast Asia. In this study, the evolution of resistance was investigated using a resistance allele [ryanodine receptor (RyR); G4946E mutation] and its flanking regions, as well as mitochondrial cytochrome c oxidase subunit I (mtCOI). The sequences of the flanking region of the G4946E and mtCOI suggested that the G4946E mutation has a key role in resistance. Furthermore, the G4946E mutation has multiple origins, and congenic resistant mutations have spread across east and southeast Asia, despite substantial geographical barriers. In addition, the susceptibility of field populations partially recovered during winter, based on the observed decrease in the G4946E (resistant allele) frequency. Finally, the resistance level indexed by the frequency of the E4946 allele was significantly lower in non-overwintering regions than in overwintering regions. The information of the present study is useful to monitor resistance using molecular markers and to develop strategies to delay the evolution of diamide resistance.

Incursion pathways of the Asian tiger mosquito (Aedes albopictus) into Australia contrast sharply with those of the yellow fever mosquito (Aedes aegypti)

Understanding pest incursion pathways is critical for preventing new invasions and for stopping the transfer of alleles that reduce the efficacy of local control methods. The mosquitoes Aedes albopictus (Skuse) and Ae. aegypti (Linnaeus) are both highly invasive disease vectors, and through a series of ongoing international incursions are continuing to colonize new regions and spread insecticide resistance alleles among established populations. This study uses high-resolution molecular markers and a set of 241 reference genotypes to trace incursion pathways of Ae. albopictus into mainland Australia, where no successful invasions have yet been observed. We contrast these results with incursion pathways of Ae. aegypti, investigated previously. Assignments successfully identified China, Japan, Singapore and Taiwan as source locations. Incursion pathways of Ae. albopictus were entirely different to those of Ae. aegypti, despite broad sympatry of these species throughout the Indo-Pacific region. Incursions of Ae. albopictus appeared to have come predominantly along marine routes from key trading locations, while Ae. aegypti was mostly linked to aerial routes from tourism hotspots. These results demonstrate how genomics can help decipher otherwise cryptic incursion pathways. The inclusion of reference genotypes from the Americas may help resolve some unsuccessful assignments. While many congeneric taxa will share common incursion pathways, this study highlights that this is not always the case, and incursion pathways of important taxa should be specifically investigated. Species differences in aerial and marine incursion rates may reflect the efficacy of ongoing control programmes such as aircraft disinsection. © 2020 Society of Chemical Industry.

Behavioural plasticity of Anopheles coluzzii and Anopheles arabiensis undermines LLIN community protective effect in a Sudanese-savannah village in Burkina Faso

BackgroundDespite the overall major impact of long-lasting insecticide treated nets (LLINs) in eliciting individual and collective protection to malaria infections, some sub-Saharan countries, including Burkina Faso, still carry a disproportionately high share of the global malaria burden. This study aims to analyse the possible entomological bases of LLIN limited impact, focusing on a LLIN-protected village in the Plateau Central region of Burkina Faso.MethodsHuman landing catches (HLCs) were carried out in 2015 for 12 nights both indoors and outdoors at different time windows during the highest biting activity phase for Anopheles gambiae (s.l.). Collected specimens were morphologically and molecularly identified and processed for Plasmodium detection and L1014F insecticide-resistance allele genotyping.ResultsAlmost 2000 unfed An. gambiae (s.l.) (54% Anopheles coluzzii and 44% Anopheles arabiensis) females landing on human volunteers were collected, corresponding to a median number of 23.5 females/person/hour. No significant differences were observed in median numbers of mosquitoes collected indoors and outdoors, nor between sporozoite rates in An. coluzzii (6.1%) and An. arabiensis (5.5%). The estimated median hourly entomological inoculation rate (EIR) on volunteers was 1.4 infective bites/person/hour. Results do not show evidence of the biting peak during night hours typical for An. gambiae (s.l.) in the absence of bednet protection. The frequency of the L1014F resistant allele (n = 285) was 66% in An. coluzzii and 38% in An. arabiensis.ConclusionsThe observed biting rate and sporozoite rates are in line with the literature data available for An. gambiae (s.l.) in the same geographical area before LLIN implementation and highlight high levels of malaria transmission in the study village. Homogeneous biting rate throughout the night and lack of preference for indoor-biting activity, suggest the capacity of both An. coluzzii and An. arabiensis to adjust their host-seeking behaviour to bite humans despite bednet protection, accounting for the maintenance of high rates of mosquito infectivity and malaria transmission. These results, despite being limited to a local situation in Burkina Faso, represent a paradigmatic example of how high densities and behavioural plasticity in the vector populations may contribute to explaining the limited impact of LLINs on malaria transmission in holo-endemic Sudanese savannah areas in West Africa.

Adaptive Introgression across Semipermeable Species Boundaries between Local Helicoverpa zea and Invasive Helicoverpa armigera Moths.

Hybridization between invasive and native species has raised global concern, given the dramatic increase in species range shifts and pest outbreaks due to anthropogenic dispersal. Nevertheless, secondary contact between sister lineages of local and invasive species provides a natural laboratory to understand the factors that determine introgression and the maintenance or loss of species barriers. Here, we characterize the early evolutionary outcomes following secondary contact between invasive Helicoverpa armigera and native H. zea in Brazil. We carried out whole-genome resequencing of Helicoverpa moths from Brazil in two temporal samples: during the outbreak of H. armigera in 2013 and 2017. There is evidence for a burst of hybridization and widespread introgression from local H. zea into invasive H. armigera coinciding with H. armigera expansion in 2013. However, in H. armigera, the admixture proportion and the length of introgressed blocks were significantly reduced between 2013 and 2017, suggesting selection against admixture. In contrast to the genome-wide pattern, there was striking evidence for adaptive introgression of a single region from the invasive H. armigera into local H. zea, including an insecticide resistance allele that increased in frequency over time. In summary, despite extensive gene flow after secondary contact, the species boundaries are largely maintained except for the single introgressed region containing the insecticide-resistant locus. We document the worst-case scenario for an invasive species, in which there are now two pest species instead of one, and the native species has acquired resistance to pyrethroid insecticides through introgression.

RDL mutations in Guangxi Anopheles sinensis populations along the China\u2013Vietnam border: distribution frequency and evolutionary origin of A296S resistance allele

BackgroundMalaria is a deadly vector-borne disease in tropical and subtropical regions. Although indigenous malaria has been eliminated in Guangxi of China, 473 confirmed cases were reported in the Northern region of neighbouring Vietnam in 2014. Considering that frequent population movement occurs across the China–Vietnam border and insecticide resistance is a major obstacle in disease vector control, there is a need to know the genotype and frequency of insecticide resistance alleles in Anopheles sinensis populations along the China–Vietnam border and to take action to prevent the possible migration of insecticide resistance alleles across the border.MethodsTwo hundred and eight adults of An. sinensis collected from seven locations in Guangxi along the China–Vietnam border were used in the investigation of individual genotypes of the AsRDL gene, which encodes the RDL gamma-aminobutyric acid (GABA) receptor subunit in An. sinensis. PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) analysis was deployed to genotype codon 345, while direct sequencing of PCR products was conducted to clarify the genotypes for codons 296 and 327 of the AsRDL gene. The genealogical relation of AsRDL haplotypes was analyzed using Network 5.0.ResultsThree putative insecticide resistance related mutations (A296S, V327I and T345S) were detected in all the seven populations of An. sinensis in Guangxi along the China–Vietnam border. The resistance-conferring A296S mutation was found to be widely distributed and present at notably high frequencies (78.8% to 100%). Relatively lower frequencies of mutations V327I (26.9% to 53.2%) and T345S (0% to 28.8%) were observed. The V327I or T345S always occurred in the presence of A296S. Evolutionary analysis of 21 AsRDL haplotypes indicated multiple origins of the A296S and V327I mutations.ConclusionThe resistance A296S allele was present at high frequencies in the An. sinensis populations along the China–Vietnam border, indicating a risk of resistance to insecticides targeting RDL. The double mutations (A296S + V327I) may have evolved from alleles carrying the A296S mutation by scaffolding the additional mutation V327I, and A296S allele may have multiple evolutionary origins. These findings will help inform strategies for vector control and malaria prevention.

Regional and seasonal detection of resistance mutation frequencies in field populations of Anopheles Hyrcanus Group and Culex pipiens complex in Korea

Pyrethroid (PYR) and organophosphate (OP) insecticides have been extensively used for mosquito control for several decades in South Korea, and has resulted in the rapid development of resistance in the field. In this study, quantitative sequencing (QS) protocols were developed for the frequency prediction of insecticide resistance alleles [e.g., the L1014F/C mutation on the voltage sensitive sodium channel as a PYR resistance allele and the G119S mutation on the acetylcholinesterase 1 as OP resistance alleles] in four regional populations of Anopheles Hyrcanus Group and Culex pipiens complex. Both of the L1014F/C and G119S mutations were observed in all examined regional populations of An. Hyrcanus Group, suggesting a wide distribution of both PYR and OP resistance. In contrast, populations of the Cx. pipiens complex were determined to possess almost no G119S mutation, but relatively higher frequencies of the L1014F mutation, showing differential resistance patterns between different mosquito groups. The mutation frequencies were also monitored throughout a mosquito season (May–October) at one collection site to determine the seasonal changes of resistance mutation frequency in mosquito populations. Dramatic decreases of both L1014F/C and G119S mutation frequencies were observed in the An. Hyrcanus Group toward the fall, with no mutations observed in the early spring, suggesting a connection between the fitness costs of overwintering and insecticide resistance. However, no apparent trends were detectable in the Cx. pipiens complex populations due to low or zero mutation frequencies.

Climate change and the genetics of insecticide resistance.

Changes in global temperature and humidity as a result of climate change are producing rapid evolutionary changes in many animal species, including agricultural pests and disease vectors, leading to changes in allele frequencies of genes involved in thermotolerance and desiccation resistance. As some of these genes have pleiotropic effects on insecticide resistance, climate change is likely to affect insecticide resistance in the field. In this review, we discuss how the interactions between adaptation to climate change and resistance to insecticides can affect insecticide resistance in the field using examples in phytophagous and hematophagous pest insects, focusing on the effects of increased temperature and increased aridity. We then use detailed genetic and mechanistic studies in the model insect, Drosophila melanogaster, to explain the mechanisms underlying this phenomenon. We suggest that tradeoffs or facilitation between adaptation to climate change and resistance to insecticides can alter insecticide resistance allele frequencies in the field. The dynamics of these interactions will need to be considered when managing agricultural pests and disease vectors in a changing climate. © 2019 Society of Chemical Industry.

Introgression between Anopheles gambiae and Anopheles coluzzii in Burkina Faso and its associations with kdr resistance and Plasmodium infection

BackgroundInsecticide resistance in Anopheles coluzzii mosquitoes has become widespread throughout West Africa including in Burkina Faso. The insecticide resistance allele (kdr or L1014F) is a prime indicator that is highly correlated with phenotypic resistance in West Africa. Studies from Benin, Ghana and Mali have suggested that the source of the L1014F is introgression of the 2L divergence island via interspecific hybridization with Anopheles gambiae. The goal of this study was to characterize local mosquito populations in the Nouna Department, Burkina Faso with respect to: (i) the extent of introgression between An. coluzzii and An. gambiae, (ii) the frequency of the L1014F mutation and (iii) Plasmodium infection rates.MethodsA total of 95 mosquitoes were collected from ten sites surrounding Nouna town in Kossi Province, Burkina Faso in 2012. The species composition, the extent of introgression in An. coluzzii mosquitoes and their Plasmodium infection rates were identified with a modified version of the “Divergence Island SNP” (DIS) genotyping assay.ResultsThe mosquito collection contained 70.5% An. coluzzii, 89.3% of which carried a 3 Mb genomic region on the 2L chromosome with L1014F insecticide resistance mutation that was introgressed from An. gambiae. In addition, 22.4% in the introgressed An. coluzzii specimens were infected with Plasmodium falciparum, whereas none of the non-introgressed (“pure”) An. coluzzii were infected.ConclusionThis paper is the first report providing divergence island SNP genotypes for natural population of Burkina Faso and corresponding Plasmodium infection rates. These observations warrant further study and could have a major impact on future malaria control strategies in Burkina Faso.

Genetic structure of Anopheles gambiae s.s populations following the use of insecticides on several consecutive years in southern Benin

BackgroundSeveral studies have reported the strong resistance of Anopheles gambiae s.l. complex species to pyrethroids. The voltage-dependent sodium channel (Vgsc) gene is the main target of pyrethroids and DDT. In Benin, the frequency of the resistant allele (L1014F) of this gene varies along the north-south transect. Monitoring the evolution of resistance is necessary to better appreciate the genetic structure of vector populations in localities subject to the intensive use of chemicals associated with other control initiatives. The purpose of this study was to map the distribution of pyrethroid insecticide resistance alleles of the Kdr gene in malaria vectors in different regions and ecological facies in order to identify the evolutionary forces that might be the basis of anopheline population dynamics.MethodsThe characterization of Anopheles gambiae s.l. populations and resistance mechanisms were performed using adult mosquitoes obtained from larvae collected in the four agroecological zones in southern Benin. Genomic DNA extraction was performed on whole mosquitoes.The extracted genomic DNA from them were used for the molecular identification of species in Anopheles gambiae s.l. complex and the identification of genotypes related to pyrethroid resistance as the Kdr gene amino acid position 1014 in sodium channel. Molecular speciation and genotyping of Kdr resistant alleles (1014) were done using PCR.Genepop software version 4.2 was used to calculate allelic and genotypic frequencies in each agroecological zone. The p value of the allelic frequency was determined using the binomial test function in R version 3.3.3. The Hardy-Weinberg equilibrium was checked for each population with Genetics software version 1.3.8.1. The observed heterozygosity and the expected heterozygosity as well as the fixation index and genetic differentiation index within and between populations were calculated using Genepop software version 4.2.ResultsDuring the study period, Anopheles coluzzii was the major species in all agroecological zones while Anopheles gambiae was scarcely represented. Regardless of the species, resistant homozygote individuals (L1014F/L1014F) were dominant in all agroecological zones, showing a strong selection of the resistant allele (L1014F). All populations showed a deficit of heterozygosity. No genetic differentiation was observed between the different populations of the two species. For Anopheles coluzzii, there was a small differentiation among the populations of the central cotton and bar-lands zones. The genetic differentiation was modest among the population of the fisheries zone (Fst = 0.1295). The genetic differentiation was very high in the population of Anopheles gambiae of the bar-lands zone (Fst = 0.2408).ConclusionThis study revealed that the use of insecticides in Benin for years has altered the genetic structure of Anopheles gambiae s.s. populations in all agroecological zones of southern Benin. It would be desirable to orientate vector control efforts towards the use of insecticides other than pyrethroids and DDT or combinations of insecticides with different modes of action.