Pyrethroid Resistance Research Articles

An observational analysis of the impact of deltamethrin + piperonyl butoxide insecticide-treated nets on malaria case incidence and entomological indicators in Ebonyi State, Nigeria, 2017–2021

BackgroundIntense pyrethroid resistance threatens the effectiveness of the primary vector control intervention, insecticide-treated nets (ITNs), in Nigeria, the country with the largest malaria burden globally. In this study, the epidemiological and entomological impact of a new type of ITN (piperonyl-butoxide [PBO] ITNs) distributed in Ebonyi State were evaluated. The epidemiological impact was also compared to the impact of standard pyrethroid-only ITNs in Cross River State.MethodsA controlled interrupted time series analysis was conducted on monthly malaria incidence data collected at the health facility level, using a multilevel mixed-effects negative binomial model. Data were analysed two years before and after the PBO ITN campaign in Ebonyi State (December 2017 to November 2021). A pre-post analysis, with no comparison group, was used to assess the impact of PBO ITNs on human biting rates and indoor resting density in Ebonyi during the high transmission season immediately before and after the PBO ITN campaign.ResultsIn Ebonyi, PBO ITNs were associated with a 46.7% decrease (95%CI: -51.5, -40.8%; p < 0.001) in malaria case incidence in the 2 years after the PBO ITN distribution compared to a modelled scenario of no ITNs distributed, with a significant decrease from 269.6 predicted cases per 1000 population to 143.6. In Cross River, there was a significant 28.6% increase (95%CI: -10.4, 49.1%; p < 0.001) in malaria case incidence following the standard ITN distribution, with an increase from 71.2 predicted cases per 1000 population to 91.6. In Ebonyi, the human biting rate was 72% lower (IRR: 0.28; 95%CI 0.21, 0.39; p < 0.001) and indoor resting density was 73% lower (IRR: 0.27; 95%CI 0.21, 0.35; p < 0.001) after the PBO ITNs were distributed.ConclusionsThe epidemiological and entomological impact of the PBO ITNs underscore the impact of these ITNs in areas with confirmed pyrethroid resistance. These findings contribute to ongoing research on the impact of new types of ITNs in Nigeria, providing critical evidence for the Nigeria National Malaria Elimination Programme and other countries for future ITN procurement decisions as part of mass ITN campaign planning and malaria programming.

Development of molecular diagnostic protocols for simultaneous identification of common bed bugs (Cimex lectularius) and tropical bed bugs (Cimex hemipterus)

BackgroundThe resurgence of two bed bug species, the common bed bug (Cimex lectularius Linnaeus, 1758) and tropical bed bug (Cimex hemipterus Fabricius, 1803), in the same geographical regions has been frequently reported recently. Consequently, the rapid identification of these species is crucial for implementing targeted capture traps and tailored pyrethroid resistance diagnosis, due to differences in genetic and physiological traits.MethodsTo develop molecular diagnostic methods, distinct protocols were established for multiplex PCR and loop-mediated isothermal amplification (LAMP) using species-specific primers based on species-specific segments of internal transcribed spacer 2 sequences. These methods were optimized for rapid and accurate identification of the two bed bug species.ResultsBoth multiplex PCR and LAMP protocols were effective in simultaneously identifying the two bed bug species, even when utilizing DNA released from dead specimens. Notably, the straightforward procedure and minimal time commitment of LAMP suggest its potential for rapid and accurate diagnosis of bed bugs in the field. The diagnostic accuracy of these methods was validated through a blind test.ConclusionsThe multiplex PCR and LAMP protocols lay the foundation for rapid and accurate field identification of bed bug species, enabling the use of appropriate traps and the detection of species-specific pyrethroid resistance mutations. This approach ensures effective management tailored to the unique characteristics of each bed bug species.Graphical

Differences in seasonal dynamics and pyrethroid resistance development among Anopheles Hyrcanus group species

BackgroundThe Anopheles Hyrcanus group, which transmits Plasmodium vivax, consists of six confirmed species in South Korea. An epidemiological study revealed differences in the seasonal occurrence patterns of each species. Pyrethroid resistance in An. sinensis dates back to the early 2000s, whereas information on pyrethroid resistance in other species is lacking despite their greater significance in malaria epidemiology.MethodsAnopheles mosquitoes were collected from two malaria-endemic regions in South Korea for 2 years and their knockdown resistance (kdr) mutations were genotyped. The larval susceptibility to λ-cyhalothrin was compared in six Anopheles species and its seasonal changes in three species were investigated. The full-length sequences of the voltage-sensitive sodium channel (VSSC) were compared across six species to evaluate potential target-site insensitivity. The contribution of the kdr mutation to phenotypic resistance was confirmed by comparing median lethal time (LT50) to λ-cyhalothrin between populations of Anopheles belenrae with distinct genotypes.ResultsThe composition and seasonal occurrence of rare species (Anopheles kleini, Anopheles lestri, and Anopheles sineroides) varied considerably, whereas An. sinensis occurs continuously throughout the season. A kdr mutation in the form of heterozygous allele was newly identified in An. belenrae, An. lesteri, An. pullus, and An. sineroides. The baseline susceptibility to λ-cyhalothrin was the highest in An. belenrae, followed by An. lesteri, An. sineroides, An. kleini, An. pullus, and An. sinensis, with median lethal concentration (LC50) values ranging from 6.0- to 73.5-fold higher than that of An. belenrae. The susceptibility of An. sinensis and An. pullus varied by season, whereas that of An. belenrae remained stable. The kdr-heterozygous An. belenare population exhibited 5.1 times higher LT50 than that of the susceptible population. Species-specific VSSC sequence differences were observed among the six species.ConclusionsOur findings suggest that the status and extent of pyrethroid resistance vary among Anopheles Hyrcanus group species. While An. sinensis, the predominant species, developed a considerable level of pyrethroid resistance through kdr mutation, the resistance levels of other species appeared to be less pronounced. Large-scale monitoring is crucial to fully understand species-specific seasonal occurrence and resistance status for effective management strategies, considering the ongoing impact of climate change on their vectorial capacity.Graphical

Barley yellow dwarf virus in winter barley: Control in light of resistance issues and loss of neonicotinoid insecticides

AbstractBarley yellow dwarf virus (BYDV) is the most widespread viral disease of cereal crops vectored by aphids, causing stunting of the crop and significant yield loss. Management recommendations for autumn sown cereals has centred on the timely control of aphid vectors, particularly their management before the crop has reached Growth Stage 31 (stem elongation). In recent years two separate but related issues have emerged, (i) the detection of pyrethroid resistance in a single Sitobion avenae SA3 clone and (ii) the withdrawal of neonicotinoid insecticide seed dressing; widely used to manage aphids in the early development of cereal crops. A series of replicated field studies were conducted from 2016 to 2019 to ascertain if the pyrethroid insecticide is still effective in light of the SA3 clones' presence in aphid populations and if withdrawal of neonicotinoid seed dressings has negatively affected aphid management. Both disease levels and yields confirmed that the neonicotinoid, clothianidin, applied as a seed treatment significantly contributed to the management of BYDV in the different trials investigated. Results of this study demonstrate application of a foliar (lambda‐cyhalothrin or a sulfoxaflor) insecticide has a significant positive impact on yield. Foliar pyrethroid insecticides were as effective in protecting yield from BYDV as the neonicotinoid seed treatments. In the absence of neonicotinoid seed dressing, alternative insecticides still offer protection for winter barley crops against the aphid vectors of BYDV and associated yield loss. Furthermore, the presence of a pyrethroid resistant Sitobion avenae SA3 clone had no observable impact on field efficacy of the pyrethroid insecticide, lambda‐cyhalothrin in these trials. However, this needs continuous monitoring because of potential further increases in resistance levels or emergence of additional resistance mechanisms, which could render pyrethroid insecticides ineffective.

Identification and functional validation of P450 genes associated with pyrethroid resistance in the malaria vector Anopheles sinensis (Diptera Culicidae)

Cytochrome P450 monooxygenases (P450s), a multifunctional protein superfamily, are one of three major classes of detoxification enzymes. However, the diversity and functions of P450 genes from pyrethroid-resistant populations of Anopheles sinensis have not been fully explored. In this study, P450 genes associated with pyrethroid resistance were systematically screened using RNA-seq in three field pyrethroid-resistant populations (AH-FR, CQ-FR, YN-FR) and one laboratory resistant strain (WX-LR) at developmental stages, tissues, and post blood-meal in comparison to the laboratory susceptible strain (WX-LS) in An. sinensis. Importantly, the expression of significantly upregulated P450s was verified using RT-qPCR, and the function of selected P450s in pyrethroid detoxification was determined with RNA interference using four laboratory pyrethroid-resistant strains (WX-LR, AH-LR, CQ-LR, YN-LR). Sixteen P450 genes were significantly upregulated in at least one field-resistant population, and 44 were significantly upregulated in different developmental stages, tissues or post blood-meal. A total of 19 P450s were selected to verify their association with pyrethroid resistance, and four of them (AsCYP6P3v1, AsCYP6P3v2, AsCYP9J10, and AsCYP9K1) demonstrated significant upregulation in laboratory pyrethroid-resistant strains using RT-qPCR. Knockdown of these four genes all significantly reduced pyrethroid resistance and increased the mortality by 57.19% (AsCYP6P3v1 and AsCYP6P3v2 knockdown group), 38.39% (AsCYP9K1 knockdown group) and 48.87% (AsCYP9J10 knockdown group) in An. sinensis by RNAi, which determined the pyrethroid detoxification function of these four genes. This study revealed the diversity of P450 genes and provided functional evidence for four P450s in pyrethroid detoxification in An. sinensis for the first time, which increases our understanding of the pyrethroid resistance mechanism, and is of potential value for pyrethroid resistance detection and surveillance.

Study on the cross-resistance of Aedes albopictus (Skuse) (Diptera: Culicidae) to deltamethrin and pyriproxyfen

BackgroundInsecticide resistance poses a significant challenge in the implementation of vector-borne disease control strategies. We have assessed the resistance levels of Aedes albopictus to deltamethrin and pyriproxyfen (PPF) in Fujian Province (China) and investigated the correlation between these resistance levels and mutations in the voltage-gated sodium channel (VGSC).MethodsThe WHO bioassay protocol was used to evaluate the resistance coefficient of Ae. albopictus to deltamethrin and PPF, comparing a susceptible population from the Foshan (FS) area with wild populations from the Sanming (SM), Quanzhou (QZ), Zhangzhou (ZZ), Putian (PT) and Fuzhou (FZ) areas in Fujian Province. Genomic DNA was analyzed by PCR and sequencing to detect knockdown resistance (kdr) in the VGSC, specifically at the pyrethroid resistance alleles V1016V, I1532I and F1534F. Molecular docking was also performed to analyze the binding interactions of PPF and its metabolite 4'-OH-PPF to cytochrome P450 (CYP) 2C19, 2C9 and 3A4 and Ae. albopictus methoprene-tolerant receptors (AeMet), respectively.ResultsThe analysis of resistance to deltamethrin and PPF among Ae. albopictus populations from the various regions revealed that except for the sensitive population in FS and the SM population, the remaining four regional populations demonstrated resistance levels ranging from 4.31- to 18.87-fold for deltamethrin and from 2.85– to 3.62-fold for PPF. Specifically, the FZ and PT populations exhibited high resistance to deltamethrin, whereas the ZZ and QZ populations approached moderate resistance levels. Also, the resistance of the FZ, PT and ZZ populations to PPF increased slowly but consistently with the increasing trend of deltamethrin resistance. Genomic analysis identified multiple non-synonymous mutations within the VGSC gene; the F1534S and F1534L mutations showed significant resistance to deltamethrin in Ae. albopictus. Molecular docking results revealed that PPF and its metabolite 4'-OH-PPF bind to the Ae. albopictus AeMet receptor and CYP2C19.ConclusionsThe wild Ae. albopictus populations of Fujian Province showed varying degrees of resistance to deltamethrin and PPF and a trend of cross-resistance to deltamethrin and PPF. Increased vigilance is needed for potential higher levels of cross-resistance, especially in the PT and FZ regions.Graphical

Genome-Wide Search for Gene Mutations Likely Conferring Insecticide Resistance in the Common Bed Bug, Cimex lectularius.

Insecticide resistance in the bed bug Cimex lectularius is poorly understood due to the lack of genome sequences for resistant strains. In Japan, we identified a resistant strain of C. lectularius that exhibits a higher pyrethroid resistance ratio compared to many previously discovered strains. We sequenced the genomes of the pyrethroid-resistant and susceptible strains using long-read sequencing, resulting in the construction of highly contiguous genomes (N50 of the resistant strain: 2.1 Mb and N50 of the susceptible strain: 1.5 Mb). Gene prediction was performed by BRAKER3, and the functional annotation was performed by the Fanflow4insects workflow. Next, we compared their amino acid sequences to identify gene mutations, identifying 729 mutated transcripts that were specific to the resistant strain. Among them, those defined previously as resistance genes were included. Additionally, enrichment analysis implicated DNA damage response, cell cycle regulation, insulin metabolism, and lysosomes in the development of pyrethroid resistance. Genome editing of these genes can provide insights into the evolution and mechanisms of insecticide resistance. This study expanded the target genes to monitor allele distribution and frequency changes, which will likely contribute to the assessment of resistance levels. These findings highlight the potential of genome-wide approaches to understand insecticide resistance in bed bugs.

Whole genome sequence analysis of population structure and insecticide resistance markers in Anopheles melas from the Bijagós Archipelago, Guinea-Bissau

BackgroundAnopheles melas is an understudied malaria vector with a potential role in malaria transmission on the Bijagós Archipelago of Guinea-Bissau. This study presents the first whole-genome sequencing and population genetic analysis for this species from the Bijagós. To our knowledge, this also represents the largest population genetic analysis using WGS data from non-pooled An. melas mosquitoes.MethodsWGS was conducted for 30 individual An. melas collected during the peak malaria transmission season in 2019 from six different islands on the Bijagós Archipelago. Bioinformatics tools were used to investigate the population structure and prevalence of insecticide resistance markers in this mosquito population.ResultsInsecticide resistance mutations associated with pyrethroid resistance in Anopheles gambiae s.s. from the Bijagós were absent in the An. melas population, and no signatures of selective sweeps were identified in insecticide resistance-associated genes. Analysis of structural variants identified a large duplication encompassing the cytochrome-P450 gene cyp9k1. Phylogenetic analysis using publicly available mitochondrial genomes indicated that An. melas from the Bijagós split into two phylogenetic groups because of differentiation on the mitochondrial genome attributed to the cytochrome C oxidase subunits COX I and COX II and the NADH dehydrogenase subunits 1, 4, 4L and 5.ConclusionsThis study identified an absence of insecticide-resistant SNPs common to An. gambiae in the An. melas population, but did identify structural variation over insecticide resistance-associated genes. Furthermore, this study presents novel insights into the population structure of this malaria vector using WGS analysis. Additional studies are required to further understand the role of this vector in malaria transmission.Graphical abstract

Whole-genome sequencing of major malaria vectors reveals the evolution of new insecticide resistance variants in a longitudinal study in Burkina Faso

BackgroundIntensive deployment of insecticide based malaria vector control tools resulted in the rapid evolution of phenotypes resistant to these chemicals. Understanding this process at the genomic level is important for the deployment of successful vector control interventions. Therefore, longitudinal sampling followed by whole genome sequencing (WGS) is necessary to understand how these evolutionary processes evolve over time. This study investigated the change in genetic structure and the evolution of the insecticide resistance variants in natural populations of Anopheles gambiae over time and space from 2012 to 2017 in Burkina Faso.MethodsNew genomic data have been generated from An. gambiae mosquitoes collected from three villages in the western part of Burkina Faso between 2012 and 2017. The samples were whole-genome sequenced and the data used in the An. gambiae 1000 genomes (Ag1000G) project as part of the Vector Observatory. Genomic data were analysed using the analysis pipeline previously designed by the Ag1000G project.ResultsThe results showed similar and consistent nucleotide diversity and negative Tajima’s D between An. gambiae sensu stricto (s.s.) and Anopheles coluzzii. Principal component analysis (PCA) and the fixation index (FST) showed a clear genetic structure in the An. gambiae sensu lato (s.l.) species. Genome-wide FST and H12 scans identified genomic regions under divergent selection that may have implications in the adaptation to ecological changes. Novel voltage-gated sodium channel pyrethroid resistance target-site alleles (V402L, I1527T) were identified at increasing frequencies alongside the established alleles (Vgsc-L995F, Vgsc-L995S and N1570Y) within the An. gambiae s.l. populations. Organophosphate metabolic resistance markers were also identified, at increasing frequencies, within the An. gambiae s.s. populations from 2012 to 2017, including the SNP Ace1-G280S and its associated duplication. Variants simultaneously identified in the same vector populations raise concerns about the long-term efficacy of new generation bed nets and the recently organophosphate pirimiphos-methyl indoor residual spraying in Burkina Faso.ConclusionThese findings highlighted the benefit of genomic surveillance of malaria vectors for the detection of new insecticide resistance variants, the monitoring of the existing resistance variants, and also to get insights into the evolutionary processes driving insecticide resistance.

Dynamics of Bactrocera dorsalis Resistance to Seven Insecticides in South China.

Bactrocera dorsalis is a highly invasive and destructive pest distributed worldwide. Chemical insecticides remain the primary measure for their control; however, this species has already developed resistance to several insecticides. In recent years, there have been several reports of monitoring B. dorsalis resistance in China, but continuous monitoring results are lacking and do not even span a decade. In this study, we monitored the dynamics of resistance to seven insecticides among 11 geographically distinct Chinese populations of B. dorsalis (2010-2013; follow-up in 2023). The 11 populations were found to adapt rapidly to antibiotic insecticides (spinosad, emamectin benzoate, and avermectin), reaching high levels of insecticide resistance in several areas. Overall, a decreasing trend in resistance to organophosphorus insecticides (chlorpyrifos and trichlorfon) was observed, whereas pyrethroid (beta-cypermethrin and cyhalothrin) resistance trends were observed to both increase and decrease. The monitoring of field resistance among different B. dorsalis populations over the duration of this study is important for improving the efficiency and sustainability of agricultural pest management, and the results provide a scientific basis for the development of more effective resistance management strategies.

Molecular Detection of kdr and superkdr Mutation Sites and Analysis of the Binding Modes of Pyrethroid Insecticides with Voltage-Gated Sodium Channels in the Plant Bug Lygus pratensis (Hemiptera: Miridae).

This study identified genetic mutations linked to resistance to pyrethroid insecticides in the plant pest Lygus pratensis. The voltage-gated sodium channel (VGSC) gene was cloned, revealing two mutations (Met918Thr and Leu1014Phe) in laboratory strains and field populations from Inner Mongolia, resulting in variable pyrethroid resistance. A 3D model of LpVGSC was created using homology modeling, and pyrethroid binding patterns were analyzed via molecular docking. Molecular dynamics simulations confirmed structural stability changes and binding stability of pyrethroids to VGSC sites. Mutation frequencies of homozygous and heterozygous genotypes did not exceed 40 and 20%, respectively. Toxicity tests showed high resistance to λ-cyhalothrin (LC50:401.31 ng/cm2). The kdr (L1014F) and superkdr (M918T) mutations weakened interaction forces, reducing pyrethroid binding. M918T and L1014F mutations are predicted to reduce Type I pyrethroid affinity, suggesting Type II pyrethroids may be more effective against resistant strains. These findings aid in resistance management and insecticide design.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are associated with insecticide resistance in the major malaria vectors Anopheles gambiae s.l. and Anopheles funestus

Malaria remains one of the highest causes of morbidity and mortality, with 249 million cases and over 608,000 deaths in 2022. Insecticides, which target the Anopheles mosquito vector, are the primary method to control malaria. The widespread nature of resistance to the most important insecticide class, the pyrethroids, threatens the control of this disease. To reverse the stall in malaria control there is urgent need for new vector control tools, which necessitates understanding the molecular basis of pyrethroid resistance. In this study we utilised multi-omics data to identify uridine-diphosphate (UDP)-glycosyltransferases (UGTs) potentially involved in resistance across multiple Anopheles species. Phylogenetic analysis identifies sequence similarities between Anopheline UGTs and those involved in agricultural pesticide resistance to pyrethroids, pyrroles and spinosyns. Expression of five UGTs was characterised in An. gambiae and An. coluzzii to determine constitutive over-expression, induction, and tissue specificity. Furthermore, a UGT inhibitor, sulfinpyrazone, restored susceptibility to pyrethroids and DDT in An. gambiae, An. coluzzii, An. arabiensis and An. funestus, the major African malaria vectors. Taken together, this study provides clear association of UGTs with pyrethroid resistance as well as highlighting the potential use of sulfinpyrazone as a novel synergist for vector control.

Transcriptome analysis of Drosophila suzukii reveals molecular mechanisms conferring pyrethroid and spinosad resistance

Drosophila suzukii lay eggs in soft-skinned, ripening fruits, making this insect a serious threat to berry production. Since its 2008 introduction into North America, growers have used insecticides, such as pyrethroids and spinosads, as the primary approach for D. suzukii management, resulting in development of insecticide resistance in this pest. This study sought to identify the molecular mechanisms conferring insecticide resistance in these populations. We sequenced the transcriptomes of two pyrethroid- and two spinosad-resistant isofemale lines. In both pyrethroid-resistant lines and one spinosad-resistant line, we identified overexpression of metabolic genes that are implicated in resistance in other insect pests. In the other spinosad-resistant line, we observed an overexpression of cuticular genes that have been linked to resistance. Our findings enabled the development of molecular diagnostics that we used to confirm persistence of insecticide resistance in California, U.S.A. To validate these findings, we leveraged D. melanogaster mutants with reduced expression of metabolic or cuticular genes that were found to be upregulated in resistant D. suzukii to demonstrate that these genes are involved in promoting resistance. This study is the first to characterize the molecular mechanisms of insecticide resistance in D. suzukii and provides insights into how current management practices can be optimized.

Sergentomyia khawi: a potential vector for Leishmania and Trypanosoma parasites affecting humans and animals and insecticide resistance status in endemic areas of Songkhla, southern Thailand

BackgroundSand flies serve as crucial vectors in various medical and veterinary diseases. Sand fly-borne diseases pose a significant public health burden globally, as the causative agents can infect a diverse range of hosts, leading to severe consequences such as leishmaniasis and sand fly fever. Additionally, the widespread use of insecticides for agricultural purposes and mosquito control is not specifically targeted at sand flies, potentially leading to resistance development. We investigated sand fly species, their potential role as vectors of various parasitic agents, and insecticide resistance in the endemic regions of Natawi and Sadao districts in Songkhla, Thailand.MethodsSand flies were collected using CDC light traps. The collected sand flies were then identified to species level using molecular techniques. Subsequent analyses included the detection of pathogens and the identification of pyrethroid resistance mutations within the voltage-sensitive sodium channel (Vgsc) domain IIS6 gene, followed by sequence analysis.ResultsThe study identified nine sand fly species belonging to the genera Phlebotomus and Sergentomyia. The DNA of Sergentomyia khawi was the only species found to test positive for one sample of Leishmania orientalis in Sadao district. This finding represents the first detection of L. orientalis in Thailand. Moreover, three samples of Leishmania martiniquensis and four samples of Trypanosoma sp. were found in the Natawi district. No I1011M, L1014F/S, V1016G, or F1020S mutations were detected in Vgsc gene.ConclusionsThe results of this study provide valuable information on sand fly species and the continuous circulation of Leishmania spp. and Trypanosoma spp. in Songkhla, southern Thailand. Moreover, the development of geo-spatial information on vectors, parasites, and insecticide resistance in sand flies has the potential to provide well-informed risk assessments and evidence-based guidance for targeted vector control in Thailand. These results can serve as a foundation for integrating the One Health approach, which is crucial for disease control, considering the diverse ecological interactions among human and/or animal reservoir hosts, parasites, and sand fly vectors.Graphic

Longitudinal survey of insecticide resistance in a village of central region of Burkina Faso reveals co-occurrence of 1014F, 1014S and 402L mutations in Anopheles coluzzii and Anopheles arabiensis

BackgroundPyrethroid resistance is one of the major threats for effectiveness of insecticide-treated bed nets (ITNs) in malaria vector control. Genotyping of mutations in the voltage gated sodium channel (VGSC) gene is widely used to easily assess the evolution and spread of pyrethroid target-site resistance among malaria vectors. L1014F and L1014S substitutions are the most common and best characterized VGSC mutations in major African malaria vector species of the Anopheles gambiae complex. Recently, an additional substitution involved in pyrethroid resistance, i.e. V402L, has been detected in Anopheles coluzzii from West Africa lacking any other resistance alleles at locus 1014. The evolution of target-site resistance mutations L1014F/S and V402L was monitored in An. coluzzii and Anopheles arabiensis specimens from a Burkina Faso village over a 10-year range after the massive ITN scale-up started in 2010.MethodsAnopheles coluzzii (N = 300) and An. arabiensis (N = 362) specimens collected both indoors and outdoors by different methods (pyrethrum spray catch, sticky resting box and human landing collections) in 2011, 2015 and 2020 at Goden village were genotyped by TaqMan assays and sequencing for the three target site resistance mutations; allele frequencies were statistically investigated over the years.ResultsA divergent trend in resistant allele frequencies was observed in the two species: 1014F decreased in An. coluzzii (from 0.76 to 0.52) but increased in An. arabiensis (from 0.18 to 0.70); 1014S occurred only in An. arabiensis and slightly decreased over time (from 0.33 to 0.23); 402L increased in An. coluzzii (from 0.15 to 0.48) and was found for the first time in one An. arabiensis specimen. In 2020 the co-occurrence of different resistance alleles reached 43% in An. coluzzii (alleles 410L and 1014F) and 32% in An. arabiensis (alleles 1014F and 1014S).ConclusionsOverall, an increasing level of target-site resistance was observed among the populations with only 1% of the two malaria vector species being wild type at both loci, 1014 and 402, in 2020. This, together with the co-occurrence of different mutations in the same specimens, calls for future investigations on the possible synergism between resistance alleles and their phenotype to implement local tailored intervention strategies.

Identification and expression patterns of voltage-gated sodium channel genes with intron retentions in different strains of Bactrocera dorsalis

Pyrethroid are the primary insecticides used for controlling of Bactricera dorsalis, a highly destructive and invasive fruit pest. Field populations have developed serious resistance, especially to β-cypermethrin. While mutations in the voltage-gated sodium channel (Vgsc) are a common mechanism of pyrethroid resistance, variations in BdVgsc associated with β-cypermethrin resistance remain unclear. Here, we reported the resistance levels of five field populations from China, with resistance ratio ranging from 1.54 to 21.34-fold. Cloning the full length of BdVgsc revealed no specific or known amino acid mutations between the most resistant population and the susceptible strain. However, three types of partial intron retention (IRE4–5, IRE19-f and IREL-24) were identified in BdVgsc transcripts, with these intron retentions containing stop codons. The expression of IRE4–5 transcripts and total BdVgsc showed different trends across developmental stages and tissues. Exposure to β-cypermethrin led to increased expression of IRE4–5. Comparison of genomic and transcriptional sequences reveled that IRE4–5 transcripts had two types (IRE4–5.5 T and IRE4–5.6 T) caused by genomic variations. Both field and congenic strains indicated that homozygotes for IRE4–5.5 T had lower IRE4–5 transcript levels than homozygotes for IRE4–5.6 T. However, congenic and field strains exhibited inconsistent results about the association of expression levels of IRE4–5 transcripts with sensitivity to β-cypermethrin. In summary, this study is the first to identify intron retention transcripts in the Vgsc gene from B. dorsalis and to examine their expression patterns across different developmental stages, tissues, and strains with varying sensitivities to β-cypermethrin. The potential role of the intron retentions of BdVgsc in insecticide toxicity is also discussed.

Common consumer residual insecticides lack efficacy against insecticide-susceptible and resistant populations of the German cockroach (Blattodea: Ectobiidae).

The German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae), is a ubiquitous pest in affordable housing. They represent a major threat to human health due to their contribution of asthma-exacerbating allergens and the potential to transfer pathogenic microorganisms indoors. Despite well-documented pyrethroid resistance, pyrethroid-based broadcast residual insecticide products are often used by residents to control cockroaches in their homes. Additionally, there is little empirical independent testing of these products. Thus, it remains unclear how effective these commonly used do-it-yourself products are at controlling German cockroaches. This study represents a comprehensive examination of the efficacy of these products with direct, limited, and continuous exposure assays on a variety of common household surfaces on field populations of cockroaches with varying levels of pyrethroid resistance. While most products performed well when applied directly to test insects, mortality was substantially lower across all surfaces with limited exposure (30min). In continuous exposure assays on a nonporous surface, products took at least 24hr to cause 100% mortality in a field population, with some products taking up to 5d to achieve 100% mortality. The findings of this study demonstrate a lack of residual efficacy from common pyrethroid-based consumer-use pesticides products. Given that it is not feasible to find and treat every cockroach in a home directly, the residuality of spray-based formulations is critical for products designed to control German cockroaches. Without residual efficacy, as shown in the consumer aerosol and spray products tested, we expect these products to add little to no value to cockroach control.

Organophosphate and carbamate susceptibility profiling of Anopheles gambiae sl. across different ecosystems in southern Benin

Background To overcome the spread of high pyrethroid resistance in the main malaria vectors and malaria disease persistence, it is crucial to look for effective and better resistance management strategies. Understanding the phenotypic profile of Anopheles gambiae sl. against alternatives insecticides like organophosphates and carbamates is crucial. Methods Anopheles larvae and pupae were collected from the breeding sites in rice fields, pineapple crop areas, and peri-urban areas. WHO susceptibility tests were conducted on unfed female mosquitoes aged 3–5 days old. Mosquitoes were exposed to malathion 5%, pirimiphos-methyl 0.25%, and bendiocarb 0.1% using the standard WHO protocol. Polymerase chain reaction (PCR) techniques were used to detect species, kdr and Ace-1 mutations. Results Anopheles gambiae sl. from Sèdjè-Dénou rice field population was resistant to bendiocarb (0.1%) with a mortality rate of 72.2% whereas Anopheles gambiae sl. populations from Zinvié-Dokomey (rice field), Zè-Tozounmè (pineapple field), and Adjagbo (peri-urban area) were suspected to be resistant with mortality rates of 90%, 93.5%, 95.4% respectively. However, all of them were susceptible to organophosphates (malathion and pirimiphos-methyl) with a mortality rate of 100%. PCR assay revealed that 100% of the mosquitoes tested were Anopheles coluzzii. The frequencies of Ace-1R mutation in all Anopheles coluzzii populations tested were low (3–27%). Conclusions Organophosphates (malathion and pirimiphos-methyl) have maintained their efficacy against Anopheles coluzzii populations from Sèdjè-Dénou (rice field), Zè Tozounmè (pineapple field), Zinvié Dokomey (rice field), or Adjagbo (peri-urban area). The good efficacy of these organophosphates against Anopheles coluzzii populations from the southern part of Benin are observed in the current study. The use of pirimiphos-methyl for IRS in this part of the country would be a successful alternative for malaria control in this area.

Durability of PBO nets (Olyset Plus®), 12 months after their distribution in Bertoua, Cameroon

BackgroundThe rapid spread of pyrethroid resistance has led to a change in strategy, going from pyrethroid-based nets to PBO + pyrethroid-treated nets. Although these new nets may significantly improve the control of pyrethroid-resistant mosquitoes, their durability in the field remain not yet well documented. This study investigates the durability and washing resistance of Olyset-Plus nets in the city centre and rural areas of Bertoua, Cameroon. In each site, a semi-structured questionnaire was administered to at least 190 households with an Olyset-Plus net. Factors such as net use, physical integrity and bioefficacy were recorded. Bioassays were conducted on the collected nets to assess their bioefficacy and resistance to washing. They were tested against wild Anopheles gambiae sensus lato (s.l.). Unused nets and the Kisumu strain were used as controls. Washing and cone testing of the nets was carried out according to standard WHO protocols. ResultsA high rate of net use by children was recorded in the urban area (89.1% (106/119)) compared to the rural area (39.7% (118/297)). The majority of Olyset-Plus nets inspected 82.2% (162/197) in the rural area and 88% (206/234) in the urban centre were in good condition (Hole Index<64). Only 5.6% and 6.8% of nets were badly torn in rural and urban sites respectively. Nets were washed more regularly in the urban centre. 88.1% of urban dwellers reported having washed their nets at least once compared to only 62% of rural dwellers. Bioefficacy tests with nets indicated a mortality rate ranging from 66% for unwashed nets to 86.7% for nets washed at least once. Bioefficacy varied significantly in the city of Bertoua according to net washing frequency, soaking time, soap type and drying location, whereas in the rural village, only washing(washed or unwashed) and soaking status (soaked or unsoaked) significantly influenced the bioefficacy of Olyset-Plus nets. ConclusionsThis study revealed different handling practices of bed nets in rural and urban settings which could significantly affect Olyset-Plus nets bio-efficacy and durability. Routine monitoring and sensitization of communities to best practices concerning bed nets usage and handling during mass distribution might enhance the net durability in the community.

Association of a rapidly selected 4.3kb transposon-containing structural variation with a P450-based resistance to pyrethroids in the African malaria vector Anopheles funestus.

Deciphering the evolutionary forces controlling insecticide resistance in malaria vectors remains a prerequisite to designing molecular tools to detect and assess resistance impact on control tools. Here, we demonstrate that a 4.3kb transposon-containing structural variation is associated with pyrethroid resistance in central/eastern African populations of the malaria vector Anopheles funestus. In this study, we analysed Pooled template sequencing data and direct sequencing to identify an insertion of 4.3kb containing a putative retro-transposon in the intergenic region of two P450s CYP6P5-CYP6P9b in mosquitoes of the malaria vector Anopheles funestus from Uganda. We then designed a PCR assay to track its spread temporally and regionally and decipher its role in insecticide resistance. The insertion originates in or near Uganda in East Africa, where it is fixed and has spread to high frequencies in the Central African nation of Cameroon but is still at low frequency in West Africa and absent in Southern Africa. A marked and rapid selection was observed with the 4.3kb-SV frequency increasing from 3% in 2014 to 98% in 2021 in Cameroon. A strong association was established between this SV and pyrethroid resistance in field populations and is reducing pyrethroid-only nets' efficacy. Genetic crosses and qRT-PCR revealed that this SV enhances the expression of CYP6P9a/b but not CYP6P5. Within this structural variant (SV), we identified putative binding sites for transcription factors associated with the regulation of detoxification genes. An inverse correlation was observed between the 4.3kb SV and malaria parasite infection, indicating that mosquitoes lacking the 4.3kb SV were more frequently infected compared to those possessing it. Our findings highlight the underexplored role and rapid spread of SVs in the evolution of insecticide resistance and provide additional tools for molecular surveillance of insecticide resistance.