Insecticide Resistance In Populations Research Articles

Complexities in the Implementation and Maintenance of Integrated Pest Management in Potato.

Integrated pest management (IPM) is an educated and systematic effort to use multiple control techniques to reduce pest damage to economically acceptable levels while minimizing negative environmental impacts. Although its benefits are widely acknowledged, IPM is not universally practiced by farmers. Potato farming, which produces one of the most important staple crops in the world, provides a good illustration of the issues surrounding IPM adoption. Potatoes are attacked by a complex of insect pests that can inflict catastrophic crop losses. Potato production has gone through the processes of consolidation and intensification, which are linked to increased pest problems, particularly selection for insecticide-resistant pest populations. While use of insecticides remains the most common method of pest control in potatoes, other techniques, including crop rotation and natural enemies, are also available. In addition, there are effective monitoring techniques for many potato pests. However, reliable economic thresholds are often lacking. Potato ecosystems are complex and diverse; therefore, the knowledge necessary for developing ecologically based pest management is not easily obtained or transferable. Furthermore, potato systems change with the arrival of new pest species and the evolution of existing pests. Modern technological advances, such as remote sensing and molecular biotechnology, are likely to improve potato IPM. However, these tools are not going to solve all problems. IPM is not just about integrating different techniques; it is also about integrating the efforts and concerns of all stakeholders. The collaboration of farmers and scientists in agricultural research is needed to foster the development of IPM systems that are appropriate for grower implementation and thus more likely to be adopted. Additional emphasis also needs to be placed on the fact that not only does IPM decrease degradation of the environment, but it also improves the economic well-being of its practitioners.

Evaluation of the efficacy of entomopathogenic nematodes on Ctenocephalides felis felis larvae (Siphonaptera: Pulicidae).

Ctenocephalides felis felis is a relevant and widely distributed ectoparasite that acts as a vector of disease-causing pathogens. Moreover, it is responsible for economic losses due the use of harmful chemicals to the environment and that favor the emergence of insecticide-resistant populations. Research on entomopathogenic nematodes may open up an alternative route to the insect chemical control. The present study aimed to evaluate the killing efficacy of Heterorhabditis bacteriophora (HP88) against C. felis larvae in 400 μL, 600 μL and 1000 μL of suspension containing 120, 160 and 200 infective juveniles/larva and 600 μL of suspension containing the same concentrations of Heterorhabditis indica (LPP30), divided into two groups (absence and presence of diet) and a control group with three replications containing only distilled water. In the bioassay with H. bacteriophora, the groups in 600 μL of suspension showed higher mortality rates than those in the other tested volumes, which were above 80% at all concentrations. On the other hand, H. indica achieved mortality rates above 70% at all concentrations used. Results indicate that flea larvae are susceptible to in vitro infection by H. bacteriophora and H. indica.

Insecticide resistant mosquitoes remain thermal stress resistant, without loss of thermal plasticity

A major component of mosquito's climate change response is their heat tolerance, and any ability to rapidly adjust to extreme environmental conditions through phenotypic plasticity. The excessive use of insecticides for the control of major mosquito species leads to resistant populations, however it is largely unclear if this concurrently impacts thermal stress resistance and their potential to adjust tolerance via phenotypic plasticity. Culex pipiens pipiens, Culex pipiens molestus and Aedes albopictus populations obtained from the same region were subjected for 12 generations to selection trials to larvicides Diflubenzuron (DFB) and Bacillus thuringiensis subsp. israelensis (Bti) to develop insecticide resistance. Adults emerging from the selected populations were acclimated at different temperatures and the upper and lower critical thermal limits (CTmax and CTmin) were estimated using dynamic thermal assays. In addition, the supercooling points (SCPs) of non-acclimated adults of resistant and control populations were determined. Our results revealed marked differences in thermal response among the three species, the different acclimation regimes and sexes. Aedes albopictus was more resistant in high than low temperatures compared to both Culex pipiens biotypes. Culex forms responded similarly to heat but differently to cold stress. In both forms, females responded better than males to all thermal stressors. Acclimation at higher and lower temperatures improves CTmax and CTmin values, respectively in both insecticide resistant and control populations of all three species. Overall, selection to insecticides did not affect the thermal performance of adults. Hence, insecticide-resistant mosquito populations perform similarly to untreated ones and are capable of readily adapting to new environmental changes rising concerns regarding their geographic range expansion and disease transmission globally.

Population dynamics and insecticide resistance in Tuta absoluta (Lepidoptera: Gelechiidae), an invasive pest on tomato in Kenya

Tuta absoluta feeds on solanaceous plants with preference on tomato. Management of the pest is mostly with chemical insecticides. This study identified insecticide resistant populations and predicted resistance to insecticides. Insecticide resistance development was modelled using system thinking, and system dynamics approaches. The model showed the pest resistance development is alarming with an exponential increase of the resistance strength mostly in recent years. Furthermore, we used seven insecticide-resistance gene markers to resolve the population structure and genetic differentiation of insecticide-resistant populations in Kenya. The genes for resistance (knockdown resistance (kdr) mutations, acetylcholinesterase (AChE) and voltage gated sodium channel (para)) were detected in all populations. Population structure analyses separated T. absoluta populations into three genetic clusters with resistant genes that are interconnected. A better insight on the population dynamics and the genetic structure T. absoluta resistant genes in Kenya will help estimate resistance strength and determine the most effective pest control strategies.

Development of rice farming: a cause of the emergence of multiple insecticide resistance in populations of Anopheles gambiae s.l and its impact on human health in Malanville, Bénin.

The rise in rice production in the district of Malanville, Northen Benin, is a present concern, as it has resulted in the widespread usage of pesticides for crop protection. This could impact human health but also life cycle of Anopheles gambiae, the main vector of malaria. Therefore, insecticide susceptibility bioassays were carried out on populations of An. gambiae s.l aged to 3-5 days old (two from areas where insecticide is highly used and other two areas of low insecticide use) and subjected to insecticide-impregnated papers (Permethrin 0.75%; deltamethrin 0.05%; DDT 4% and bendiocarb 0.1%) following WHO protocol. Polymerase Chain Reactions (PCRs) were used for the detection of Acethlylcholinestrase (Ace-1) and the knock down resistance (kdr) L1014F mutations in An. gambiae populations. Finally, indirect bioassays were conducted for the investigating on the factors affecting the life cycle of An. gambiae due to the use of pesticides. An. gambiae from the four sites were resistant to DDT (6 to 8% and 10 to 14% respectively from areas of high and low dose), pyrethroids (22 to 26% and 30 to 36% for permethrin, from areas of high and low dose respectively and 66 to 70% and 72 to 80% for deltamethrin, from high and low dose) but susceptible to carbamate. The kdr L1014F mutation was detected in An. gambiae populations (0.88 to 0.90 and 0.84 to 0.88 from high and low dose, respectively). The ace-1 was detected at low frequencies (<0.002). Bioassays on the impacts of the use of pesticides in the life cycle of An. gambiae showed that soil substrates with pesticides residues have a negative impact on the life cycle eggs of An. gambiae. These findings confirmed the negative impacts of pesticides use in rice farming and its impacts on the life cycle of An. gambiae.

Cuticular profiling of insecticide resistant Aedes aegypti

Insecticides have made great strides in reducing the global burden of vector-borne disease. Nonetheless, serious public health concerns remain because insecticide-resistant vector populations continue to spread globally. To circumvent insecticide resistance, it is essential to understand all contributing mechanisms. Contact-based insecticides are absorbed through the insect cuticle, which is comprised mainly of chitin polysaccharides, cuticular proteins, hydrocarbons, and phenolic biopolymers sclerotin and melanin. Cuticle interface alterations can slow or prevent insecticide penetration in a phenomenon referred to as cuticular resistance. Cuticular resistance characterization of the yellow fever mosquito, Aedes aegypti, is lacking. In the current study, we utilized solid-state nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry, and transmission electron microscopy to gain insights into the cuticle composition of congenic cytochrome P450 monooxygenase insecticide resistant and susceptible Ae. aegypti. No differences in cuticular hydrocarbon content or phenolic biopolymer deposition were found. In contrast, we observed cuticle thickness of insecticide resistant Ae. aegypti increased over time and exhibited higher polysaccharide abundance. Moreover, we found these local cuticular changes correlated with global metabolic differences in the whole mosquito, suggesting the existence of novel cuticular resistance mechanisms in this major disease vector.

Recombinant expression and characterization of GSTd3 from a resistant population of Anopheles arabiensis and comparison of DDTase activity with GSTe2

The development of insecticide resistance in malaria vectors is a challenge for the global effort to control and eradicate malaria. Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the detoxification of many classes of insecticides. For mosquitoes, it is known that overexpression of an epsilon GST, GSTe2, confers resistance towards DDT and pyrethroids. In addition to GSTe2, consistent overexpression of a delta class GST, GSTd3, has been observed in insecticide resistant populations of different malaria vector species. However, the functional role of GSTd3 towards DDT resistance has not yet been investigated. Here, we recombinantly expressed both GSTe2 and GSTd3 from Anopheles arabiensis and compared their metabolic activities against DDT. Both AaGSTd3 and AaGSTe2 exhibited CDNB-conjugating and glutathione peroxidase activity and DDT metabolism was observed for both GSTs. However, the DDT dehydrochlorinase activity exhibited by AaGSTe2 was much higher than for AaGSTd3, and AaGSTe2 was also able to eliminate DDE although the metabolite could not be identified. Molecular modeling revealed subtle differences in the binding pocket of both enzymes and a better fit of DDT within the H-site of AaGSTe2. The overexpression but much lower DDT metabolic activity of AaGSTd3, might suggest that AaGSTd3 sequesters DDT. These findings highlight the complexity of insecticide resistance in the major malaria vectors and the difficulties associated with control of the vectors using DDT, which is still used for indoor residual spraying.

Lethal and sublethal effects of orally delivered double-stranded RNA on the cabbage stem flea beetle, Psylliodes chrysocephala.

The cabbage stem flea beetle (Psylliodes chrysocephala) is one of the most important insect pests of oilseed rape (Brassica napus) in northern Europe. The emergence of insecticide-resistant populations and the ban on neonicotinoid seed treatments have made the management of this pest challenging and research is needed to develop alternative strategies such as RNA interference (RNAi). We investigated lethal and sublethal effects of orally delivered double-stranded (ds)RNAs targeting P. chrysocephala orthologs of Sec23 and vacuolar adenosine triphosphatase subunit G (VatpG), which are involved in endoplasmic reticulum-Golgi transport and organelle acidification, respectively. Feeding bioassays on P. chrysocephala adults showed that the highest concentration (200 ng/leaf disk) of dsSec23 caused mortalities of 76% and 56% in pre-aestivating and post-aestivating beetles, respectively, while the same concentration of dsVatpG led to mortality rates of ~34% in both stages. Moreover, sublethal effects, such as decreased feeding rates and attenuated locomotion were observed. Small RNA sequencing and gene expression measurements following the delivery of dsRNAs demonstrated the generation of ~21 nucleotide-long small interfering RNAs and a systemic RNAi response in P. chrysocephala. We demonstrate that P. chrysocephala is a promising candidate for developing RNAi-based pest management strategies. Further research is necessary to identify more effective target genes and to assess potential non-target effects. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Low frequency of knockdown resistance mutation (L1014F) and the efficacy of PBO synergist in multiple insecticide-resistant populations of Anopheles gambiae in Ikorodu, Lagos State, Nigeria.

We evaluated the susceptibility status of Anopheles gambiae in two communities of Ikorodu, Lagos, Nigeria to DDT, deltamethrin, lambda cyhalothrin and bendiocarb. Anopheles immature stages were collected from their habitats in the surveyed community and allowed to emerge before exposure adult females to discriminating doses of WHO insecticides including DDT, deltamethrin, lambda cyhalothrin, bendiocarb and malathion. PBO synergistic bioassay was conducted for insecticides where the mosquito samples showed resistance. PCR assay was used for the detection of kdr mutation in the mosquitoes. Resistance to DDT (40% and 86%) and lambda cyhalothrin (75% and 84%) in Oke-Ota and Majidun respectively. Suspected resistance to deltamethrin (94.9%) and bendiocarb (93.5%) was recorded in Oke-Ota community and the mosquitoes were susceptible to malathion in both communities. KDR mutation (L1014F) from resistance samples from both locations though with a low frequency that significantly departs from Hardy-Weinberg's probability (P> 0.01). PBO synergized bioassay was able to increase knockdown, percentage mortality and restore full susceptibility to deltamethrin and bendiocarb. Results from this study indicates that the metabolic resistance mechanism is highly implicated in the resistance to different classes of insecticide in Ikorodu and this should be taken into consideration when implementing vector control activities in this area.

Larvicidal, growth inhibitory and biochemical effects of soil bacterium, Pseudomonas sp. EN4 against Spodoptera litura (Fab.) (Lepidoptera: Noctuidae)

BackgroundSpodoptera litura (Fabricius) (Lepidoptera: Noctuidae) also known as tobacco caterpillar, is one of the most serious polyphagous pests that cause economic losses to a variety of commercially important agricultural crops. Over the past few years, many conventional insecticides have been used to control this pest. However, the indiscriminate use of these chemicals has led to development of insecticide resistant populations of S. litura in addition to harmful effects on environment. Due to these ill effects, the emphasis is being laid on alternative eco-friendly control measures. Microbial control is one of the important components of integrated pest management. Thus, in search for novel biocontrol agents, the current work was carried out with the aim to evaluate the insecticidal potential of soil bacteria against S. litura.ResultsAmong the tested soil bacterial isolates (EN1, EN2, AA5, EN4 and R1), maximum mortality (74%) was exhibited by Pseudomonas sp. (EN4). The larval mortality rate increased in a dose-dependent manner. Bacterial infection also significantly delayed the larval development, reduced adult emergence, and induced morphological deformities in adults of S. litura. Adverse effects were also detected on various nutritional parameters. The infected larvae showed a significant decrease in relative growth and consumption rate as well as efficiency of conversion of ingested and digested food to biomass. Histopathological studies indicated damage to the midgut epithelial layer of larvae due to the consumption of bacteria treated diet. The infected larvae also showed a significantly decreased level of various digestive enzymes. Furthermore, exposure to Pseudomonas sp. also caused DNA damage in the hemocytes of S. litura larvae.ConclusionAdverse effects of Pseudomonas sp. EN4 on various biological parameters of S. litura indicate that this soil bacterial strain may be used as an effective biocontrol agent against insect pests.

Development of insecticide resistance in Hyalella azteca

Hyalella azteca are epibenthic amphipods that have developed resistance to pyrethroid and organophosphate insecticides due to single amino acid substitutions in the voltage-gated sodium channel and the acetylcholinesterase-1 gene, respectively. Aquatic systems are often contaminated with several different types of insecticides, therefore there is a possibility that H. azteca have also developed resistance to other classes of insecticides. The aims of the current study were to verify that pyrethroid- and organophosphate-resistant H. azteca have retained their resistance after being cultured in the absence of selective pressure for 5 years (Escondido Creek population) and 9 years (Mosher Slough population), to determine if these populations have cross-resistance to carbaryl (carbamate) and 4,4′-dichlorodiphenyltrichloroethane (DDT; organochlorine), and determine whether previous field exposure to fipronil (phenylpyrazole) and imidacloprid (neonicotinoid) caused resistance in cultured pyrethroid- and organophosphate-resistant H. azteca populations. Escondido Creek and Mosher Slough H. azteca populations both maintained high tolerances for bifenthrin due to L925I and I936F amino acid substitutions. Resistance was also found for chlorpyrifos in the Escondido Creek and Mosher Slough populations with lower genotype frequencies of the G119S substitution, indicating that additional factors may be responsible for organophosphate resistance in this study. Mosher Slough H. azteca were moderately resistant to DDT, and Escondido Creek and Mosher Slough H. azteca were moderately resistant to carbaryl, suggesting cross-resistance. No differences were observed in acute toxicity values across the three populations of H. azteca for fipronil and imidacloprid, and this is possibly due to the lack of exposure to toxic concentrations of these insecticides in the field and lack of similar modes of action to pyrethroids and organophosphates. Resistance is known to be associated with fitness costs that can place insecticide-resistant populations at risk for decline through decreased survival and reduced fecundity.

Cross-resistance and biochemical mechanism in an insecticide-resistant population of Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae) and its parasitizing efficiency against invasive fall armyworm Spodoptera frugiperda (J.E. Smith)

Cross-resistance and biochemical mechanism in an insecticide-resistant population of Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae) and its parasitizing efficiency against invasive fall armyworm Spodoptera frugiperda (J.E. Smith)

ANALYSIS OF INSECTICIDE SUSCEPTIBILITY DATA FOR DENGUE VECTORS USING INSECTICIDE RESISTANCE MANAGEMENT SYSTEM (IRMS)

Laboratory testing generates data, and data analysis contributes to the production of results that aid in problem resolution. As more samples are tested, more data is generated, and the complexity of data management increases. Surveillance monitoring and real-time assessment are crucial in managing insecticide resistance; consequently, manual management of insecticide resistance data requires digital transformation. The Insecticide Resistance Management System (IRMS) was developed in this study to analyse and manage 244 insecticide susceptibility data. This system permits immediate analysis following the entry of laboratory data. Red dots dominated green dots on a map displayed in IRMS depicting the prevalence of insecticide-resistant mosquito populations in both Aedes species. In addition, the heatmap revealed that the majority of Aedes aegypti were resistant to all tested insecticides during all monitoring cycles. This system circumvents the time-consuming limitations of manual data analysis by enabling rapid result visualisation and report generation.

Multi-centre discriminating concentration determination of broflanilide and potential for cross-resistance to other public health insecticides in Anopheles vector populations

Novel insecticides are urgently needed to control insecticide-resistant populations of Anopheles malaria vectors. Broflanilide acts as a non-competitive antagonist of the gamma-aminobutyric acid receptor and has shown prolonged effectiveness as an indoor residual spraying product (VECTRON T500) in experimental hut trials against pyrethroid-resistant vector populations. This multi-centre study expanded upon initial discriminating concentration testing of broflanilide, using six Anopheles insectary colonies (An. gambiae Kisumu KCMUCo, An. gambiae Kisumu NIMR, An. arabiensis KGB, An. arabiensis SENN, An. coluzzii N’Gousso and An. stephensi SK), representing major malaria vector species, to facilitate prospective susceptibility monitoring of this new insecticide; and investigated the potential for cross-resistance to broflanilide via the A296S mutation associated with dieldrin resistance (rdl). Across all vector species tested, the discriminating concentration for broflanilide ranged between LC99 × 2 = 1.126–54.00 μg/ml or LC95 × 3 = 0.7437–17.82 μg/ml. Lower concentrations of broflanilide were required to induce complete mortality of An. arabiensis SENN (dieldrin-resistant), compared to its susceptible counterpart, An. arabiensis KGB, and there was no association between the presence of the rdl mechanism of resistance and survival in broflanilide bioassays, demonstrating a lack of cross-resistance to broflanilide. Study findings provide a benchmark for broflanilide susceptibility monitoring as part of ongoing VECTRON T500 community trials in Tanzania and Benin.

Integrating Trap Cropping and Entomopathogenic Nematode Foliar Sprays to Manage Diamondback Moth and Imported Cabbage Worm

Diamondback moth (DBM), Plutella xylostella, and imported cabbage worm (ICW), Pieris rapae, are destructive pests of crucifers worldwide. Although several insecticides are effective against ICW, pesticide management against DBM is challenged by insecticide resistant populations. The objective of this study was to explore the potential of integrating foliar sprays of the entomopathogenic nematode (EPN) Steinernema feltiae with trap cropping using kai choi (Brassica juncea) planted as an intercrop for the management of DBM and ICW. Four 2 × 2 (trap crop × EPN) factorial designed field trials were conducted with 2 trials on head cabbage (Brassica oleraceae var capitata) and 2 on kale (Brassica oleraceae var acephala). In the first head cabbage trial, trap cropping reduced DBM abundance by 46% and ICW abundance by 73%. Leaf damage by DBM and ICW were reduced by 45% and 33%, respectively. In the second head cabbage trial, DBM populations were reduced by 19% whereas ICW was reduced by 65%. No effects were observed on leaf damage. Trap cropping suppressed DBM abundance by 50% and DBM leaf damage by 19% in the first kale trial. No significant effects were observed on ICW. In the second kale trial, trap cropping reduced ICW leaf damage by 13%. In the first head cabbage trial, adding EPN foliar sprays further reduced DBM populations in plots with trap crops and ICW in plots without trap crops. In the second kale trial, EPNs suppressed DBM populations entirely. No effects from EPNs were observed in the second head cabbage trial or the first kale trial. It is concluded that trap cropping with kai choi did not improve the efficacy of EPN foliar sprays consistently. EPNs were most successful at suppressing DBM and ICW populations when the average pest pressure was below 0.5/plant whereas trap crops worked more effectively at insect populations above 0.5/plant. Although the use of trap cropping reduced pest abundance and leaf damage, the weight of head cabbage and kale was lower when planted 30 cm or closer to kai choi plants. This was resolved by leaving a distance of 60 cm between cash and trap crops. With further optimization, the use of trap cropping and EPN foliar sprays can be beneficial to an integrated pest management program to control DBM and ICW in cruciferous crops.

Field screening of wild cotton, Gossypium hirsutum, landraces for resistance to thrips (Thysanoptera: Thripidae)

Thrips (Thysanoptera) are the most prevalent early season pests of cotton and are often detrimental to the establishing crop. Heavy reliance on prophylactic insecticides, primarily used as seed treatments, has led to insecticide resistance in populations of Frankliniella fusca Hinds. Sustainable, alternative management tactics are needed to protect seedling cotton from thrips. We evaluated day-neutral, exotic landraces (N = 164) of Upland cotton (Gossypium hirsutum L.) for thrips-resistance traits in three field trials using insecticide-treated and -untreated plots. The cotton landraces were chosen for their diverse backgrounds, unique genotypes, and day-neutrality, allowing them to easily cross with USA breeding lines. The trials were conducted at the North Carolina State University Sandhills Research Station in Jackson Springs, NC (NC2018), and the Clemson University Pee Dee Research and Education Center in Florence, SC (SC2018 and SC2020). A selection index was created to quantify thrips resistance based on densities of thrips and ratings of injury by thrips at the first and third true-leaf stages in untreated plots, and genotypic differences in above-ground dry biomass between treated and untreated plots at 42 days after planting. Subsamples of adult thrips were collected for species identification. Dominant thrips species were F. fusca in NC2018 and SC2020 and Frankliniella tritici in SC2018. Eight putatively thrips-resistant genotypes (TX-1109, TX-1975, TX-2320, TX-2383, TX-101, TX-2347, TX-2362, TX-251) and four putatively thrips-susceptible genotypes (TX-203, TX-1212, TX-1094, TX-2403-2) were identified in the field trials. Further investigation of the putatively thrips-resistant and -susceptible genotypes should focus on mechanisms of thrips resistance to enhance future breeding efforts.

Modeling the Resistance Evolution to Insecticides Driven by Lepidopteran Species Competition in Cotton, Soybean, and Corn Crops.

Simple SummaryLepidopteran species commonly interact in the same niches in multiple crops. Interspecific competition has been neglected as a pressure selection agent in insecticide resistance studies. Our results showed that competition may act as an agent to speed up the evolution of diamide resistance in Spodoptera frugiperda and Helicoverpa armigera.Intra- and interspecific competition is considered a fundamental phenomenon in ecology. It acts as one of the most powerful selective forces that drives ecological diversity, the spatiotemporal distribution of organisms, fitness, and evolutionary aspects. Spodoptera frugiperda and Helicoverpa armigera are devastating pests and can co-occur in systems consisting of multiple agricultural crops and compete for food resources. Insecticide resistance in populations of these species has been a major threat to the sustainability of agroecosystems. No study to date has shown the effect of intra- and interspecific competition as a selective pressure agent on the evolution of insecticide resistance in lepidopteran pests in an experimental and theoretical way. Our study developed a parameterized computational model with experimental results for S. frugiperda and H. armigera competition. We simulated the behavior of heterozygous individuals with a competition capacity 100% equal to homozygous individuals resistant (100 RR) or susceptible to insecticides (00 RR), and intermediate between them (50 RR). Competition involving strains of these insect species can accelerate the evolution of their resistance to insecticides in agricultural crops. We found that competitive processes can result in a high probability of competitive exclusion for individuals with the susceptibility allele of these lepidopteran species. The results of this study are of paramount importance for understanding the impact of ecological factor competition on the evolution of insecticide resistance in lepidopteran pests, which until now has been neglected in these types of evolutionary dynamics studies.

Spatial distribution of insecticide resistant populations of Aedes aegypti and Ae. albopictus and first detection of V410L mutation in Ae. aegypti from Cameroon

BackgroundDengue (DENV), chikungunya (CHIKV) and Zika virus (ZIKV), are mosquito-borne viruses of medical importance in most tropical and subtropical regions. Vector control, primarily through insecticides, remains the primary method to prevent their transmission. Here, we evaluated insecticide resistance profiles and identified important underlying resistance mechanisms in populations of Aedes aegypti and Ae. albopictus from six different regions in Cameroon to pesticides commonly used during military and civilian public health vector control operations.MethodsAedes mosquitoes were sampled as larvae or pupae between August 2020 and July 2021 in six locations across Cameroon and reared until the next generation, G1. Ae. aegypti and Ae. albopictus adults from G1 were tested following World Health Organization (WHO) recommendations and Ae. aegypti G0 adults screened with real time melting curve qPCR analyses to genotype the F1534C, V1016I and V410L Aedes kdr mutations. Piperonyl butoxide (PBO) assays and real time qPCR were carried out from some cytochrome p450 genes known to be involved in metabolic resistance. Statistical analyses were performed using Chi-square test and generalized linear models.ResultsLoss of susceptibility was observed to all insecticides tested. Mortality rates from tests with 0.25% permethrin varied from 24.27 to 85.89% in Ae. aegypti and from 17.35% to 68.08% in Ae. albopictus. Mortality rates for 0.03% deltamethrin were between 23.30% and 88.20% in Ae. aegypti and between 69.47 and 84.11% in Ae. albopictus. We found a moderate level of resistance against bendiocarb, with mortality rates ranging from 69.31% to 90.26% in Ae. aegypti and from 86.75 to 98.95% in Ae. albopictus. With PBO pre-exposure, we found partial or fully restored susceptibility to pyrethroids and bendiocarb. The genes Cyp9M6F88/87 and Cyp9J10 were overexpressed in Ae. aegypti populations from Douala sites resistant to permethrin and deltamethrin. Cyp6P12 was highly expressed in alphacypermethrin and permethrin resistant Ae. albopictus samples. F1534C and V1016I mutations were detected in A. aegypti mosquitoes and for the first time V410L was reported in Cameroon.ConclusionsThis study revealed that Ae. aegypti and Ae. albopictus are resistant to multiple insecticide classes with multiple resistance mechanisms implicated. These findings could guide insecticide use to control arbovirus vectors in Cameroon.

Rapid detection of kdr mutation F1534C in Aedes aegypti using recombinase polymerase amplification and lateral flow dipsticks

Insecticide resistance monitoring is essential in assessing the efficacy of vector control measures. However, gold standard PCR-based molecular analyses for insecticide resistance detection are often hindered by time-consuming sample processing, as well as considerable infrastructure and resourcing requirements. In this study, we combined a novel one-step sample preparation reagent with a rapid isothermal molecular test that detects a knock down resistance (kdr) mutation (F1534C) that enables pyrethroid resistance in Aedes aegypti mosquitoes. We trialled the rapid F1534C pyrethroid resistance test using insecticide resistant Ae. aegypti mosquito bodies and compared results to a conventional, allele-specific quantitative PCR (AS-qPCR) coupled with melt curve genotyping in corresponding mosquito heads. From a strain of Ae. aegypti established from an insecticide resistant population in Merida, Mexico (n = 27), all the mosquito bodies (n = 27) tested positive with the rapid F1534C test regardless of whether they were homozygous or heterozygous. To assess diagnostic test specificity, we confirmed that F1534 was not detected in laboratory-reared, fully susceptible Ae. aegypti mosquito bodies (n = 28) using the rapid F1534C test or the conventional AS-qPCR melt curve analysis. All corresponding mosquito heads (n = 28) were homozygous wild-type FF1534. The rapid F1534C test thus demonstrated 100% diagnostic sensitivity (95% CI: 87.23% to 100%) and 100% diagnostic specificity (95% CI: 87.66% to 100.00%) for detection of the F1534C pyrethroid resistant single nucleotide polymorphism (SNP) in both heterozygous and homozygous Ae. aegypti. In the collection of mutant mosquitoes from Mexico, CC1534 homozygous mutants occurred at a frequency of 74.1% (n = 20) and FC heterozygous mutants at a frequency of 25.9% (n = 7). The rapid F1534C test significantly reduced the sample processing and testing time from approximately 6 h for the AS-qPCR melt curve analysis to only 25 min. These results demonstrate significant potential for our approach to resistance testing as a field-based, low-resource, rapid alternative to time-consuming and expensive laboratory-based detection.

Functional characterization of CYP6G4 from the house fly in propoxur metabolism and resistance

The house fly (Musca domestica L.) (Diptera: Muscidae) is a global vector that can transmit >250 human and animal diseases. The control of house flies has heavily relied on the application of various chemical insecticides. The carbamate insecticide propoxur has been widely used for the control of house flies, and resistance to propoxur has been documented in many house fly populations worldwide. Previous studies have identified several propoxur resistance-conferring mutations in the target protein acetylcholinesterase; however, the molecular basis for metabolic resistance to propoxur remains unknown. In this study, we investigated the involvement of CYP6G4, a cytochrome P450 overexpressed in many insecticide resistant populations of Musca domestica, in propoxur metabolism and resistance by using combined approaches of recombinant protein-based insecticide metabolism and the Drosophila GAL4/UAS transgenic system. The recombinant CYP6G4 and its redox partners (NADPH-dependent cytochrome P450 reductase and cytochrome b5) were functionally expressed in Escherichia coli. Metabolism experiments showed that CYP6G4 was able to transform propoxur with a turnover rate of around 0.79 min−1. Six metabolites were putatively identified, suggesting that CYP6G4 could metabolize propoxur via hydroxylation, O-depropylation and N-demethylation. Moreover, bioassay results showed that ectopic overexpression of CYP6G4 in fruit flies significantly increased their tolerance to propoxur. Our in vivo and in vitro data convincingly demonstrate that CYP6G4 contributes to propoxur metabolism and resistance.