Susceptible Laboratory Strain Research Articles

Functional characterization of cytochrome P450s associated with pyrethroid resistance in the olive fruit fly Bactrocera oleae

Resistance to pyrethroid insecticides has evolved in Bactrocera oleae populations in Greece, threatening the efficacy of control interventions based on this insecticide class. Here we report the collection of populations from Crete, with resistance levels reaching up to 132-folds, compared to susceptible laboratory strains and show that pyrethroid resistance is substantially suppressed by the PBO synergist, suggesting the involvement of detoxification enzymes. To identify specific candidate genes implicated in resistance, we performed comparative transcriptomic analysis, between the pyrethroid resistant populations from Crete and the susceptible laboratory strains, using both whole bodies and Malpighian tubules. Several genes were found differentially transcribed between resistant and susceptible flies in each comparison, with P450s being among the most highly over-expressed detoxification genes in pyrethroid resistant populations. Four of the over-expressed P450s (Cyp6A61, Cyp6G6, Cyp4P6 and Cyp6G28) were recombinantly expressed in Escherichia coli and in vitro metabolism assays revealed that CYP6A61 is capable of metabolizing alpha-cypermethrin, while CYP6G6, CYP4P6 and CYP6G28 are capable of metabolizing deltamethrin. No metabolism of neonicotinoid insecticides was recorded. We further silenced CYP6G6 in vivo, via RNAi, which led to a small, but significant increase in deltamethrin toxicity. The study provides valuable information towards the development of molecular diagnostics and evidence-based insecticide resistance management strategies.

The sublethal and transgenerational effects of pyridaben on fitness and induced tolerance in Panonychus citri

Panonychus citri is a dominant citrus pest mite in the world. Pesticide-induced population resurgence is a concern for environmental and resistance risk assessment in mite control. To evaluate the effective mite management strategies, the sublethal and transgenerational effects of pyridaben on P. citri were assessed using life-table parameters. Two segregated pyridaben-resistant and susceptible strains (previously obtained from the crossing between laboratory susceptible strain and pyridaben-resistant population) were used in our study. After exposure to LC30 pyridaben, the fecundity of Pyr_Control and Pyr_Rs strains were both significantly reduced in the parental generation (F0). Life-table results showed that LC30 pyridaben significantly induced the fecundity of both strains in F1 generation (egg stage was prolonged as well). Interestingly, this effect also stimulated the fecundity of F2 generation in Pyr_Control strain. But no significant effects were observed in Pyr_Rs strain. While detoxification enzyme assays indicated that only the activity of P450s was significantly activated by LC30 pyridaben in Pyr_Control and Pyr_Rs strains (1.242- fold and 1.355-fold increase, respectively). The expressions of P450 gene (CYP4CL2) and reproduction-related gene (Pc_Vg) were determined and the results showed that the expression of Pc_Vg of pyridaben treated mites in F0 generation was significantly reduced in both strains. In Pyr_Control and Pyr_Rs strains, the significant upregulation CYP4CL2 (2.029-fold and 2.516-fold increase, respectively) and PcVg (1.771-fold and 2.924-fold increase, respectively) of F1 generation suggested there were delayed hormesis effects on reproduction and developed tolerance to pyridaben. No significant difference was found between LC30 treatments and controls in F2 generation of both strains. These results provided evidence that transgenerational hormesis effects of low concentration of pyridaben might lead to mite population outbreak and resistance risk in the field through stimulating the reproduction and developing tolerance to pyridaben. Furthermore, LC30 pyridaben could affect the offspring populations of susceptible strain in the longer-term as compared to resistant strain.

Selection for resistance to pirimiphos-methyl, permethrin and spinosad in a field strain of Sitophilus oryzae: resistance risk assessment, cross-resistance potential and synergism of insecticides.

Indiscriminate use of insecticides in food storage facilities for controlling insect pests has deleterious effects on the environment and non-targeted organisms in the premises. Continuous use of insecticides may result in resistance development in insect pests, which compel the stakeholders to increase the dosage of insecticides to manage resistant insect pests. The increased dosage of insecticides ultimately may result in contamination of stored food stuff that affects human health. The present study was planned to generate data that will be helpful to delay resistance development and to reduce environmental pollution. A field strain of Sitophilus oryzae, one of the most common insect pests of stored foodstuff, was selected separately with pirimiphos-methyl, permethrin, or spinosad for five consecutive generations. The selected strains were studied for resistance risk assessment, time taken to develop resistance to insecticides after continuous exposure in the selection process, preliminary mechanism of resistance, and whether the development of resistance due to the selection with a particular insecticide could develop cross-resistance to other insecticide or not. In comparison to a laboratory susceptible reference strain, the insecticide-selected strains revealed rapid development of resistance against insecticides as a result of selection process: 31.05-fold resistance to pirimiphos-methyl, 156.49-fold resistance to permethrin, and 65.6-fold resistance to spinosad. The selected strains did not show cross-resistance to insecticides to with these strains were not exposed during selection experiments, i.e., strain selected with pirimiphos-methyl did not show cross-resistance to spinosad and permethrin. In the synergism bioassays, the synergists (S,S,S-tributyl phosphorotrithioate and piperonyl butoxide) significantly reduced resistance of the selected strain against insecticides to with these were selected, revealing the probability of metabolic mechanism of resistance. The present study revealed high risks of resistance development to pirimiphos-methyl, spinosad, and permethrin under consistent selection pressure. Lack of cross-resistance among insecticides provides an opportunity to use insecticides in rotation instead of increasing dosages to manage resistant insects that will ultimately pollute the environment.

Resistance to Multiple Insecticide Classes in the Vinegar Fly Drosophila melanogaster (Diptera: Drosophilidae) in Michigan Vineyards.

Vinegar flies are vectors of pathogens causing fruit rots of grapes, so control of these insects is important for preventing vineyard yield loss. Recent outbreaks of sour rots may be linked to greater challenges controlling vinegar flies, so we investigated the insecticide susceptibility of populations collected from commercial vineyards across Michigan. We first determined the discriminating concentration for phosmet, malathion, methomyl, and zeta-cypermethrin using a laboratory susceptible (Canton-S) strain of D. melanogaster females. The discriminating concentrations were determined as 252.08, 2.58, 0.96, and 1.68 ppm of the four insecticides, respectively. These concentrations were first tested in 2020 against populations from the two major counties for grape production. In 2021, we expanded monitoring to twenty-three populations collected from vineyards across six counties. All populations had significantly lower sensitivity to all four insecticides compared with Canton-S strain, with up to 98.8% lower mortality for phosmet. The LC50, LC90, and LC99 values of the four insecticides for the two populations tested in 2020 were 7-1,157-fold higher than the Canton-S strain. For the twenty-three populations collected in 2021, mortality ranged from 56.3 to 100% when the flies were screened using a 10x concentration of the discriminating concentration of the insecticides, whereas it ranged from 82.4 to 100% when the flies were screened using a 20x concentration. Our results suggest variable levels of resistance to insecticides from multiple chemical classes in D. melanogaster populations in Michigan vineyards, highlighting the need to implement integrated sour rot management approaches that are less dependent on insecticides for control of this species.

Lack of fitness cost and inheritance of resistance to abamectin based on the establishment of a near-isogenic strain of Tetranychus urticae

Many populations of the two-spotted spider mite, Tetranychus urticae Koch, have developed high levels of resistance to the pesticide abamectin in China and other countries. This study developed a near-isogenic line to understand better the inheritance, cross-resistance, and fitness costs associated with abamectin resistance in the field population of T. urticae in China. We introduced the trait that confers extremely high abamectin resistance in a field-collected population of T. urticae into a susceptible laboratory strain (IPP-SS) to generate an abamectin-resistant near-isogenic line (NIL-Aba). This process was carried out through multiple backcrossing to IPP-SS and via parthenogenesis and abamectin screening. Compared with IPP-SS, the NIL-Aba strain had a 25 147-fold resistance to abamectin and a high level of cross-resistance to bifenthrin (288.17-fold), an intermediate level to emamectin benzoate (42.57-fold), and low levels to bifenazate, chlorfenapyr, cyflumetofen, cyenopyrafen, and cyetpyrafen with resistance ranging from 3.18- to 9.31-fold. But it had no cross-resistance to profenofos. The resistance to abamectin in NIL-Aba was autosomal, incompletely dominant, and polygenic. Based on two sex life table parameters, no fitness cost was found in NIL-Aba. Establishing the NIL-Aba strain provides a reliable basis for an in-depth study of abamectin resistance in T. urticae. New information on toxicological characteristics and fitness cost should facilitate the management of abamectin resistance in field populations of T. urticae.

Formulation of abamectin and plant oil-based nanoemulsions with efficacy against the two-spotted spider mite Tetranychus urticae (Acari: Tetranychidae) under laboratory and field conditions

Due to the harmful effects of synthetic chemical acaricides on ecosystems and human health, there is increasing interest in the use of nanotechnology to fabricate eco-friendly nanoemulsions based on plant oils in the field of spider mite control. In this study, nanoemulsions of abamectin, garlic, and neem oils were prepared by a high-energy approach and characterized by transmission electron microscopy. The droplet sizes of all tested nanoemulsions were less than 100 nm. The acaricidal activities of the prepared nanoemulsions compared to abamectin were evaluated against a susceptible laboratory strain of Tetranychus urticae Koch (Acari: Tetranychidae) under laboratory and field conditions. The results showed that abamectin nanoemulsion was the most toxic compound against adult females of T. urticae followed by abamectin emulsion. Neem nanoemulsion had moderate toxicity and garlic nanoemulsion had the lowest toxicity. The effects of tested compound residues on egg deposition and egg hatching in descending order were as follows: abamectin nanoemulsion > abamectin emulsion > neem oil nanoemulsion < garlic oil nanoemulsion. In the field experiment, all tested compounds were effective in reducing the population density of T. urticae in the motile stage, with mean reductions ranging between 66.08% and 95.24% for all compounds. The most effective compound was abamectin nanoemulsion. The results of the present study demonstrate that nanoemulsion enhanced the biological activity of abamectin. Further, neem and garlic oil nanoemulsions have potential utility as environmentally friendly acaricides in integrated pest management programs.

Resistance risk assessment in diamondback moth, Plutella xylostella (L.) to fluxametamide

The diamondback moth Plutella xylostella (L.) (Lepidoptera: Plutellidae) is a devastating pest that infests several cruciferous vegetable crops worldwide and has developed insecticide resistance in most chemical classes. Fluxametamide is a novel neuroactive isoxazoline compound that binds to a unique site on the γ-aminobutyric acid (GABA) receptor and has an exceptional insecticidal activity on a range of lepidopteran caterpillars. The present study aimed to assess the risk of resistance in P. xylostella to fluxametamide under laboratory conditions. After the repeated exposure to fluxametamide for successive 18 generations of P. xylostella, there was 3.095-fold increase in the LC50. When the realized heritability (h2) of P. xylostella to fluxametamide in the open field was assumed to be the laboratory-estimated value (h2 = 0.180) and the mortality was 70–90%, only 22.9–33.1 generations were expected to be required to obtain a 10-fold increase in fluxametamide resistance. Compared with the laboratory susceptible strain (F0), the Flux-SEL (F18) strain exhibited a very low level of cross-resistance to emamectin benzoate (5.17-fold) and no significant cross-resistance to fipronil or flubendiamide. With the F0 and the Flux-SEL test strains (F12, F15 and F18), the specific activities of cytochrome P450 and glutathione S-transferase showed a remarkable increase during selection, while the esterase activity did not differ significantly. The results indicated that a potential risk of resistance development to fluxametamide exists in P. xylostella after continuous exposure. These pieces of information will be crucial in formulating rational fluxametamide application and resistance management guidelines for controlling P. xylostella under field condition.

Microbe-mediated activation of indoxacarb in German cockroach (Blattella germanica L.)

The German cockroach (Blattella germanica L.) is a major urban pest worldwide and is notorious for its ability to detoxify and resist insecticides. German cockroaches have generalist feeding habits that expose them to a range of potential hazardous substances and host a wide variety of unique microbial species, which may potentially facilitate unique detoxification capabilities. Since field German cockroach populations are routinely exposed to both bait and spray insecticide treatments, we hypothesized whether these unique gut microbes might play roles in toxicological processes of the host insect. The goals of this research were to understand the metabolic processes inside the German cockroach gut after treatment with kanamycin, a broad-ranging antibiotic, and indoxacarb, an oxadiazine pro-insecticide used in cockroach bait products. In these experiments, two resistant cockroach strains were obtained from field populations in Danville, IL and compared to a susceptible laboratory strain that had no previous exposure to insecticides (J-wax). Roaches provided kanamycin-infused water had lower median mortality to indoxacarb compared to the control treatment in feeding bioassays regardless of strain, but in vial (surface contact) bioassays, only susceptible cockroaches experienced a shift in mortality apparently due to their greater susceptibility. When frass extracts of indoxacarb-fed cockroaches were analyzed, less of the active, hydrolytic metabolite DCJW (N-decarbomethoxyllated JW062) was produced relative to the parent compound indoxacarb with the antibiotic KAN. This result was further corroborated by hydrolase activity assays of whole homogenized cockroach guts. Taken together these results provide novel evidence of microbe-mediated pro-insecticide activation in the cockroach gut.

Toxicities and Cross-Resistance of Imidacloprid, Acetamiprid, Emamectin Benzoate, Spirotetramat, and Indoxacarb in Field Populations of Culex quinquefasciatus (Diptera: Culicidae).

Simple SummaryCulex quinquefasciatus is a major vector of several human and animal diseases. This species’ ability to develop resistance to synthetic insecticides can lead to failure to control it. Therefore, we conducted toxicity bioassays of imidacloprid, acetamiprid, emamectin benzoate, spirotetramat, and indoxacarb on five field populations of Cx. Quinquefasciatus from Pakistan. These five populations showed susceptibility to high resistance against imidacloprid, susceptibility to moderate resistance against acetamiprid, susceptibility to emamectin benzoate, susceptibility to spirotetramat, and low–high resistance against indoxacarb. Correlation analyses revealed a significant positive correlation between imidacloprid, acetamiprid, and spirotetramat median lethal concentration values, indicating the possibility of cross-resistance. Meanwhile, there were no significant correlations among the median lethal concentration values of other tested insecticides, indicating the possible absence of cross-resistance. Our findings provide a useful background for the public health authorities, medical entomologists, and pest managers to control Cx. quinquefasciatus.Culex quinquefasciatus is a major vector of several pathogens and is capable of breeding in various aquatic habitats. The extensive and injudicious use of synthetic chemicals against the mosquito species has led to the problem of insecticide resistance. To explore this resistance in detail, toxicity bioassays of imidacloprid, acetamiprid, emamectin benzoate, spirotetramat, and indoxacarb were performed on five Cx. quinquefasciatus field populations from Pakistan in addition to a laboratory susceptible strain. Compared with the susceptible strain, results for the five Cx. quinquefasciatus field populations were as follows: susceptibility to high resistance against imidacloprid (resistance ratio (RR): 0.09–11.18), susceptibility to moderate resistance against acetamiprid (RR: 0.39–8.00), susceptibility to emamectin benzoate (RR: 0.002–0.020), susceptibility to spirotetramat (RR: 0.01–0.07), and low to high resistance against indoxacarb (RR: 3.00–118.00). Correlation analyses revealed a significant positive correlation between imidacloprid, acetamiprid, and spirotetramat median lethal concentration (LC50) values, indicating the possibility of cross-resistance. In contrast, there were no significant correlations between the LC50 values of other tested insecticides, indicating the possible absence of cross-resistance. These results can assist public health authorities, medical entomologists, and pest managers to manage the insecticide resistance of Cx. quinquefasciatus as well as the associated pollution and human health issues.

Role of social aggregation in the fitness cost of pyrethroid‐resistant German cockroaches

AbstractLiving in aggregations likely gives fitness benefits to resistant German cockroaches. We first investigated the trade‐offs in life‐history traits between six field pyrethroid‐resistant German cockroach strains and a laboratory susceptible strain (SUS‐1). Second, each strain was placed in groups of 2, 10, 20, 30 and 40 individuals with an equal sex ratio in each group to investigate the effect of social aggregation on fitness and overall population parameters. All resistant strains exhibited reduced nymphal survival (50%–83%) and a prolonged development period in both male (49–63 days) and female (47–62 days) nymphs when compared to SUS‐1 individuals (nymphal development period: 39 ± 2 days for male and 40 ± 3 days for female). Such fitness costs were minimized when the resistant cockroaches were in aggregations. Specifically, male (54 ± 3 days) and female nymphal development periods (52 ± 3 days) of German cockroach significantly decreased to 44 ± 1 days in the largest group. The nymphal survival was substantially enhanced (more than 80% survival) in German cockroaches with elevated cytochrome P450 monooxygenases, or both cytochrome P450 monooxygenases and esterases activities in the largest group. The effect of pyrethroid resistance on the life‐history traits of adult German cockroaches was limited; only adult male longevity was significantly prolonged. Group living did not significantly extend adult longevity and increase the number of offspring per ootheca. Contrary to expectations, the number of nymphs produced by a female and the oothecal hatchability were inversely proportional to the number of individuals in aggregations. Moreover, a lower proportion of viable oothecae were produced in German cockroaches when living in aggregations. Because of low fecundity, population parameters of German cockroaches living in aggregations (net reproductive rate and intrinsic rate of increase) were significantly lower than the parameters of those living in pairs.

The Cross-Resistance Pattern and the Metabolic Resistance Mechanism of Acetamiprid in the Brown Planthopper, Nilaparvata lugens (Stål).

Acetamiprid is widely used in paddy fields for controlling Nilaparvata lugens (Stål). However, the risk of resistance development, the cross-resistance pattern and the resistance mechanism of acetamiprid in this pest remain unclear. In this study, an acetamiprid-resistant strain (AC-R) was originated from a field strain (UNSEL) through successive selection with acetamiprid for 30 generations, which reached 60.0-fold resistance when compared with a laboratory susceptible strain (AC-S). The AC-R strain (G30) exhibited cross-resistance to thiamethoxam (25.6-fold), nitenpyram (21.4-fold), imidacloprid (14.6-fold), cycloxaprid (11.8-fold), dinotefuran (8.7-fold), sulfoxaflor (7.6-fold) and isoprocarb (8.22-fold), while there was no cross-resistance to etofenprox, buprofezin and chlorpyrifos. Acetamiprid was synergized by the inhibitor piperonyl butoxide (2.2-fold) and the activity of cytochrome P450 monooxygenase was significantly higher in the AC-R strain compared with the AC-S strain, suggesting the critical role of P450. The gene expression results showed that the P450 gene CYP6ER1 was significantly overexpressed in AC-R compared with the AC-S and UNSEL strains. In addition, the RNA interference (RNAi) of CYP6ER1 significantly increased the susceptibility of AC-R to acetamiprid. Molecular docking predicted that acetamiprid and CYP6ER1 had close binding sites, and the nitrogen atoms had hydrogen bond interactions with CYP6ER1. These results demonstrated that the overexpression of CYP6ER1 contributed to acetamiprid resistance in N. lugens.

An experimental hut study evaluating the impact of pyrethroid-only and PBO nets alone and in combination with pirimiphos-methyl-based IRS in Ethiopia

BackgroundPyrethroid resistance observed in populations of malaria vectors is widespread in Ethiopia and could potentially compromise the effectiveness of insecticide-based malaria vector control interventions. In this study, the impact of combining indoor residual spraying (IRS) and insecticide-treated nets (ITNs) on mosquito behaviour and mortality was evaluated using experimental huts.MethodsA Latin Square Design was employed using six experimental huts to collect entomological data. Human volunteers slept in huts with different types of nets (pyrethroid-only net, PBO net, and untreated net) either with or without IRS (Actellic 300CS). The hut with no IRS and an untreated net served as a negative control. The study was conducted for a total of 54 nights. Both alive and dead mosquitoes were collected from inside nets, in the central rooms and verandah the following morning. Data were analysed using Stata/SE 14.0 software package (College Station, TX, USA).ResultsThe personal protection rate of huts with PermaNet® 2.0 alone and PermaNet® 3.0 alone was 33.3% and 50%, respectively. The mean killing effect of huts with PermaNet® 2.0 alone and PermaNet® 3.0 alone was 2% and 49%, respectively. Huts with PermaNet® 2.0 alone and PermaNet® 3.0 alone demonstrated significantly higher excito-repellency than the control hut. However, mosquito mortality in the hut with IRS + untreated net, hut with IRS + PermaNet® 2.0 and hut with IRS + PermaNet® 3.0 were not significantly different from each other (p > 0.05). Additionally, pre-exposure of both the susceptible Anopheles arabiensis laboratory strain and wild Anopheles gambiae sensu lato to PBO in the cone bioassay tests of Actellic 300CS sprayed surfaces did not reduce mosquito mortality when compared to mortality without pre-exposure to PBO.ConclusionMosquito mortality rates from the huts with IRS alone were similar to mosquito mortality rates from the huts with the combination of vector control intervention tools (IRS + ITNs) and mosquito mortality rates from huts with PBO nets alone were significantly higher than huts with pyrethroid-only nets. The findings of this study help inform studies to be conducted under field condition for decision-making for future selection of cost-effective vector control intervention tools.Graphical

Genetic mapping of a bioethanol yeast strain reveals new targets for hydroxymethylfurfural- and thermotolerance

Current technology that enables bioethanol production from agricultural biomass imposes harsh conditions for Saccharomyces cerevisiae’s metabolism. In this work, the genetic architecture of industrial bioethanol yeast strain SA-1 was evaluated. SA-1 segregant FMY097 was previously described as highly aldehyde resistant and here also as thermotolerant: two important traits for the second-generation industry. A Quantitative Trait Loci (QTL) mapping of 5-hydroxymethylfurfural (HMF) -resistant segregants of hybrid FMY097/BY4742 disclosed a region in chromosome II bearing alleles with uncommon non-synonymous (NS) single nucleotide polymorphisms (SNPs) in FMY097: MIX23, PKC1, SEA4, and SRO77. Allele swap to susceptible laboratory strain BY4742 revealed that SEA4FMY097 enhances robustness towards HMF, but the industrial fitness could not be fully recovered. The genetic network arising from the causative genes in the QTL window suggests that intracellular signaling TOR (Target of Rapamycin) and CWI (Cell Wall Integrity) pathways are regulators of this phenotype in FMY097. Because the QTL mapping did not result in one major allelic contribution to the evaluated trait, a background effect in FMY097′s HMF resistance is expected. Quantification of NADPH - cofactor implied in endogenous aldehyde detoxification reactions - supports the former hypothesis, given its high availability in FMY097. Regarding thermotolerance, SEA4FMY097 grants BY4742 ability to grow in temperatures as high as 38 ºC in liquid, while allele PKC1FMY097 allows growth up to 40 ºC in solid medium. Both SEA4FMY097 and PKC1FMY097 encode rare NS SNPs, not found in other > 1013S. cerevisiae. Altogether, these findings point towards crucial membrane and stress mediators for yeast robustness.

An impact assessment of insecticides application on the non-targeted mosquito Aedes albopictus (Skuse) in Punjab rice fields, Pakistan.

Insecticidal control of insect pests of rice crop may influence the environment and nontarget species in rice fields. Aedes albopictus, one of the most common nontarget species present in rice fields, received lethal and sublethal exposures to insecticides used in rice cultivated fields. The present work explores the effects of insecticides in six non-targeted Ae. albopictus strains collected from rice fields with a history of insecticidal usage in comparison with a laboratory susceptible reference strain (REF) and a strain (LHR) collected from a rice field with no, or minimal, history of insecticidal usage. Two types of effects, the resistance development and performance of biological traits, were studied by selecting seven commonly used insecticides in rice fields in Punjab, Pakistan. The results revealed that the strains collected from the rice fields with histories of insecticidal usage exhibited significant levels of resistance to flonicamid, chlorantraniliprole, gamma-cyhalothrin, fipronil, monomehypo, triazophos, and carbofuran, when compared with REF and LHR strains. In addition, Ae. albopictus strains revealed a significantly weaker performance of biological traits (rate of pupae formation, survival of male and female adults (except females of OKR and MTN strains), and ovipositing females) than those of the REF and LHR strains. However, the fecundity of all field strains was only significantly different with that of the REF strain. In conclusion, the results highlight the problem of the negative effects of insecticidal usage in rice fields on nontarget species present in the same environment, and emphasize the need to adopt pest management activities that are safe for the environment.

Reduced performance of community bednets against pyrethroid-resistant Anopheles funestus and Anopheles gambiae, major malaria vectors in Cameroon

BackgroundLong-lasting insecticidal nets (LLINs) are a vital tool in the fight against malaria vectors. However, their efficacy in the field can be impacted by several factors, including patterns of usage, net age, mosquito resistance and the delayed mortality effect, all of which could influence malaria transmission. We have investigated the effectiveness of the various brands of LLINs available in markets and households in Cameroon on pyrethroid-resistant mosquitoes and assessed their post-exposure effect.MethodsFollowing quality control assessment on a susceptible laboratory mosquito strain, we evaluated the immediate and delayed mortality effects of exposure to LLINs (both newly bough LLINst and used ones collected from households in Elende village, Cameroon, in 2019) using standard WHO cone tests on Anopheles gambiae and Anopheles funestus populations collected from the Centre region of Cameroon. Alive female mosquitoes were genotyped for various resistance markers at different time points post-exposure to evaluate the impact of insecticide resistance on the efficacy of bednets.ResultsThe laboratory-susceptible strain experienced high mortality rates when exposed to all pyrethroid-only brands of purchased nets (Olyset® Net, Super Net, PermaNet® 2.0, Yorkool®, Royal Sentry®) (Mean±SEM: 68.66 ± 8.35% to 93.33 ± 2.90%). However, low mortality was observed among wild An. funestus mosquitoes exposed to the bednets (0 ± 0 to 28 ± 6.7%), indicating a reduced performance of these nets against field mosquitoes. Bednets collected from households also showed reduced efficacy on the laboratory strain (mortality: 19–66%), as well as displaying a significant loss of efficacy against the local wild strains (mortality: 0 ± 0% to 4 ± 2.6% for An. gambiae sensu lato and 0 ± 0% to 8 ± 3.2% for An. funestus). However, compared to the unexposed group, mosquitoes exposed to bednets showed a significantly reduced longevity, indicating that the efficacy of these nets was not completely lost. Mosquitoes with the CYP6P9a-RR and L119F-GSTe2 mutations conferring pyrethroid resistance showed greater longevity after exposure to the Olyset net than their susceptible counterparts, indicating the impact of resistance on bednet efficacy and delayed mortality.ConclusionThese findings show that although standard bednets drastically lose their efficacy against pyrethroid-resistant field mosquitoes, they still are able to induce delayed mortality in exposed populations. The results of this study also provide evidence of the actual impact of resistance on the quality and efficacy of LLINs in use in the community, with mosquitoes carrying the CYP6P9a-RR and L119F-GSTe2 mutations conferring pyrethroid resistance living longer than their susceptible counterparts. These results highlight the need to use new-generation nets that do not rely solely on pyrethroids.Graphical

Resistance in Spodoptera litura (F.) to Insecticides and Detoxification Enzymes

Standard leaf dip bioassay experiments were conducted to study the insecticide resistance in Spodoptera litura (F.) in soybean. Chlorantraniliprole was the most toxic while, cypermethrin and organophosphates (profenophos and triazophos) recorded least toxicity against S. litura populations. Field population of S. litura showed the highest resistance to cypermethrin with resistance ratios (RRs) ranging from 244 to 376. The field populations collected from three locations of Southern Rajasthan exhibited low resistance to newer insecticides, chlorantraniliprole and spinetoram with Lab-SS strain. All the enzyme activities were significantly higher in the whole larval extracts and midgut extracts of S. litura larvae when compared to the Lab-SS strain. Analysis of the carboxyl esterase enzyme in the native PAGE assay revealed that the electrophoretic profiles showed presence of different band regions (EST 1 to EST 7) with esterase activity in the field populations along with the laboratory susceptible strain. The susceptibility of three field populations to different group of insecticides along with the profile of detoxification enzymes indicate the need to formulate a region-specific insecticide resistance management (IRM).

Determination of the Discriminating Concentration Towards Permethrin for Surveying Resistance in Amblyomma americanum.

Amblyomma americanum Linnaeus (Ixodida: Ixodidae) is ubiquitously present throughout the southeastern United States and is capable of vectoring several pathogens. White-tailed deer are the main host for adult A. americanum. However, this tick species is a generalist that will feed on most vertebrates, including humans, deer, livestock, and pets. Management of this species can be challenging due to a lack of cost-effective strategies. Acaricides are often utilized, however, this may lead to pesticide resistance. The Food and Agriculture Organization of the United Nations (FAO) larval packet test (LPT) was performed on susceptible A. americanum to determine the lethal concentration (LC) and discriminating concentration (DC) values for permethrin. The FAO LPT was used at these pre-established values to compare levels of resistance in ticks collected from a captive deer farm and wild areas representing high and low permethrin exposure settings, respectively. Resistance ratios (RR) calculated from the LC values for the ticks collected from farmed and wild deer ranged between 1 and 2. A. americanum collected from farmed and wild deer were not found to be resistant, however, some samples had slightly elevated RRs as compared to the susceptible laboratory strain, which may suggest tolerance development. Although the A. americanum sampled in this study were not resistant to permethrin, the DC calculated in this study will allow for rapid evaluation of resistance in a permethrin resistance monitoring program such that alternate management strategies can be adopted if resistance is detected.

Durability of three types of dual active ingredient long-lasting insecticidal net compared to a pyrethroid-only LLIN in Tanzania: methodology for a prospective cohort study nested in a cluster randomized controlled trial

BackgroundProgress achieved by long-lasting insecticidal nets (LLINs) against malaria is threatened by widespread selection of pyrethroid resistance among vector populations. LLINs with non-pyrethroid insecticides are urgently needed. This study aims to assess the insecticide and textile durability of three classes of dual-active ingredient (A.I.) LLINs using techniques derived from established WHO LLIN testing methods to set new standards of evaluation.MethodsA WHO Phase 3 active ingredients and textile durability study will be carried out within a cluster randomized controlled trial in 40 clusters in Misungwi district, Tanzania. The following treatments will be evaluated: (1) Interceptor®G2 combining chlorfenapyr and the pyrethroid alpha-cypermethrin, (2) Royal Guard® treated with pyriproxyfen and alpha-cypermethrin, (3) Olyset™ Plus which incorporates a synergist piperonyl butoxide and the pyrethroid permethrin, and (4) a reference standard alpha-cypermethrin only LLIN (Interceptor®). 750 nets will be followed in 5 clusters per intervention arm at 6, 12, 24 and 36 months post distribution for survivorship and hole index assessment. A second cohort of 1950 nets per net type will be identified in 10 clusters, of which 30 LLINs will be withdrawn for bio-efficacy and chemical analysis every 6 months up to 36 months and another 30 collected for experimental hut trials every year. Bio-efficacy will be assessed using cone bioassays and tunnel tests against susceptible and resistant laboratory strains of Anopheles gambiae sensu stricto. Efficacy of field-collected nets will be compared in six experimental huts. The main outcomes will be Anopheles mortality up to 72 h post exposure, blood feeding and egg maturation using ovary dissection to assess impact on fecundity.ConclusionsStudy findings will help develop bio-efficacy and physical durability criteria for partner A.I., in relation to the cRCT epidemiological and entomological outcomes, and refine preferred product characteristics of each class of LLIN. If suitable, the bioassay and hut outcomes will be fitted to transmission models to estimate correlation with cRCT outcomes.Trial registration number: NCT03554616.

Insecticide resistance in Australian Spodoptera frugiperda (J.E. Smith) and development of testing procedures for resistance surveillance.

Spodoptera frugiperda (J.E. Smith) is a highly invasive noctuid pest first reported in northern Australia during early 2020. To document current status of resistance in S. frugiperda in Australia, insecticide toxicity was tested in field populations collected during the first year of establishment, between March 2020 and March 2021. Dose-response was measured by larval bioassay in 11 populations of S. frugiperda and a susceptible laboratory strain of Helicoverpa armigera. Emamectin benzoate was the most efficacious insecticide (LC50 0.023μg/ml) followed by chlorantraniliprole (LC50 0.055μg/ml), spinetoram (LC50 0.098μg/ml), spinosad (LC50 0.526μg/ml), and methoxyfenozide (1.413μg/ml). Indoxacarb was the least toxic selective insecticide on S. frugiperda (LC50 3.789μg/ml). Emamectin benzoate, chlorantraniliprole and methoxyfenozide were 2- to 7-fold less toxic on S. frugiperda compared with H. armigera while spinosyns were equally toxic on both species. Indoxacarb was 28-fold less toxic on S. frugiperda compared with H. armigera. There was decreased sensitivity to Group 1 insecticides and synthetic pyrethroids in S. frugiperda compared with H. armigera: toxicity was reduced up to 11-fold for methomyl, 56 to 199-fold for cyhalothrin, and 44 to 132-fold for alpha cypermethrin. Synergism bioassays with metabolic inhibitors suggest involvement of mixed function oxidase in pyrethroid resistance. Recommended diagnostic doses for emamectin benzoate, chlorantraniliprole, spinetoram, spinosad, methoxyfenozide and indoxacarb are 0.19, 1.0, 0.75, 6, 12 and 48μg/μl, respectively.

Emerging Mosquito Resistance to Piperonyl Butoxide-Synergized Pyrethroid Insecticide and Its Mechanism.

Piperonyl butoxide (PBO)-synergized pyrethroid products are widely available for the control of pyrethroid-resistant mosquitoes. To date, no study has examined mosquito resistance after pre-exposure to PBO and subsequent enzymatic activity when exposed to PBO-synergized insecticides. We used Culex quinquefasciatus Say (Diptera: Culicidae), an important vector of arboviruses and lymphatic filariasis, as a model to examine the insecticide resistance mechanisms of mosquitoes to PBO-synergized pyrethroid using modified World Health Organization tube bioassays and biochemical analysis of metabolic enzyme expressions pre- and post-PBO exposure. Mosquito eggs and larvae were collected from three cities in Orange County in July 2020 and reared in insectary, and F0 adults were used in this study. A JHB susceptible strain was used as a control. Mosquito mortalities and metabolic enzyme expressions were examined in mosquitoes with/without pre-exposure to different PBO concentrations and exposure durations. Except for malathion, wild strain Cx quinquefasciatus mosquitoes were resistant to all insecticides tested, including PBO-synergized pyrethroids (mortality range 3.7 ± 4.7% to 66.7 ± 7.7%). Wild strain mosquitoes had elevated levels of carboxylesterase (COE, 3.8-fold) and monooxygenase (P450, 2.1-fold) but not glutathione S-transferase (GST) compared to susceptible mosquitoes. When wild strain mosquitoes were pre-exposed to 4% PBO, the 50% lethal concentration of deltamethrin was reduced from 0.22% to 0.10%, compared to 0.02% for a susceptible strain. The knockdown resistance gene mutation (L1014F) rate was 62% in wild strain mosquitoes. PBO pre-exposure suppressed P450 enzyme expression levels by 25~34% and GST by 11%, but had no impact on COE enzyme expression. Even with an optimal PBO concentration (7%) and exposure duration (3h), wild strain mosquitoes had significantly higher P450 enzyme expression levels after PBO exposure compared to the susceptible laboratory strain. These results further demonstrate other studies that PBO alone may not be enough to control highly pyrethroid-resistant mosquitoes due to multiple resistance mechanisms. Mosquito resistance to PBO-synergized insecticide should be closely monitored through a routine resistance management program for effective control of mosquitoes and the pathogens they transmit.