Pyrethroids Permethrin Research Articles

Insecticide resistance status of malaria vectors in the malaria endemic states of India: implications and way forward for malaria elimination

BackgroundIn 2012, the World Health Organization (WHO) released the Global Plan for Insecticide Resistance Management in malaria vectors to stress the need to address insecticide resistance. In a prospective multi-centric study commissioned by the Indian Council of Medical Research (ICMR), we assessed the insecticide susceptibility status of the primary malaria vectors in India from 2017 through 2019. MethodsThe insecticide susceptibility status of the prevalent primary malaria vectors – An. culicifacies, An. fluviatilis, An. stephensi, An. minimus and An. baimaii and secondary malaria vectors - An. aconitus, An. annularis and An. philippinensis/nivepes from 328 villages in 79 districts of 15 states of India were assessed following the WHO method mainly to insecticides used in vector control, organochlorine (DDT), organophosphate (malathion), and other pyrethroids (alpha-cypermethrin, cyfluthrin, lambda-cyhalothrin and permethrin). The study sites were selected as suggested by the National Vector Borne Disease Control Programme. ResultsThe primary malaria vector An. culicifacies showed resistance to DDT (50/50 districts including two districts of Northeastern India), malathion (27/44 districts), and deltamethrin (17/44 districts). This species was resistant to DDT alone in 19 districts, double resistant to DDT-malathion in 16 districts, double resistant to DDT-deltamethrin in 6 districts, and triple resistant to DDT-malathion-deltamethrin in 9 districts. An. minimus and An. baimaii were susceptible in Northeastern India while An. fluviatilis and the secondary malaria vector An. annularis was resistant to DDT in Jharkhand. ConclusionIn this study we report that among the primary vectors An. culicifacies is predominantly resistant to multiple insecticides. Our data suggest that periodic monitoring of insecticide susceptibility is vital. The national malaria program can take proactive steps for insecticide resistance management to continue its push toward malaria elimination in India.

Deep eutectic solvent as the acceptor phase in three-phase hollow fiber liquid-phase microextraction for the determination of pyrethroid insecticides from environmental water samples prior to HPLC.

In this study, a deep eutectic solvent as the acceptor phase was applied in three-phase hollow fiber liquid-phase microextraction for the microextraction of two pyrethroids (permethrin as well as deltamethrin) from environmental water samples prior to HPLC-UV. A deep eutectic solvent was synthesized of tetrabutylammonium bromide-decanoic acid (in a ratio of 1:2) as an acceptor phase and 1-decanol was applied as a supported liquid membrane. Some main variables affecting the extraction recoveries, comprising the types/contents of extraction solvent and acceptor phase, stirring speed, sample phase pH and extraction time, were checked and optimized. Under optimal conditions, the detection limits and limits of quantitation determined were 0.09-0.12 and 0.29-0.39 μgl-1 for deltamethrin and permethrin, respectively. The enrichment factors were 627 and 613, while the relative standard deviations (n= 5) were 4.8 and 5.7%, for deltamethrin and permethrin, respectively. The created technique was assessed as satisfactory to ascertain the two pyrethroid poisons (permethrin and deltamethrin) in environmental water samples.

Insecticide Susceptibility Status of Lab and Field Populations of Aedes albopictus from Gainesville, Florida, to Organophosphates and Pyrethroids.

Previous unpublished results from 2 open-field studies in South Florida evaluated the effectiveness of 4 ultra-low volume malathion formulations, applied by ground application against a USDA (Gainesville) laboratory-colonized strain of Aedes albopictus. Field results of the bioassays indicated that the species was highly resistant to malathion compared with other insecticide-susceptible mosquito species concurrently tested. Unfortunately, when the Ae. albopictus colony was initially started in 2003 from a field collection in Gainesville, FL, susceptibility to insecticides was not determined. Since that time, this species has been in constant production without exposure to pesticides by the Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL. In order to determine its susceptibility level to malathion and compare it with 3 current field-collected Ae. albopictus populations, 3 organophosphates (malathion, chlorpyrifos, naled) and 2 pyrethroids (permethrin, deltamethrin) were tested in the laboratory, using the Centers for Disease Control and Prevention bottle bioassays. Results of the study confirmed that the USDA-Gainesville Ae. albopictus was still highly resistant to malathion even after being maintained in the laboratory for 16 years without exposure to pesticides. The 3 field-collected populations were also resistant to malathion but slightly less so than the USDA-Gainesville population. Rotation of pyrethroid products 16 years ago did not result in any significant reduction of malathion resistance in the field populations. Neither the USDA-Gainesville nor the field-collected Ae. albopictus populations showed cross-resistance to chlorpyrifos and naled. Lower mortalities were observed in the 10-min diagnostic time from the 3 field-collected populations against permethrin but rose significantly at 15-min exposure. Resistance was not detected for deltamethrin in any of the 4 Gainesville Ae. albopictus populations.

Sequential phase I metabolism of pyrethroids by duplicated CYP6P9 variants results in the loss of the terminal benzene moiety and determines resistance in the malaria mosquito Anopheles funestus

Pyrethroid resistance in Anopheles funestus is threatening the eradication of malaria. One of the major drivers of pyrethroid resistance in An. funestus are cytochrome P450 monooxygenases CYP6P9a and CYP6P9b, which are found upregulated in resistant An. funestus populations from Sub-Saharan Africa and are known to metabolise pyrethroids. Here, we have functionally expressed CYP6P9a and CYP6P9b variants and investigated their interactions with azole-fungicides and pyrethroids. Some azole fungicides such as prochloraz inhibited CYP6P9a and CYP6P9b at nanomolar concentrations, whereas pyrethroids were weak inhibitors (>100 μM). Amino acid sequence comparisons suggested that a valine to isoleucine substitution at position 310 in the active site cavity of CYP6P9a and CYP6P9b, respectively, might affect substrate binding and metabolism. We therefore swapped the residues by site directed mutagenesis to produce CYP6P9aI310V and CYP6P9bV310I. CYP6P9bV310I produced stronger metabolic activity towards coumarin substrates and pyrethroids, particularly permethrin. The V310I mutation was previously also detected in a pyrethroid resistant field population of An. funestus in Benin. Additionally, we found the first metabolite of permethrin and deltamethrin after hydroxylation, 4′OH permethrin and 4′OH deltamethrin, were also suitable substrates for CYP6P9-variants, and were depleted by both enzymes to a higher extent than as their respective parent compounds (approximately 20% more active). Further, we found that both metabolites were toxic against An. funestus FANG (pyrethroid susceptible) but not towards FUMOZ-R (pyrethroid resistant) mosquitoes, the latter suggesting detoxification by overexpressed CYP6P9a and CYP6P9b. We confirmed by mass-spectrometric analysis that CYP6P9a and CYP6P9b are capable of cleaving phenoxybenzyl-ethers in type I pyrethroid permethrin and type II pyrethroid deltamethrin and that both enzymes preferentially metabolise trans-permethrin. This provides new insight into the metabolism of pyrethroids and a greater understanding of the molecular mechanisms of pyrethroid resistance in An. funestus.

Molecular Drivers of Multiple and Elevated Resistance to Insecticides in a Population of the Malaria Vector Anopheles gambiae in Agriculture Hotspot of West Cameroon.

(1) Background: Malaria remains a global public health problem. Unfortunately, the resistance of malaria vectors to commonly used insecticides threatens disease control and elimination efforts. Field mosquitoes have been shown to survive upon exposure to high insecticide concentrations. The molecular mechanisms driving this pronounced resistance remain poorly understood. Here, we elucidated the pattern of resistance escalation in the main malaria vector Anopheles gambiae in a pesticide-driven agricultural hotspot in Cameroon and its impact on vector control tools; (2) Methods: Larval stages and indoor blood-fed female mosquitoes (F0) were collected in Mangoum in May and November and forced to lay eggs; the emerged mosquitoes were used for WHO tube, synergist and cone tests. Molecular identification was performed using SINE PCR, whereas TaqMan-based PCR was used for genotyping of L1014F/S and N1575Y kdr and the G119S-ACE1 resistance markers. The transcription profile of candidate resistance genes was performed using qRT-PCR methods. Characterization of the breeding water and soil from Mangoum was achieved using the HPLC technique; (3) Results: An. gambiae s.s. was the only species in Mangoum with 4.10% infection with Plasmodium. These mosquitoes were resistant to all the four classes of insecticides with mortality rates <7% for pyrethroids and DDT and <54% for carbamates and organophophates. This population also exhibited high resistance intensity to pyrethroids (permethrin, alpha-cypermethrin and deltamethrin) after exposure to 5× and 10× discriminating doses. Synergist assays with PBO revealed only a partial recovery of susceptibility to permethrin, alpha-cypermethrin and deltamethrin. Only PBO-based nets (Olyset plus and permaNet 3.0) and Royal Guard showed an optimal efficacy. A high amount of alpha-cypermethrin was detected in breeding sites (5.16-fold LOD) suggesting ongoing selection from agricultural pesticides. The 1014F-kdr allele was fixed (100%) whereas the 1575Y-kdr (37.5%) and the 119S Ace-1R (51.1%) were moderately present. Elevated expression of P450s, respectively, in permethrin and deltamethrin resistant mosquitoes [CYP6M2 (10 and 34-fold), CYP6Z1(17 and 29-fold), CYP6Z2 (13 and 65-fold), CYP9K1 (13 and 87-fold)] supports their role in the observed resistance besides other mechanisms including chemosensory genes as SAP1 (28 and 13-fold), SAP2 (5 and 5-fold), SAP3 (24 and 8-fold) and cuticular genes as CYP4G16 (6 and 8-fold) and CYP4G17 (5 and 27-fold). However, these candidate genes were not associated with resistance escalation as the expression levels did not differ significantly between 1×, 5× and 10× surviving mosquitoes; (4) Conclusions: Intensive and multiple resistance is being selected in malaria vectors from a pesticide-based agricultural hotspot of Cameroon leading to loss in the efficacy of pyrethroid-only nets. Further studies are needed to decipher the molecular basis underlying such resistance escalation to better assess its impact on control interventions.

Maternal and fetal tissue distribution of α-cypermethrin and permethrin in pregnant CD-1 mice.

Pyrethroid insecticides are widely used throughout agriculture and household products. Recent studies suggest that prenatal exposure to these insecticides may adversely affect fetal development; however, little is known about the distribution of these chemicals in pregnant animals. The present study aimed to address this gap in knowledge by investigating the distribution of two commonly used pyrethroid insecticides, permethrin and α-cypermethrin, in maternal and fetal tissues of pregnant CD-1 mice. Dams were dosed from gestational days 6 to 16 via oral gavage with permethrin (1.5, 15, and 50 mg/kg), α-cypermethrin (0.3, 3, and 10 mg/kg), or corn oil vehicle. Pyrethroid levels were determined in gestational day 16 tissues collected 90 min after the final dose was administered. Across maternal tissues, levels of both pyrethroids were the highest in maternal ovaries, followed by liver and brain, respectively. In addition, levels of both pyrethroids in maternal tissues and placenta were significantly higher than those in the fetal body and amniotic fluid, suggesting that these compounds may exhibit low transfer across the mouse placenta. While additional toxicokinetic studies are needed to verify the time course of pyrethroids in the fetal compartment, these findings support investigation into indirect modes of action relevant to the effects of pyrethroids on mammalian fetal development.

Resistant status and intensity of resistance of Anopheles gambiae sensu lato to pyrethroid and organophosphate in Lafia, Nasarawa State Nigeria

Monitoring and understanding the trend and dynamics of insecticide resistance are key to devising efficient control strategies. This study was carried out to investigate the resistance status of Anopheles gambiae sl mosquitoes in Lafia, Nasarawa State Nigeria to three pyrethroid (deltamethrin, Alphacypermethrin and permethrin) with concentration of 12.5 µg/bottle each and an organophosphate (pirimiphose-methyl 20 µg/bottle). Larvae and pupae of Anopheles mosquito were sampled within the metropolis and reared to adults in the insectary of Nasarawa State University, Keffi Nasarawa State. Emerged adults of 2 to 5 days old and non-blood fed were tested for resistance using CDC bottle bioassay susceptibility protocol. Results showed that the local mosquitoes species were resistant to all the three pyrethroid (deltamethrin, Alphacypermethrin and permethrin) but showed possible resistant to organophosphate (pirimiphose-methyl) with mortalities of 88, 84, 54, and 95% respectively. Exposure to piperonyl butoxide (PBO) as a synergist resulted in the susceptibility (100 and 98%) of mosquitoes to deltamethrin and Alphacypermethrin respectively but resistant (81%) to permethrin. The intensity of resistance of the vector population to pyrethroid was high especially with permethrin. The mosquitoes were only susceptible (100%) to deltamethrin and Alphacypermethrin at X5 concentration and permethrin at X10 concentration. Insecticide based intervention strategy remain the principal vector control measure in malaria endemic countries like Nigeria. Based on the result of this study, future vector control programme should take into consideration the deployment of nets that are treated with alphacypermethrin or deltamethrin combined with PBO (PBO based nets) in the study area or increase the concentration of the pyrethoid insecticides used in this study on long lasting nets after their safety to human is assured.

Mitochondrial bioenergetics and redox dysfunction in nephrotoxicity induced by pyrethroid permethrin are ameliorated by flavonoid-rich fraction.

The present study was designed to evaluate in vitro and in vivo the potential anti-inflammatory and nephroprotective potential of ethyl acetate fraction extracted from Fumaria officinalis (EAF) against permethrin (PER). Male wistar rats were treated daily by gavage during 7days as follows: group C: negative control rats received 2mL/kg bw of corn oil, group EAF: positive control rats received EAF at a dose of 200mg/kg bw dissolved in water, group PER: rats received PER at a dose of 34.05mg/kg bw and group (PER + EAF): rats received PER (34.05mg/kg bw) and EAF (200mg/kg bw). In vitro study showed the ability of EAF to inhibit protein denaturation and heat-induced hemolysis confirming its anti-inflammatory activity. In vivo, PER treatment decreased calcium (Ca) and phosphorus (P) levels and increased lactate dehydrogenase (LDH) activity in plasma. It induced oxidative stress objectified by an increase in the lipid peroxidation and protein oxidation and a perturbation of antioxidant system in kidney and mitochondria. The activities of NADH-ubiquinone reductase, ubiquinol-cytochrome C reductase and cytochrome C oxidase activities were reduced. These alterations were confirmed by histopathological studies. Co-treatment with EAF improved the antioxidant status and mitochondrial bioenergetics. The nephroprotective effects of EAF could be attributed to its modulation of detoxification enzymes and/or free radical scavenging actions.

First evidence of pyrethroid resistance in Italian populations of West Nile virus vector Culex pipiens.

Culex pipiens (Linnaeus), one of the most abundant mosquito species in Europe, plays a crucial role in the endemic transmission of West Nile virus and caused the large outbreak with >1600 human cases in 2018. Although evidence of resistance to pyrethroids has been reported for Cx. pipiens populations from Spain and Greece, resistance monitoring has been largely neglected in Italy. Herein, we investigate susceptibility of Italian Cx. pipiens populations to the pyrethroids permethrin and deltamethrin. Results from WHO‐tube‐bioassays revealed mortalities ranging from 14–54%, indicating high levels of resistance, in four out of 10 populations exposed to permethrin (0.75%) and of 63% in one of three populations exposed to deltamethrin (0.05%). Reduced susceptibility (mortality<98%) was detected in almost all other populations. A clear association is shown between the resistant phenotype and the presence of kdr‐alleles in position 1014 of the VSSC, strongly suggesting its role in reducing susceptibility. The study provides the first evidence of pyrethroid‐resistance in Italian Cx. pipiens populations and reports levels of resistance paralleled in the European region only in Turkey. This highlights the urgent need to implement insecticide‐resistance management plans to restore the efficacy of the nowadays only chemical weapon available to control arbovirus transmission in Europe.

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.

Quantification and risk assessment of pyrethroid residues in seafood based on nanoparticle-extraction approach

Seafood is a good source of essential nutrients, but the presence of pyrethroid contaminants may pose risks to consumers' health and its intricate matrices obstruct chemical hazard detection. In this study, a method for detecting residues of four pyrethroids (cypermethrin, deltamethrin, permethrin, and bifenthrin) in fish and finfish was optimized and validated. The proposed method performed with low-temperature cleanup (−20 °C) followed by nanoparticle-extraction and HPLC-PDA. The critical parameters of the method's performance were studied to demonstrate the optimum conditions. The method was verified using mackerel fillet, and its application was extended to determine pyrethroid residues in commonly consumed seafood (salmon, sea bass, threadfin fish, tiger prawn, vannamei prawn, shrimp, squid, grand jackknife clam, and oyster) obtained from local supermarkets in Singapore. The proposed method provided satisfactory recoveries of 75.95–96.81% and achieved LOQs of 0.54–0.85 ng/g, presenting the accomplishment for pyrethroid determination in seafood. Among tested seafood samples, at least one of the target pyrethroids was monitored (0.28–11.28 ng/g); however, the residue was below the level with safety concern. Estimated hazard indexes (≤0.12%) from risk assessment indicate a negligible risk of exposure to four target pyrethroids via consumption of seafood in Singapore. • Five impacted parameters for nanoparticles extraction were optimized and validated. • The proposed method showed satisfactory recoveries and high sensitivity. • At least one pyrethroid was found in tested seafood samples. • HIs (≤0.12%) show a negligible risk of pyrethroids exposure from tested samples.

Covalent triazine frameworks/cobalt composites for magnetic solid phase extraction of pyrethroids from food samples followed by gas chromatography-flame ionization detection

Magnetic composite was synthesized by hydrazine hydrate reduction of cobalt ions on covalent triazine frameworks (CTFs). The CTFs/Co composites are chemically stable in organic solvents; the porous structure and magnetic property contribute to fast adsorption/desorption dynamics, phase separation and high extraction efficiency (52–67%) for interest pyrethroids. Accordingly, CTFs/Co composites were employed for the extraction and pre-concentration of five pyrethroids (bifenthrin, fenpropathrin, cypermethrin, permethrin and fenvalerate) from food samples, followed by gas chromatography (GC)-flame ionization detector (FID) measurement. Under the optimized conditions, the limits of detection for interest pyrethroids were 0.34–0.84 μg/L, with the enrichment factor of 156–201. The developed method was validated by analyzing quality control samples (apple juice, CFAPA-QC198A-1), fruit juice, honey and vegetables (recovery of 73.0–118% for spiked samples). This method is simple, sensitive, selective, environment friendly, and could be employed for other aromatic compounds analysis in food samples.

Characterization of resistance profile (intensity and mechanisms) of Anopheles gambiae in three communes of northern Benin, West Africa

BackgroundThe selection and the spread of insecticide resistance in malaria vectors to the main classes of insecticides used in vector control tools are a major and ongoing challenge to malaria vector control programmes. This study aimed to determine the intensity of vector resistance to insecticides in three regions of Benin with different agro-ecological characteristics.MethodsLarvae of Anopheles gambiae sensu lato (s.l.) were collected from September to November 2017 in different larval sites in three northern Benin communes: Parakou, Kandi and Malanville. Two to five-day-old, non-blood-fed, female mosquitoes were exposed to papers impregnated with deltamethrin, permethrin and bendiocarb at dosages of 1 × the diagnostic dose, 5 × and 10 × to determine the intensity of resistance in these vectors. Molecular frequencies of the kdr L1014F and ace-1R G119S insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled at each study site.ResultsResistance to pyrethroids (permethrin and deltamethrin) was recorded in all three communes with mortality rates below 60% using the diagnostic dose (1x). The results obtained after exposure of An. gambiae to permethrin 10 × were 99% in Kandi, 98% in Malanville and 99% in Parakou. With deltamethrin 10x, mortality rates were 100% in Kandi, 96% in Malanville and 73% in Parakou. For the diagnostic dose of bendiocarb, suspected resistance was recorded in the communes of Malanville (97%) and Kandi (94%) while sensitivity was observed in Parakou (98%).Using the 10 × dose, mortality was 98% in Kandi, 100% in Malanville and 99% in Parakou. The frequencies of the kdr L1014F allele varied between 59 and 83% depending on the sites and species of the An. gambiae complex, while the frequency of the ace-1R G119S gene varied between 0 and 5%. Biochemical tests showed high levels of oxidase and esterase activity compared to the susceptible colony strain of An. gambiae sensu stricto (Kisumu strain).ConclusionAnopheles gambiae showed a generalized loss of susceptibility to permethrin and deltamethrin but also showed moderate to high intensity of resistance in different regions of Benin. This high intensity of resistance is a potential threat to the effectiveness of vector control.

Susceptibility of Anopheles gambiae sensu lato to different insecticide classes and mechanisms involved in the South-North transect of Benin

Monitoring vector resistance to insecticides is a major component of resistance management. Adults of Anopheles gambiae s.l. from larvae collected at thirteen sites were tested with papers impregnated with bendiocarb, permethrin; deltamethrin; pirimiphos-methyl and induced bottles of insecticide including bendiocarb; deltamethrin, permethrin and pirimiphos-methyl. Molecular analyses were performed and detoxification enzyme levels were determined for each study site. This study revealed a generalized distribution of resistance of An. gambiae s.l. to pyrethroids (permethrin and deltamethrin) and a clear resistance to carbamates (bendiocarb) especially in the northern region of the country. This vector is still susceptible to organophosphates, especially pirimiphos-methyl, but resistance is a dynamic phenomenon and it would be necessary to monitor the susceptibility of these anopheles to pyrimiphos-methyl and to find alternative vector control methods to slow down the spread of resistance genes.

Knockdown Resistance (kdr) Mutations I1532T and F1534S Were Identified in Aedes albopictus Field Populations in Zhejiang Province, Central China.

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

Molecular bases of P450-mediated resistance to the neonicotinoid insecticide imidacloprid in the mosquito Ae. aegypti.

Resistance to chemical insecticides including pyrethroids, the main insecticide class used against mosquitoes, has re-kindled interest in the use of neonicotinoids. In this context, the present study aimed to characterize the molecular basis of neonicotinoid resistance in the mosquito Aedes aegypti. Resistance mechanisms were studied by combining transcriptomic and genomic data obtained from a laboratory strain selected at the larval stage after 30 generations of exposure to imidacloprid (Imida-R line). After thirty generations of selection, larvae of the Imida-R line showed an 8-fold increased resistance to imidacloprid and a significant cross-tolerance to the pyrethroids permethrin and deltamethrin. Cross-resistance to pyrethroids was only observed in adults when larvae were previously exposed to imidacloprid suggesting a low but inducible expression of resistance alleles at the adult stage. Resistance of the Imida-R line was associated with a slower larval development time in females. Multiple detoxification enzymes were over-transcribed in larvae in association with resistance including the P450s CYP6BB2, CYP9M9 and CYP6M11 previously associated with pyrethroid resistance. Some of them together with their redox partner NADPH P450 reductase were also affected by non-synonymous mutations associated with resistance. Combining genomic and transcriptomic data allowed identifying promoter variations associated with the up-regulation of CYP6BB2 in the resistant line. Overall, these data confirm the key role of P450s in neonicotinoid resistance in Ae. aegypti and their potential to confer cross-resistance to pyrethroids, raising concerns about the use of neonicotinoids for resistance management in this mosquito species.

The contribution of detoxification pathways to pyrethroid resistance in Hyalella azteca

Chronic exposure to pyrethroid insecticides can result in strong selective pressures on non-target species in aquatic systems and drive the evolution of resistance and population-level changes. Characterizing the underlying mechanisms of resistance is essential to better understanding the potential consequences of contaminant-driven microevolution. The current study found that multiple mechanisms enhance the overall tolerance of Hyalella azteca to the pyrethroid permethrin. In H. azteca containing mutations in the voltage-gated sodium channel (VGSC), both adaptation and acclimation played a role in mitigating the adverse effects of pyrethroid exposures. Pyrethroid resistance is primarily attributed to the heritable mutation at a single locus of the VGSC, resulting in reduced target-site sensitivity. However, additional pyrethroid tolerance was conferred through enhanced enzyme-mediated detoxification. Cytochrome P450 monooxygenases (CYP450) and general esterases (GE) significantly contributed to the detoxification of permethrin in H. azteca. Over time, VGSC mutated H. azteca retained most of their pyrethroid resistance, though there was some increased sensitivity from parent to offspring when reared in the absence of pyrethroid exposure. Permethrin median lethal concentrations (LC50s) declined from 1809 ng/L in parent (P0) individuals to 1123 ng/L in the first filial (F1) generation, and this reduction in tolerance was likely related to alterations in acclimation mechanisms, rather than changes to target-site sensitivity. Enzyme bioassays indicated decreased CYP450 and GE activity from P0 to F1, whereas the VGSC mutation was retained. The permethrin LC50s in resistant H. azteca were still two orders-of-magnitude higher than non-resistant populations indicating that the largest proportion of resistance was maintained through the inherited VGSC mutation. Thus, the noted variation in tolerance in H. azteca is likely associated with inducible traits controlling enzyme pathways. A better understanding of the mechanistic and genomic basis of acclimation is necessary to more accurately predict the ecological and evolutionary consequences of contaminant-driven change in H. azteca.

Genome-Wide Transcriptional Analysis and Functional Validation Linked a Cluster of Epsilon Glutathione S-Transferases with Insecticide Resistance in the Major Malaria Vector Anopheles funestus across Africa.

Resistance is threatening the effectiveness of insecticide-based interventions in use for malaria control. Pinpointing genes associated with resistance is crucial for evidence-based resistance management targeting the major malaria vectors. Here, a combination of RNA-seq based genome-wide transcriptional analysis and RNA-silencing in vivo functional validation were used to identify key insecticide resistance genes associated with DDT and DDT/permethrin cross-resistance across Africa. A cluster of glutathione-S-transferase from epsilon group were found to be overexpressed in resistant populations of Anopheles funestus across Africa including GSTe1 [Cameroon (fold change, FC: 2.54), Ghana (4.20), Malawi (2.51)], GSTe2 [Cameroon (4.47), Ghana (7.52), Malawi (2.13)], GSTe3 [Cameroon (2.49), Uganda (2.60)], GSTe4 in Ghana (3.47), GSTe5 [Ghana (2.94), Malawi (2.26)], GSTe6 [Cameroun (3.0), Ghana (3.11), Malawi (3.07), Uganda (3.78)] and GSTe7 (2.39) in Ghana. Validation of GSTe genes expression profiles by qPCR confirmed that the genes are differentially expressed across Africa with a greater overexpression in DDT-resistant mosquitoes. RNAi-based knock-down analyses supported that five GSTe genes are playing a major role in resistance to pyrethroids (permethrin and deltamethrin) and DDT in An. funestus, with a significant recovery of susceptibility observed when GSTe2, 3, 4, 5 and GSTe6 were silenced. These findings established that GSTe3, 4, 5 and 6 contribute to DDT resistance and should be further characterized to identify their specific genetic variants, to help design DNA-based diagnostic assays, as previously done for the 119F-GSTe2 mutation. This study highlights the role of GSTes in the development of resistance to insecticides in malaria vectors and calls for actions to mitigate this resistance.

Acute toxicity of typical ant control agents to the red imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae)

The red imported fire ant Solenopsis invicta Buren (Hymenoptera: Formicidae), is a serious invasive alien ant around the world and has expanded its invasive range to the Pacific Rim since the early 2000s. It was first reported in Japan in 2017, and its entry through cargo has been reported numerous times in many ports. Colonies have been found in Tokyo Port since 2019, and now it is an urgent issue to prevent further invasion and establishment. Chemical control is the best tested method of insect control, but we have little information on the efficacy of insecticides against S. invicta in Japan. Here, we conducted acute toxicity assays of six quick-acting pyrethroids (transfluthrin, prallethrin, phenothrin, permethrin, metofluthrin, and pyrethrin) for killing adults and five new-type insecticides (fipronil, thiamethoxam, indoxacarb, imidacloprid, and hydramethylnon) for controlling colonies with toxic baits. We found that the LD50 from six pyrethroids were comparable to each other. The ED50 causing abnormal behaviors were smaller than LD50, but some ants recovered from paralysis within 12 h. Fipronil showed the lowest LD50 suggesting this chemical is the most promising agent for controlling S. invicta. Our results promise to develop a method for the chemical control of S. invicta.

Pyrethroid bioaccumulation in field-collected insecticide-resistant Hyalella azteca.

Wild-type Hyalella azteca are highly sensitive to pyrethroid insecticides and typically do not survive exposure; however, pyrethroid bioaccumulation by insecticide-resistant H. azteca is an important potential risk factor for the transfer of pyrethroids to higher trophic species in aquatic systems. In the current study, four populations of pyrethroid-resistant H. azteca with corresponding sediment samples were sampled throughout the year, and nine-current use pyrethroids (tefluthrin, fenpropathrin, bifenthrin, cyhalothrin, permethrin, cyfluthrin, cypermethrin, esfenvalerate and deltamethrin) were measured. Bifenthrin was detected in every pyrethroid-resistant H. azteca tissue sample, up to 813 ng/g lipid, while cyhalothrin and permethrin were detected in fewer (18 and 28%, respectively) samples. Concurrent sampling of the sediment showed total pyrethroid concentrations exceeding toxic unit thresholds for non-resistant H. azteca survival, and confirmed the ubiquitous presence of bifenthrin at each site and sampling event. Bifenthrin concentrations in H. azteca tended to be higher in samples collected in winter months, and seasonal factors, such as temperature and rainfall, may have contributed to the noted differences in bioaccumulation. Finally, the bifenthrin and permethrin biota-sediment accumulation factors (BSAF) for pyrethroid-resistant H. azteca were similar to the BSAF values for less sensitive invertebrates, and therefore the development of resistance may enable an additional pathway for trophic transfer of pyrethroids in species that would otherwise be too sensitive to survive the exposure.