Toxicity of isocycloseram, an isoxazoline insecticide, against laboratory and field-collected German cockroaches (Blattodea: Ectobiidae).

Isocycloseram is a new insecticide in the isoxazoline class that targets insect GABA-gated chloride channels. In this study, we evaluated a cockroach gel bait formulation containing 1% isocycloseram against a susceptible strain (UCR) and 5 field-collected strains (WM, RG386, Ryan, CDR, and SY) of the German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae), and compared it with several commercial insecticide baits in the laboratory. Using the Ebeling choice box method, we also tested a residual deposit of an SC formulation of isocycloseram against the UCR, RG386, and Ryan strains. The isocycloseram bait was among the fastest-performing treatments against adult males (mean survival time: 0.9-2.7 days) and mixed stages and sexes (mean survival time: 1.4-5.4 days) across all strains. Secondary transfer effects of the bait were demonstrated in the UCR strain by exposing new adult males to individuals killed by direct bait treatment. Physiological resistance was not detected in the WM, CDR, and RG386 strains with topical treatment of a diagnostic dose (3× LD95) of isocycloseram developed using the UCR strain. However, topical assays revealed resistance ratios (RR50) of 1.6 and 3.0× in the Ryan and SY strains, respectively. The performance of a 0.05% isocycloseram residual application against the Ryan strain was improved with the addition of piperonyl butoxide.

Time of exposure and assessment influence the mortality induced by insecticides against metabolic resistant mosquitoes.

BACKGROUND: Increasing metabolic resistance in malaria vector mosquitoes resulted in the development of insecticide-treated nets (ITNs) with active ingredients (AI) that target them. Bioassays that accurately measure the mortality induced by these AIs on ITNs are needed. Mosquito metabolic enzyme expression follows a circadian rhythm. Thus, this study assessed (i) influence of the time of day of mosquito exposure and (ii) timing of assessment of mortality post exposure (24 and 72 h) to ITNs against vectors that are susceptible to pyrethroids and those with metabolic and knockdown resistance mechanisms. METHODS: Two cone bioassay experiments were conducted following World Health Organization (WHO) guidelines. Firstly, on ITNs incorporated with 2 g AI/kg of deltamethrin (DM) alone, or combined with 8 g AI/kg piperonyl butoxide (PBO) synergist, during the day (9:00-14:00 h) and repeated in the evening (18:00-20:00 h). This was followed by a confirmatory experiment during the afternoon (12:00-14:00 h) and repeated in the night (22:00-24:00 h) using mosquitoes unexposed or pre-exposed to PBO for 1 h before exposure to DM ITNs. Each net piece was tested with a minimum of eight cones per time (N = 24). The outcome was mortality after 24 h (M24) or 72 h (M72) of holding. RESULTS: The cone bioassays performed using metabolic resistant mosquitoes during the evening showed significantly lower M24 than those performed in the day for DM: odds ratio (OR) 0.14 [95% confidence interval (CI) 0.06-0.30, p < 0.0001] and DM PBO [OR 0.29 (95% CI 0.18-0.49, p < 0.0001). M72 was higher than M24 for metabolic resistant mosquitoes exposed to DM [OR 1.44 (95% CI 1.09-1.88), p = 0.009] and DM PBO [OR 1.82 (95% CI 1.42-2.34), p < 0.0001]. An influence of hour of experiment and time of assessment was not observed for mosquitoes that had knockdown resistance or that were pyrethroid-susceptible. CONCLUSIONS: Time of day of experiment and hour of assessment of delayed mortality after exposure of mosquitoes are important considerations in evaluating insecticides that interact with mosquito metabolism to counter metabolic resistant mosquitoes. This is important when evaluating field-aged ITNs that may have lower concentrations of AI.

Mechanisms of piperonyl butoxide cytotoxicity and its enhancement with imidacloprid and metals in Chinese hamster ovary cells.

The widespread use of chemicals and the presence of chemical and metal residues in various foods, beverages, and other consumables have raised concerns about the potential for enhanced toxicity. This study assessed the cytotoxic effects of Piperonyl butoxide (PBO) and its enhancement by combination with major contamination chemicals including Imidacloprid and metals, using different cytotoxic and genotoxic assays in Chinese hamster ovary (CHO) cells. PBO exhibited elevated cytotoxic effects in poly (ADP-ribose) polymerase (PARP) deficient CHO mutants but not in Glutathione S-transferase deficient CHO mutants. PBO cytotoxicity was enhanced by PARP inhibitor, Olaparib. PBO cytotoxicity was also enhanced with co-exposure to Imidacloprid, Lead Chloride, or Sodium Selenite. PBO induces γH2AX foci formation and apoptosis. The induction of DNA damage markers was elevated with PARP deficiency and co-exposure to Imidacloprid, Lead Chloride, or Sodium Selenite. Moreover, PBO triggers to form etch pits on plastic surfaces. These results revealed novel mechanisms of PBO cytotoxicity associated with PARP and synergistic effects with other environmental pollutants. The toxicological mechanisms underlying exposure to various combinations at different concentrations, including concentrations below the permitted limit of intake or the level of concern, require further study.

Biological activities of nitromethylene analogues of imidacloprid with a 4,5-dimethylated imidazolidine ring.

Four nitromethylene analogues of imidacloprid (CH-IMIs) having a 4,5-dimethylated (diMe) imidazolidine ring were stereospecifically synthesized to evaluate their affinity for the nicotinic acetylcholine receptors of the housefly Musca domestica. Among the analogues, the 4S,5R-diMe analogue showed the highest receptor affinity (Ki=0.39 nM). The insecticidal activity against M. domestica of the synthesized compounds was also measured under synergistic and nonsynergistic conditions. Under nonsynergistic conditions, the insecticidal activity of the 4S,5R-diMe analogue was the highest. The order of the insecticidal potency of the four diMe-CH-IMIs (4S,5R->4R,5S-=4R,5R->4S,5S-diMe analogues) was the same as that of the receptor affinity. Piperonyl butoxide (PBO) did not synergize with the test compounds, but both PBO and NIA16388 applications strengthened the activity of analogues other than the 4S,5S-diMe analogue. This suggests that the configuration of the substituents on the imidazolidine ring should influence the metabolism process of CH-IMI in houseflies.

Community evaluation of the physical and insecticidal durability of DuraNet® Plus, an alpha-cypermethrin and piperonyl butoxide incorporated mosquito net: protocol for a multi-country study in West, Central and East Africa.

BACKGROUND: Pyrethroid-PBO nets have demonstrated improved impact against clinical malaria transmitted by pyrethroid resistant mosquito vectors and are being scaled up across Africa. However very little is known about their physical and insecticidal durability under operational conditions. This study will investigate the attrition, fabric integrity, insecticide content and bioefficacy of DuraNet® Plus, a new WHO prequalified alphacypermethrin and PBO incorporated net developed by Shobikaa Impex Private Limited over 3 years of field use in communities in Benin, Cameroon and Tanzania. METHODS: The study will be conducted in parallel in selected villages in Zakpota District in Benin, Mbalmayo, District in Cameroon and Muheza District in Tanzania. In each country, ~ 1800 households will be recruited and randomised to receive DuraNet® Plus or DuraNet® (a WHO prequalified alphacypermethrin-only ITN). Follow up surveys will be performed at 1 month post distribution to investigate adverse events and subsequently every 6-12 months to assess ITN attrition and fabric integrity following standard WHO procedures. A second cohort of nets will be withdrawn every 6-12 months and assessed for alpha-cypermethrin and PBO content and for entomological activity in laboratory bioassays (cone bioassays and tunnel tests). Alpha-cypermethrin bioefficacy will be monitored using the susceptible Anopheles gambiae Kisumu strain in cone bioassays while PBO bioefficacy will be monitored using pyrethroid resistant strains with overexpressed P450 enzymes in tunnel tests to determine the proportion of efficacious nets (≥ 95% knockdown, ≥ 80% mortality or ≥ 90% blood feeding inhibition in tunnels) at each time point. Nets withdrawn at 12, 24 and 36 months from each country will also be tested in experimental hut trials against wild free-flying pyrethroid resistant Anopheles gambiae sl in Côvè Benin to investigate the superiority of DuraNet® Plus over DuraNet® at each time point under semi field conditions. CONCLUSION: This large-scale multi country trial will provide useful information on the durability of a pyrethroid-PBO net (DuraNet® Plus) in 3 different regions in sub-Saharan Africa. The methods proposed for bioefficacy testing could also contribute towards the development of new standardised guidelines for monitoring the insecticidal efficacy of pyrethroid-PBO nets under operational conditions.

Effectiveness of piperonyl butoxide and pyrethroid-treated long-lasting insecticidal nets (LLINs) versus pyrethroid-only LLINs with and without indoor residual spray against malaria infection: third year results of a cluster, randomised controlled, two-by-two factorial design trial in Tanzania.

BACKGROUND: After decades of success in reducing malaria through the scale-up of pyrethroid long-lasting insecticidal nets (LLINs), the decline in the malaria burden has stalled, coinciding with the rapid spread of pyrethroid resistance. In a previously reported study, nets treated with a pyrethroid and a synergist, piperonyl butoxide (PBO), demonstrated superior efficacy compared to standard pyrethroid LLINs (std-LLINs) against malaria. Evidence was used to support the public health recommendation of PBO-Pyrethroid-LLIN by the World Health Organization in 2018. This study looks at the third year of rollout of these nets in Muleba district, Tanzania to inform whether policy guidelines need to be updated. METHODS: A four-group cluster randomized trial (CRT) using a two-by-two factorial design was carried out between January 2014 and December 2017. A total of 48 clusters, were randomized in a 1:1:1:1 ratio to the following treatment groups, each intervention being provided once in 2015: 1/std-LLIN; 2/PBO-pyrethroid LLIN; 3/std-LLIN + Indoor Residual Spraying (IRS) and 4/PBO-Pyrethroid-LLIN + IRS. During the third year follow-up, malaria infection prevalence in 80 children per cluster, aged 6 months to 14 years, was measured at 28- and 33-months post-intervention and analysed as intention-to-treat (ITT) and per protocol (PP). Mosquito collections were performed monthly in all clusters, using CDC light traps in 7 randomly selected houses per cluster. RESULTS: At 28 and 33 months, study net usage among household participants was only 47% and 31%, respectively. In ITT analysis, after 28 months malaria infection prevalence among 7471 children was 80.9% in the two std-LLIN groups compared to 69.3% in the two PBO-Pyrethroid-LLIN (Odds Ratio: 0.45, 95% Confidence Interval: 0.21-0.95, p-value: 0.0364). After 33 months the effect was weaker in the ITT analysis (prevalence 59.6% versus 49.9%, OR: 0.60, 95%CI:0.32-1.13, p-value: 0.1131) but still evident in the PP analysis (57.2% versus 44.2%, OR: 0.34, 95%CI: 0.16-0.71, p-value: 0.0051). Mean number of Anopheles per night collected per house was similar between PBO-Pyrethroid-LLIN groups (5.48) and std-LLIN groups (5.24) during the third year. CONCLUSIONS: Despite low usage of PBO- Pyrethroid LLIN, a small impact of those nets on malaria infection prevalence was still observed in the 3rd year with the most protection offered to children still using them. To maximize impact, it is essential that net re-distribution cycles are aligned with this LLIN lifespan to maintain maximum coverage. TRIAL REGISTRATION: The trial was registered with ClinicalTrials.gov (registration number NCT02288637).

Pharmacokinetic analysis of acute and dietary exposure to piperonyl butoxide in the mouse.

Piperonyl butoxide (PBO) is a popular insecticide synergist present in thousands of commercial, agricultural, and household products. PBO inhibits cytochrome P450 activity, impairing the ability of insects to detoxify insecticides. PBO was recently discovered to also inhibit Sonic hedgehog signaling, a pathway required for embryonic development, and rodent studies have demonstrated the potential for in utero PBO exposure to cause structural malformations of the brain, face, and limbs, or more subtle neurodevelopmental abnormalities. The current understanding of the pharmacokinetics of PBO in mice is limited, particularly with respect to dosing paradigms associated with developmental toxicity. To establish a pharmacokinetic (PK) model for oral exposure, PBO was administered to female C57BL/6J mice acutely by oral gavage (22-1800 mg/kg) or via diet (0.09 % PBO in chow). Serum and adipose samples were collected, and PBO concentrations were determined by HPLC-MS/MS. The serum concentrations of PBO were best fit by a linear one-compartment model. PBO concentrations in visceral adipose tissue greatly exceeded those in serum. PBO concentrations in both serum and adipose tissue decreased quickly after cessation of dietary exposure. The elimination half-life of PBO in the mouse after gavage dosing was 6.5 h (90 % CI 4.7-9.5 h), and systemic oral clearance was 83.3 ± 20.5 mL/h. The bioavailability of PBO in chow was 41 % that of PBO delivered in olive oil by gavage. Establishment of this PK model provides a foundation for relating PBO concentrations that cause developmental toxicity in the rodent models to Sonic hedgehog signaling pathway inhibition.

LLIN Evaluation in Uganda Project (LLINEUP) - Plasmodium infection prevalence and genotypic markers of insecticide resistance in Anopheles vectors from 48 districts of Uganda.

Background: In 2017-2019, we conducted a large-scale, cluster-randomised trial (LLINEUP) to evaluate long-lasting insecticidal nets (LLINs) treated with a pyrethroid insecticide plus the synergist piperonyl butoxide (PBO LLINs), as compared to conventional, pyrethroid-only LLINs across 104 health sub-districts (HSDs) in Uganda. In LLINEUP, and similar trials in Tanzania, PBO LLINs were found to provide greater protection against malaria than conventional LLINs, reducing parasitaemia and vector density. In the LLINEUP trial, cross-sectional entomological surveys were carried out at baseline and then every 6 months for two years. In each survey, ten households per HSD were randomly selected for indoor household entomological collections. Results: Overall, 5395 female Anopheles mosquitoes were collected from 5046 households. The proportion of mosquitoes infected with Plasmodium falciparum did not change significantly over time, while infection with non-falciparum malaria decreased in An. gambiae s.s, but not An. funestus. The frequency of genetic markers associated with pyrethroid resistance increased significantly over time, but the rate of change was not different between the two LLIN types. The knock-down resistance (kdr) mutation Vgsc-995S declined over time as Vgsc-995F, the alternative resistance mutation at this codon, increased. Vgsc-995F appears to be spreading into Uganda. Conclusions: Distribution of LLINs in Uganda was associated with reductions in parasite prevalence and vector density, but the proportion of infective mosquitoes remained stable, suggesting that the potential for transmission persisted. The increased frequency of markers of pyrethroid resistance indicates that LLIN distribution favoured the evolution of resistance within local vectors and highlights the potential benefits of resistance management strategies.Trial registration:: This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395.

Can the performance of pyrethroid-chlorfenapyr nets be reduced when combined with pyrethroid-piperonyl butoxide (PBO) nets?

BACKGROUND: Pyrethroid-chlorfenapyr (CFP) and pyrethroid-piperonyl butoxide (PBO) nets are being scaled across endemic countries to improve control of malaria transmitted by pyrethroid-resistant mosquitoes. CFP is a pro-insecticide requiring activation by mosquito cytochrome P450 monooxygenase enzymes (P450s) while PBO improves pyrethroid potency by inhibiting the action of these enzymes in pyrethroid-resistant mosquitoes. The inhibitory action of PBO against P450s may thus reduce the efficacy of pyrethroid-CFP nets when applied inside the same household as pyrethroid-PBO nets. METHODS: Two experimental hut trials were performed to evaluate the entomological impact of two different types of pyrethroid-CFP ITN (Interceptor® G2, PermaNet® Dual) when applied alone and in combination with pyrethroid-PBO ITNs (DuraNet® Plus, PermaNet® 3.0) against a pyrethroid-resistant vector population in southern Benin. In both trials, all net types were tested as single and double net treatments. Bioassays were also performed to assess the resistance profile of the vector population at the hut site and investigate interactions between CFP and PBO. RESULTS: The vector population was susceptible to CFP but exhibited a high intensity of pyrethroid resistance that was overcame by PBO pre-exposure. Vector mortality was significantly lower in huts with combinations of pyrethroid-CFP nets plus pyrethroid-PBO nets compared to huts with two pyrethroid-CFP nets (74% vs. 85% for Interceptor® G2 and 57% vs. 83% for PermaNet® Dual, p < 0.001). PBO pre-exposure reduced the toxicity of CFP in bottle bioassays suggesting this effect may be partly attributable to antagonism between CFP and PBO. Higher levels of vector mortality were observed in huts with net combinations that included pyrethroid-CFP nets compared to those that did not and highest mortality was achieved when pyrethroid-CFP nets were applied alone as two nets together (83-85%). CONCLUSIONS: This study shows evidence of a reduced performance of pyrethroid-CFP nets when combined with pyrethroid-PBO ITNs compared to when applied alone and higher efficacy with net combinations that included pyrethroid-CFP nets. These findings suggest that in similar contexts, prioritizing distribution of pyrethroid-CFP nets over other net types would maximize vector control impact.

Simultaneous Determination of Pyrethrins, Pyrethroids, and Piperonyl Butoxide in Animal Feeds by Liquid Chromatography-Tandem Mass Spectrometry.

The presence of insecticides like pyrethrins and synthetic pyrethroids, combined with the synergist piperonyl butoxide, in animal feeds can pose a risk to both animal and human health by contaminating the food chain. In this study, a simple and fast method was developed for the simultaneous determination of these compounds in contaminated animal feeds using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample preparation was carried out using a QuEChERS-based approach, and the method was validated with acceptable accuracy ranging from 84 to 115% and precision below 10%. The limit of detection (LOD) and limit of quantification (LOQ) were between 0.15 and 3 and 1 and 10 µg/kg, respectively. The method detected insecticide contaminations in various livestock and poultry feeds. Furthermore, the method was applied to a toxicology case, where it identified and quantified piperonyl butoxide and deltamethrin in the submitted horse feed sample. These results demonstrate that the method can be a valuable tool in animal health and food safety diagnostic applications, as well as veterinary toxicology investigations concerning pyrethrin-related feed contamination.

Pesticide contamination in an intensive insect predator of honey bees.

Pesticides used for plant protection can indirectly affect target and non-target organisms and are identified as a major cause of insect decline. Depending on species interactions, pesticides can be transferred into the environment from plants to preys and predators. While the transfer of pesticides is often studied through vertebrate and aquatic exposure, arthropod predators of insects may represent valuable bioindicators of environmental exposure to pesticides. A modified QuEChERS extraction coupled with HPLC-MS/MS analysis was used to address the question of the exposure to pesticides of the invasive hornet Vespa velutina, a specialist predator of honey bees. This analytical method allows the accurate quantification of nanogram/gram levels of 42 contaminants in a sample weight that can be obtained from single individuals. Pesticide residues were analyzed in female workers from 24 different hornet nests and 13 different pesticides and 1 synergist, piperonyl butoxide, were identified and quantified. In 75 % of the explored nests, we found at least one compound and in 53 % of the positive samples we could quantify residues ranging from 0.5 to 19.5 ng.g-1. In this study, hornets from nests located in sub-urban environments were the most contaminated. Pesticide residue analysis in small and easy to collect predatory insects opens new perspectives for the study of environmental contamination and the transfer of pesticides in terrestrial trophic chains.

Insecticide resistance status of Aedes aegypti in southern and northern Ghana.

BACKGROUND: Outbreaks of Aedes-borne arboviral diseases are becoming rampant in Africa. In Ghana, there is no organized arboviral control programme with interventions restricted to mitigate outbreaks. Insecticide application is a crucial part of outbreak responses and future preventative control measures. Thus, knowledge of the resistance status and underlying mechanisms of Aedes populations is required to ensure optimal insecticide choices. The present study assessed the insecticide resistance status of Aedes aegypti populations from southern Ghana (Accra, Tema and Ada Foah) and northern Ghana (Navrongo) respectively. METHODS: Phenotypic resistance was determined with WHO susceptibility tests using Ae. aegypti collected as larvae and reared into adults. Knockdown resistance (kdr) mutations were detected using allele-specific PCR. Synergist assays were performed with piperonyl butoxide (PBO) to investigate the possible involvement of metabolic mechanisms in resistance phenotypes. RESULTS: Resistance to DDT was moderate to high across sites (11.3 to 75.8%) and, for the pyrethroids deltamethrin and permethrin, moderate resistance was detected (62.5 to 88.8%). The 1534C kdr and 1016I kdr alleles were common in all sites (0.65 to 1) and may be on a trajectory toward fixation. In addition, a third kdr mutant, V410L, was detected at lower frequencies (0.03 to 0.31). Pre-exposure to PBO significantly increased the susceptibility of Ae. aegypti to deltamethrin and permethrin (P < 0.001). This indicates that in addition to kdr mutants, metabolic enzymes (monooxygenases) may be involved in the resistance phenotypes observed in the Ae. aegypti populations in these sites. CONCLUSION: Insecticide resistance underpinned by multiple mechanisms in Ae. aegypti indicates the need for surveillance to assist in developing appropriate vector control strategies for arboviral disease control in Ghana.

Reduced synergistic efficacy of piperonyl butoxide in combination with alpha-cypermethrin in vitro in an insecticide-resistant strain of the sheep blowfly, Lucilia cuprina.

Control of flystrike on sheep relies on the use of insecticides. The present study used in vitro assays to examine the potential for increasing the efficacy of synthetic pyrethroids against sheep blowfly larvae using the synergist piperonyl butoxide (PBO). We examined the potency of alpha-cypermethrin (ACP) / PBO combinations against a reference insecticide-susceptible strain (LS) and a field-derived strain showing resistance to dicyclanil and imidacloprid. Co-treatment of the insecticide-susceptible strain with ACP/PBO resulted in increasing levels of synergism as the PBO concentration was increased, with synergism ratios (SRs) of up to 114-fold. Treatment with PBO/ACP combinations at ratios of 20:1 and 5:1 resulted in significant levels of synergism: SRs of 13.5- and 7.6-fold, respectively. However, the levels of synergism were significantly less for the insecticide-resistant strain: SRs of 4.6- and 2.6-fold for the 20:1 and 5:1 ratios, respectively. The resistant strain showed no resistance to ACP when administered alone, however, was 2-fold less sensitive than the LS strain to the toxic effects of PBO alone. This insensitivity to PBO was removed by co-treatment with the P450 inhibitor aminobenzotriazole, suggesting an increased level of P450-mediated metabolism of the PBO in this strain compared to the LS strain, and hence providing a likely explanation for the reduced synergistic efficacy of PBO on ACP toxicity in the resistant strain. While PBO was able to synergise ACP with both of the blowfly strains examined here, the reduced synergistic efficacy observed with the field-derived insecticide-resistant strain lessens the potential usefulness of such a combination for blowfly control in the field.

Characterizing pyrethroid resistance and mechanisms in Anopheles gambiae (s.s.) and Anopheles arabiensis from 11 districts in Uganda.

Insecticide resistance threatens recent progress on malaria control in Africa. To characterize pyrethroid resistance in Uganda, Anopheles gambiae (s.s.) and Anopheles arabiensis were analyzed from 11 sites with varied vector control strategies. Mosquito larvae were collected between May 2018 and December 2020. Sites were categorized as receiving no indoor-residual spraying ('no IRS', n â€‹= â€‹3); where IRS was delivered from 2009 to 2014 and in 2017 and then discontinued ('IRS stopped', n â€‹= â€‹4); and where IRS had been sustained since 2014 ('IRS active', n â€‹= â€‹4). IRS included bendiocarb, pirimiphos methyl and clothianidin. All sites received long-lasting insecticidal nets (LLINs) in 2017. Adult mosquitoes were exposed to pyrethroids; with or without piperonyl butoxide (PBO). Anopheles gambiae (s.s.) and An. arabiensis were identified using PCR. Anopheles gambiae (s.s.) were genotyped for Vgsc-995S/F, Cyp6aa1, Cyp6p4-I236M, ZZB-TE, Cyp4j5-L43F and Coeae1d, while An. arabiensis were examined for Vgsc-1014S/F. Overall, 2753 An. gambiae (s.l.), including 1105 An. gambiae (s.s.) and 1648 An. arabiensis were evaluated. Species composition varied by site; only nine An. gambiae (s.s.) were collected from 'IRS active' sites, precluding species-specific comparisons. Overall, mortality following exposure to permethrin and deltamethrin was 18.8% (148/788) in An. gambiae (s.s.) and 74.6% (912/1222) in An. arabiensis. Mortality was significantly lower in An. gambiae (s.s.) than in An. arabiensis in 'no IRS' sites (permethrin: 16.1 vs 67.7%, P â€‹< â€‹0.001; deltamethrin: 24.6 vs 83.7%, P â€‹< â€‹0.001) and in 'IRS stopped' sites (permethrin: 11.3 vs 63.6%, P â€‹< â€‹0.001; deltamethrin: 25.6 vs 88.9%, P â€‹< â€‹0.001). When PBO was added, mortality increased for An. gambiae (s.s.) and An. arabiensis. Most An. gambiae (s.s.) had the Vgsc-995S/F mutation (95% frequency) and the Cyp6p4-I236M resistance allele (87%), while the frequency of Cyp4j5 and Coeae1d were lower (52% and 55%, respectively). Resistance to pyrethroids was widespread and higher in An. gambiae (s.s.). Where IRS was active, An. arabiensis dominated. Addition of PBO to pyrethroids increased mortality, supporting deployment of PBO LLINs. Further surveillance of insecticide resistance and assessment of associations between genotypic markers and phenotypic outcomes are needed to better understand mechanisms of pyrethroid resistance and to guide vector control.

Multi-country evaluation of the durability of pyrethroid plus piperonyl-butoxide insecticide-treated nets: study protocol.

BACKGROUND: Mass distributions of long-lasting insecticidal nets (LLINs) have contributed to large reductions in the malaria burden. However, this success is in jeopardy due in part to the increasing pyrethroid-resistant mosquito population as well as low LLINs coverage in various areas because the lifespan of LLINs is often shorter than the interval between replenishment campaigns. New insecticide-treated nets (ITNs) containing pyrethroid and piperonyl-butoxide (PBO) have shown a greater reduction in the incidence of malaria than pyrethroid LLINs in areas with pyrethroid-resistant mosquitoes. However, the durability (attrition, bio-efficacy, physical integrity and chemical retainment) of pyrethroid-PBO ITNs under operational settings has not been fully characterized. This study will measure the durability of pyrethroid-PBO ITNs to assess whether they meet the World Health Organization (WHO) three years of operational performance criteria required to be categorized as "long-lasting". METHODS: A prospective household randomized controlled trial will be conducted simultaneously in Tanzania, India and Côte d'Ivoire to estimate the field durability of three pyrethroid-PBO ITNs (Veeralin®, Tsara® Boost, and Olyset® Plus) compared to a pyrethroid LLIN: MAGNet®. Durability monitoring will be conducted up to 36 months post-distribution and median survival in months will be calculated. The proportion of ITNs: (1) lost (attrition), (2) physical integrity, (3) resistance to damage score, (4) meeting WHO bio-efficacy (≥ 95% knockdown after 1 h or ≥ 80% mortality after 24 h for WHO cone bioassay, or ≥ 90% blood-feeding inhibition or ≥ 80% mortality after 24 h for WHO Tunnel tests) criteria against laboratory-reared resistant and susceptible mosquitoes, and insecticidal persistence over time will be estimated. The non-inferiority of Veeralin® and Tsara® Boost to the first-in-class, Olyset® Plus will additionally be assessed for mortality, and the equivalence of 20 times washed ITNs compared to field aged ITNs will be assessed for mortality and blood-feeding inhibition endpoints in the Ifakara Ambient Chamber Test, Tanzania. CONCLUSION: This will be the first large-scale prospective household randomized controlled trial of pyrethroid-PBO ITNs in three different countries in East Africa, West Africa and South Asia, simultaneously. The study will generate information on the replenishment intervals for PBO nets.

Utilization of piperonyl butoxide and 1-aminobenzotriazole for metabolic studies of toxic chemicals in Daphnia magna and Chironomus yoshimatsui.

Daphnids and chironomids have been used to assess the ecological effects of chemicals released into water bodies; however, the toxicity mechanisms in organisms are generally difficult to identify. Here, we developed a system capable of estimating the contribution of cytochrome P450 (CYP) to the metabolism of test substances in Daphnia magna and Chironomus yoshimatsui based on toxicity differences in the absence and presence of the CYP inhibitors piperonyl butoxide (PBO) and 1-aminobenzotriazole (ABT). The optimum concentrations of PBO and ABT that could effectively reduce the toxicity of diazinon, which is toxic after oxidative metabolism in vivo, were determined as 0.5 and 0.6 mg/L for D. magna, and 2.0 and 40.0 mg/L for C. yoshimatsui, respectively. Acute immobilization tests of 15 insecticides were conducted for D. magna and C. yoshimatsui, with and without the optimum concentrations of PBO or ABT. In the presence of either inhibitor, chlorpyrifos and chlorfenapyr toxicity was reduced in both organisms, whereas those of thiocyclam, nereistoxin, and silafluofen were enhanced in C. yoshimatsui. Liquid chromatography-mass spectrometry analysis of D. magna and C. yoshimatsui samples exposed to chlorfenapyr confirmed that the level of the active metabolite produced by CYP was decreased by PBO or ABT in both organisms. The system to which the test substance was co-exposed to PBO or ABT will be valuable for estimating the contribution of CYPs to metabolism and elucidating the toxicity mechanism in daphnids and chironomids.

Wild bees are exposed to low levels of pesticides in urban grasslands and community gardens.

Globally documented wild bee declines threaten sustainable food production and natural ecosystem functioning. Urban environments are often florally abundant, and consequently can contain high levels of pollinator diversity compared with agricultural environments. This has led to the suggestion that urban environments are an increasingly important habitat for pollinators. However, pesticides, such as commercial bug sprays, have a range of lethal and sub-lethal impacts on bees and are widely available for public use, with past work indicating that managed bees (honeybees and bumblebees) are exposed to a range of pesticides in urban environments. Despite this, we still have a poor understanding of (i) whether wild bees foraging in urban environments are exposed to pesticides and (ii) if exposure differs between genera. Here we assessed pesticide exposure in 8 bee genera foraging across multiple urban landscapes. We detected 13 different pesticides, some at concentrations known to have sub-lethal impacts on pollinators. Both the likelihood of pesticides being detected, and the concentrations observed, were higher for larger bees, likely due to their greater foraging ranges. Our results suggest that restricting agrochemical use in urban environments, where the economic benefits are limited, is a simple way to reduce anthropogenic stress on wild bees.

Knowledge, attitudes, and practices regarding malaria control among communities living in the south Cameroon forest region.

Objective: This study assessed knowledge, attitudes, and practices (KAP) regarding malaria among communities living in the equatorial forest region of south Cameroon. Methods: The study was conducted in Olama and Nyabessan. Interviews were undertaken using a semi-structured questionnaire for data collection on KAP, while malaria rapid diagnostic testing, using SD BIOLINE kits, was employed for malaria parasite detection. Results: In total, 186 heads of households (HoH), comprising 105 (56.45%) males and 81 (43.45%) females, were interviewed. The majority of HoH demonstrated good knowledge of malaria (86.56%; n = 161) and control measures, with a high proportion of long-lasting insecticidal net (LLIN) ownership (96.8%; n = 180). More than two-thirds (81.1%; n = 151) of households owned at least one LLIN for two people. The majority of HoH (85.40%) declared visiting hospitals or clinics in cases of suspected malaria. Malaria parasite prevalence was high in the two study sites (63.9% in Nyabessan and 48.65% in Olama), and varied according to age, house type, and sleeping time. Conclusion: The study indicated that despite good knowledge of malaria, high possession and utilization of control measures by population, transmission of malaria still persist in the area. The study stress the need for implementing additional control measures to improve the fight against malaria in the area.

Relationships between biological age, distance from aquatic habitats and pyrethroid resistance status of Anopheles funestus mosquitoes in south-eastern Tanzania.

BACKGROUND: Malaria transmission can be highly heterogeneous between and within localities, and is influenced by factors such as survival and biting frequencies of Anopheles mosquitoes. This study investigated the relationships between the biological age, distance from aquatic habitats and pyrethroid resistance status of Anopheles funestus mosquitoes, which currently dominate malaria transmission in south-east Tanzania. The study also examined how such relationships may influence malaria transmission and control. METHODS: Female An. funestus were collected in houses located 50-100 m, 150-200 m or over 200 m from the nearest known aquatic habitats. The mosquitoes were exposed to 1×, 5× and 10× the diagnostic doses of deltamethrin or permethrin, or to the synergist, piperonyl butoxide (PBO) followed by the pyrethroids, then monitored for 24 h-mortality. Ovaries of exposed and non-exposed mosquitoes were dissected to assess parity as a proxy for biological age. Adults emerging from larval collections in the same villages were tested against the same insecticides at 3-5, 8-11 or 17-20 days old. FINDINGS: Mosquitoes collected nearest to the aquatic habitats (50-100 m) had the lowest mortalities compared to other distances, with a maximum of 51% mortality at 10× permethrin. For the age-synchronized mosquitoes collected as larvae, the insecticide-induced mortality assessed at both the diagnostic and multiplicative doses (1×, 5× and 10×) increased with mosquito age. The highest mortalities at 1× doses were observed among the oldest mosquitoes (17-20 days). At 10× doses, mortalities were 99% (permethrin) and 76% (deltamethrin) among 8-11 day-olds compared to 80% (permethrin) and 58% (deltamethrin) among 3-5 day-olds. Pre-exposure to PBO increased the potency of both pyrethroids. The proportion of parous females was highest among mosquitoes collected farthest from the habitats. CONCLUSION: In this specific setting, older An. funestus and those collected farthest from the aquatic habitats (near the centre of the village) were more susceptible to pyrethroids than the younger ones and those caught nearest to the habitats. These findings suggest that pyrethroid-based interventions may remain at least moderately effective despite widespread pyrethroid-resistance, by killing the older, less-resistant and potentially-infective mosquitoes. Further studies should investigate how and whether these observations could be exploited to optimize malaria control in different settings.

Disruption of CYP6DF1 and CYP6DJ2 increases the susceptibility of Dendroctonus armandi to (+)-α-pinene.

Bark beetles rely on detoxifying enzymes to resist the defensive oleoresin terpenes of the host tree. Insect cytochrome P450 (CYPs) plays a key role in the detoxification of plant allelochemicals and pesticides. CYP6 family is unique to Insecta, and its biochemical function is basically related to catabolize heterologous substances. In this study, two Dendroctonus armandi CYP6 genes, CYP6DF1 and CYP6DJ2, were characterized. Spatiotemporal expression profiling revealed that CYP6DF1 and CYP6DJ2 expressions were higher in larvae and adult stages of D. armandi than in egg and pupae stages, and that two genes predominantly expressed in brain, midgut, fat body, or Malpighian tubules. Moreover, CYP6DF1 and CYP6DJ2 expressions were significantly induced after exposure to (+)-α-pinene. Importantly, silencing CYP6DF1 and CYP6DJ2 significantly inhibited the CYP activity and increased the mortality in the adults fumigated with (+)-α-pinene. Additionally, piperonyl butoxide exposure to adults also increase the sensitivity after treatment with (+)-α-pinene, which led to a significant reduction of the CYP activity, resulting a significant increase in adult mortality. These results suggest that the CYP6 family plays a key role in determining the susceptibility of D. armandi to (+)-α-pinene, which may have implications for the development of novel therapeutics to control this important pest.