Physical and insecticidal durability of Interceptor®, Interceptor® G2, and PermaNet® 3.0 insecticide-treated nets in Burkina Faso: results of durability monitoring in three sites from 2019 to 2022.

BACKGROUND: National Malaria Programmes (NMPs) monitor the durability of insecticide-treated nets (ITNs) to inform procurement and replacement decisions. This is crucial for new dual active ingredients (AI) ITNs, for which less data is available. Pyrethroid-only ITN (Interceptor®) and dual AI (Interceptor® G2, and PermaNet® 3.0) ITNs were assessed across three health districts over 36 months in southern Burkina Faso to estimate median ITN survival, insecticidal efficacy, and to identify factors contributing to field ITN longevity. METHODS: Durability was monitored through a prospective study of a cohort of nets distributed during the 2019 mass campaign. Three health districts were selected for their similar pyrethroid-resistance, environmental, epidemiological, and population profiles. Households were recruited after the mass campaign, with annual household questionnaire follow-ups over three years. Each round, ITNs were withdrawn for bioassays and chemical residue testing. Key measures were the percentage of cohort ITNs in serviceable condition, insecticidal effectiveness, and chemical residue content against target dose. Cox proportional hazard models were used to identify determinants influencing ITN survival. RESULTS: At endline, the median useful life was 3.2 (95% CI 2.5-4.0) years for PermaNet® 3.0 ITNs in Orodara, 2.6 (95% CI 1.9-3.2) years for Interceptor® G2 ITNs in Banfora and 2.4 (95% CI 1.9-2.9) years for Interceptor® ITNs in Gaoua. Factors associated with ITN survival included cohort ITNs from Orodara (adjusted hazard ratio (aHR) = 0.58, p = 0.026), households seeing less rodents (aHR = 0.66, p = 0.005), female-headed households (aHR = 0.66, p = 0.044), exposure to social behavior change (SBC) messages (aHR = 0.52, ≤ 0.001) and folding nets when not in use (aHR = 0.47, p < 0.001). At endline, PermaNet® 3.0 ITN recorded 24-h mortality of 26% against resistant mosquitos on roof panels, with an 84% reduction in PBO content. Interceptor® G2 ITN 72-h mortality was 51%, with a 67% reduction in chlorfenapyr content. Interceptor® ITN 24-h mortality was 71%, with an 84% reduction in alpha-cypermethrin content. CONCLUSION: Only PermaNet® 3.0 ITNs surpassed the standard three-year survival threshold. Identified protective factors should inform SBC messaging. Significant decreases in chemical content and resulting impact on bioefficacy warrant more research in other countries to better understand dual AI ITN insecticidal performance.

Bioaccumulation of Deltamethrin and Piperonyl butoxide in Labeo rohita fish.

Deltamethrin (DLM), in combination with the synergist piperonyl butoxide (PBO), is extensively used in pest control programs due to its potent pesticidal properties and appreciable safety margin. However, various research studies report their adverse effects on non-target organisms. In this study, we investigated the toxicity of DLM, PBO, and a DLM-PBO (3:1) combination on Labeo rohita (L. rohita) fish fingerlings. Fish behavior and mortality rates were recorded at different time intervals up to 96 h for concentrations of 0.003, 0.007, 0.015, 0.031, and 0.062 µg/mL, respectively. Biochemical, hematological, and histopathological studies were carried out. High-performance liquid chromatography (HPLC) was used to detect and quantify residues in fish samples. The LC50 values after 48 h for DLM, PBO, and DLM-PBO exposed fish fingerlings were found to be 0.028, 0.066, and 0.007 µg/mL, respectively. At a concentration of 0.003 µg/mL of DLM, PBO, and DLM-PBO, the treated fish fingerlings exhibited similar behavior to the control group. Hematological parameters, such as red blood cell (RBC) and white blood cell (WBC) counts, were reduced in the treated groups compared to the control. Biochemical parameters showed increased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), while total serum protein levels decreased in DLM, PBO, and DLM-PBO treated fingerlings. Histopathological examination of liver, gill, and heart tissues revealed lesions with hydropic degeneration in the liver and fusions of gill lamellae in the treated tissues. Fish fingerlings exposed to the DLM-PBO combination appeared highly prone to toxicity compared to those treated with DLM and PBO separately.

Piperonyl butoxide elicits a robust transcriptional response in adult Drosophila melanogaster.

While much attention has been devoted to understanding the transcriptional changes underlying resistance to insecticides, comparatively little is known about the transcriptional response of naive insects to agrochemicals. In this study, we analyze the transcriptomic response of an insecticide susceptible strain of Drosophila melanogaster to nine agrochemicals using a robust method that goes beyond classical replication standards. Our findings demonstrate that exposure to piperonyl butoxide (PBO), but not to eight other compounds, elicits a robust transcriptional response in a wild-type strain of Drosophila melanogaster. PBO exposure leads to the upregulation of a subset of Cyps, GSTs, UGTs and EcKls. This response is both time and concentration-dependent, suggesting that the degree of inhibition of P450 activity correlates with the magnitude of the transcriptional response. Furthermore, the upregulation of these enzymes is excluded from reproductive organs. Additionally, different sets of genes are regulated in the digestive/secretory tract and the carcass. Our results suggest that P450s play a role in metabolizing yet unidentified endogenous compounds and are involved in an as-yet-unknown physiological regulatory feedback loop.

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).

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.

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.

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.

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.

A quasi-experimental study estimating the impact of long-lasting insecticidal nets with and without piperonyl butoxide on pregnancy outcomes.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the main vector control tool for pregnant women, but their efficacy may be compromised, in part, due to pyrethroid resistance. In 2017, the Ugandan Ministry of Health embedded a cluster randomized controlled trial into the national LLIN campaign, where a random subset of health subdistricts (HSDs) received LLINs treated with piperonyl butoxide (PBO), a chemical synergist known to partially restore pyrethroid sensitivity. Using data from a small, non-randomly selected subset of HSDs, this secondary analysis used quasi-experimental methods to quantify the overall impact of the LLIN campaign on pregnancy outcomes. In an exploratory analysis, differences between PBO and conventional (non-PBO) LLINs on pregnancy outcomes were assessed. METHODS: Birth registry data (n = 39,085) were retrospectively collected from 21 health facilities across 12 HSDs, 29 months before and 9 months after the LLIN campaign (from 2015 to 2018). Of the 12 HSDs, six received conventional LLINs, five received PBO LLINs, and one received a mix of conventional and PBO LLINs. Interrupted time-series analyses (ITSAs) were used to estimate changes in monthly incidence of stillbirth and low birthweight (LBW; <2500 g) before-and-after the campaign. Poisson regression with robust standard errors modeled campaign effects, adjusting for health facility-level differences, seasonal variation, and time-varying maternal characteristics. Comparisons between PBO and conventional LLINs were estimated using difference-in-differences estimators. RESULTS: ITSAs estimated the campaign was associated with a 26% [95% CI: 7-41] reduction in stillbirth incidence (incidence rate ratio (IRR) = 0.74 [0.59-0.93]) and a 15% [-7, 33] reduction in LBW incidence (IRR=0.85 [0.67-1.07]) over a 9-month period. The effect on stillbirth incidence was greatest for women delivering 7-9 months after the campaign (IRR=0.60 [0.41-0.87]) for whom the LLINs would have covered most of their pregnancy. The IRRs estimated from difference-in-differences analyses comparing PBO to conventional LLINs was 0.78 [95% CI: 0.52, 1.16] for stillbirth incidence and 1.15 [95% CI: 0.87, 1.52] for LBW incidence. CONCLUSIONS: In this region of Uganda, where pyrethroid resistance is high, this study found that a mass LLIN campaign was associated with reduced stillbirth incidence. Effects of the campaign were greatest for women who would have received LLINs early in pregnancy, suggesting malaria protection early in pregnancy can have important benefits that are not necessarily realized through antenatal malaria services. Results from the exploratory analyses comparing PBO and conventional LLINs on pregnancy outcomes were inconclusive, largely due to the wide confidence intervals that crossed the null. Thus, future studies with larger sample sizes are needed.

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.

BACKGROUND: Progress 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. METHODS: A 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. CONCLUSIONS: Study 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.

Comparative efficacy of two pyrethroid-piperonyl butoxide nets (Olyset Plus and PermaNet 3.0) against pyrethroid resistant malaria vectors: a non-inferiority assessment.

BACKGROUND: Pyrethroid-PBO nets were conditionally recommended for control of malaria transmitted by mosquitoes with oxidase-based pyrethroid-resistance based on epidemiological evidence of additional protective effect with Olyset Plus compared to a pyrethroid-only net (Olyset Net). Entomological studies can be used to assess the comparative performance of other brands of pyrethroid-PBO ITNs to Olyset Plus. METHODS: An experimental hut trial was performed in Cové, Benin to compare PermaNet 3.0 (deltamethrin plus PBO on roof panel only) to Olyset Plus (permethrin plus PBO on all panels) against wild pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) following World Health Organization (WHO) guidelines. Both nets were tested unwashed and after 20 standardized washes compared to Olyset Net. Laboratory bioassays were also performed to help explain findings in the experimental huts. RESULTS: With unwashed nets, mosquito mortality was higher in huts with PermaNet 3.0 compared to Olyset Plus (41% vs. 28%, P < 0.001). After 20 washes, mortality declined significantly with PermaNet 3.0 (41% unwashed vs. 17% after washing P < 0.001), but not with Olyset Plus (28% unwashed vs. 24% after washing P = 0.433); Olyset Plus induced significantly higher mortality than PermaNet 3.0 and Olyset Net after 20 washes. PermaNet 3.0 showed a higher wash retention of PBO compared to Olyset Plus. A non-inferiority analysis performed with data from unwashed and washed nets together using a margin recommended by the WHO, showed that PermaNet 3.0 was non-inferior to Olyset Plus in terms of mosquito mortality (25% with Olyset Plus vs. 27% with PermaNet 3.0, OR = 1.528, 95%CI = 1.02-2.29) but not in reducing mosquito feeding (25% with Olyset Plus vs. 30% with PermaNet 3.0, OR = 1.192, 95%CI = 0.77-1.84). Both pyrethroid-PBO nets were superior to Olyset Net. CONCLUSION: Olyset Plus outperformed PermaNet 3.0 in terms of its ability to cause greater margins of improved mosquito mortality compared to a standard pyrethroid net, after multiple standardized washes. However, using a margin of non-inferiority defined by the WHO, PermaNet 3.0 was non-inferior to Olyset Plus in inducing mosquito mortality. Considering the low levels of mortality observed and increasing pyrethroid-resistance in West Africa, it is unclear whether either of these nets would demonstrate the same epidemiological impact observed in community trials in East Africa.

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.

Transcriptomic analysis of s-methoprene resistance in the lesser grain borer, Rhyzopertha dominica, and evaluation of piperonyl butoxide as a resistance breaker.

BACKGROUND: The lesser grain borer, Rhyzopertha dominica is a serious pest of stored grains. Fumigation and contact insecticides play a major role in managing this pest globally. While insects are developing genetic resistance to chemicals, hormonal analogues such as s-methoprene play a key role in reducing general pest pressure as well as managing pest populations that are resistant to fumigants and neurotoxic contact insecticides. However, resistance to s-methoprene has been reported in R. dominica with some reports showing a remarkable high resistance, questioning the use of this compound and other related analogues in grain protection. The current study attempts to identify possible molecular mechanisms that contribute in resistance to s-methoprene in R. dominica. RESULTS: Transcriptome analysis of resistant and susceptible strains of this pest species identified a set of differentially expressed genes related to cytochrome P450s, indicating their potential role in resistance to s-methoprene. Laboratory bioassays were performed with s-methoprene treated wheat grains in presence and absence of piperonyl butoxide (PBO), a cytochrome P450 inhibitor. The results indicate that PBO, when applied alone, at least at the concentration tested here, had no effect on R. dominica adult emergence, but has a clear synergistic effect to s-methoprene. The number of produced progeny decreased in presence of the inhibitor, especially in the resistant strain. In addition, we also identified CYP complement (CYPome) of R. dominica, annotated and analysed phylogenetically, to understand the evolutionary relationships with other species. CONCLUSIONS: The information generated in current study suggest that PBO can effectively be used to break resistance to s-methoprene in R. dominica.

Evidence supporting deployment of next generation insecticide treated nets in Burkina Faso: bioassays with either chlorfenapyr or piperonyl butoxide increase mortality of pyrethroid-resistant Anopheles gambiae.

BACKGROUND: Pyrethroid resistance poses a major threat to the efficacy of insecticide-treated nets (ITNs) in Burkina Faso and throughout sub-Saharan Africa, particularly where resistance is present at high intensity. For such areas, there are alternative ITNs available, including the synergist piperonyl butoxide (PBO)-based ITNs and dual active ingredient ITNs such as Interceptor G2 (treated with chlorfenapyr and alpha-cypermethrin). Before deploying alternative ITNs on a large scale it is crucial to characterize the resistance profiles of primary malaria vector species for evidence-based decision making. METHODS: Larvae from the predominant vector, Anopheles gambiae sensu lato (s.l.) were collected from 15 sites located throughout Burkina Faso and reared to adults for bioassays to assess insecticide resistance status. Resistance intensity assays were conducted using WHO tube tests to determine the level of resistance to pyrethroids commonly used on ITNs at 1×, 5 × and 10 × times the diagnostic dose. WHO tube tests were also used for PBO synergist bioassays with deltamethrin and permethrin. Bottle bioassays were conducted to determine susceptibility to chlorfenapyr at a dose of 100 µg/bottle. RESULTS: WHO tube tests revealed high intensity resistance in An. gambiae s.l. to deltamethrin and alpha-cypermethrin in all sites tested. Resistance intensity to permethrin was either moderate or high in 13 sites. PBO pre-exposure followed by deltamethrin restored full susceptibility in one site and partially restored susceptibility in all but one of the remaining sites (often reaching mortality greater than 80%). PBO pre-exposure followed by permethrin partially restored susceptibility in 12 sites. There was no significant increase in permethrin mortality after PBO pre-exposure in Kampti, Karangasso-Vigué or Mangodara; while in Seguenega, Orodara and Bobo-Dioulasso there was a significant increase in mortality, but rates remained below 50%. Susceptibility to chlorfenapyr was confirmed in 14 sites. CONCLUSION: High pyrethroid resistance intensity in An. gambiae s.l. is widespread across Burkina Faso and may be a predictor of reduced pyrethroid ITN effectiveness. PBO + deltamethrin ITNs would likely provide greater control than pyrethroid nets. However, since susceptibility in bioassays was not restored in most sites following pre-exposure to PBO, Interceptor G2 may be a better long-term solution as susceptibility was recorded to chlorfenapyr in nearly all sites. This study provides evidence supporting the introduction of both Interceptor G2 nets and PBO nets, which were distributed in Burkina Faso in 2019 as part of a mass campaign.

Efficacy evaluation of Veeralin LN, a PBO-incorporated alpha-cypermethrin long-lasting insecticidal net against Anopheles culicifacies in experimental huts in Odisha State.

BACKGROUND: The success of malaria control using long-lasting insecticidal nets (LLINs) is threatened by pyrethroid resistance developed by the malaria vectors, worldwide. To combat the resistance, synergist piperonyl butoxide (PBO) incorporated LLINs is one of the available options. In the current phase II hut trial, the efficacy of Veeralin®LN (an alpha-cypermethrin and PBO-incorporated net) was evaluated against Anopheles culicifacies, a pyrethroid resistant malaria vector. METHODS: The performance of Veeralin®LN was compared with MAGNet®LN and untreated net in reducing the entry, induced exit, mortality and blood feeding inhibition of target vector species. RESULTS: The performance of Veeralin was equal to MAGNet in terms of reducing hut entry, inhibiting blood feeding and inducing exophily, and with regard to causing mortality Veeralin was better than MAGNet. When compared to untreated net, a significant reduction in hut entry and blood feeding and an increase in exophily and mortality were observed with Veeralin. In cone bioassays, unwashed Veeralin caused > 80% mortality of An. culicifacies. CONCLUSIONS: Veeralin performed equal to (entry, exit, feeding) or better than (mortality in huts and cone bioassays) MAGNet and could be an effective tool against pyrethroid resistant malaria vectors.

Evaluation of DawaPlus 3.0 and DawaPlus 4.0, deltamethrin-PBO combination nets against pyrethroid-resistant Anopheles culicifacies in experimental huts in India.

BACKGROUND: The development of resistance in vectors is one of the major impediments for malaria control. Adding synergists to insecticides has proven to be an alternative choice for controlling resistant mosquitoes. DawaPlus 3.0 and DawaPlus 4.0 are new long-lasting insecticidal nets (LLINs) in which deltamethrin and a synergist, piperonyl butoxide (PBO) are added into filaments and their efficacy was tested against resistant malaria vector, Anopheles culicifacies in experimental huts in India. METHODS: The performance of two trial nets in terms of deterrence induced exiting, blood-feeding inhibition and mortality of An. culicifacies was compared with DawaPlus 2.0 and untreated net. RESULTS: There was a significant reduction in entry, blood feeding and mortality (p < 0.05) and increase in exit rates of An. culicifacies in the treatment arms compared to untreated arm. But, both candidate LNs washed 20 times could not perform better than the washed reference net (DawaPlus 2.0). Cone bioassay results showed that all the treatment arms (both washed and unwashed) produced < 80% mortality of An. culicifacies before and after hut evaluation. CONCLUSIONS: DawaPlus 3.0 and DawaPlus 4.0 with their current specification may not be as effective as required to control the resistant vector, An. culicifacies, in east-central India.

Susceptibility of Anopheles gambiae from Côte d'Ivoire to insecticides used on insecticide-treated nets: evaluating the additional entomological impact of piperonyl butoxide and chlorfenapyr.

BACKGROUND: Pyrethroid-treated mosquito nets are currently the mainstay of vector control in Côte d'Ivoire. However, resistance to pyrethroids has been reported across the country, limiting options for insecticide resistance management due to the paucity of alternative insecticides. Two types of insecticide-treated nets (ITNs), ITNs with pyrethroids and the synergist piperonyl butoxide (PBO), and Interceptor®G2 nets, a net treated with a combination of chlorfenapyr and alpha-cypermethrin, are believed to help in the control of pyrethroid-resistant mosquitoes. METHODS: The susceptibility of Anopheles gambiae sensu lato (s.l.) to pyrethroid insecticides with and without pre-exposure to PBO as well as to chlorfenapyr was investigated in fifteen sites across the country. Susceptibility tests were conducted on 2- to 4-day old adult female An. gambiae s.l. reared from larval collections. The resistance status, intensity, and effects of PBO on mortality after exposure to different concentrations of deltamethrin, permethrin and alpha-cypermethrin were determined using WHO susceptibility test kits. In the absence of a WHO-recommended standard protocol for chlorfenapyr, two interim doses (100 and 200 µg/bottle) were used to test the susceptibility of mosquitoes using the CDC bottle assay method. RESULTS: Pre-exposure to PBO did not result in full restoration of susceptibility to any of the three pyrethroids for the An. gambiae s.l. populations from any of the sites surveyed. However, PBO pre-exposure did increase mortality for all three pyrethroids, particularly deltamethrin (from 4.4 to 48.9%). Anopheles gambiae s.l. from only one site (Bettie) were susceptible to chlorfenapyr at the dose of 100 µg active ingredient (a.i.)/bottle. At the dose of 200 µg (a.i.)/bottle, susceptibility was only recorded in 10 of the 15 sites. CONCLUSION: Low mosquito mortality was found for pyrethroids alone, and while PBO increased mortality, it did not restore full susceptibility. The vector was not fully susceptible to chlorfenapyr in one third of the sites tested. However, vector susceptibility to chlorfenapyr seems to be considerably higher than for pyrethroids alone or with PBO. These data should be used cautiously when making ITN procurement decisions, noting that bioassays are conducted in controlled conditions and may not fully represent field efficacy where the host-seeking behaviours, which include free-flying activity are known to enhance pro-insecticide chlorfenapyr intoxication to mosquitoes.

Molecular Pathological Differences in Global Gene Expression between Two Sustained Proliferative Lesions, Nodular Regenerative Hepatocellular Hyperplasia and Hepatocellular Adenoma, in Mice.

Long-term exposure to piperonyl butoxide (PBO) induces multiple nodular masses along with hepatocellular tumors in the liver of mice. The histopathological features of the nodules led to our diagnosis of nodular regenerative hepatocellular hyperplasia (NRH). However, because of the lack of data on the biological characteristics of NRH, whether this lesion is truly nonneoplastic remains unknown. In this study, the molecular characteristics of NRH were compared with those of hepatocellular adenoma (HCA) by global gene expression analysis. Six-week-old male ICR mice were fed a diet containing 6,000 ppm PBO for 43 weeks to induce NRH and HCA development. Complementary DNA microarray analysis was performed using messenger RNA extracted from NRH and HCA frozen sections collected by laser microdissection. Hierarchical cluster analysis showed that all NRH samples clustered together but were separate from the HCA cluster. Pathway analysis revealed activation of the cell cycle and Delta-Notch signaling in both lesions, but the latter was more upregulated in HCA. Downregulation of cytochrome p450 enzymes was observed in NRH, but not in HCA. These results imply that NRH differs from HCA in terms of not only morphological but also molecular characteristics.

Simultaneous determination of spinosad, temephos, and piperonyl butoxide in animal-derived foods using LC-MS/MS.

Pesticides, which are used as plant protection products, can enter the food chain, and exposure to these xenobiotics can cause a wide array of health problems in humans. Therefore, the objective of the present study was to develop an analytical method for the simultaneous determination of residual spinosad (sum of spinosyn A and D), temephos and piperonyl butoxide in porcine muscle, egg, milk, eel, flatfish and shrimp (sampling period: February to June 2018) using liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS/MS). The target analytes were extracted with a combination of acidified acetonitrile and ethyl acetate and subsequently purified with original QuEChERS kits (composed of magnesium sulfate and sodium chloride) as well as n-hexane. All analytes were separated on a reversed-phase analytical column using a mobile phase of (A) 0.1% formic acid containing 10 mm ammonium formate in distilled water and (B) methanol. Good linearity (R2 ≥ 0.980) was achieved over the tested concentration range (3.5-35 µg/kg for spinosyn A; 1.5-15 µg/kg for spinosyn D; 5-50 µg/kg for temephos and piperonyl butoxide) in matrix-matched standard calibrations. Fortified samples at three spiking levels yielded recoveries in the range of 71-105% with relative standard deviations ≤9.2%. The applicability of the method was evaluated via evaluating samples collected from a large wholesale market located in Seoul, and none of the samples contained any of the target analytes. In conclusion, the current approach is simple, efficient and reliable and can successfully determine the residual levels of spinosad, temephos and piperonyl butoxide in complex animal-derived food products.

Effects of piperonyl butoxide on exploratory behaviour in female mice.

Previous studies reported that piperonyl butoxide (PBO) induces adverse effects on exploratory behaviour in male mice. However, no consistent effects of PBO treatment were observed in female mice. This study aimed to evaluate PBO's neurobehavioral effects in female mice. Female mice were exposed to PBO through diet to provide levels of 0 (control), 0.025%, 0.1%, and 0.4% from 5 to 12 weeks of age, and selected behavioural parameters were measured. The average female body weight showed no significant effect from PBO treatment through the experimental periods. Regarding multiple-T water maze performance at 10 weeks of age, no significant effect caused by PBO treatment was observed. Exploratory behaviour examination of 8-week-old female mice indicated that the average speed declined in a significant dose-related manner, and the longitudinal pattern indicated a significant difference between the control and high-dose groups. For exploratory behaviour examination at 11 weeks of age, the total exploration distance shortened in a significant dose-related manner, and the average speed declined similarly. These longitudinal patterns showed significant differences between the control and high-dose groups. The PBO dose levels in this study produced several adverse effects on exploratory behaviour in female mice.