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.

Transcriptomics and Metabolomics for Co-Exposure to a Cocktail of Neonicotinoids and the Synergist Piperonyl Butoxide.

Here, the transcriptomics and metabolomics on a model of exposure to a cocktail of neonicotinoids (Neo) containing seven commercial compounds and a synergist piperonyl butoxide (PBO) were established. The results showed that Neo and PBO disrupted mRNA and metabolite levels in a dose-dependent manner. Neo caused tryptophan pathway-related neurotoxicity, reduced lipolysis, and promoted fat mass accumulation in the liver, while PBO induced an increase in inflammatory factors and damage to intercellular membranes. Co-exposure enhanced Neo-induced liver steatosis, focal necrosis, and oxidative stress by inhibiting oxidative phosphorylation (OXPHOS). Furthermore, diglycerides and metabolic biomarkers demonstrated that the activation of insulin signaling is associated with restricted OXPHOS, which commonly leads to a high risk of non-alcoholic fatty liver disease (NAFLD) and Alzheimer's disease (AD) as the result of over-synthesis of lipids, low energy supply, and high thermogenesis. The study demonstrates that chronic disease can be induced by Neo and the synergist PBO at the molecular level.

Promoting Efficacy and Environmental Safety of Pesticide Synergists via Non-Ionic Gemini Surfactants with Short Fluorocarbon Chains.

Improving the utilization rate of pesticides is key to achieve a reduction and synergism, and adding appropriate surfactant to pesticide preparation is an effective way to improve pesticide utilization. Fluorinated surfactants have excellent surface activity, thermal and chemical stability, but long-chain linear perfluoroalkyl derivatives are highly toxic, obvious persistence and high bioaccumulation in the environment. Therefore, new strategies for designing fluorinated surfactants which combine excellent surface activity and environmental safety would be useful. In this study, four non-ionic gemini surfactants with short fluorocarbon chains were synthesized. The surface activities of the resulting surfactants were assessed on the basis of equilibrium surface tension, dynamic surface tension, and contact angle. Compared with their monomeric counterparts, the gemini surfactants had markedly lower critical micelle concentrations and higher diffusivities, as well as better wetting abilities. We selected a single-chain surfactant and a gemini surfactant with good surface activities as synergists for the glyphosate water agent. Both surfactants clearly improved the efficacy of the herbicide, but the gemini surfactant had a significantly greater effect than the single-chain surfactant. An acute toxicity test indicated that the gemini surfactant showed slight toxicity to rats.

Occurrence of L1014F and L1014S mutations in insecticide resistant Culex quinquefasciatus from filariasis endemic districts of West Bengal, India.

INTRODUCTION: Lymphatic filariasis causes long term morbidity and hampers the socio-economic status. Apart from the available treatments and medication, control of vector population Culex quinquefasciatus Say through the use of chemical insecticides is a widely applied strategy. However, the unrestrained application of these insecticides over many decades has led to resistance development in the vectors. METHODS: In order to determine the insecticide susceptibility/resistance status of Cx. quinquefasciatus from two filariasis endemic districts of West Bengal, India, wild mosquito populations were collected and assayed against six different insecticides and presence of L1014F; L1014S kdr mutations in the voltage-gated sodium channel gene was also screened along with the use of synergists to evaluate the role of major detoxifying enzymes in resistance development. RESULTS: The collected mosquito populations showed severe resistance to insecticides and the two synergists used-PBO (piperonyl butoxide) and TPP (triphenyl phosphate), were unable to restore the susceptibility status of the vector thereupon pointing towards a minor role of metabolic enzymes. kdr mutations were present in the studied populations in varying percent with higher L1014F frequency indicating its association with the observed resistance to pyrethroids and DDT. This study reports L1014S mutation in Cx. quinquefasciatus for the first time.

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.

An Adaptive Intervention Trial Design for Finding the Optimal Integrated Strategies for Malaria Control and Elimination in Africa: A Model Simulation Study.

There are a number of available and emerging malaria intervention tools that require innovative trial designs to find the optimal combinations at given epidemiologic settings. We simulated intervention strategies based on adaptive interventions, which included long-lasting insecticidal nets (LLINs), piperonyl butoxide-treated LLINs (PBO-LLINs), indoor residual spraying (IRS), and long-lasting microbial larviciding (LLML). The aims were to determine if PBO-LLINs or LLIN+IRS combination is more effective for initial interventions than LLINs and to identify the most effective intervention. We used a clustered, randomized adaptive trial design with malaria infection prevalence (MIP) as the outcome variable. The results indicate that during the initial stage of interventions, compared with regular LLINs, PBO-LLINs (relative reduction [RR]: 29.3%) and LLIN plus IRS with alternative-insecticide (RR: 26.8%) significantly reduced MIP. In the subsequent interventions, adding alternative insecticide IRS (RR: 23.8%) or LLML (RR: 31.2%) to existing PBO-LLIN was effective in further reducing MIP. During the next stage of interventions, adding LLML on top of PBO-LLIN+IRS (with alternative insecticides) had a significant impact on MIP (RR: 39.2%). However, adding IRS (with alternative insecticides) on top of PBO-LLIN+LLML did not significantly reduce MIP (11.6%). Overall, in clusters initiated with PBO-LLIN, adding LLML would be the most effective strategy in reducing MIP; in clusters initiated with LLIN+IRS, replacing LLIN+IRS with PBO-LLIN and LLML would be the most effective in reducing MIP. This study provides a new pathway for informing the optimal integrated malaria vector interventions, and the new strategy can be tested in field trials.

Piperonyl butoxide (PBO) combined with pyrethroids in insecticide-treated nets to prevent malaria in Africa.

BACKGROUND: Pyrethroid long-lasting insecticidal nets (LLINs) have been important in the large reductions in malaria cases in Africa, but insecticide resistance in Anopheles mosquitoes threatens their impact. Insecticide synergists may help control insecticide-resistant populations. Piperonyl butoxide (PBO) is such a synergist; it has been incorporated into pyrethroid-LLINs to form pyrethroid-PBO nets, which are currently produced by five LLIN manufacturers and, following a recommendation from the World Health Organization (WHO) in 2017, are being included in distribution campaigns. This review examines epidemiological and entomological evidence on the addition of PBO to pyrethroid nets on their efficacy. OBJECTIVES: To compare effects of pyrethroid-PBO nets currently in commercial development or on the market with effects of their non-PBO equivalent in relation to: 1. malaria parasite infection (prevalence or incidence); and 2. entomological outcomes. SEARCH METHODS: We searched the Cochrane Infectious Diseases Group (CIDG) Specialized Register, CENTRAL, MEDLINE, Embase, Web of Science, CAB Abstracts, and two clinical trial registers (ClinicalTrials.gov and WHO International Clinical Trials Registry Platform) up to 25 September 2020. We contacted organizations for unpublished data. We checked the reference lists of trials identified by these methods. SELECTION CRITERIA: We included experimental hut trials, village trials, and randomized controlled trials (RCTs) with mosquitoes from the Anopheles gambiae complex or the Anopheles funestus group. DATA COLLECTION AND ANALYSIS: Two review authors assessed each trial for eligibility, extracted data, and determined the risk of bias for included trials. We resolved disagreements through discussion with a third review author. We analysed data using Review Manager 5 and assessed the certainty of evidence using the GRADE approach. MAIN RESULTS: Sixteen trials met the inclusion criteria: 10 experimental hut trials, four village trials, and two cluster-RCTs (cRCTs). Three trials are awaiting classification, and four trials are ongoing.  Two cRCTs examined the effects of pyrethroid-PBO nets on parasite prevalence in people living in areas with highly pyrethroid-resistant mosquitoes (< 30% mosquito mortality in discriminating dose assays). At 21 to 25 months post intervention, parasite prevalence was lower in the intervention arm (odds ratio (OR) 0.79, 95% confidence interval (CI) 0.67 to 0.95; 2 trials, 2 comparisons; moderate-certainty evidence). In highly pyrethroid-resistant areas, unwashed pyrethroid-PBO nets led to higher mosquito mortality compared to unwashed standard-LLINs (risk ratio (RR) 1.84, 95% CI 1.60 to 2.11; 14,620 mosquitoes, 5 trials, 9 comparisons; high-certainty evidence) and lower blood feeding success (RR 0.60, 95% CI 0.50 to 0.71; 14,000 mosquitoes, 4 trials, 8 comparisons; high-certainty evidence). However, in comparisons of washed pyrethroid-PBO nets to washed LLINs, we do not know if PBO nets had a greater effect on mosquito mortality (RR 1.20, 95% CI 0.88 to 1.63; 10,268 mosquitoes, 4 trials, 5 comparisons; very low-certainty evidence), although the washed pyrethroid-PBO nets did decrease blood-feeding success compared to standard-LLINs (RR 0.81, 95% CI 0.72 to 0.92; 9674 mosquitoes, 3 trials, 4 comparisons; high-certainty evidence). In areas where pyrethroid resistance is moderate (31% to 60% mosquito mortality), mosquito mortality was higher with unwashed pyrethroid-PBO nets compared to unwashed standard-LLINs (RR 1.68, 95% CI 1.33 to 2.11; 751 mosquitoes, 2 trials, 3 comparisons; moderate-certainty evidence), but there was little to no difference in effects on blood-feeding success (RR 0.90, 95% CI 0.72 to 1.11; 652 mosquitoes, 2 trials, 3 comparisons; moderate-certainty evidence). For washed pyrethroid-PBO nets compared to washed standard-LLINs, we found little to no evidence for higher mosquito mortality or reduced blood feeding (mortality: RR 1.07, 95% CI 0.74 to 1.54; 329 mosquitoes, 1 trial, 1 comparison, low-certainty evidence; blood feeding success: RR 0.91, 95% CI 0.74 to 1.13; 329 mosquitoes, 1 trial, 1 comparison; low-certainty evidence). In areas where pyrethroid resistance is low (61% to 90% mosquito mortality), studies reported little to no difference in the effects of unwashed pyrethroid-PBO nets compared to unwashed standard-LLINs on mosquito mortality (RR 1.25, 95% CI 0.99 to 1.57; 948 mosquitoes, 2 trials, 3 comparisons; moderate-certainty evidence), and we do not know if there was any effect on blood-feeding success (RR 0.75, 95% CI 0.27 to 2.11; 948 mosquitoes, 2 trials, 3 comparisons; very low-certainty evidence). For washed pyrethroid-PBO nets compared to washed standard-LLINs, we do not know if there was any difference in mosquito mortality (RR 1.39, 95% CI 0.95 to 2.04; 1022 mosquitoes, 2 trials, 3 comparisons; very low-certainty evidence) or on blood feeding (RR 1.07, 95% CI 0.49 to 2.33; 1022 mosquitoes, 2 trials, 3 comparisons; low-certainty evidence). In areas where mosquito populations are susceptible to insecticides (> 90% mosquito mortality), there may be little to no difference in the effects of unwashed pyrethroid-PBO nets compared to unwashed standard-LLINs on mosquito mortality (RR 1.20, 95% CI 0.64 to 2.26; 2791 mosquitoes, 2 trials, 2 comparisons; low-certainty evidence). This is similar for washed nets (RR 1.07, 95% CI 0.92 to 1.25; 2644 mosquitoes, 2 trials, 2 comparisons; low-certainty evidence). We do not know if unwashed pyrethroid-PBO nets had any effect on the blood-feeding success of susceptible mosquitoes (RR 0.52, 95% CI 0.12 to 2.22; 2791 mosquitoes, 2 trials, 2 comparisons; very low-certainty evidence). The same applies to washed nets (RR 1.25, 95% CI 0.82 to 1.91; 2644 mosquitoes, 2 trials, 2 comparisons; low-certainty evidence). In village trials comparing pyrethroid-PBO nets to LLINs, there was no difference in sporozoite rate (4 trials, 5 comparisons) nor in mosquito parity (3 trials, 4 comparisons). AUTHORS' CONCLUSIONS: In areas of high insecticide resistance, pyrethroid-PBO nets have greater entomological and epidemiological efficacy compared to standard LLINs, with sustained reduction in parasite prevalence, higher mosquito mortality and reduction in mosquito blood feeding rates 21 to 25 months post intervention. Questions remain about the durability of PBO on nets, as the impact of pyrethroid-PBO nets on mosquito mortality was not sustained over 20 washes in experimental hut trials, and epidemiological data on pyrethroid-PBO nets for the full intended three-year life span of the nets is not available. Little evidence is available to support greater entomological efficacy of pyrethroid-PBO nets in areas where mosquitoes show lower levels of resistance to pyrethroids.

Personal protection with PBO-pyrethroid synergist-treated nets after 2 years of household use against pyrethroid-resistant Anopheles in Tanzania.

BACKGROUND: The spread of pyrethroid resistance in malaria vectors threatens the effectiveness of standard long-lasting insecticidal nets (LLIN). Synergist nets combine pyrethroid (Py) and piperonyl-butoxide (PBO) to enhance potency against resistance mediated by mono-oxygenase mechanisms. Our project assessed personal protection of the World Health Organization first-in-class PBO-Py LLIN (Olyset Plus) versus the standard LLIN (Olyset net) against pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) and An. funestus in North-West Tanzania after 20 months of household use. METHODS: From a household survey, 39 standard Olyset net and 39 Olyset Plus houses were selected. The physical integrity and hole index (HI) of the nets were assessed, and resting mosquitoes were collected from inside nets and from room walls. The indoor abundance was estimated using CDC light traps and species identified using PCR. The bioefficacy of PBO and standard LLINs against wild Anopheles was assessed using 30-minute cylinder bioassays. RESULTS: Of 2397 Anopheles collected, 8.9% (n = 213) were resting inside standard Olyset nets, while none were found inside Olyset Plus nets (PBO-Py LLINs) of any HI category. Resting density of blood-fed mosquitoes was higher on walls of sleeping rooms with Olyset nets compared to Olyset Plus (0.62 vs 0.10, density ratio [DR]: 0.03, 95% CI 0.01-0.13, p < 0.001). Mosquitoes were found inside Olyset nets of all WHO HI categories, but more were collected inside the more damaged nets (HI ≥ 643) than in less damaged (HI 0-64) nets (DR: 6.4, 95% CI 1.1-36.0, p = 0.037). In bioassay, mortality of An. gambiae s.l. was higher with Olyset Plus than with Olyset nets for new nets (76.8% vs 27.5%) and nets used for 20 months (56.8% vs 12.8%); similar trends were observed with An. funestus. CONCLUSION: The PBO-Py LLINs provided improved protection after 20 months of household use, as demonstrated by the higher bioassay mortality and absence of pyrethroid-resistant An. gambiae sensu stricto (s.s.) and An. funestus collected from inside Olyset Plus nets, irrespective of HI category, as compared to Olyset nets.

Control of Rhipicephalus annulatus resistant to deltamethrin by spraying infested cattle with synergistic eucalyptus essential oil-thymol-deltamethrin combination.

The current study investigated the synergistic effect of combinations containing deltamethrin (D), Eucalyptus essential oil (E), and the thyme essential oil component thymol (T), against a field population of Rhipicephalus annulatus in Egypt that was characterized to be resistant to D. Solutions of T, E, or TE at concentrations of 1.25-5% were combined with 5% deltamethrin at different dilutions (0.25-2 mL/L). Results of the adult immersion test used to estimate the in vitro acaricidal activity of these combinations at 5% yielded LC50 values for D, E-D, T-D, and TE-D of 3.87 mL/L, 3.89 mL/L, 0.14 mL/L, and 0.05 mL/L, respectively. Biochemical analyses using whole-body homogenate of ticks from the in vitro tests revealed that the lowest acetylcholinesterase and glutathione peroxidase activity, and the maximum lipid peroxidation were recorded in ticks treated with 5% TE-D. Glutathione content significantly decreased (p ≤ 0.05) in all treated ticks. Three groups, each containing five cross breed cattle naturally infested with R. annulatus from the same area where resistance to D was detected, were sprayed twice at two-week intervals using 1 mL/L of 5% solutions of D, T-D, or TE-D. Overall efficacy of the D, T-D, and TE-D sprays by day 30 post-treatment was 21.6, 88.3, and 95 %, respectively. Ticks collected from infested cattle three days after treatment with the D spray deposited egg masses that were able to hatch, deposited small masses of eggs unable to hatch when exposed to the T-D spray, and laid few eggs that didn't hatch when sprayed with the TE-D combination. Values for liver and kidney function parameters were comparable in cattle before and after treatment with the combination sprays tested. The TE-D spray overcame the insensitivity to D of this R. annulatus population in Egypt, which also highlighted the significant synergistic effect of thymol on the acaricidal activity of deltamethrin observed in vitro. Acaricidal activity of the TE-D combination apparently has deleterious effects on multiple tick systems involving inhibition of acetylcholinesterase, increased lipid peroxidation, and oxidative stress. These findings document that combinations of natural and synthetic products can be part of integrated management solutions to the problem with widespread resistance to pyrethroids like deltamethrin in populations of cattle ticks, including R. annulatus, around the world.

Piperonyl butoxide, a synergist of pesticides can elicit male-mediated reproductive toxicity.

A semi-synthetic methylenedioxyphenyl compound piperonyl butoxide (PBO) has been used as a ubiquitous synergist to increase the insecticidal effect of pesticides for agricultural and household use. Despite previously demonstrated effects of PBO, the detailed mechanism of PBO in spermatozoa and reproductive toxic effects on male germ cells have not been fully elucidated. Therefore, this study evaluated the effects of PBO on various sperm functions during capacitation and clarified the mechanisms of reproductive toxic effects on male fertility at different concentrations of PBO (0.1, 1, 10, and 100 µM). Sperm motility and kinematics were assessed using computer-assisted sperm analysis and the status of capacitation was evaluated using combined H33258/chlortetracycline (CTC) staining. Intracellular adenosine triphosphate (ATP) and cell viability levels were also measured. In addition, protein kinase A (PKA) activity and protein tyrosine phosphorylation were evaluated. In addition, in vitro fertilization was performed to determine the effects of PBO on cleavage and blastocyst formation rates. We found that PBO significantly decreased sperm motility, kinematics, and acrosome-reacted and capacitated spermatozoa. In addition, PBO suppressed the intracellular ATP levels and directly affected cell viability. Moreover, PBO detrimentally decreased the activation of PKA and altered the levels of tyrosine-phosphorylated proteins. Consequently, cleavage and blastocyst formation rates were significantly reduced in a dose-dependent manner. In line with our observations, the synergist of pesticides PBO may directly and/or indirectly cause disorder in male fertility. Hence, we suggest that careful attention is made to consider reproductive toxicity when using PBO as a synergist.

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.

First report of the lethal activity and synergism between deltamethrin, amitraz and piperonyl butoxide against susceptible and pyrethroid-resistant nymphs of Triatoma infestans.

In South America, Triatoma infestans (Hemiptera: Reduviidae) is the main vector of the parasite Trypanosoma cruzi, etiological agent of Chagas disease. The main strategy for vector control is to spray domestic structures with pyrethroids. Reports of populations of T. infestans with varying degrees of resistance to pyrethroids have made the search for alternative molecules for vector control necessary. In the first stage of this work we investigated the lethal activity of amitraz and deltamethrin against susceptible and pyrethroid-resistant nymphs of Triatoma infestans. Lethal dose at 50% (LD50) of susceptible nymphs were compared with those recorded in pyrethroid-resistant nymphs and the resistance ratio (RR50) was obtained. The RR50 of deltamethrin was approximately 300. In the case of amitraz, we observed similar triatomicidal activity in the two nymph populations (RR50: 0.7). In a second stage of the work, we determined the synergistic effect of amitraz and piperonyl butoxide (PBO) on the lethal activity of deltamethrin. The strong synergistic effect of PBO on the lethal activity of deltamethrin in resistant nymphs produced a decrease in RR50 to almost one third of the RR50 reported in absence of the synergist. Amitraz plus PBO lethal activity was similarly increased in pyrethroid susceptible and resistant nymphs. Our data indicate that deltamethrin synergism by amitraz was higher against resistant than to susceptible nymphs (Synergist ratio (SR50) of: 7.2- and 4.1-fold, respectively). In pyrethroid resistant nymphs, the highest level of synergism was obtained combining deltamethrin with amitraz and PBO (SR50: 26.7-fold). These results indicate that this combination could be considered an effective alternative for the control of T. infestans.

Assessment of Resistance to Organophosphates and Pyrethroids in Aedes aegypti (Diptera: Culicidae): Do Synergists Affect Mortality?

Aedes aegypti (L.) is the primary vector of Zika, dengue, yellow fever, and chikungunya viruses. Insecticides used in mosquito control can help prevent the spread of vector-borne diseases. However, it is essential to determine insecticide resistance (IR) status before control measures are undertaken. Only the most effective insecticides should be used to avoid ineffective control and/or promotion of IR. Pyrethroids and organophosphates are the most commonly used insecticides for mosquito control. Here, the efficacy of two active ingredients (AIs; permethrin [pyrethroid], chlorpyrifos [organophosphate]), two formulated products (FPs; Biomist [AI: permethrin]) and (Mosquitomist [AI: chlorpyrifos]), and three synergists (piperonyl butoxide, diethyl maleate, S-S-S-tributyl phosphorotrithioate) was evaluated in two Ae. aegypti colonies (pyrethroid resistant and susceptible). Mosquitomist was most effective against the pyrethroid-resistant colony (100% mortality at diagnostic time). Pre-exposure to synergists did not increase the efficacy of AIs against the pyrethroid-resistant colony. Further research is needed to discover how synergists may affect the efficacy of insecticides when used on pyrethroid-resistant mosquitoes.

Effect of long-lasting insecticidal nets with and without piperonyl butoxide on malaria indicators in Uganda (LLINEUP): a pragmatic, cluster-randomised trial embedded in a national LLIN distribution campaign.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the primary malaria prevention tool, but their effectiveness is threatened by pyrethroid resistance. We embedded a pragmatic cluster-randomised trial into Uganda's national LLIN campaign to compare conventional LLINs with those containing piperonyl butoxide (PBO), a synergist that can partially restore pyrethroid susceptibility in mosquito vectors. METHODS: 104 health sub-districts, from 48 districts in Uganda, were randomly assigned to LLINs with PBO (PermaNet 3.0 and Olyset Plus) and conventional LLINs (PermaNet 2.0 and Olyset Net) by proportionate randomisation using an iterative process. At baseline 6, 12, and 18 months after LLIN distribution, cross-sectional surveys were done in 50 randomly selected households per cluster (5200 per survey); a subset of ten households per cluster (1040 per survey) were randomly selected for entomological surveys. The primary outcome was parasite prevalence by microscopy in children aged 2-10 years, assessed in the as-treated population at 6, 12, and 18 months. This trial is registered with ISRCTN, ISRCTN17516395. FINDINGS: LLINs were delivered to households from March 25, 2017, to March 18, 2018, 32 clusters were randomly assigned to PermaNet 3.0, 20 to Olyset Plus, 37 to PermaNet 2.0, and 15 to Olyset Net. In the as-treated analysis, three clusters were excluded because no dominant LLIN was received, and four clusters were reassigned, resulting in 49 PBO LLIN clusters (31 received PermaNet 3.0 and 18 received Olyset Plus) and 52 non-PBO LLIN clusters (39 received PermaNet 2.0 and 13 received Olyset Net). At 6 months, parasite prevalence was 11% (386/3614) in the PBO group compared with 15% (556/3844) in the non-PBO group (prevalence ratio [PR] adjusted for baseline values 0·74, 95% CI 0·62-0·87; p=0·0003). Parasite prevalence was similar at month 12 (11% vs 13%; PR 0·73, 95% CI 0·63-0·85; p=0·0001) and month 18 (12% vs 14%; PR 0·84, 95% CI 0·72-0·98; p=0·029). INTERPRETATION: In Uganda, where pyrethroid resistance is high, PBO LLINs reduced parasite prevalence more effectively than did conventional LLINs for up to 18 months. This study provides evidence needed to support WHO's final recommendation on use of PBO LLINs. FUNDING: The Against Malaria Foundation, UK Department for International Development, Innovative Vector Control Consortium, and Bill and Melinda Gates Foundation.

Piperonyl butoxide: Mode of action analysis for mouse liver tumour formation and human relevance.

In a 79 week bioassay the pesticide synergist piperonyl butoxide (PBO) was shown to significantly increase the incidence of hepatocellular adenoma (but not hepatocellular carcinoma) in male CD-1 mice at dietary levels of 100 and 300 mg/kg/day PBO and in female mice at a dietary level of 300 mg/kg/day. As PBO is not a genotoxic agent, a series of investigative studies were undertaken to elucidate the mode of action (MOA) for PBO-induced mouse liver tumour formation. Male CD-1 mice were fed diets to provide intakes of 0 (control), 30, 100 and 300 mg/kg/day PBO and for purposes of comparison 500 ppm sodium phenobarbital (NaPB), a known constitutive androstane receptor (CAR) activator, for 7 and 14 days. Treatment with 100 and 300 mg/kg/day PBO and 500 ppm NaPB increased relative liver weight which was associated with hepatocyte hypertrophy, with hepatocyte replicative DNA synthesis (RDS) being increased after 7 days treatment. The treatment of CD-1 mice with 30-300 mg/kg/day PBO for 14 days resulted in significant dose-dependent increases in hepatic microsomal cytochrome P450 (CYP) content and 7-pentoxyresorufin O-depentylase (PROD) activity and in hepatic Cyp2b10 mRNA levels. In contrast, PBO produced a biphasic effect on markers of activation of the peroxisome proliferator-activated receptor alpha (PPARα), with small increases in microsomal lauric acid 12-hydroxylase activity and hepatic Cyp4a10 mRNA levels being observed in mice given 100 mg/kg/day with PBO, with either no increase or a significant inhibition being observed in mice given 300 mg/kg/day PBO. The hepatic effects of PBO in male CD-1 mice were generally similar to those produced by NaPB and were reversible after the cessation of treatment for 28 days. Studies were also performed in male C57BL/6J (wild type) mice and in hepatic CAR and pregnane X receptor (PXR) knockout mice (CAR KO/PXR KO mice), where in the CAR KO/PXR KO mice PBO had little effect on markers of CAR activation, but produced some increases in markers of PPARα activation. The treatment of male CD-1 mouse hepatocytes for 4 days with 5-50 µM PBO, 10-1000 µM NaPB and 25 ng/mL epidermal growth factor (EGF) resulted in significant increases in hepatocyte RDS. While treatment of hepatocytes from one male and one female human donor with 5-500 µM PBO and 10-1000 µM NaPB for 4 days had no effect on hepatocyte RDS, treatment with EGF resulted in significant increases in RDS in both human hepatocyte preparations. In summary, PBO is predominantly a hepatic CAR activator at carcinogenic dose levels in CD-1 mice, with activation of hepatic CAR resulting in a suppression of the effect of PBO on hepatic PPARα. A robust MOA for PBO-induced mouse liver tumour formation has been established, this MOA being similar to that previously identified for NaPB and some other rodent liver CAR activators. Based on the lack of effect of PBO on RDS in human hepatocytes, it is considered that the MOA for PBO-induced mouse liver tumour formation is qualitatively not plausible for humans.

Physical integrity and survivorship of long-lasting insecticidal nets distributed to households of the same socio-cultural community in Benin, West Africa.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are designed to survive and sustain their physical barrier for 3 years in household conditions. However, studies have shown that most of these nets are usually torn or no longer present in the households in less than 3 years. This study was initiated in Benin to compare the survivorship and physical integrity of seven types of LLINs in a same socio-geographic area. METHODS: In August 2017, 1890 households were selected in 9 villages in the municipality of Zagnanado in central Benin. Each one of the selected households received one of the seven LLIN products: Aspirational®, DawaPlus® 2.0, OlysetNet®, PermaNet® 2.0, PermaNet® 3.0, Royal Sentry® and Yorkool®. Overall, 270 LLINs of each type were freely distributed in Zagnanado, at a rate of 30 LLINs per type per village. These bed nets have been monitored and evaluated every 6 months to identify the most resilient and preferred LLINs in the community. Net survivorship was assessed using the rate of net loss and physical condition. RESULTS: The survivorship of all types of LLIN was estimated at 92% (95% CI 90.33-92.96) after 6 months and 70% (95% CI 67.25-71.81) after a year of use. At 12 months, all bed nets monitored were below the NetCalc model threshold of 92.8% for an LLIN with a lifespan of 3 years. Only 1.73% of all types of LLIN had a visible loss of integrity after 6 months with a median proportionate hole index (PHI) estimated at zero. The percentage significantly increased after 12 months with 10.41% of damaged nets (all types of LLINs). The median PHI for each brand of net was 23, 196, 141, 23, 23, 121 and 72, respectively for Aspirational®, DawaPlus® 2.0, OlysetNet®, PermaNet® 2.0, PermaNet® 3.0, Royal Sentry® and Yorkool®. A significant difference was noted between the PHI at 6 and 12 months (p < 0.0001). After 12 months, the DawaPlus®2.0, OlysetNet® and Royal Sentry® suffered significantly more damage compared to the others (p < 0.001). CONCLUSION: The results of this study showed that after a year of use, the survivorship of the 7 LLIN products in households was lower than expected. However, all the LLIN products successfully met WHO standards for physical integrity after 12 months of use. The monitoring continues. The next steps will help to identify the most sustainable LLINs.

Screening for Enhancement of Permethrin Toxicity by Plant Essential Oils Against Adult Females of the Yellow Fever Mosquito (Diptera: Culicidae).

Aedes aegypti L. (Diptera: Culicidae) is one of the most medically important mosquito species, due to its ability to spread viruses of yellow fever, dengue fever, and Zika in humans. In this study, the insecticidal activity of 17 plant essential oils was evaluated via topical application against two strains of Ae. aegypti mosquito, Orlando (insecticide-susceptible) and Puerto Rico (pyrethroid-resistant). Initial screens with the Orlando strain showed that cucumber seed oil (2017 sample) was the most toxic, followed by sandalwood and thyme oil. When the essential oils were mixed with permethrin, they failed to show any significant synergism of insecticidal activity. Sandalwood and thyme oils displayed consistently high mortality against the resistant Puerto Rico strain, with low resistance ratios of 2.1 and 1.4, respectively. In contrast, cucumber seed oil showed significantly less activity against Puerto Rico mosquitoes, with a resistance ratio of 45. Bioactivity-guided fractionation of the 2017 sample of cucumber seed oil sample via flash column chromatography produced 11 fractions, and gas-chromatography/mass spectrometry analysis revealed that the three active fractions were contaminated with 0.33, 0.36, and 0.33% of chlorpyrifos-methyl, an organophosphorus insecticide, whereas inactive fractions did not show any trace of it. These results suggested that the insecticidal activity of cucumber seed oil was probably due to the presence of the insecticide, later confirmed with a clean batch of cucumber seed oil obtained in 2018, which showed negligible insecticidal activity. These findings demonstrate clearly the need for essential oil analysis to confirm purity before any claims are made about pesticidal potency.

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.

Piperonyl Butoxide Enhances the Insecticidal Toxicity of Nanoformulation of Imidacloprid on Culex pipiens (Diptera: Culicidae) Mosquito.

The use of conventional pesticides becomes a complicated issue as more species of insect pests become resistant to them. Nanopesticides suit new approaches in pest control. Herein, we tested the toxicological efficacy of imidacloprid compared with three of its nanoformulations (IMD01, IMD02, and IMD03) on second and fourth instar of Culex pipiens larvae. Furthermore, we assessed the synergistic actions of piperonyl butoxide (PBO) on imidacloprid and its nanoformulations against second and fourth instar of C. pipiens. The nanoformulation (IMD03) was the most potent insecticide (LC50 = 14, 6, and 2 ng/mL after 24, 48, and 72 h of exposure, respectively), whereas the lowest toxic nanoformulation was IMD01. However, imidacloprid had the lowest toxicity among the tested compounds (LC50 = 1015, 705, and 621 ng/mL after 24, 48, and 72 h of exposure, respectively). PBO significantly synergized imidacloprid and its nanoformulations. However, the most synergistic effects were on IMD03 and the lowest was imidacloprid itself. Based on our results, nanopesticides are currently the most promising tool to control C. pipiens mosquitoes. However, further semifield and field studies should be done to illustrate the efficacy of imidacloprid and its nanoformulations on C. pipiens mosquitoes.

Evaluation of an alpha-cypermethrin + PBO mixture long-lasting insecticidal net VEERALIN® LN against pyrethroid resistant Anopheles gambiae s.s.: an experimental hut trial in M'bé, central Côte d'Ivoire.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the primary method of malaria prevention. However, the widespread resistance to pyrethroids among major malaria vector species represents a significant threat to the continued efficacy of pyrethroid LLIN. Piperonyl butoxide (PBO) is a synergist that inhibits the activity of metabolic enzymes of the cytochrome P450 family known to detoxify insecticides including pyrethroids. Synergist LLIN incorporating PBO and a pyrethroid may provide improved control compared to pyrethroid-only LLIN. METHODS: The efficacy of VEERALIN® LN (VKA polymers Pvt Ltd, India), an alpha-cypermethrin PBO synergist net was evaluated in experimental huts in M'bé, central Côte d'Ivoire against wild pyrethroid resistant Anopheles gambiae s.s. Comparison was made with a standard alpha-cypermethrin-treated net (MAGNet® LN, VKA polymers Pvt Ltd, India). Nets were tested unwashed and after 20 standardized washes. RESULTS: VEERALIN® LN demonstrated improved efficacy compared to MAGNet® LN against wild free-flying pyrethroid-resistant An. gambiae s.s. Before washing, VEERALIN® LN produced mortality of An. gambiae s.s. (51%) significantly higher than the standard pyrethroid-only net (29%) (P < 0.0001). Although there was a significant reduction in mortality with both LLINs after 20 washes, VEERALIN® LN remained superior in efficacy to MAGNet® LN (38 vs 17%) (P < 0.0001). Blood-feeding was significantly inhibited with both types of insecticide-treated nets relative to the untreated control net (P < 0.0001). Unwashed VEERALIN® LN induced significantly higher blood-feeding inhibition of An. gambiae s.s. (62.6%) compared to MAGNet® LN (35.4%) (P < 0.001). The difference persisted after washing, as there was no indication that either LLIN lost protection against biting or blood-feeding. The level of personal protection derived from the use of VEERALIN® LN was high (87%) compared to MAGNet® LN (66-69%) whether unwashed or washed. The AI content of VEERALIN® LN after 20 washes decreased from 6.75 to 6.03 g/kg for alpha-cypermethrin and from 2.95 to 2.64 g/kg for PBO, corresponding to an overall retention of 89% for each compound. CONCLUSIONS: The addition of the synergist PBO to pyrethroid net greatly improved protection and control of pyrethroid-resistant An. gambiae s.s. The pyrethroid-PBO VEERALIN® LN has the potential to reduce transmission in areas compromised by pyrethroid resistance.