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.

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.

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.

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 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.

Effects of disruption of the peritrophic membrane on larval susceptibility to Bt toxin Cry1Ac in cabbage loopers.

The insect midgut peritrophic membrane (or peritrophic matrix) (PM) is an extracellular structure, lining the midgut epithelium. The PM facilitates the food digestion process and plays important roles in insect-microbe interactions as a barrier against microbial pathogens. The soil bacterium, Bacillus thuringiensis (Bt), and its proteinaceous toxins are widely used for insect control. To understand the protective role of PM in insects against Bt toxins, the effect of PM on larval susceptibility to Bt toxin Cry1Ac was examined in Cry1Ac-susceptible and -resistant strains of the cabbage looper, Trichoplusia ni. The PM in T. ni was disrupted, using a baculovirus enhancin (TnGV enhancin) to degrade the major PM mucin protein IIM and a chitin binding chemical, Calcofluor, to inhibit the binding of PM proteins to chitin. Bioassays of the susceptibility of T. ni larvae to Cry1Ac with treatment of TnGV enhancin showed significantly increased larval mortality in both the Cry1Ac susceptible and resistant strains, confirming that the PM is a protective barrier to the passage of Cry1Ac and plays a protective role against the toxin. However, treatment of T. ni larvae with Calcofluor significantly reduced the larval susceptibility to Cry1Ac. The level of mortality reduction by treatment with Calcofluor was more significant in the resistant T. ni strains than in the susceptible strain. The mechanism for the decrease of susceptibility to Cry1Ac in T. ni treated with Calcofluor needs to be understood. It may result from binding of the toxin to the over expressed PM proteins, preventing the Cry1Ac from reaching the midgut receptor for the toxin or from potential binding of Calcofluor to the midgut receptor for Cry1Ac, leading to inhibition of the toxicity of Cry1Ac in larvae.

Evaluation of Resistance to Different Insecticides and Metabolic Detoxification Mechanism by Use of Synergist in the Common Bed Bug (Heteroptera: Cimicidae).

Bed bugs have become a common urban pest with consequences on human health and economic costs to the hotel and tourism sectors. Insecticide resistance is considered an important factor in the current bed bug resurgence, and multiple resistance mechanisms could be working in the resistant bed bug populations. In the present study, we determined the resistance profile to four insecticides with a different mode of action in Cimex lectularius L. (Heteroptera: Cimicidae) field-collected colonies from Argentina. Furthermore, the synergism effect of piperonyl butoxide (PBO) with deltamethrin was investigated to explore the contribution of detoxification metabolism to resistance. Our results showed that most of the field-collected colonies are extremely resistant to deltamethrin and propoxur, much more than to azametiphos and imidacloprid. The differences in resistance ratios among field-collected colonies could be associated with different modes of action of insecticides used in control pest and the mechanisms involved in the resistance. PBO pretreatment led to a significantly decreased RR in pyrethroid-resistant colonies, suggesting an upturn of monooxygenase activity for deltamethrin detoxification. However, the high RR detected could involve other mechanisms as part of the whole resistant phenotype in colonies of C. lectularius resistant to pyrethroids.

LLIN Evaluation in Uganda Project (LLINEUP): a cross-sectional survey of species diversity and insecticide resistance in 48 districts of Uganda.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the principal tool for malaria control in Africa and are presently treated with a single class of insecticide; however, increasing levels of insecticide resistance threaten their success. In response to this threat nets have been developed that incorporate the synergist, piperonyl butoxide (PBO), which inhibits the activity of cytochrome P450s which is one main mechanisms of insecticide resistance, allowing resistance to pyrethroids to be reversed. However, data on the value and cost effectiveness of these nets is lacking. A large-scale cluster randomised trial of conventional LLINs and PBO-LLINs was conducted in Uganda in 104 health sub-districts (HSDs) in 2017-2019. Prior to the mass distribution of LLINs, a baseline entomological survey was carried out, the results of which are reported herein. Ten households from each HSD were randomly selected for entomological surveillance at baseline which included household mosquito collections. RESULTS: Prior to LLIN distribution entomological collections were carried out in 1029 houses across the 104 HSDs. Anopheles gambiae (s.l.) was the principal vector in all but 9 of the 71 HSDs that yielded vector species. Molecular analysis found An. gambiae (s.s.) to be the predominant vector collected. Plasmodium falciparum was detected in 5.5% of An. gambiae (s.s.) and in 4.0% of An. funestus (s.s.) examined. Infection rates of other plasmodium species (P. vivax, P. ovale and P. malariae) were lower with infection rates of 1.2% and 1.7% for An. gambiae (s.s.) and An. funestus (s.s.), respectively. The knockdown resistance (kdr) mutation Vgsc-L1014S was found at very high frequency in An. gambiae (s.s.) with the Vgsc-L1014F mutation at low frequency and the wild-type allele virtually absent. In An. arabiensis the wild-type allele was predominant. The resistance-associated alleles, Cyp4j5-L43F and Coeae1d were found at moderate frequencies which varied across the study site. Vgsc-N1575Y mutation was not found in any samples examined. CONCLUSIONS: No significant differences between planned intervention arms was observed in vector densities, sporozoite infection rate or insecticide resistance marker frequency across the study site prior to the distribution of LLINs. Very high levels of kdr resistance were observed in all areas; however, the resistance-associated markers Cyp4j5-L43F and Coeae1d were found at varying frequencies across the study site which may have implications for the effectiveness of standard LLINs. Trial registration This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395.

Resistance to insecticides and synergistic and antagonistic effects of essential oils on dimefluthrin toxicity in a field population of Culex quinquefasciatus Say.

In this work, we firstly tested five spatial repellent pyrethroids, meperfluthrin, dimefluthrin, heptafluthrin, metofluthrin and transfluthrin, to determine the susceptibility of pyrethroids to field strains of Culex quinquefasciatus using adult topical bioassay. The results showed that though field strains exhibited the highest resistance to dimefluthrin among the selected five pyrethroids, it still can be considered low resistance in the scale of Cui et al. (2006; 2007). Then, the aim of this study was to optimise the synergistic efficacy of essential oils combined with dimefluthrin and explore the major contribution composition of eucalyptus oil, basil oil and cinnamon oil as natural synergist of dimefluthrin against the field populations of C. quinquefasciatus. GC-MS analysis showed 1,8-cineole, eugenol and trans-cinnamaldehyde were the main chemical components of eucalyptus oil, basil oil and cinnamon oil, respectively. The results of bioactivity showed that eucalyptus oil and 1,8-cineole have highly fumigant knock-down activity to the adults, showing KT50 (the median knockdown time) of 5.76 and 4.27 min at the concentration of 24.2 µL/L; basil oil and eugenol, cinnamon oil and trans-cinnamaldehyde have highly fumigant toxicity to the adults, showing LD50 of 1.00 and 0.79, 1.26 and 1.03 µL/L, respectively. Three effective main essential oil components were selected to prepare binary mixtures, which combined with dimefluthrin against the field population of Culex quinquefasciatus. 1,8-cineole+eugenol (9:1, w/w), 1,8-cineole+trans-cinnamaldehyde (1:1, w/w) and trans-cinnamaldehyde+eugenol (9:1, w/w) combined with dimefluthrin (10:1, w/w) were the most synergistic interaction, showed SR (synergistic ratio) values of 1.2471, 1.5709 and 1.1969; KT50 of 11.68, 9.51 and 10.67 min respectively, by quadrate box method. In addition, to validate the stable synergistic interaction of 1,8-cineole+trans-cinnamaldehyde (1:1, w/w) combined with dimefluthrin (10:1, w/w), the SR values were about 1.3, and KT50 values were 38.72-50.26 min by simulated house method. Overall, our results pointed out the promising potential of these essential oils to increase the efficacy of dimefluthrin. It might be expected that these essential oils could be developed to a useful botanical synergist of dimefluthrin for the control of the field populations of C. quinquefasciatus.

Enhanced mortality in deltamethrin-resistant Aedes aegypti in Thailand using a piperonyl butoxide synergist.

Aedes aegypti is the primary vector of dengue viruses in Thailand. Control of this mosquito continues to rely heavily on use of insecticides in various forms and applications. The synergistic effect of piperonyl butoxide (PBO), combined with deltamethrin against eight populations of Ae. aegypti collected from different regions in Thailand is presented. The standard WHO adult contact bioassays found all populations with low to moderate levels of resistance to deltamethrin alone (using a 0.05% discriminating concentration), with final mortalities ranging from 15.6 to 70%, while a laboratory strain was fully susceptible (100% mortality). Pre-exposure of female mosquitoes to 4% PBO for 1 h, followed immediately by exposure to deltamethrin for 1 h, significantly increased mortality in seven populations (64.8-98.1%) with the exception of mosquitoes derived from Lampang Province. The knockdown time (KDT) synergist ratios between deltamethrin only and PBO + deltamethrin ranged from 1.7 to 2.8 for KDT50 and 1.9 to 4.0 for KDT95. Between deltamethrin alone and mosquitoes exposed to PBO + deltamethrin, all resistant populations produced significant differences (P < 0.05) in final 24-h mortality, except marginally for Lampang (P = 0.053). The synergistic effects of PBO with deltamethrin-resistant Ae. aegypti suggest a combination of this synergist with deltamethrin or other pyrethroid compounds can significantly enhance the effectiveness of these insecticides against pyrethroid-resistant Ae. aegypti found commonly in Thailand.

Insecticide resistance status, frequency of L1014F Kdr and G119S Ace-1 mutations, and expression of detoxification enzymes in Anopheles gambiae (s.l.) in two regions of northern Benin in preparation for indoor residual spraying.

BACKGROUND: This study aims to provide baseline data on the resistance status to insecticides, the frequency of mechanisms involved and the impact of the association with the synergist piperonyl butoxide (PBO) on resistant Anopheles gambiae (s.l.) populations in two regions of northern Benin, prior to an indoor residual spraying campaign and introduction of next generation long-lasting insecticidal nets (LLINs) incorporating PBO. METHODS: Adult Anopheles gambiae (s.l.) originating from larvae collected in two study regions (Alibori within the Kandi-Gogounou-Segbana districts and Donga within the Djougou-Copargo-Ouake districts) were tested with impregnated papers (bendiocarb 0.1%, pirimiphos-methyl 0.25%, permethrin 0.75% and deltamethrin 0.05%). The synergist PBO was used to check for the involvement of detoxification enzymes in pyrethroid resistant populations. Molecular analyses were performed for the identification of species within the Anopheles gambiae (s.l.) complex and kdr L1014F and G119S Ace-1 mutations. Biochemical assays assessed the activity of detoxification enzymes. RESULTS: Anopheles gambiae (s.l.) was resistant to pyrethroids, with a mortality range of 25-83% with deltamethrin and 6-55% with permethrin. A significant increase in mortality was observed after pre-exposure to PBO for both deltamethrin (63-99%) and permethrin (56-99%). With bendiocarb, An. gambiae (s.l.) were susceptible in Kandi (99% mortality), with possible resistance (92-95%) recorded in Djougou, Copargo, Gogounou, Ouake and Segbana. All study populations were fully susceptible to pirimiphos-methyl. The frequencies of resistant mutations varied according to species and sites: 0.67-0.88 for L1014F kdr and 0-0.06 for G119S Ace-1. Three study locations (Djougou, Gogounou and Kandi) showed high oxidase activity and four sites (Djougou, Ouake, Copargo and Kandi) showed elevated esterase activity. CONCLUSIONS: This study confirms resistance to pyrethroids and suggests emerging bendiocarb resistance in An. gambiae (s.l.) populations in northern Benin. However, recovery of susceptibility to pyrethroids after PBO exposure, and susceptibility to organophosphates in the An. gambiae (s.l.) populations indicate that next generation LLINs incorporating PBO synergist combined with an indoor residual spraying (IRS) campaign with organophosphate insecticides may be regarded as alternative control tools.

QSAR modelling of synergists to increase the efficacy of deltamethrin against pyrethroid-resistant Aedes aegypti mosquitoes$.

Space spraying of deltamethrin allows the control of adult Aedes (Stegomyia) aegypti mosquitoes. Unfortunately, many vector control programs are threatened by the development of resistances that decrease the efficacy of this adulticide. Faced with this situation, we can either try to use another insecticide presenting a different mechanism of action or find a strategy that brings back the efficacy of the insecticide at a satisfying level to pursue its use in vector control. Restoration of the efficacy of an insecticide can be obtained by means of a synergist. In this context, QSAR modelling was used to find synergists to combine with deltamethrin for increasing its efficacy against resistant strains of Ae. aegypti. Seventy-four structurally diverse chemicals with their 24-hour LD50 values, obtained under the same experimental conditions on Ae. aegypti females, were used. Molecules were described by means of autocorrelation vectors encoding lipophilicity, molar refractivity, H-bonding acceptor and donor ability. A three-layer perceptron (TLP) was employed as statistical tool. The performances of the models were evaluated through the analysis of the prediction results obtained on the different training and test sets (80%/20%) as well as from an out-sample test set. A 6/4/1 TLP computed with the Broyden-Fletcher-Goldfarb-Shanno second-order training algorithm led to the best prediction results. The convergence was obtained in 132 cycles. The sum of squares was used as error function. The hidden and output activation functions were tanh and exponential, respectively. Various chemical structures were identified as potential synergists and searched for their commercial availability. Molecules of interest were tested in vivo on Ae. aegypti by using the susceptible reference Bora Bora strain and two resistant strains from Martinique island. This led to the identification of the PSM-05 molecule that shows interesting synergistic activity.

Acaricidal toxicities and synergistic activities of Salvia lavandulifolia oil constituents against synanthropic mites.

BACKGROUND: The acaricidal activity of Salvia lavandulifolia oil and its major components was evaluated using contact and vapor bioassays. Synergistic interactions among components contained in S. lavandulifolia oil were studied. RESULTS: The 50% lethal dose (LD50 ) of S. lavandulifolia oil was 3.66, 3.37, and 5.04 µg cm-3 , respectively, in the vapor bioassay against Dermatophagoides farinae, Dermatophagoides pteronyssinus and Tyrophagus putrescentiae. The major components of S. lavandulifolia oil were (-)-camphor, camphene, 1,8-cineole, (±)-limonene, and α-pinene (79.11% combined). Several combinations of these components exhibited synergistic effects against D. farinae, D. pteronyssinus, and T. putrescentiae, particularly (-)-camphor, camphene, and α-pinene. (-)-Camphor usually had synergistic interactive effects in the mixtures. For the vapor action, the mixture of (-)-camphor, camphene and α-pinene was the most potent combination against D. farinae (R = 2.34), D. pteronyssinus (R = 2.75), and T. putrescentiae (R = 2.30) when used at their naturally occurring ratio. CONCLUSION: This study is the first report on the acaricidal activity of S. lavandulifolia oil and the synergistic interactive effects of its components against D. farinae, D. pteronyssinus and T. putrescentiae. The oil may be an alternative tool for the control of synanthropic mites. © 2018 Society of Chemical Industry.

Resistance status to deltamethrin pyrethroid of Culex pipiens pipiens (Diptera: Culicidae) collected from three districts of Tunisia.

OBJECTIVES: The aim of the present study was to determine the susceptibility status of Culex pipiens pipiens populations against deltamehtrin insecticide. METHODS: Larvae of Culex pipiens pipiens were collected from three breeding places in Northern and Southern Tunisia between 2003 and 2005. Early third and late fourth instars were tested against deltamethrin pyrethroid insecticide. Cross-resistance with DDT resistance was evaluated in studied samples to estimate the role of target site insensitivity and two synergists including piperonyl butoxide (Pb) and S,S,S-tributyl phosphorotrithioate (DEF) were used to estimate the role of detoxification enzymes. RESULTS: Our results revealed that the level of deltamehtrin resistance ranged from 0.67 to 31.4. We also showed the non-involvement of kdr resistance in pyrethroid resistance and no cross-resistance with DDT resistance was detected in all studied populations including the most resistant one. Synergists study on the resistant population (sample # 1) showed the involvement of CYP450 in the recorded resistance to the deltamethrin insecticide. CONCLUSION: The results obtained from this study should be considered in the current control programs to combat mosquitoes in Tunisia.

Characterization and functional analysis of a carboxylesterase gene associated with chlorpyrifos resistance in Nilaparvata lugens (Stål).

The widespread and extensive application of insecticides have promoted the development of resistance in the brown planthopper Nilaparvata lugens (Stål), one of the most important rice pests in Asia. To better understand the underlying molecular mechanisms of metabolic resistance to insecticides, a chlorpyrifos-resistant (CR) strain of N. lugens was selected and its possible resistance mechanism was investigated. Synergistic tests using carboxylesterases (CarEs) inhibitor triphenyl phosphate (TPP) decreased the resistance of N. lugens to chlorpyrifos, and CarE activities could be induced by low concentrations of chlorpyrifos. Subsequently, a gene putatively encoding CarE, namely NlCarE, predominant in the midgut and ovary was isolated and characterized. The expression levels of NlCarE were detected and compared between the CR and a susceptible (SS) strain of N. lugens. Consistent with the increased CarE activity, this gene was overexpressed in the CR strain compared to the SS strain. The transcript levels of NlCarE were up-regulated by chlorpyrifos exposure, showing dose- and time-dependent expression patterns. Furthermore, RNA interference (RNAi)-mediated knockdown of NlCarE followed by insecticide application significantly increased the susceptibility of N. lugens to chlorpyrifos. These results demonstrate that NlCarE plays an important role in chlorpyrifos detoxification and its overexpression may be involved in chlorpyrifos resistance in N. lugens.

WHO cone bio-assays of classical and new-generation long-lasting insecticidal nets call for innovative insecticides targeting the knock-down resistance mechanism in Benin.

BACKGROUND: To increase the effectiveness of insecticide-treated nets (ITN) in areas of high resistance, new long-lasting insecticidal nets (LLINs) called new-generation nets have been developed. These nets are treated with the piperonyl butoxide (PBO) synergist which inhibit the action of detoxification enzymes. The effectiveness of the new-generation nets has been proven in some studies, but their specific effect on mosquitoes carrying detoxification enzymes and those carrying both detoxification enzymes and the knock-down resistance gene in Benin is not well known. Thus, the objective of this study is to evaluate the efficacy of LLINs treated with PBO on multi-resistant Anopheles gambiae s.l. METHODS: The study occurred in seven cities in Benin, Abomey, Cotonou, Porto-Novo, Zangnanado, Parakou, Malanville and Tanguiéta, and included ten locations selected on a north-south transect. Mosquito larvae were collected from these sites, and adult females from these larvae were exposed to single-pyrethroid-treated nets (LifeNet, PermaNet 2.0, Olyset Net) and bi-treated nets (PermaNet 3.0 and Olyset Plus) based on their level of resistance and using WHO cone tests following WHO guidelines. RESULTS: The different LLINs showed 100% mortality of the susceptible laboratory strain Kisumu and the resistant strain Ace-1R Kisumu. However, with the resistant laboratory strain kdr-Kisumu, mortality was low (16-32%) for all LLINs except PermaNet 3.0 (82.9%). The mortality of local strains carrying only the kdr mechanism varied from 0 to 47% for the single-pyrethroid-treated LLINs and 9 to 86% for bi-treated LLINs. With local strains carrying several mechanisms of resistance (kdr + detoxification enzymes), the observed mortality with different LLINs was also low except for PermaNet 3.0, which induced significantly higher mortality, usually greater than 75% (p < 0.001), with multi-resistant strains. The inhibition of the mortalities induced by the LLINs (11-96%) on multi-resistant field populations was similar to the inhibition observed with the laboratory strain carrying only the knock-down resistance mechanism (kdr-Kisumu) (p > 0.05). CONCLUSION: This study showed that the new-generation LLINs treated with pyrethroids and PBO showed better efficacy compared to conventional LLINs. Although the addition of PBO significantly increased the mortality of mosquitoes, the significant role of the kdr resistance gene in the low efficacy of LLINs calls for LLIN technology innovation that specifically targets this mechanism.