Lemongrass essential oil and its major component citronellol: evaluation of larvicidal activity and acetylcholinesterase inhibition against Anopheles sinensis.

Mosquito-borne diseases, such as malaria, dengue fever, and the Zika virus, pose significant global health challenges, affecting millions annually. Due to increasing insecticide resistance, there is a growing interest in natural alternatives for mosquito control. Lemongrass essential oil, derived from Cymbopogon citratus, has shown promising repellent and larvicidal properties against various mosquito species. In this study, we investigated the larvicidal effect of lemongrass oil and its major compounds on Anopheles sinensis, the primary malaria vector in China. GC-MS analysis identified the major compounds of lemongrass oil as ( +)-citronellal (35.60%), geraniol (21.84%), and citronellol (13.88%). Lemongrass oil showed larvicidal activity against An. sinensis larvae, with an LC50 value of 119.20 ± 3.81 mg/L. Among the major components, citronellol had the lowest LC50 value of 42.76 ± 3.18 mg/L. Moreover, citronellol demonstrated inhibitory effects on acetylcholinesterase (AChE) activity in An. sinensis larvae, assessed by homogenizing larvae at different time points following treatment. Molecular docking studies further elucidated the interaction between citronellol and AChE, revealing the formation of hydrogen bonds and Pi-Sigma bonds. Aromatic amino acid residues such as Tyr71, Trp83, Tyr370, and Tyr374 played a pivotal role in these interactions. These findings may contribute to understanding lemongrass oil's larvicidal activity against An. sinensis and the mechanisms underlying these effects.

Larvicidal potential of Trachyspermum ammi essential oil and Delphinium speciosum extract against malaria, dengue, and filariasis mosquito vectors.

Mosquito-borne diseases, such as malaria, dengue, and Zika, pose major public health challenges globally, affecting millions of people. The growing resistance of mosquito populations to synthetic insecticides underscores the critical need for effective and environmentally friendly larvicides. Although chemical pesticides can initially be effective, they often lead to negative environmental consequences and health hazards for non-target species, including humans. This study aimed to evaluate the larvicidal effects of Trachyspermum ammi essential oil and Delphinium speciosum extract on the larvae of three major mosquito species: Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus. Mosquito larvae of Ae. aegypti, An. stephensi, and Cx. quinquefasciatus were reared under controlled laboratory conditions. The larvicidal activity of T. ammi essential oil and D. speciosum extract was evaluated through standard bioassays, using various concentrations of essential oils (10, 20, 40, 80, and 160 ppm) and extracts (160, 320, 640, 1280, and 2560 ppm) to determine the lethal concentration (LC50) values after 24 h of exposure. Fresh plant materials were collected, with the essential oil extracted via hydro-distillation, and the extract prepared using methanol solvent extraction. The chemical composition of T. ammi essential oil was examined using gas chromatography-mass spectrometry (GC-MS). Additionally, the preliminary analysis of the chemical compounds in D. speciosum extract was carried out using thin layer chromatography (TLC) and nuclear magnetic resonance spectroscopy (NMR) techniques. The results indicated that the essential oil of T. ammi exhibited more effective larvicidal activity compared to the D. speciosum extract. Specifically, the essential oil demonstrated LC50 values of 18 ppm for Cx. quinquefasciatus and 19 ppm for Ae. aegypti. In contrast, the D. speciosum extract showed the strongest larvicidal effect against An. stephensi, with an LC50 of 517 ppm. Concentrations of 40 ppm of the essential oil and 1280 ppm of the extract resulted in 100% mortality across all three species. Both the essential oil of T. ammi and the D. speciosum extract exhibited concentration-dependent larvicidal activity, and these results were statistically significant (p < 0.001) compared to the no-treatment group. GC-MS analysis revealed thymol (88.95%), o-cymen-5-ol (4.11%), and γ-terpinene (2.10%) as the major constituents of the T. ammi essential oil. Additionally, TLC verified the presence of alkaloids in both chloroform and methanolic extracts. Proton NMR identified a diterpene structure for these alkaloids. These findings suggest that T. ammi essential oil is a promising candidate for natural mosquito control strategies. Given its efficacy, further research is warranted to explore its potential in integrated vector management programs.

Longitudinal survey of insecticide resistance in a village of central region of Burkina Faso reveals co-occurrence of 1014F, 1014S and 402L mutations in Anopheles coluzzii and Anopheles arabiensis.

BACKGROUND: Pyrethroid resistance is one of the major threats for effectiveness of insecticide-treated bed nets (ITNs) in malaria vector control. Genotyping of mutations in the voltage gated sodium channel (VGSC) gene is widely used to easily assess the evolution and spread of pyrethroid target-site resistance among malaria vectors. L1014F and L1014S substitutions are the most common and best characterized VGSC mutations in major African malaria vector species of the Anopheles gambiae complex. Recently, an additional substitution involved in pyrethroid resistance, i.e. V402L, has been detected in Anopheles coluzzii from West Africa lacking any other resistance alleles at locus 1014. The evolution of target-site resistance mutations L1014F/S and V402L was monitored in An. coluzzii and Anopheles arabiensis specimens from a Burkina Faso village over a 10-year range after the massive ITN scale-up started in 2010. METHODS: Anopheles coluzzii (N = 300) and An. arabiensis (N = 362) specimens collected both indoors and outdoors by different methods (pyrethrum spray catch, sticky resting box and human landing collections) in 2011, 2015 and 2020 at Goden village were genotyped by TaqMan assays and sequencing for the three target site resistance mutations; allele frequencies were statistically investigated over the years. RESULTS: A divergent trend in resistant allele frequencies was observed in the two species: 1014F decreased in An. coluzzii (from 0.76 to 0.52) but increased in An. arabiensis (from 0.18 to 0.70); 1014S occurred only in An. arabiensis and slightly decreased over time (from 0.33 to 0.23); 402L increased in An. coluzzii (from 0.15 to 0.48) and was found for the first time in one An. arabiensis specimen. In 2020 the co-occurrence of different resistance alleles reached 43% in An. coluzzii (alleles 410L and 1014F) and 32% in An. arabiensis (alleles 1014F and 1014S). CONCLUSIONS: Overall, an increasing level of target-site resistance was observed among the populations with only 1% of the two malaria vector species being wild type at both loci, 1014 and 402, in 2020. This, together with the co-occurrence of different mutations in the same specimens, calls for future investigations on the possible synergism between resistance alleles and their phenotype to implement local tailored intervention strategies.

A review of selective indoor residual spraying for malaria control.

BACKGROUND: Indoor residual spraying (IRS) is one of the most effective malaria control tools. However, its application has become limited to specific contexts due to the increased costs of IRS products and implementation programmes. Selective spraying-selective spray targeted to particular areas/surfaces of dwellings-has been proposed to maintain the malaria control and resistance-management benefits of IRS while decreasing the costs of the intervention. METHODS: A literature search was conducted to find (1) studies that assessed the resting behaviour of Anopheles mosquitoes and (2) studies that evaluated the impact of selective spraying on entomological and malaria outcomes. Additional articles were identified through hand searches of all references cited in articles identified through the initial search. A cost model was developed from PMI VectorLink IRS country programmes, and comparative cost analysis reports to describe the overall cost benefits of selective IRS. RESULTS: In some studies, there appeared to be a clear resting preference for certain Anopheles species in terms of the height at which they rested. However, for other species, and particularly the major African malaria vectors, a clear resting pattern was not detected. Furthermore, resting behaviour was not measured in a standardized way. For the selective spray studies that were assessed, there was a wide range of spray configurations, which complicates the comparison of methods. Many of these spray techniques were effective and resulted in reported 25-68% cost savings and reduced use of insecticide. The reported cost savings in the literature do not always consider all of the IRS implementation costs. Using the IRS cost model, these savings ranged from 17 to 29% for programs that targeted Anopheles spp. and 18-41% for programmes that targeted Aedes aegypti. CONCLUSIONS: Resting behaviour is generally measured in a simplistic way; noting the resting spot of mosquitoes in the morning. This is likely an oversimplification, and there is a need for better monitoring of resting mosquitoes. This may improve the target surface for selective spray techniques, which could reduce the cost of IRS while maintaining its effectiveness. Reporting of cost savings should be calculated considering the entire implementation costs, and a cost model was provided for future calculations.

Evaluating trends in damage to attractive targeted sugar baits (ATSBs) deployed during the second year of a two-year Phase III trial in Western Zambia.

BACKGROUND: Attractive Targeted Sugar Baits (ATSBs) are a proposed new vector control tool for malaria that contain sugar and an ingestion toxicant, and are designed to attract and kill sugar-feeding mosquitoes. During a two-arm cluster randomized Phase III trial conducted in Zambia to test the efficacy of ATSB stations on malaria incidence, ATSB stations deployed on eligible household structures within intervention clusters were routinely monitored to ensure their good physical condition and high coverage. This study investigates trends in prevalence and rate of damage to ATSB stations during year 2 of the two-year trial. METHODS: The analysis was conducted using monitoring data collected in year 2, which included types of damage observed, location, and date of removal and/or replacement of ATSB stations. The study evaluated temporal trends in the prevalence of overall damage and different damage types among 68,299 ATSB stations deployed. A profile of all ATSB stations installed on each structure was constructed, and spatial analyses conducted on overall damage and different damage types observed on 18,890 structures. Mixed effects regression analyses were conducted to investigate drivers of damage to ATSB stations on these structures. RESULTS: Prevalence of overall damage and different damage types was temporally and spatially heterogeneous. Among damaged ATSB stations observed during monitoring, tears and mold had the highest prevalences on average, with tears maintaining above 50.0% prevalence through most of the monitoring period, while mold prevalence increased steadily during the first few months, peaking in February. Overall, 45.6% of structures had at least one damaged ATSB station, however this varied spatially across the trial site. Both structure characteristics and environmental factors significantly impacted the odds and rate of damage to ATSB stations on structures, including: ATSB stations' level of protection from rainfall and sunshine; roof and wall material of the structure; night-time temperature; rainfall; enhanced vegetation index, and land cover. CONCLUSION: Damage to ATSB stations in this setting was common and was temporally and spatially heterogeneous. This has implications on operational feasibility, sustainability, and cost of future deployment. Further research is required to understand the mechanisms of damage, and to minimize prevalence and rate of damage to ATSB stations.

Larvicidal activity of ß-Citral: An In-vitro and In-silico study to understand its potential against mosquito.

Tropical and subtropical regions face millions of deaths from mosquito-borne illnesses yearly. Insecticides prevent transmission but pose health risks like dermatitis and allergies. The primary objective was to mitigate the recurring dependence on synthetic insecticides, thereby curbing the development of mosquito resistance. Leaves of Cymbopogon flexuosus (lemongrass) was collected from Mayurbhanj, India, processed, then extracted by steam distillation for essential oils & analyzed spectroscopically. Larvicidal assays were performed across varying concentrations, revealing the significant mortality induced by the Cymbopogon flexuosus extract against Anopheles stephensi larvae. 3D structure was modelled by using G protein-coupled receptors (GPCR) sequence and structural stability was also validated. After docking the binding free energy was determined from GPCR protein with ß-citral complex. Molecular dynamics (MD) study was conducted on the docked pose that displayed an optimal interactome profile. The larvicidal assay at the 12th and 24th hour revealed the highest LC50 (lethal concentration) of 23.493 ppm and 19.664 ppm . ß-Citral has a high binding affinity and an identifiable binding site, which suggests that it may play a larvicidal role in regulating the receptor's function by creating stable complexes with it. ß-Citral from lemongrass oils has potential larvicidal activity and effective against GPCR family 1 of mosquito and highly effective repellents against mosquito-borne diseases.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are associated with insecticide resistance in the major malaria vectors Anopheles gambiae s.l. and Anopheles funestus.

Malaria remains one of the highest causes of morbidity and mortality, with 249 million cases and over 608,000 deaths in 2022. Insecticides, which target the Anopheles mosquito vector, are the primary method to control malaria. The widespread nature of resistance to the most important insecticide class, the pyrethroids, threatens the control of this disease. To reverse the stall in malaria control there is urgent need for new vector control tools, which necessitates understanding the molecular basis of pyrethroid resistance. In this study we utilised multi-omics data to identify uridine-diphosphate (UDP)-glycosyltransferases (UGTs) potentially involved in resistance across multiple Anopheles species. Phylogenetic analysis identifies sequence similarities between Anopheline UGTs and those involved in agricultural pesticide resistance to pyrethroids, pyrroles and spinosyns. Expression of five UGTs was characterised in An. gambiae and An. coluzzii to determine constitutive over-expression, induction, and tissue specificity. Furthermore, a UGT inhibitor, sulfinpyrazone, restored susceptibility to pyrethroids and DDT in An. gambiae, An. coluzzii, An. arabiensis and An. funestus, the major African malaria vectors. Taken together, this study provides clear association of UGTs with pyrethroid resistance as well as highlighting the potential use of sulfinpyrazone as a novel synergist for vector control.

Scavenger Receptor C1 Mediates Toxicity of Binary Toxin from Lysinibacillus sphaericus to Ag55 Cells.

Lysinibacillus sphaericus harboring Binary (BinA and BinB) toxins is highly toxic against Anopheles and Culex mosquito larvae. The Anopheles Ag55 cell line is a suitable model for investigating the mode of Bin toxin action. Based on the low-levels of α-glycosidase Agm3 mRNA in Ag55 cells and the absence of detectable Agm3 proteins, we hypothesized that a scavenger receptor could be mediating Bin cytotoxicity. Preliminary RNA interference knockdown of the expressed scavenger receptors, combined with Bin cytotoxicity assays, was conducted. The scavenger Receptor C1 (SCRC1) became the focus of this study, as a putative receptor for Bin toxins in Ag55 cells, and SCRBQ2 was selected as a negative control. Open reading frames encoding SCRC1 and SCRBQ2 were cloned and expressed in vitro, and polyclonal antibodies were prepared for immunological analyses. The RNAi silencing of SCRC1 and SCRBQ2 resulted in the successful knockdown of both SCRC1 and SCRBQ2 transcripts and protein levels. The cytolytic toxicity of Bin against Ag55 cells was severely reduced after the SCRC1-RNAi treatment. The phagocytic receptor protein SCRC1 mediates endocytosis of the Bin toxin into Ag55 cells, thereby facilitating its internal cytological activity. The results support a mechanism of the Bin toxin entering Ag55 cells, possibly via SCRC1-mediated endocytosis, and encourage investigations into how Bin is transferred from its bound form on the midgut epithelial cells into the epithelial endocytic system.

Impact of blood meals taken on ivermectin-treated livestock on survival and egg production of the malaria vector Anopheles coluzzii under laboratory conditions.

Treatment of livestock with endectocides such as ivermectin is viewed as a complementary vector control approach to address residual transmission of malaria. However, efficacy of this treatment may vary between animal species. Hence, our purpose was to investigate the effects of ivermectin treatments of common livestock species on life history traits of the opportunistic malaria vector Anopheles coluzzii. Sheep, goats and pigs were treated using injectable veterinary ivermectin formulation at the species-specific doses (recommended dose for all species and high dose in pig). Mosquito batches were exposed to treated and control (not injected) animals at different days after treatment. Daily mosquito mortality was recorded and fecundity assessed through the count of gravid females and the number of eggs they developed. The recommended dose of ivermectin induced a significant decrease in mosquito survival for up to 7 days after injection (DAI), with a decrease of 89.7%, 66.7%, and 48.4% in treated pigs, goats and sheep, respectively, compared to control animals. In treated pigs, the triple therapeutic dose decreased mosquito survival of 68.97% relatively to controls up to 14 DAI. The average number in gravid females Anopheles that survived after feeding on treated animals were reduced when blood-meals were taken on sheep (2.57% and 42.03% at 2 and 7 DAI), or on goats (decrease of the 28.28% and 73.64% respectively at 2 and 7 DAI). This study shows that ivermectin treatments to animals negatively impacts An. coluzzii life history traits and could reduce vector densities in areas where livestock live near humans. However, due to short-term efficacy of single dose treatments, repeated treatments and potentially increased dosages would be required to span the transmission season. The use of long-acting ivermectin formulations is discussed as a mean for extending efficacy while remaining cost effective.

Functional Validation of Endogenous Redox Partner Cytochrome P450 Reductase Reveals the Key P450s CYP6P9a/-b as Broad Substrate Metabolizers Conferring Cross-Resistance to Different Insecticide Classes in Anopheles funestus.

The versatility of cytochrome P450 reductase (CPR) in transferring electrons to P450s from other closely related species has been extensively exploited, e.g., by using An. gambiae CPR (AgCPR), as a homologous surrogate, to validate the role of An. funestus P450s in insecticide resistance. However, genomic variation between the AgCPR and An. funestus CPR (AfCPR) suggests that the full metabolism spectrum of An. funestus P450s might be missed when using AgCPR. To test this hypothesis, we expressed AgCPR and AfCPR side-by-side with CYP6P9a and CYP6P9b and functionally validated their role in the detoxification of insecticides from five different classes. Major variations were observed within the FAD- and NADP-binding domains of AgCPR and AfCPR, e.g., the coordinates of the second FAD stacking residue AfCPR-Y456 differ from that of AgCPR-His456. While no significant differences were observed in the cytochrome c reductase activities, when co-expressed with their endogenous AfCPR, the P450s significantly metabolized higher amounts of permethrin and deltamethrin, with CYP6P9b-AfCPR membrane metabolizing α-cypermethrin as well. Only the CYP6P9a-AfCPR membrane significantly metabolized DDT (producing dicofol), bendiocarb, clothianidin, and chlorfenapyr (bioactivation into tralopyril). This demonstrates the broad substrate specificity of An. funestus CYP6P9a/-b, capturing their role in conferring cross-resistance towards unrelated insecticide classes, which can complicate resistance management.

Susceptibility status and synergistic activity of DDT and Lambda-cyhalothrin on Anopheles gambiae and Aedes aegypti in Delta State, Nigeria.

The detection of insecticide resistance in male mosquitoes has been treated with less importance in monitoring insecticide resistance spread in mosquitoes. There are no studies on the susceptibility and synergistic activity of DDT and lambda-cyhalothrin on male Anopheles gambiae and Aedes aegypti in Delta State, Nigeria. Even though studies have extensively reported resistance in female mosquitoes, the susceptibility of male mosquitoes to insecticide classes should be ascertained. In this study, we tested the susceptibility status and synergistic activity of DDT and Lambda-cyhalothrin on An. gambiae and Ae. aegypti in Delta State, Nigeria, in order to ascertain the level of resistance and knockdown. In addition, we modelled the knockdown time using Probit analysis model. WHO bioassay method was used to expose two days old adult mosquitoes to 4% DDT and 0.05% lambda-cyhalothrin. The results showed that An. gambiae mosquitoes exposed to DDT and lambda-cyhalothrin were confirmed resistant (61% and 53% respectively). However, pre-exposing the resistant mosquito population to piperonyl butoxide (4%) showed an increase in mortality to 90% (possible resistance) in DDT and 98% (susceptible) in lambda-cyhalothrin. Ae. aegypti mosquitoes exposed to DDT were susceptible (98%) while those exposed to lambda-cyhalothrin were confirmed resistant (87%) and this increased to complete mortality (100%) in PBO+lambda-cyhalothrin population. Furthermore, the results showed that the knockdown time (KDT50 and KDT95) in An. gambiae exposed to DDT was 39.5-71.2 minutes and 124.5-146.4 minutes respectively, while that of lambda-cyhalothrin was 33.0-81.8 minutes and 64.0-124.4 minutes respectively. In Ae. aegypti, KDT50 and KDT95 was 23.9 and 61.7minutes for DDT exposure whereas it was 5.6-15.3 minutes and 36.1-72.3 minutes for lambda-cyhalothrin exposure. It can be concluded that male An. gambiae mosquitoes exposed to the insecticides were resistant and the causes may be linked to certain resistant genes in the mosquitoes. The chances of transferring resistance are possible in wild species and molecular-based studies on the resistant gene in male mosquitoes as well as the tendencies of transfer are required to establish this focus.

Laboratory evaluation of the regeneration time, efficacy and wash-resistance of PermaNet Dual (a deltamethrin-chlorfenapyr net) against susceptible and pyrethroid-resistant strains of Anopheles gambiae sensu lato.

Pyrethroid-chlorfenapyr nets have been recommended for malaria control by the World Health Organisation (WHO) after an alpha-cypermethrin-chlorfenapyr net showed improved impact in epidemiological trials. PermaNet® Dual is a new deltamethrin-chlorfenapyr net developed by Vestergaard Sàrl to expand options to control programmes. A series of laboratory studies were performed according to WHO guidelines to assess the regeneration time, efficacy and wash-resistance of PermaNet® Dual. Regeneration time was determined by subjecting net pieces to cone bioassays and tunnel tests before and 0, 1, 2, 3, 5 and 7 days after washing. The wash-resistance of PermaNet® Dual was evaluated compared to WHO-prequalified pyrethroid-only (PermaNet® 2.0) and pyrethroid-chlorfenapyr (Interceptor® G2) nets by testing net pieces washed 0, 1, 3, 5, 10, 15 and 20 times in cone bioassays and tunnel tests. Tests were performed with susceptible and pyrethroid-resistant strains of Anopheles gambiae to assess the pyrethroid and chlorfenapyr components separately. Net pieces were also analysed to determine insecticide content. In regeneration time studies, the biological activity of the deltamethrin and chlorfenapyr components of PermaNet® Dual regenerated within one day after washing and a 1-day washing interval was adopted for wash-resistance studies. PermaNet® Dual induced high mortality (98%) and blood-feeding inhibition (98%) of the susceptible strain after 20 washes fulfilling WHO efficacy criteria in tunnel tests (≥80% mortality, ≥90% blood-feeding inhibition). Similar results were obtained with PermaNet® 2.0 (99% mortality, 99% blood-feeding inhibition) and Interceptor® G2 (99% mortality, 98% blood-feeding inhibition) washed 20 times. In wash-resistance tunnel tests against the pyrethroid-resistant strain, PermaNet® Dual washed 20 times induced high mortality (91%) and blood-feeding inhibition (73%), which was similar to Interceptor® G2 (87% mortality, 79% blood-feeding inhibition) and superior to PermaNet® 2.0 (47% mortality, 68% blood-feeding inhibition). PermaNet® Dual fulfilled WHO efficacy criteria in laboratory bioassays and showed potential to improve control of pyrethroid-resistant malaria vectors.

Substrate promiscuity of key resistance P450s confers clothianidin resistance while increasing chlorfenapyr potency in malaria vectors.

Novel insecticides were recently introduced to counter pyrethroid resistance threats in African malaria vectors. To prolong their effectiveness, potential cross-resistance from promiscuous pyrethroid metabolic resistance mechanisms must be elucidated. Here, we demonstrate that the duplicated P450s CYP6P9a/-b, proficient pyrethroid metabolizers, reduce neonicotinoid efficacy in Anopheles funestus while enhancing the potency of chlorfenapyr. Transgenic expression of CYP6P9a/-b in Drosophila confirmed that flies expressing both genes were significantly more resistant to neonicotinoids than controls, whereas the contrasting pattern was observed for chlorfenapyr. This result was also confirmed by RNAi knockdown experiments. In vitro expression of recombinant CYP6P9a and metabolism assays established that it significantly depletes both clothianidin and chlorfenapyr, with metabolism of chlorfenapyr producing the insecticidally active intermediate metabolite tralopyril. This study highlights the risk of cross-resistance between pyrethroid and neonicotinoid and reveals that chlorfenapyr-based control interventions such as Interceptor G2 could remain efficient against some P450-based resistant mosquitoes.

Efficacy of Interceptor G2, Royal Guard and PermaNet 3.0 against pyrethroid-resistant Anopheles gambiae s.l. from Za-Kpota, southern Benin: an experimental hut trial.

BACKGROUND: The widespread use of insecticide-treated nets (ITNs) has significantly contributed to the reduction in malaria cases and deaths observed across Africa. Unfortunately, this control strategy is threatened by the rapid spread of pyrethroid resistance in malaria vectors. Dual-active-ingredient insecticidal nets are now available to mitigate the impact of pyrethroid resistance. To facilitate evidence-based decisions regarding product selection in specific use settings, data are needed on the efficacy of these different nets against local mosquito populations. METHODS: Two experimental hut trials were performed in Za-Kpota, southern Benin in 2021 to evaluate the performance of Interceptor G2 (BASF), Royal Guard (Disease Control Technologies) and PermaNet 3.0 (Vestergaard Frandsen), all dual-active-ingredient bednets, in comparison to untreated or standard pyrethroid-treated bednets, against free-flying wild Anopheles gambiae mosquitoes. The performance of some of these next-generation nets was compared to the same type of nets that have been in use for up to 2 years. Mosquitoes collected in the huts were followed up after exposure to assess the sublethal effects of treatments on certain life-history traits. RESULTS: The predominant species in the study site was Anopheles gambiae sensu stricto (An. gambiae s.s.). Both Anopheles coluzzii and An. gambiae s.s. were resistant to pyrethroids (deltamethrin susceptibility was restored by piperonyl butoxide pre-exposure). In the experimental hut trials, the highest blood-feeding inhibition (5.56%) was recorded for the Royal Guard net, relative to the standard PermaNet 2.0 net (44.44% inhibition). The highest 72-h mortality rate (90.11%) was recorded for the Interceptor G2 net compared to the PermaNet 2.0 net (56.04%). After exposure, the risk of death of An. gambiae sensu lato (An. gambiae s.l.) was 6.5-fold higher with the Interceptor G2 net and 4.4-fold higher with the PermaNet 3.0 net compared to the respective untreated net. Lower mosquito mortality was recorded with an aged Interceptor G2 net compared to a new Interceptor G2 net. Oviposition rates were lower in mosquitoes collected from huts containing ITNs compared to those of untreated controls. None of the mosquitoes collected from huts equipped with Royal Guard nets laid any eggs. CONCLUSIONS: The Royal Guard and Interceptor G2 nets showed a potential to significantly improve the control of malaria-transmitting vectors. However, the PermaNet 3.0 net remains effective in pyrethroid-resistant areas.

Non-contact detection of pyrethroids widely used in vector control by Anopheles mosquitoes.

Pyrethroids are the most widely used insecticides to control vector borne diseases including malaria. Physiological resistance mechanisms to these insecticides have been well described, whereas those for behavioral resistance remain overlooked. Field data suggest the presence of spatial sensory detection by Anopheles mosquitoes of the pyrethroid molecules used in insecticide-based control tools, such as long-lasting insecticide nets or insecticide residual spraying. This opens the way to the emergence of a wide range of behavioral adaptations among malaria vectors. However, the spatial sensory detection of these molecules is controversial and needs to be demonstrated. The goal of this study was to behaviorally characterize the non-contact detection of three of the most common pyrethroids used for malaria vector control: permethrin, deltamethrin an ⍺-cypermethrin. To reach this goal, we recorded the behavior (takeoff response) of Anopheles gambiae pyrethroid-sensitive and resistant laboratory strains, as well as field collected mosquitoes from the Gambiae Complex, when exposed to the headspace of bottles containing different doses of the insecticides at 25 and 35°C, in order to represent a range of laboratory and field temperatures. We found the proportion of laboratory susceptible and resistant female mosquitoes that took off was, in all treatments, dose and the temperature dependent. Sensitive mosquitoes were significantly more prone to take off only in the presence of ⍺-cypermethrin, whereas sensitive and resistant mosquitoes showed similar responses to permethrin and deltamethrin. Field-collected mosquitoes of the Gambiae Complex were also responsive to permethrin, independently of the species identity (An. gambiae, An. coluzzii and An. arabiensis) or their genotypes for the kdr mutation, known to confer resistance to pyrethroids. The observed ability of Anopheles spp. mosquitoes to detect insecticides without contact could favor the evolution of behavioral modifications that may allow them to avoid or reduce the adverse effect of insecticides and thus, the development of behavioral resistance.

Community end user perceptions of hessian fabric transfluthrin vapour emanators for protecting against mosquitoes under conditions of routine use in Port-au-Prince, Haiti.

BACKGROUND: A treated fabric device for emanating the volatile pyrethroid transfluthrin was recently developed in Tanzania that protected against night-biting Anopheles and Culex mosquitoes for several months. Here perceptions of community end users provided with such transfluthrin emanators, primarily intended to protect them against day-active Aedes vectors of human arboviruses that often attack people outdoors, were assessed in Port-au-Prince, Haiti. METHODS: Following the distribution of transfluthrin emanators to participating households in poor-to-middle class urban neighbourhoods, questionnaire surveys and in-depth interviews of end-user households were supplemented with conventional and Photovoice-based focus group discussions. Observations were assessed synthetically to evaluate user perceptions of protection and acceptability, and to solicit advice for improving and promoting them in the future. RESULTS: Many participants viewed emanators positively and several outlined various advantages over current alternatives, although some expressed concerns about smell, health hazards, bulkiness, unattractiveness and future cost. Most participants expressed moderate to high satisfaction with protection against mosquitoes, especially indoors. Protection against other arthropod pests was also commonly reported, although satisfaction levels were highly variable. Diverse use practices were reported, some of which probably targeted nocturnal Culex resting indoors, rather than Aedes attacking them outdoors during daylight hours. Perceived durability of protection varied: While many participants noted some slow loss over months, others noted rapid decline within days. A few participants specifically attributed efficacy loss to outdoor use and exposure to wind or moisture. Many expressed stringent expectations of satisfactory protection levels, with even a single mosquito bite considered unsatisfactory. Some participants considered emanators superior to fans, bedsheets, sprays and coils, but it is concerning that several preferred them to bed nets and consequently stopped using the latter. CONCLUSIONS: The perspectives shared by Haitian end-users are consistent with those from similar studies in Brazil and recent epidemiological evidence from Peru that other transfluthrin emanator products can protect against arbovirus infection. While these encouraging sociological observations contrast starkly with evidence of essentially negligible effects upon Aedes landing rates from parallel entomological assessments across Haiti, Tanzania, Brazil and Peru, no other reason to doubt the generally encouraging views expressed herein by Haitian end users could be identified.

Preliminary monitoring of knockdown resistance (kdr) mutation in Anopheles stephensi: insights from a malarious area in Southeastern Iran.

BACKGROUND: Anopheles stephensi is recognized as the main malaria vector in Iran. In recent years, resistance to several insecticide classes, including organochlorine, pyrethroids, and carbamate compounds, has been reported for this medically important malaria vector. The main objective of the present study was to evaluate the insecticide susceptibility status of An. stephensi collected from the southern part of Iran, and to clarify the mechanism of resistance, using bioassay tests and molecular methods comparing the sequence of susceptible and resistant mosquitoes. METHODS: Mosquito larvae were collected from various larval habitats across six different districts (Gabrik, Sardasht, Tidar, Dehbarez, Kishi and Bandar Abbas) in Hormozgan Provine, located in the southern part of Iran. From each district standing water areas with the highest densities of Anopheles larvae were selected for sampling, and adult mosquitoes were reared from them. Finally, the collected mosquito species were identified using valid keys. Insecticide susceptibility of An. stephensi was tested using permethrin 0.75%, lambdacyhalothrin 0.05%, deltamethrin 0.05%, and DDT 4%, following the World Health Organization (WHO) test procedures for insecticide resistance monitoring. Additionally, knockdown resistance (kdr) mutation in the voltage-gated sodium channel (vgsc) gene was sequenced and analysed among resistant populations to detect possible molecular mechanisms of observed resistance phenotypes. RESULTS: The susceptibility status of An. stephensi revealed that resistance to DDT and permethrin was found in all districts. Furthermore, resistance to all tested insecticides in An. stephensi was detected in Gabrik, Sardasht, Tidar, and Dehbarez. Analysis of knockdown resistance (kdr) mutations at the vgsc did not show evidence for the presence of this mutation in An. stephensi. CONCLUSION: Based on the results of the current study, it appears that in An. stephensi from Hormozgan Province (Iran), other resistance mechanisms such as biochemical resistance due to detoxification enzymes may be involved due to the absence of the kdr mutation or non-target site resistance. Further investigation is warranted in the future to identify the exact resistance mechanisms in this main malaria vector across the country.

Key gene modules and hub genes associated with pyrethroid and organophosphate resistance in Anopheles mosquitoes: a systems biology approach.

Indoor residual spraying (IRS) and insecticide-treated nets (ITNs) are the main methods used to control mosquito populations for malaria prevention. The efficacy of these strategies is threatened by the spread of insecticide resistance (IR), limiting the success of malaria control. Studies of the genetic evolution leading to insecticide resistance could enable the identification of molecular markers that can be used for IR surveillance and an improved understanding of the molecular mechanisms associated with IR. This study used a weighted gene co-expression network analysis (WGCNA) algorithm, a systems biology approach, to identify genes with similar co-expression patterns (modules) and hub genes that are potential molecular markers for insecticide resistance surveillance in Kenya and Benin. A total of 20 and 26 gene co-expression modules were identified via average linkage hierarchical clustering from Anopheles arabiensis and An. gambiae, respectively, and hub genes (highly connected genes) were identified within each module. Three specific genes stood out: serine protease, E3 ubiquitin-protein ligase, and cuticular proteins, which were top hub genes in both species and could serve as potential markers and targets for monitoring IR in these malaria vectors. In addition to the identified markers, we explored molecular mechanisms using enrichment maps that revealed a complex process involving multiple steps, from odorant binding and neuronal signaling to cellular responses, immune modulation, cellular metabolism, and gene regulation. Incorporation of these dynamics into the development of new insecticides and the tracking of insecticide resistance could improve the sustainable and cost-effective deployment of interventions.

In vitro larvicidal efficacy of Lantana camara essential oil and its nanoemulsion and enzyme inhibition kinetics against Anopheles culicifacies.

Mosquitoes are important vectors for the transmission of several infectious diseases that lead to huge morbidity and mortality. The exhaustive use of synthetic insecticides has led to widespread resistance and environmental pollution. Using essential oils and nano-emulsions as novel insecticides is a promising alternative approach for controlling vector borne diseases. In the current study, Lantana camara EO and NE were evaluated for their larvicidal and pupicidal activities against Anopheles culicifacies. The inhibitory effect of EO and NE on AChE, NSE (α/ß), and GST was also evaluated and compared. GC-MS analysis of oil displayed 61 major peaks. The stable nano-emulsion with an observed hydrodynamic diameter of 147.62 nm was formed using the o/w method. The nano-emulsion exhibited good larvicidal (LC50 50.35 ppm and LC90 222.84 ppm) and pupicidal (LC50 54.82 ppm and LC90 174.58 ppm) activities. Biochemical evaluations revealed that LCEO and LCNE inhibited AChE, NSE (α/ß), and GST, displaying LCNE to be a potent binder to AChE and NSE enzyme, whereas LCEO showed higher binding potency towards GST. The nano-emulsion provides us with novel opportunities to target different mosquito enzymes with improved insecticidal efficacy. Due to its natural origin, it can be further developed as a safer and more potent larvicide/insecticide capable of combating emerging insecticide resistance.

Indoor residual spraying of experimental huts in Cameroon highlights the potential of Fludora® Fusion to control wild pyrethroid-resistant malaria vectors.

Elevated resistance to pyrethroids in major malaria vectors has led to the introduction of novel insecticides including neonicotinoids. There is a fear that efficacy of these new insecticides could be impacted by cross-resistance mechanisms from metabolic resistance to pyrethroids. In this study, after evaluating the resistance to deltamethrin, clothianidin and mixture of clothianidin + deltamethrin in the lab using CDC bottle assays, the efficacy of the new IRS formulation Fludora® Fusion was tested in comparison to clothianidin and deltamethrin applied alone using experimental hut trials against wild free-flying pyrethroid-resistant Anopheles funestus from Elende and field An. gambiae collected from Nkolondom reared in the lab and released in the huts. Additionally, cone tests on the treated walls were performed each month for a period of twelve months to evaluate the residual efficacy of the sprayed products. Furthermore, the L1014F-kdr target-site mutation and the L119F-GSTe2 mediated metabolic resistance to pyrethroids were genotyped on a subset of mosquitoes from the EHT to assess the potential cross-resistance. All Anopheles species tested were fully susceptible to clothianidin and clothianidin + deltamethrin mixture in CDC bottle assay while resistance was noted to deltamethrin. Accordingly, Fludora® Fusion (62.83% vs 42.42%) and clothianidin (64.42% vs 42.42%) induced significantly higher mortality rates in EHT than deltamethrin (42.42%) against free flying An. funestus from Elende in month 1 (M1) and no significant difference in mortality was observed between the first (M1) and sixth (M6) months of the evaluation (P > 0.05). However, lower mortality rates were recorded against An. gambiae s.s from Nkolondom (mortality rates 50%, 45.56% and 26.68%). In-situ cone test on the wall showed a high residual efficacy of Fludora® Fusion and clothianidin on the susceptible strain KISUMU (> 12 months) and moderately on the highly pyrethroid-resistant An. gambiae strain from Nkolondom (6 months). Interestingly, no association was observed between the L119F-GSTe2 mutation and the ability of mosquitoes to survive exposure to Fludora® Fusion, whereas a trend was observed with the L1014F-kdr mutation. This study highlights that Fludora® Fusion, through its clothianidin component, has good potential of controlling pyrethroid-resistant mosquitoes with prolonged residual efficacy. This could be therefore an appropriate tool for vector control in several malaria endemic regions.