1 Abstract In 2024–2025 a multi-centre study involving seven international laboratories was conducted with the support of the World Health Organization (WHO). The aim of the study was to establish and validate discriminating concentrations (DCs) in WHO bottle bioassays for monitoring susceptibility to broflanilide and isocycloseram in Anopheles gambiae s . s ., An. funestus, An. stephensi and Aedes aegypti . The following values are recommended for adoption as DCs for broflanilide: 10 µg/bottle for An. gambiae and Ae. aegypti , 15 µg/bottle for An. funestus , and 25 µg/bottle for An. stephensi . The recommended DCs for isocycloseram are 15 µg/bottle for Ae. aegypti , 30 µg/bottle for An. gambiae , 50 µg/bottle for An. stephensi , and 60 µg/bottle for An. funestu s. Based on the experiences of conducting this study, which represents the application of a generic protocol for establishing discriminating concentrations produced by the WHO, technical recommendations are made on the generation and analysis of DC data for insecticides in future.

IntroductionDiscriminating concentrations (DCs) underpin routine monitoring of insecticide susceptibility, but none had been established for broflanilide or isocycloseram, two new active ingredients entering vector control. This study aimed to establish and validate DCs in WHO bottle bioassays for monitoring susceptibility to broflanilide and isocycloseram in Anopheles gambiae s.s., An. funestus, An. stephensi, and Aedes aegypti.MethodsIn 2024–2025, a multi-centre study involving seven international laboratories was conducted with the support of the World Health Organization (WHO), applying the generic WHO protocol for establishing discriminating concentrations to susceptible reference strains of each species.ResultsThe following values are recommended for adoption as DCs for broflanilide: 10 μg/bottle for An. gambiae and Ae. aegypti, 15 μg/bottle for An. funestus, and 25 μg/bottle for An. stephensi. The recommended DCs for isocycloseram are 15 μg/bottle for Ae. aegypti, 30 μg/bottle for An. gambiae, 50 μg/bottle for An. stephensi, and 60 μg/bottle for An. funestus.DiscussionBased on the experience gained from conducting this study, technical recommendations are made to support the generation and analysis of DC data for insecticides in the future.

Abstract Testing of insecticides and insecticide-based vector control tools relies on the use of bioassays whereby insects are exposed and mortality, or another relevant entomological endpoint, is measured. To be able to compare results between products and across time and space standard laboratory strains are used. Insecticide-susceptible strains are often used as comparators in studies involving insecticide-resistant populations. ‘Kisumu’ is a long-established, widely-used insecticide-susceptible strain of Anopheles gambiae . In this study, 16 facilities across different countries exposed their Kisumu colonies aged 3-5 days to four insecticides (permethrin, alpha-cypermethrin, DDT and pirimiphos-methyl) at a range of concentrations in WHO tube assays to assess susceptibility. Insecticide-treated filter papers were made at the Liverpool School of Tropical Medicine and sent to testing centres, along with a standard protocol and testing conditions requirements, to standardise testing as much as possible. A first observation was that DDT results were affected by the use of Risella oil, the WHO-recommended carrier oil but immiscible in acetone, leading to uneven coating of papers and increased variability in bioassay results (40x variance between colonies). Bioassay data generated by the testing facilities suggest a wide range of susceptibility to different insecticides between colonies of Kisumu: LC 50 values between colonies differed, though not significantly, by up to 4x for permethrin, 6x for pirimiphos-methyl, and 15x for alpha-cypermethrin. Some colonies are classified as insecticide resistant based on the WHO definition of <98% mortality on exposure to a discriminating concentration. The assumption that Kisumu is a standard susceptible strain, consistent between facilities is challenged, which has implications when comparing results between studies. We propose genotypic analysis of the colonies to identify signs of resistance markers indicative of contamination from resistant strains or wild mosquitoes, and generation of guidance to support the maintenance and monitoring of susceptible mosquito strains.

The "South American Research Network for the Surveillance and Control of Insecticide-Resistance in Arthropod Vectors" (WINSA), established in 2024 by the IRD and FIOCRUZ with support from the US-CDC VecNet initiative and WHO-TDR, aims to coordinate research on insecticide resistance in arthropod vectors in South America, provide a platform for regional collaboration, and develop effective mitigation strategies. WINSA brings together leading technical experts representing research institutions from 14 countries and territories located in South America, the USA, and France to promote collaboration and information exchange, identify research gaps and priorities, enhance technical capacity in insecticide resistance monitoring, and support national and regional programs on vector resistance issues. This network seeks to contribute to the reduction and elimination of vector-borne diseases in South America.

Malathion is a widely used pesticide with potentially oncogenic properties and may have deleterious effects on organism health and fitness. Although malathion use is now restricted in the European Union, it remains widely used for public health campaigns in other parts of the world, particularly for mosquito control. Understanding its sublethal and long-term effects is thus essential, both for evaluating its ecotoxicological impacts and for anticipating resistance mechanisms. However, empiric data on its effects in wild organisms - especially in invertebrates - remain limited. Here, we quantitatively investigated whether larval exposure to environmentally realistic concentrations of malathion could affect mosquito tissue structure and gene expression profiles of adult Aedes aegypti (yellow fever mosquitoes), using both RNA-seq and histological approaches. Results show no neoplastic or pre-neoplastic lesions in adults exposed to malathion during larval development, contrary to previous studies in other organisms showing carcinogenic effects of malathion. However, our differential gene expression analyses revealed significant changes in genes related to mitochondrial function, energy metabolism, and detoxification pathways, suggesting significant physiological impacts of malathion in adults after early-life pesticide exposure. Notably, females exhibited stronger transcriptomic responses than males, including the upregulation of genes involved in detoxification (e.g., P450 cytochromes), olfactory perception, and stress response, with potential consequences for resistance mechanisms. Our findings underscore the ability of mosquitoes to mount transient molecular responses to environmental pollutants, potentially contributing to the long-term selection of metabolic resistance traits - an outcome with important implications for vector control strategies.

Aedes mosquito-borne diseases remain a significant global health threat, necessitating effective control strategies. This study introduces monoterpenes-based nanohydrogels for potential use as repellents against Aedes aegypti, the primary dengue vector worldwide. We formulated hydrogels using cymene- and myrcene-based nanoemulsions with different polymers: chitosan, carboxymethylcellulose (CMC), and carbopol®. Our evaluations of rheological, texture, and bioadhesive properties identified CMC hydrogel as the most promising gelling agent for topical application, exhibiting sustained monoterpene release over 12 h with low skin permeation and high retention in the stratum corneum. Myrcene-loaded CMC hydrogel achieved a 57% feeding deterrence compared to 47% with cymene hydrogel in the mosquito membrane-feeding model. Molecular docking studies revealed interactions between myrcene and an essential amino acid (Ile116) in the Ae. aegypti odorant-binding protein 22 (AeOBP22), corroborating its higher repellent efficacy. These findings suggest that myrcene-loaded CMC hydrogels offer a promising, minimally invasive strategy for personal protection against Ae. aegypti and warrant further investigation to optimize monoterpene concentrations for vector control.

The development of insecticide resistance in mosquitoes of public health importance has encouraged extensive research into innovative vector control methods. Terpenes are the largest among Plants Secondary Metabolites and have been increasingly studied for their potential as insecticidal control agents. Although promising, terpenes are insoluble in water, and they show low residual life which limits their application for vector control. In this study, we developed and evaluated the performances of terpenoid-based nanoemulsions (TNEs) containing myrcene and p-cymene against the dengue vector Aedes aegypti and investigated their potential toxicity against non-target organisms. Our results showed that myrcene and p-cymene showed moderate larvicidal activity against mosquito larvae compared to temephos an organophosphate widely used for mosquito control. However, we showed similar efficacy of TNEs against both susceptible and highly insecticide-resistant mosquitoes from French Guyana, hence suggesting an absence of cross-resistance with conventional insecticides. We also showed that TNEs remained effective for up to 45 days in laboratory conditions. The exposure of zebrafish to TNEs triggered behavioral changes in the fish at high doses but they did not alter the normal functioning of zebrafish organs, suggesting a good tolerability of non-target organisms to these molecules. Overall, this study provides new insights into the insecticidal properties and toxicity of terpenes and terpenoid-based formulations and confirms that TNE may offer interesting prospects for mosquito control as part of integrated vector management.

Abstract Background The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both. Methods A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species (Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids (metofluthrin, prallethrin and transfluthrin), neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration–response curves for each insecticide–species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population (LC50 and LC99, respectively) and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively. Results Overall, about 200,000 mosquitoes were tested with the new bottle bioassay, and LC50/LC99 or OI50/OI99 values were determined for all insecticides. Variation was seen between laboratories in estimates for some mosquito species–insecticide combinations, while other test results were consistent. The variation was generally greater with transfluthrin and flupyradifurone than with the other compounds tested, especially against Anopheles species. Overall, the mean within-bioassay variability in mortality and oviposition inhibition were < 10% for most mosquito species-insecticide combinations. Conclusion Our findings, based on the largest susceptibility dataset ever produced on mosquitoes, showed that the new WHO bottle bioassay is adequate for evaluating mosquito susceptibility to new and promising public health insecticides currently deployed for vector control. The datasets presented in this study have been used recently by the WHO to establish 17 new insecticide discriminating concentrations (DCs) for either Aedes spp. or Anopheles spp. The bottle bioassay and DCs can now be widely used to monitor baseline insecticide susceptibility of wild populations of vectors of malaria and Aedes-borne diseases worldwide. Graphical abstract

In response to the widespread use of control strategies such as Insecticide Treated Nets (ITN), Anopheles mosquitoes have evolved various resistance mechanisms. Kdr is a mutation that provides physiological resistance to the pyrethroid insecticides family (PYR). In the present study, we investigated the effect of the Kdr mutation on the ability of female An. gambiae to locate and penetrate a 1cm-diameter hole in a piece of netting, either treated with insecticide or untreated, to reach a bait in a wind tunnel. Kdr homozygous, PYR-resistant mosquitoes were the least efficient at penetrating an untreated damaged net, with about 51% [39-63] success rate compared to 80% [70-90] and 78% [65-91] for homozygous susceptible and heterozygous respectively. This reduced efficiency, likely due to reduced host-seeking activity, as revealed by mosquito video-tracking, is evidence of a recessive behavioral cost of the mutation. Kdr heterozygous mosquitoes were the most efficient at penetrating nets treated with PYR insecticide, thus providing evidence for overdominance, the rarely-described case of heterozygote advantage conveyed by a single locus. The study also highlights the remarkable capacity of female mosquitoes, whether PYR-resistant or not, to locate holes in bed-nets.

The olon tree, Zanthoxylum heitzii (syn. Fagara heitzii) is commonly found in the central-west African forests. In the Republic of Congo (Congo-Brazzaville) its bark is anecdotally reported to provide human protection against fleas. Here we assess the insecticidal activities of Z. heitzii stem bark, seed and leaf extracts against Anopheles gambiae s.s, the main malaria vector in Africa. Extracts were obtained by Accelerated Solvent Extraction (ASE) using solvents of different polarity and by classical Soxhlet extraction using hexane as solvent. The insecticidal effects of the crude extracts were evaluated using topical applications of insecticides on mosquitoes of a susceptible reference strain (Kisumu [Kis]), a strain homozygous for the L1014F kdr mutation (kdrKis), and a strain homozygous for the G119S Ace1R allele (AcerKis). The insecticidal activities were measured using LD50 and LD95 and active extracts were characterized by NMR spectroscopy and HPLC chromatography. Results show that the ASE hexane stem bark extract was the most effective compound against An. gambiae (LD50 = 102 ng/mg female), but was not as effective as common synthetic insecticides. Overall, there was no significant difference between the responses of the three mosquito strains to Z. heitzii extracts, indicating no cross resistance with conventional pesticides.

Background Dengue fever is reemerging on the island of Martinique and is a serious threat for the human population. During dengue epidemics, adult Aedes aegypti control with pyrethroid space sprays is implemented in order to rapidly reduce transmission. Unfortunately, vector control programs are facing operational challenges with the emergence of pyrethroid resistant Ae. aegypti populations. Methodology/Principal Findings To assess the impact of pyrethroid resistance on the efficacy of treatments, applications of deltamethrin and natural pyrethrins were performed with vehicle-mounted thermal foggers in 9 localities of Martinique, where Ae. aegypti populations are strongly resistant to pyrethroids. Efficacy was assessed by monitoring mortality rates of naturally resistant and laboratory susceptible mosquitoes placed in sentinel cages. Before, during and after spraying, larval and adult densities were estimated. Results showed high mortality rates of susceptible sentinel mosquitoes treated with deltamethrin while resistant mosquitoes exhibited very low mortality. There was no reduction of either larval or adult Ae. aegypti population densities after treatments. Conclusions/Significance This is the first documented evidence that pyrethroid resistance impedes dengue vector control using pyrethroid-based treatments. These results emphasize the need for alternative tools and strategies for dengue control programs.

BACKGROUND:The main malaria vector Anopheles gambiae and the urban pest nuisance Culex quinquefasciatus are increasingly resistant to pyrethroids in many African countries. There is a need for new products and strategies. Insecticide paint Inesfly 5A IGR™, containing two organophosphates (OPs), chlorpyrifos and diazinon, and insect growth regulator (IGR), pyriproxyfen, was tested under laboratory conditions for 12 months following WHOPES Phase I procedures.METHODS:Mosquitoes used were laboratory strains of Cx. quinquefasciatus susceptible and resistant to OPs. The paint was applied at two different doses (1 kg/6 m2 and 1 kg/12 m2) on different commonly used surfaces: porous (cement and stucco) and non-porous (softwood and hard plastic). Insecticide efficacy was studied in terms of delayed mortality using 30-minute WHO bioassay cones. IGR efficacy on fecundity, fertility and larval development was studied on OP-resistant females exposed for 30 minutes to cement treated and control surfaces.RESULTS:After treatment, delayed mortality was high (87-100%) even against OP-resistant females on all surfaces except cement treated at 1 kg/12 m2. Remarkably, one year after treatment delayed mortality was 93-100% against OP-resistant females on non-porous surfaces at both doses. On cement, death rates were low 12 months after treatment regardless of the dose and the resistance status. Fecundity, fertility and adult emergence were reduced after treatment even at the lower dose (p < 10(-3)). A reduction in fecundity was still observed nine months after treatment at both doses (p < 10(-3)) and adult emergence was reduced at the higher dose (p < 10(-3)).CONCLUSIONS:High mortality rates were observed against laboratory strains of the pest mosquito Cx. quinquefasciatus susceptible and resistant to insecticides. Long-term killing remained equally important on non-porous surfaces regardless the resistance status for over 12 months. The paint's effect on fecundity, fertility and adult emergence may continue to provide an additional angle of attack in reducing overall population densities when the lethal effect of OPs diminishes over time. Some options on how to deal with porous materials are given. Implications in vector control are discussed.

P>ObjectiveTo define the insecticide resistance status of Triatoma infestans to deltamethrin (pyrethroid), malathion (organophosphate) and bendiocarb (carbamate) in Bolivia.MethodsFifty populations of T. infestans were sampled in Bolivian human dwellings. Quantal response data were obtained by topical applications of 0.2 mu l of insecticide-acetone solutions on nymphs N1 of the F1 generations. For most populations, dose-mortality relationships and resistance ratios (RR) were analysed. Discriminating concentrations were established for each insecticide with a susceptible reference strain and used on the other field populations. A tarsal-contact diagnostic test using insecticide impregnated papers was designed to rapidly identify deltamethrin-resistant populations in the field.ResultsDiscriminating concentrations for topical applications were 5, 70 and 120 ng active ingredient per insect for deltamethrin, bendiocarb and malathion, respectively. The diagnostic concentration for deltamethrin was 0.30% for the 1-h exposure by tarsal contact. All populations sampled in human dwellings exhibited significant levels of resistance to deltamethrin, from 6 to 491 and varied among regions. Resistant populations did not recover complete susceptibility to deltamethrin when the synergist piperonyl butoxide (PBO) was used. None of the sampled populations exhibited significant resistance to bendiocarb (all RR(50) < 1.8) or malathion (all RR(50) < 2.2).ConclusionIn Bolivia, most 'domestic'T. infestans populations are resistant to deltamethrin. Because insecticide vector control is the only selection pressure, resistance likely originates from it. Switching from pyrethroids to organophosphates or carbamates could be a short-term solution to control this vector, but other alternative integrated control strategies should also be considered in the long term.

Background With the spread of pyrethroid resistance in mosquitoes, the combination of an insecticide (carbamate or organophosphate) with a repellent (DEET) is considered as a promising alternative strategy for the treatment of mosquito nets and other relevant materials. The efficacy of these mixtures comes from the fact that they reproduce pyrethroid features and that positive interactions occur between insecticides and repellent. To better understand the mechanisms involved and assess the impact of detoxifying enzymes (oxidases and esterases) in these interactions, bioassays were carried out in the laboratory against the main dengue vector Aedes aegypti . Methods Topical applications of DEET and propoxur (carbamate), used alone or as a mixture, were carried out on female mosquitoes, using inhibitors of the two main detoxification pathways in the insect. PBO, an inhibitor of multi-function oxidases, and DEF, an inhibitor of esterases, were applied one hour prior to the main treatment. Results Results showed that synergism between DEET and propoxur disappeared in the presence of PBO but not with DEF. This suggests that oxidases, contrary to esterases, play a key role in the interactions occurring between DEET and cholinesterase inhibitors in mosquitoes. Conclusion These findings are of great interest for the implementation of "combination nets" in the field. They support the need to combine insecticide with repellent to overcome insecticide resistance in mosquitoes of public health importance.

Pyrethrum is a natural mixture of six insecticidal esters, recognized for low mammalian toxicity and limited persistence in the environment. In this study, World Health Organization standard bioassays were used to evaluate the performance of pyrethrum against both susceptible and pyrethroid-resistant Anopheles gambiae s.s. The results showed that the intrinsic activity of pyrethrum was similar to that of permethrin but lower than that of deltamethrin against susceptible mosquitoes. However, pyrethrum was less affected by the presence of the kdr mutation than synthetic pyrethroids (with lower resistance ratios) and showed good knock-down effect, repellency, and blood-feeding inhibition against the pyrethroid-resistant strain. In laboratory condition, mosquito nets treated with 500-1,000 mg/m2 (pyrethrum) remained effective, i.e., > 80% mortality and/or > 95% KD effect, for 9 mo. Conversely, the efficacy and residual activity of pyrethrum (Pynet 5% EC) on substrates was not conclusive, especially concerning mud, which is a porous substrate (mortality < 80% after 3 mo at 2 g/m2). These findings suggested that pyrethrum may be a potential alternative candidate for the impregnation of mosquito nets and textiles in areas where resistance to pyrethroids has become problematic.

BACKGROUND:The evolutionary dynamics of xenobiotic resistance depends on how resistance mutations influence the fitness of their bearers, both in the presence and absence of xenobiotic selection pressure. In cases of multiple resistance, these dynamics will also depend on how individual resistance mutations interact with one another, and on the xenobiotics applied against them. We compared Culex quinquefasciatus mosquitoes harbouring two resistance alleles ace-1R and KdrR (conferring resistance to carbamate and pyrethroid insecticides, respectively) to mosquitoes bearing only one of the alleles, or neither allele. Comparisons were made in environments where both, only one, or neither type of insecticide was present.RESULTS:Each resistance allele was associated with fitness costs (survival to adulthood) in an insecticide-free environment, with the costs of ace-1R being greater than for KdrR. However, there was a notable interaction in that the costs of harbouring both alleles were significantly less than for harbouring ace-1R alone. The two insecticides combined in an additive, synergistic and antagonistic manner depending on a mosquito's resistance status, but were not predictable based on the presence/absence of either, or both mutations.CONCLUSION:Insecticide resistance mutations interacted to positively or negatively influence a mosquito's fitness, both in the presence or absence of insecticides. In particular, the presence of the KdrR mutation compensated for the costs of the ace-1R mutation in an insecticide-free environment, suggesting the strength of selection in untreated areas would be less against mosquitoes resistant to both insecticides than for those resistant to carbamates alone. Additional interactions suggest the dynamics of resistance will be difficult to predict in populations where multiple resistance mutations are present or that are subject to treatment by different xenobiotics.

In order to evaluate whether criteria for LLIN field performance (phase III) set by the WHO Pesticide Evaluation Scheme are met, first and second generations of one of these products, PermaNet®, a polyester net using the coating technology were tested.

The diagnostic dose for temephos susceptibility test was established based on Aedes aegypti, the susceptible Bora (French Polynesia) strain, for practical and routine use. The diagnostic dose was subsequently used to evaluate the susceptibility/resistance status in F1 progenies of field-collected samples from Bangkok and various parts of Thailand. It appeared that Ae. aegypti mosquitoes of one collection site each in Bangkok, Nakhon Sawan (northcentral), and Nakhon Ratchasrima (northeast) were resistant to temephos, with mortality ranging from 50.5 to 71.4%. Moreover, there was a trend of resistance to temephos among Ae. aegypti populations of all studied districts of Nakorn Ratchasima and most areas of Nakhon Sawan, of which those in one area were susceptible. However, various levels of temephos susceptibility were found in Bangkok populations, including resistance and incipient resistance. In Chonburi Province (eastern), all mosquitoes were susceptible to temephos with an indication of tolerance in one sample. Additionally, mosquitoes from Songkhla (south), Chiang Rai (north), Kanchanaburi (west), and Chanthaburi (east) remained susceptible to temephos during the sample collecting period. Bioassay tests on Aedes albopictus populations collected in this study from Nakhon Sawan, Nakorn Ratchasima, Songkhla, and Kanchanaburi revealed high susceptibility to temephos. Although the use of temephos seems to be potentially effective in many areas of the country, a noticeable trend of resistance indicated that alternative vector control methods should be periodically applied.