Discriminating concentrations are the unsung heroes of resistance surveillance. These are standard doses of an insecticide used in bioassays to determine whether a wild mosquito population is susceptible or resistant to a given compound. Field programmes typically test their local mosquitoes annually, exposing them to a known concentration and measuring mortality after 24 hours. If susceptible mosquitoes die but a significant proportion of the local population survives, that is an early warning sign that resistance is emerging. The concept is elegantly simple: the discriminating concentration is traditionally defined as twice the LC99 value, the dose that kills 99 percent of individuals in a fully susceptible laboratory strain, as determined by a log-probit statistical model. The approach is cost-effective, efficient, and has been widely adopted for monitoring resistance in mosquitoes and other disease vectors worldwide.

The urgency of this study stems from the arrival of two entirely new modes of action in vector control. Broflanilide and isocycloseram both belong to class 30 of the Insecticide Resistance Action Committee classification, functioning as meta-diamides and GABA-gated chloride channel allosteric modulators. Their mode of action is fundamentally distinct from those of pyrethroids, carbamates, organophosphates, and organochlorines, the older classes that have dominated vector control for decades. This novelty is precisely their value: because they act on a target site different from established insecticide classes, limited cross-resistance with pyrethroids and other older compounds is expected, making them powerful tools for deployment against resistant mosquito populations. Both compounds are now incorporated into vector control products prequalified by the WHO for public health use, which means national programmes need reliable methods to track susceptibility as these products roll out.

The study followed a rigorous, three-step protocol formalized by the WHO as a generic approach for determining discriminating concentrations. In Step 1, preliminary bottle bioassays screened approximately 10 to 12 serial concentrations of each insecticide to establish a full range of mortality from 0 to 100 percent in susceptible mosquitoes. Step 2 involved multi-centre concentration-response testing, with each centre completing three validated replicates for each concentration of each compound against its assigned species. The validity criteria were strict: each test needed at least two concentrations producing less than 50 percent mortality, one producing approximately 50 percent, two producing more than 50 percent, and one producing approximately 100 percent mortality. Each bioassay used at least 100 mosquitoes per concentration, with different batches tested on different days, and impregnated bottles were used only once. Step 3 then validated the tentative discriminating concentrations across at least three independent laboratories against the same well-characterised susceptible strains.

The technical execution demanded careful attention to the chemical properties of the two compounds. Both broflanilide and isocycloseram are highly lipophilic, adhering to bottle surfaces and dissolving poorly without help. The solution was to add a surfactant, rapeseed oil methyl ester, at 1,500 parts per million for Aedes aegypti and 800 parts per million for Anopheles species, following the manufacturers’ recommendations and the WHO bottle bioassay standard operating procedure. Broflanilide was supplied by Mitsui Chemicals Crop and Life Solutions, and isocycloseram by Syngenta Crop Protection AG. Aliquots of each insecticide and of the surfactant were distributed centrally from Liverpool to all testing centres, ensuring a single source and identical concentrations across the entire study. Three- to five-day-old, non-blood-fed female mosquitoes were exposed to treated bottles for one hour, then held at 27 degrees Celsius with 75 percent relative humidity until the 24-hour mortality assessment.

The resulting discriminating concentrations, now recommended for adoption by the WHO, vary by species and compound in ways that reflect real biological differences in baseline sensitivity. For broflanilide, the recommended values are 10 micrograms per bottle for Anopheles gambiae and Aedes aegypti, 15 micrograms per bottle for Anopheles funestus, and 25 micrograms per bottle for Anopheles stephensi. For isocycloseram, the values are 15 micrograms per bottle for Aedes aegypti, 30 micrograms per bottle for Anopheles gambiae, 50 micrograms per bottle for Anopheles stephensi, and 60 micrograms per bottle for Anopheles funestus. Lethal concentrations were generally lower for Anopheles gambiae and Aedes aegypti than for Anopheles funestus and Anopheles stephensi, suggesting differences in body size, cuticular penetration, metabolic detoxification capacity, or target-site sensitivity between species. The study was not designed to attribute these differences to specific mechanisms, but the authors note that resolving their relative contributions would require dedicated biochemical and genetic characterisation in future work.

The statistical framework underpinning the analysis was equally sophisticated. Concentration-response data for each insecticide-species combination were analysed using a Bayesian binomial modelling approach, fitting a five-parameter logistic regression on square-root-transformed concentration scales. Models ran on four Markov chains for up to 10,000 iterations, with convergence assessed using the Gelman-Rubin statistic, requiring a value below 1.01 with zero divergent iterations. All models that met these criteria produced steep slopes with narrow confidence intervals at higher mortality levels, indicating consistent concentration-response relationships. Within-bioassay variability, measured as the mean absolute deviation of observed mortality from the model’s best-fit line, was remarkably low: the median was approximately 4 to 5 percent mortality, and 28 of 30 site-species-compound combinations fell below 10 percent, confirming that the WHO bottle bioassay is reproducible within sites.

Not everything went perfectly smoothly, and the study’s honest accounting of its challenges is itself instructive. The unforeseen withdrawal of one testing centre left only the Liverpool School of Tropical Medicine able to test Anopheles stephensi. The London School of Hygiene and Tropical Medicine held an independent colony of the same SDA500 strain and provided mosquitoes to Liverpool, allowing testing to proceed across three sites, but the lack of fully multi-centre data restricted inter-laboratory comparison and reduced the strength of evidence for validation. Seven rather than nine replicate tests were possible for this species. Some datasets, notably those from the Centre for Research in Infectious Diseases for Anopheles gambiae and Anopheles funestus, could not be fitted to the model because mortality was at or near 100 percent across all tested concentrations, meaning the dose-response curve could not be adequately resolved. In each case, however, the discriminating concentration was anchored on fitted curves from other centres and subsequently confirmed in Step 3 validation, where the proposed values produced 100 percent mortality.

One of the most valuable contributions of the study is the set of technical recommendations distilled from the experience of running it. The authors highlight that estimating an LC99 is inherently difficult because it depends on observations very close to, but not reaching, 100 percent mortality. A common misconception is that a full dose-response curve with a wide spread of mortality values is required; in fact, rich information near the target value matters far more than accurately defining the shape of the lower parts of the curve. The authors also question whether the traditional multiplier of two, applied to the LC99 to derive the discriminating concentration, is truly optimal, noting that the choice appears arbitrary and that a multiplier set too high risks missing early signs of resistance. They propose that a conservative approach would use the lower bound of the 95 percent confidence interval when defining the discriminating concentration, and that intermediate values such as LC80 or LC90 may offer a more realistic compromise between biological relevance and statistical confidence.

The implications for global health are immediate and profound. With these benchmarks now validated, national malaria and dengue programmes can integrate broflanilide and isocycloseram into their routine resistance monitoring activities, testing wild mosquito populations against standardized doses and detecting emerging resistance before it undermines control efforts. As field use of these new insecticides expands, monitoring with the discriminating concentrations established here will allow the expectation of limited cross-resistance with older classes to be tested in real-world conditions. The study also validates the WHO’s generic protocol for establishing discriminating concentrations, providing a template for future multi-centre studies as additional novel active ingredients enter the pipeline. In a field where resistance can emerge silently and spread rapidly, having reliable, internationally agreed benchmarks for the newest tools in the arsenal is nothing short of essential.

Subject of Research: Multi-centre laboratory study to determine discriminating concentrations for broflanilide and isocycloseram resistance monitoring in mosquitoes

Article Title: Multi-centre laboratory study to determine discriminating concentrations for broflanilide and isocycloseram resistance monitoring in mosquitoes

Article References: Praulins, G., Mechan, F., Harvey, G., Brooke, B. D., Corbel, V., Duchon, S., Kaiser, M. L., Moore, S., Mpelepele, A. B., Oliver, S. V., Singh, H., Stevenson, J., Toguem, Y. G. F., Verma, V., Wondji, C., & Lees, R. S. (2026). Multi-centre laboratory study to determine discriminating concentrations for broflanilide and isocycloseram resistance monitoring in mosquitoes. Frontiers in Malaria, 4, Article 1874255. https://doi.org/10.3389/fmala.2026.1874255

Image Credits: AI Generated

DOI: 10.3389/fmala.2026.1874255

Keywords: insecticide resistance, mosquito vectors, WHO bottle bioassay, discriminating concentration, broflanilide, isocycloseram, malaria, dengue, vector control, Multi-centre, laboratory, determine