DETERMINING A DIAGNOSTIC DOSE OF PIRIMIPHOS-METHYL FOR AEDES AEGYPTI USING TREATED BOTTLES¹

Insecticide-based interventions are broadly used to control Aedes aegypti (L.), the primary vector of dengue, chikungunya, and Zika, and are termed Aedes-borne viruses (ABVs). This has led to the global emergence and rapid propagation of insecticide resistance (Moyes et al. 2017, Vazquez-Prokopec et al. 2017a). Insecticide resistance and the limited efficacy of commonly used interventions, such as peridomestic thermal fogging and ultra-low volume (ULV) applications of insecticides (Esu et al. 2010), are challenging the efforts to contain and prevent ABV transmission, highlighting the need for innovative and more effective strategies. Residual insecticide applications, consisting of the application of an insecticide to a surface using compression sprayers, generally lasted several months on the treated surfaces (WHO 2007) and were historically used to control Ae. aegypti. The successful Ae. aegypti eradication campaign in the Americas used the perifocal residual spraying of dichlorodiphenyltrichloroethane around peridomestic larval habitats (Soper 1965). After the dismantlement of the eradication programs, residual insecticide applications were replaced by the operationally faster application of peridomestic thermal fogging and ULV sprays. Although ULV kills peridomestic Ae. aegypti, it often fails to enter the indoor environment where most human–mosquito contacts occur (Perich et al. 2000, Chadee 2013, Dzul-Manzanilla et al. 2017).

Recent investigations of Ae. aegypti resting behavior have shown that the mosquito prefers to rest in rooms on surfaces below 1.5 m (Dzul-Manzanilla et al. 2017, Facchinelli et al. 2023, Seang-Arwut et al. 2023), leading to the development of targeted indoor residual spraying (TIRS) as a rational option for applying insecticides indoors. Targeted indoor residual spraying applies insecticides to key Ae. aegypti resting sites indoors, such as exposed walls below 1.5 m and under beds and furniture, with minimum preparation of houses (Vazquez-Prokopec et al. 2017b, Manrique-Saide et al. 2020). Evidence from Cairns, Queensland, Australia, showed that TIRS could prevent up to 86–96% of dengue illness (Vazquez-Prokopec et al. 2017b). Mathematic models have confirmed such effectiveness estimates (Hladish et al. 2018, 2020) and also pointed to the important epidemiologic gains of using insecticides that have long (5+ months) residual efficacy (Hladish et al. 2018).

The next-generation and longer-lasting residual insecticide formulations developed for malaria control could also be used by TIRS for Ae. aegypti control. A clinical trial quantifying the epidemiologic impact of TIRS on ABVs in the city of Mérida, Mexico (Manrique-Saide et al. 2020) used pirimiphos-methyl (Actellic® 300CS, Syngenta, Basel, Switzerland) as the active ingredient for preventive Ae. aegypti control. A prior study that showed that Actellic 300CS reduced Ae. aegypti density for up to 7 months (Vazquez-Prokopec et al. 2022) guided the choice of insecticide for the trial. Over the trial, the susceptibility of the local Ae. aegypti population to pirimiphos-methyl was monitored using the Centers for Disease Control and Prevention (CDC) bottle bioassay.

Here, we present a diagnostic dose and time for pirimiphos-methyl using the CDC bottle assay method. We applied this to determine the susceptibility of the local Ae. aegypti populations to pirimiphos-methyl prior to the implementation of the TIRS trial.

Two independent laboratories (CDC, Atlanta, GA, and Collaborative Unit for Entomological Bioassays, UCBE, Mérida, Mexico) tested 8 concentrations of pirimiphos-methyl, eliciting a range of mortality between 0% and 100% in an insecticide-susceptible reference strain (Rockefeller) of Ae. aegypti. The selection of the 8 doses was independent for each laboratory, but both used the same batch of technical-grade pirimiphos-methyl (N-13064; ChemService, Inc., West Chester, PA), provided by the CDC. Pirimiphos-methyl, an active ingredient, becomes unstable when exposed to light and can be affected by high temperatures (PubChem 2004, FAO 2016). Both laboratories carried out bioassays with similar temperature conditions (average 25 ± 1°C). The insecticides were diluted in acetone, and for each insecticide concentration, 3 bottles were treated, and 20 3-day-old female Ae. aegypti were exposed for 2 h. A bottle coated with acetone was used as the negative control. The number of alive and dead Ae. aegypti was monitored every 10 min. After agreeing on a diagnostic time of 30 min, both laboratories used a log–probit model (R package ecotox; Hlina et al. 2021) to estimate the lethal dose 50 (LD₅₀) and 99 (LD₉₉) and to plot dose–response curves. The diagnostic dose for the results generated by each lab was calculated as 2×LD₉₉.

Results were very similar at both testing locations and were predicted by a probit regression (Fig. 1). Using the fitted parameters of the probit regression, we estimated an average LD₅₀ of 6.2 μg/ml and an average LD₉₉ of 12.5 μg/ml, resulting in a diagnostic dose for pirimiphos-methyl of 25 μg/ml at a 30-min diagnostic time (Table 1).

The similarities in the LDs generated between laboratories strengthens our confidence in the estimates. As residual insecticide treatments containing pirimiphos-methyl are being considered for Ae. aegypti control, monitoring the susceptibility of Ae. aegypti populations is critical. Evidence from malaria control shows that resistance to pirimiphos-methyl is already occurring in Anopheles gambiae Giles s.l. (Olatunbosun-Oduola et al. 2019, Grau-Bove et al. 2021, Kitungulu et al. 2022), so having timely and reliable information about the susceptibility status of Ae. aegypti is critical to inform insecticide resistance management plans and the choice of vector control products.

This project received support from the National Institutes of Health, National Institute of Allergy and Infectious Disease (U01AI148069; G. Vazquez-Prokopec, principal investigator).