Developing reliable and standardized methods for testing mosquito swarm susceptibility to insecticides is crucial for understanding the effectiveness of new active ingredients or formulations. Methods for testing mosquito swarm susceptibility to contact insecticides or products (such as those promoted in public health programs) are well established and standardized. However, testing methods for volatile or aerosol insecticides used in household products are difficult to implement effectively. Based on World Health Organization recommendations for household insecticides, we developed a standardized and high-throughput method for testing aerosol products using caged mosquitoes and an effective disinfection method conducted in a Peet-Grady test chamber (PG test chamber). We validated the effectiveness of this new method using populations of insecticide-resistant and susceptible Aedes and Anopheles mosquitoes. A novel feature of this method is the inclusion of a chamber directed at the mosquito cages, allowing for real-time quantitative assessment of mosquito kill rates after insecticide exposure. Swab disinfection effectively removes residual pyrethroid-containing aerosol oil from the test chamber surface, with mortality rates of less than 2% for susceptible mosquitoes tested directly on the chamber surface. No spatial heterogeneity in kill or mortality rates among caged mosquitoes was observed in the PG chamber. Our dual-cage method provides eight times higher throughput than the free-flight method, enabling simultaneous testing of different mosquito strains and effective discrimination between susceptible and resistant mosquito populations tested in parallel.
To date, aerosol insecticides have been primarily used in the home for personal protection, with limited use in public health programs. However, recent studies have shown widespread household insecticide use in areas where vector-borne diseases are prevalent. Whether the motivation is mosquito repellent or disease prevention, there is a pressing need for standardized and easy-to-use methods for screening endemic mosquito populations for susceptibility to household insecticides. This is crucial for predicting insecticide effectiveness against local vectors and understanding how household insecticide use influences the evolutionary selection for insecticide resistance.
Supplemental Method 1 provides detailed step-by-step instructions for conducting our aerosol insecticide testing program.
Although WHO guidelines recommend the use of automated nebulizers, they do not provide specific technical specifications. The use of automated nebulizers is crucial, as manual nebulization in a propylene glycol chamber is not only labor-intensive but can also cause spatial inconsistencies and variations in nebulization duration.
The reaction chamber must be sterilized after each test, but the internal cleaning method recommended in WHO Guideline involves applying water from a hose. In our daily work, this method is the most labor-intensive step in operating bioanalytical equipment, so we developed and tested a swab-based sterilization procedure.
The removable parts of the fan are treated as described above, and the blades and frame of the fan are cleaned with a sponge soaked in a 5% solution of Decon 90.
Based on the relationship between spray duration and product delivery rate, our aerosol dispenser also demonstrated good accuracy in controlling the aerosol dosage ratio, at least over the tested range of 1 to 4 times . As shown in Fig. 3b, this characteristic is particularly important for characterizing the dose-response relationship of new aerosol formulations or determining the identification dose for detecting insecticide resistance.
We demonstrate that our revised protocol for evaluating household aerosol insecticides, using swab disinfection, double cages, remotely controlled sprayers, and biometric recording from action cameras, is a more effective and feasible alternative to current WHO recommendations. The swab disinfection method, requiring only 20 minutes, significantly saves time compared to the existing protocol (which typically requires one hour per test chamber). It also reduces the time operators spend donning full personal protective equipment (e.g., respiratory helmets and antistatic work clothing). Furthermore, this method generates less contaminated liquid and clothing for treatment than a complete cleaning of the test chamber, thereby minimizing the potential for contamination of the room housing the test chamber. The swab disinfection method is also suitable for disinfection of semi-permanent test rooms requiring minimal furniture placement in a variety of room layouts.
A key issue explored in this study and others is the standardization of exposure doses of insecticides applied in the environment across different testing protocols. As shown in Figure 2b, despite a fixed spray duration, the spray volume varied across aerosol can types, potentially reflecting differences in manufacturing processes (e.g., internal pressure, propellant use, nozzle structure, etc.). Furthermore, the current lack of commercially available remote spraying devices with the required flexibility in spray duration limits their use in assessing the dose-response relationship for mosquito control. Manual spraying through test hatches or access hatches (if available) can lead to variations in exposure doses. In fact, our results highlight the need and importance of reducing these sources of variation. For resistant Aedes aegypti populations, we observed a correlation between the aerosol dose and the final determination of susceptibility or resistance (Figure 3b). Ideally, aerosol doses should be standardized in grams of aerosolized substance rather than in duration of aerosolization to facilitate comparisons between different studies.
RCAD offers an alternative approach for future research that minimizes the impact of process variations. Although we found that standardization of aerosol sprays is not feasible, we demonstrated that the mass of aerosol delivered through different aerosol cans can be reproducibly estimated by calibrating the spray length (Figures 2b, 3a). Such standardization of aerosol concentration in any test chamber is crucial for improving the reproducibility of results.
Based on our experience and that of other research groups , the recommendations contained in current Guideline regarding the use of aerosol detection methods for testing free-flying mosquitoes pose significant logistical challenges for laboratory and semi-field studies. For example, free-flying mosquito detection methods have very low throughput (including labor-intensive recapture of surviving free-flying mosquitoes) and suffer from a number of technical limitations, such as difficulties in determining kill rates in real time.
Although our validated double-cage experiment addresses the issue of flow limitations and is a feasible method for screening mosquito susceptibility to aerosol insecticides, it should be noted that mortality rates of Cayman Islands mosquitoes were significantly lower in the cage experiment than in the free-flight experiment (Fig. 5c, Table 1). This difference may reflect a reduction in the insecticide dose inside the cage, as fewer aerosol droplets penetrate the mesh and enter the cage. Future studies could use larger-mesh fabrics and cage designs with higher fan airflow rates (e.g., cylindrical designs ) to further validate the results obtained with the different experimental methods.
Post time: Feb-02-2026