Designing a simple bioassay to assess repellents against Aedes aegypti (Diptera: Culicidae): investigating stimuli sources and exposure duration on mosquito behavior

Repellents are effective in preventing mosquito contact with the skin and in vitro test modules are widely used in the research of potential repellents against mosquito bites. This study evaluated different stimuli and exposure times to develop a new cost-effective bioassay protocol to test repellents against Aedes aegypti. The test module was designed to present different source of stimuli, i.e. human blood, artificial blood meal (SkitoSnack), and human forearm (standard stimulus) and exposure times, i.e., 2, 5, and 10 min. Ten females were introduced in the module, i.e., 480 mL polypropylene adapted cage, and the number of landings and engorgements on stimuli sources was recorded. Subsequently, the Complete Protection Time (CPT) of DEET was evaluated. A. aegypti mosquitoes consistently landed on the module, with an average of 4.3 mosquitoes in the first min, 5.3 in the second min, and 6.5 in the third min, after which the number decreased after eight min. Similarly, engorgement increased from 3.0 mosquitoes after two min to 7.7 mosquitoes after ten min, across all tested conditions, regardless of the stimulus source. However, prolonged exposure to the stimuli led to an increase in both landings and feedings. The CPT of DEET when using alternative stimulus sources (≈ 48 min) differed from that of the human source (75 min), yet both demonstrated similar antifeeding outcomes. Additionally, both SkitoSnack and blood, when offered through an artificial feeder with a 5-min exposure time, are effective for use in experiments with our module for repellent testing.