Prior to conducting wind tunnel experiments, mosquitoes must be prepared for testing. Important factors and state-dependent processes of the mosquito-like the sex, age, infection status, reproductive status, or nutritional status-should be evaluated and motivated by questions and hypotheses one seeks to address. Other critical external factors that can impact the mosquitoes' behavior and should be controlled for both in the colony and in the room where the wind tunnel experiments take place include the circadian rhythm, room temperature, light intensity, and relative humidity. Together, the internal and external factors, and wind tunnel design, ultimately control the behavior of the mosquito and, hence, the success of the experiments. In the present protocol, we describe methods using a standard wind tunnel design in which the fan pulls the air through the working section of the wind tunnel and the mosquito behavior is recorded by a multicamera system. Variations around the camera tracking system can be adapted according to the research questions being asked and include real-time tracking for both closed-loop and open-loop control of the stimulus environment or recording of the videos for off-line digitization and analysis. Within the working section, the sensory environment (odor, visual, wind) can be controlled to test the mosquito responses to different stimuli, and below we include different equipment and tools for modifying the stimuli the mosquito experiences during flight. Finally, the methods described here are applicable to multiple mosquito species, although the experiment parameters may need to be changed (e.g., ambient luminosity).
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