A novel methodology for recording wing beat frequencies of untethered male and female Aedes aegypti

Aedes aegypti is a vector of many significant arboviruses worldwide including dengue, Zika, chikungunya and yellow fever viruses. With vector control methodology pivoting towards rearing and releasing large numbers of insects for either population suppression or virus-blocking, economical remote (sentinel) surveillance methods for release tracking become increasingly necessary. Recent steps in this direction include advances in optical sensors that identify and classify insects based on their wing beat frequency (WBF). As these traps are being developed, there is a strong need to better understand the environmental and biological factors influencing mosquito WBFs. Here, we developed new untethered-subject methodology to detect changes in WBFs of male and female Ae. aegypti. This new methodology involves directing an ultrasonic transducer at a free-flying subject and measuring the Doppler shift of the reflected ultrasonic continuous wave signal. This system’s utility was assessed by determining its ability to confirm previous reports on the effect of temperature, body size and age on the WBFs generated from acoustic or optical-based experiments. The presented ultrasonic method successfully detected expected trends for each factor for both male and female Ae. aegypti without the need for subject manipulation and potential impediment of natural flight dynamics due to tethering. As a result, this ultrasonic methodology provides a new method for understanding the environmental and physiological determinants of male and female WBFs which can inform the design of remote mosquito surveillance systems