Assessing learning in mosquito larvae using video-tracking

Mosquito larvae display a stereotyped escape response when they rest attached to the water surface. It consists in detaching from the surface and diving, to return to the surface after a brief time. It has been shown that this response can be evoked several times, by repeatedly presenting a moving shadow. Diving triggered by a potential danger revealed as a simple bioassay for investigating behavioural responses in mosquito larvae, in particular their ability to learn. In the present work, we describe an automated system, based on video-tracking individuals, and extracting quantitative data of their movements. We validated our system, by reinvestigating the habituation response of larvae of Aedes aegypti reared in the laboratory, and providing original data on field-collected larvae of genera Culex and Anopheles. Habituation could be demonstrated to occur in all the species, even though it was not possible to induce dishabituation in Culex and Anopheles mosquitoes. In addition to non-associative learning, we characterised motor activity in the studied species, thanks to the possibility offered by the tracking system to extract multiple variables. The here-described system and algorithms can be easily adapted to multiple experimental situations and variables of interest.