Figure 4. Behavioural assay to investigate thermal avoidance behaviour of A. gambiae mosquitoes following a blood meal.

a, Diagram of the behavioural assay. The apparatus comprises two clear Perspex tubes joined by a sliding gate. One tube (the holding tube) is wrapped in plastic piping through which water is circulated. Infected or uninfected blood-fed mosquitoes (blood fed at 06:00h [ZT0]) are introduced into the holding tube and after a period of acclimation, the water is gradually heated from 28-36°C, and the sliding gate opened. The rate at which the mosquitoes leave the holding tube and enter the adjacent escape tube is recorded. For a control, the water is maintained at constant 28°C to measure baseline movement rates across the assay period for both infected and uninfected mosquitoes. b, Cumulative escape rate of infected and uninfected A. gambiae mosquitoes (error bars = 95% confidence intervals) in relation to temperature in the holding tube. The black line shows mean temperature with standard deviation (grey lines) in the holding tube in the ramping temperature treatment from three replicate runs. There were six replicates in total for each of the four mosquito groups (infected or uninfected, with either ramping temperature or constant temperature). The data reveal that mosquitoes were unresponsive to temperatures around 33°C, and only exhibited strong escape responses as temperatures was ramped up to 35°C and beyond. Control mosquitoes showed negligible movement across the assay period. These patterns were consistence whether mosquitoes had taken an infected or uninfected blood meal. Log-rank test was used to compare escape probability between the treatment groups (ns, not significant at P = 0.05).