(A–F) Heat-seeking at different ambient temperatures (n = 5–6 trials per condition): 21°C (blue), 26°C (gray), 31°C (orange). Data in A, B, D, and E are plotted as mean ± s.e.m. (A,D) Percent of mosquitoes on Peltier during seconds 90–180 of stimuli of indicated temperature, normalized to stimulus 10°C above ambient (A, open circle) or 40°C stimulus (D, open circle). (B,E) Same data as in (A) and (D), respectively, plotted using differential between ambient and Peltier temperature. For each ambient temperature, arrowheads indicate the lowest temperature stimulus found to elicit a significant increase in heat-seeking compared to an ambient temperature stimulus (A,B) or a reduction in heat-seeking compared to a 40°C stimulus (D,E) (p < 0.05; repeated-measures ANOVA with Bonferroni correction). For each ambient temperature, linear regressions (A, B, 21°C: 10.6/°C, R2 = 0.98, 26°C: 12/°C, R2 = 0.99, 31°C: 9.5/°C, R2 = 0.97) or variable slope sigmoidal dose–response curves (D, E, 21°C: IC50 = 55.4°C, R2 = 0.87, 26°C: IC50 = 52.5°C, R2 = 0.92, 31°C: IC50 = 50.5°C, R2 = 0.91) are plotted. (C,F) Heat maps showing mean mosquito occupancy on the Peltier (red square) and surrounding area, during seconds 90–180 of each stimulus period. Bold borders indicate stimuli with responses significantly different from an ambient-temperature stimulus (C) in (A,B), or significantly different from a 40°C stimulus (F) in (D,E) (p < 0.05; repeated-measures ANOVA with Bonferroni correction). (G,H) Analysis of mosquito responses to cooling from data in (Figure 1D). (G) Mean percent of mosquitoes on Peltier during thermal stimuli 31–40°C. Dashed line indicates the end of the stimulus period. (H) Post-stimulus time at which the percent of mosquitoes on Peltier has decayed to one half of the mean during seconds 90–180 of the stimulus period from (Figure 1E). Each replicate is indicated by a dot, mean ± s.e.m. by lines (NS, not significant; one-way ANOVA with Bonferroni correction).