To determine if flies were sensitive to ethanol dose in ethanol sedation assays, we tested control w1118 flies in the presence of water vapor or vapor from 30–50% ethanol. Neither females nor males became sedated in the presence of water vapor (Figure 2A, 2B, 3A, 3B and S5A). In contrast, exposure to vapor from increasing concentrations of ethanol progressively hastened time-dependent sedation (females, Figure 2A; males, 2B) and therefore also decreased ST50s (females, Figure 2C; males, 2D). Exposure to vapor from increasing concentrations of ethanol also increased the internal ethanol content of flies (females, Figure 2E; males, 2F), demonstrating that sedation in the ethanol sedation assay is dose-dependent. Although in some of our initial studies we noticed that ST50s in w1118 females and males appeared to be different when tested on different days in separate experiments (e.g. Figure 2C and 2D), we found that males and females performed indistinguishably when tested on the same day side-by-side (Figure S5A and S5B). Therefore, comparisons were made only between groups tested on the same day. Flies in ethanol sedation assays lost a comparable
