Figure 1. An optogenetic behavior assay reports on sensory valence.

(A) Side view schematic (top) showing air flow path and overview (bottom) of the WALISAR assay. Individual flies were placed in chambers and given a choice between no light or red-light illumination. Air flow was used in some experiments. (B) A representative path of a fly displaying strong light avoidance. The white line represents the location of a Gr66a-Gal4> UAS-CsChrimson fly in the chamber throughout an experiment. The light preference of each fly was calculated by how much time it spent in the illuminated zones after the initial encounter with light (yellow arrows). (C-E) Trace plots representing the mean location of controls (w¹¹¹⁸; Gr66a-Gal4 and w¹¹¹⁸; UAS-CsChrimson), and test flies (Gr66a-Gal4 > UAS-CsChrimson) throughout an experiment at 14, 42, and 70 μW/mm² light intensities. The blue and orange ribbons indicate 95% CIs for the control and test flies, respectively. (F) An estimation plot presents the individual preference (upper axes) and valence (lower) of flies with activatable Gr66a+ neurons in the WALISAR assay. In the upper panel, each dot indicates a preference score (wTSALE) for an individual fly: w¹¹¹⁸; Gr66a-Gal4 and w¹¹¹⁸; UAS-CsChrimson flies are colored blue; and Gr66a-Gal4 > UAS-CsChrimson flies are in orange. The mean and 95% CIs associated with each group are shown by the adjacent broken line. In the bottom panel, the black dots indicate the mean difference (ΔwTSALE) between the relevant two groups: the valence effect size. The black whiskers span the 95% CIs, and the orange curve represents the distribution of the mean difference.

Figure 1—figure supplement 1. An optogenetic behavior assay reports on Gr66a-induced avoidance.

(A) In each experiment, the flies were subjected to 12 consecutive epochs: three light intensities that were applied in an increasing order (14, 42, 70 μW/mm²); with and without airflow; and the illumination side was flipped in alternating epochs. (B) The light preferences of control flies across the 12 epochs, as estimated by weighted time spent after light encounter (wTSALE). Each line represents a single fly; the green lines indicate w¹¹¹⁸; Gr66a-Gal4 flies, and the orange lines indicate w¹¹¹⁸; UAS-CsChrimson flies. The black dots represent the mean wTSALE scores of all control flies per condition; the whiskers indicate the 95% Cis. (C) The light preference of Gr66a-Gal4 > UAS-CsChrimson flies across the epochs. (D) The optogenetic effect sizes, calculated by taking the difference of test and control preferences (ΔwTSALE).

Figure 1—figure supplement 2. Occupancy analysis of the WALISAR chambers during Gr66a-neuron activation.

(A) The number of occurrences of the control flies (w¹¹¹⁸; Gr66a-Gal4 and w¹¹¹⁸; UAS-CsChrimson, N 104) across the WALISAR chambers are drawn. The brown curves present the total number, the y axis represents the chamber, and the x axis is the frequency of occurrence (ranging between 0 and 2000). The red shading indicates the three light intensities; and the labels from S1 to S12 represent the 12 experimental epochs (Figure 1C). (B) The number of occurrences of the experimental flies (Gr66a-Gal4>UAS-CsChrimson, N 52) across the WALISAR chambers are summed and drawn for the 12 epochs. The blue curves represent the total number, the y axis represents the chamber, and the x axis is the frequency of occurrence (ranging between 0 and 1500).

Figure 1—figure supplement 3. The effect sizes in WALISAR and previously reported optogenetic valence assays are comparable.

(A) A comparison of the Gr66a-mediated aversion (Cohen’s d) between this study at different light intensities and Shao et al. at 5 µW/mm² of blue light, both in still-air conditions (Shao et al., 2017). (B) The magnitude of the attraction (standardized effect size, Cohen’s d) produced by Orco-neuron activation in the present study is compared to that of two previous studies (Bell and Wilson, 2016; Suh et al., 2007). The light intensity is shown in µW/mm², while ‘On’ and ‘Off’ indicate the presence of wind.