Upper panels show pseudocolored graphs of calcium recordings of individual neuronal responses to stimulation with an ascr#2/#3/#8 mixture. Each animal underwent three rounds of stimulation; each row depicts a single response, sorted by peak activity. The pseudocolor scale represents GCaMP3 ΔF (red highest, blue lowest). Middle panels show average ΔF intensities ± SEM for the recordings depicted in the upper panels. Gray shading indicates the period of ascaroside stimulation. Lower panels show peak ΔF values for each recording; dots represent individual responses to each pulse. (A) Wild-type males showed robust calcium transients in ADF upon ascaroside stimulation. The strength of this response was significantly reduced in ADF-feminized males. The modest reduction in mean response (central panel) likely reflects a bimodal distribution of responses arising from variable effects of the ADF feminization transgene, more clearly apparent in the lower panel. (B) In wild-type hermaphrodites, ADF appeared insensitive to ascaroside stimulation. Masculinization of ADF in an otherwise wild-type hermaphrodite was sufficient to confer robust ascaroside sensitivity to ADF. (C) unc-13 mutants maintained a marked sex difference in the response of ADF to ascaroside stimulation. n = 9–66 trials (3–22 animals with 3 trials per animal). Groupwise comparison of peak ΔF values was carried out using Kruskal-Wallis analysis and Dunn’s posthoc test with Bonferroni correction. **p ≤ 0.01, ***p ≤ 0.001. See also Figure S3.