Figure 5. Male-specific tuning of ADF is necessary for males to use ascarosides to locate mates.

(A) A modified quadrant assay was used to measure the ability of males to distinguish between immobilized ascaroside-producing (unc-13, red) and ascaroside-lacking (unc-13; daf-22, pink) hermaphrodites. (B) Wild-type males exhibited a marked preference for associating with ascaroside-producing over ascaroside-lacking hermaphrodites, while wild-type hermaphrodites showed no bias toward either. ADF feminization reduced this preference in males, and ADF ablation nearly eliminated it. Neither ADF masculinization nor ADF ablation had noticeable effects in hermaphrodites. Kruskal-Wallis analysis with Holm-corrected Dunn’s tests were used to assess significance. (C, D) Male mate-searching assays. Upper, diagrams of assay conditions. Gray regions represent lawns of E. coli. The starting points of males (blue) and hermaphrodites (red) are shown. Lower, cumulative rates with which males locate and initiate copulation with hermaphrodites. (C) Compared to wild-type males (blue line), feminization of ADF (solid red line) compromised the ability of males to locate and copulate with hermaphrodites. Feminization of ASK or ADL (dashed or dotted red lines) had no detectable effect. (D) Wild-type males took longer to find and copulate with daf-22 hermaphrodites (dashed blue line) compared to wild-type hermaphrodites (solid blue line). Feminization of ADF (solid red line) impaired males’ ability to find mates; it also eliminated the ability of males to use hermaphrodite-produced ascarosides as mate-location cues (compare solid red to dashed red lines). In (C) and (D), Kaplan-Meier survival curves were generated and compared using log-rank tests with the Holm correction. Numbers of males assayed n is shown in the figure. ⁽*⁾p = 0.093, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. See also Figure S5.