Attraction of males to females in the fourth larval stage (L4) or as adults

Data from figure 3d. To quantify the degree of attraction of worms to conditioned medium produced by different sexes, we used chemotaxis assays. Conditioned medium (henceforth referred to as ‘supernatant’) was generated by placing 5-10 worms in 100 µL of M9 buffer for 12 h at room temperature. The supernatant was stored in 10 µL aliquots at -20 °C until required. The assay was performed either on a microscope slide with a layer of 1.5% agar placed on top of an empty plate, or on a 6 cm NGM plate. 3 µL of supernatant was added on one side of the slide and 3 cm apart from the control drop that just contained M9. 15 young-adults were placed on the midline between the spots. After 30 min, worms were scored on the basis of their location. The values for the chemotaxis index (CI) shown in figures 3a, 3d, 3e (in x axis) and S1 were calculated with at least ten replicates for each condition. To calculate the CI, the number of worms attracted to the test spot were subtracted by the number of worms in the control spot, and divided by the total number of males assayed. A CI of one indicates that all worms are attracted to the chemical in the test spot, whereas a CI close to zero indicates no attraction.