Synchronized C. elegans (N2 Bristol) were grown on S-complete medium supplemented with E. coli (strain HB101) to desired life stage, washed with M9 buffer to remove bacteria and incubated for 1 h in double-diluted water (ddH2O) to collect worm-secreted metabolites. Metabolite samples thus produced were tested for mating activity, chromatographically fractionated and analysed using NMR spectroscopy and mass spectrometry (see Methods and Supplementary Information for details).
Mating assays were performed as described previously5 (link) but were population based. All assays were conducted on plates containing nematode growth medium with a thin film of E. coli (OP50) spread throughout the plate as a food source. The worms were given a choice of worm metabolite fraction (or synthetic ascaro-sides) and control water, and the amount of time spent in each region was measured (see Methods and Supplementary Methods for details). To analyse locomotory behaviour of worms in presence of the ascarosides, standard nematode-growth-medium plates were prepared with the different concentrations of the ascarosides, and worm movement was monitored using an automated tracker to calculate parameters of locomotion16 .
Ascr#1, ascr#2 and ascr#3 were synthesized from L-rhamnose and (2R)-propylene oxide (ascr#1, ascr#3) or (2R,5R)-hexanediol (ascr#2) as described previously7 (link), and ascr#4 was subsequently prepared from acetobromo-α-D-glucose and ascr#2 (see Supplementary Methods for details). For NMR spectroscopic comparisons of daf-22 and wild-type-derived metabolite mixtures, two-week-old liquid cultures of daf-22 or wild-type (N2) worms raised on E. coli (OP50) were extracted and the resulting metabolite samples directly prepared for NMR spectroscopic analyses by means of double-quantum filtered correlation spectroscopy as previously described7 (link). For comparison of worm-derived and bacterial metabolites, E. coli (OP50) cultures were extracted and subsequently analysed by NMR spectroscopy using the same protocol.