In many organisms, population-density sensing and sexual attraction rely on small-molecule-based signalling systems1,2. In the nematode Caenorhabditis elegans, population density is monitored through specific glycosides of the dideoxysugar ascarylose (the `ascarosides') that promote entry into an alternative larval stage, the non-feeding and highly persistent dauer stage3,4. In addition, adult C. elegans males are attracted to hermaphrodites by a previously unidentified small-molecule signal5,6. Here we show, by means of combinatorial activity-guided fractionation of the C. elegans metabolome, that the mating signal consists of a synergistic blend of three dauer-inducing ascarosides, which we call ascr#2, ascr#3 and ascr#4. This blend of ascarosides acts as a potent male attractant at very low concentrations, whereas at the higher concentrations required for dauer formation the compounds no longer attract males and instead deter hermaphrodites. The ascarosides ascr#2 and ascr#3 carry different, but overlapping, information, as ascr#3 is more potent as a male attractant than ascr#2, whereas ascr#2 is slightly more potent than ascr#3 in promoting dauer formation7. We demonstrate that ascr#2, ascr#3 and ascr#4 are strongly synergistic, and that two types of neuron, the amphid single-ciliated sensory neuron type K (ASK) and the male-specific cephalic companion neuron (CEM), are required for male attraction by ascr#3. On the basis of these results, male attraction and dauer formation in C. elegans appear as alternative behavioural responses to a common set of signalling molecules. The ascaroside signalling system thus connects reproductive and developmental pathways and represents a unique example of structure- and concentration-dependent differential activity of signalling molecules.

In the model organism Caenorhabditis elegans, a family of endogenous small molecules, the ascarosides, function as key regulators of developmental timing and behavior that act upstream of conserved signaling pathways. The ascarosides are based on the dideoxysugar ascarylose, which is linked to fatty acid-like side chains of varying lengths derived from peroxisomal β-oxidation. Despite their importance for many aspects of C. elegans biology, knowledge of ascaroside structures, biosynthesis, and homeostasis remains incomplete. We used an MS/MS-based screen to profile ascarosides in C. elegans wild type and mutant metabolomes, which revealed a much greater structural diversity of ascaroside derivatives than previously reported. Comparison of the metabolomes from wild type and a series of peroxisomal β-oxidation mutants showed that the enoyl CoA-hydratase MAOC-1 serves an important role in ascaroside biosynthesis and clarified the functions of two other enzymes, ACOX-1 and DHS-28. We show that following peroxisomal β-oxidation the ascarosides are selectively derivatized with moieties of varied biogenetic origin and that such modifications can dramatically affect biological activity, producing signaling molecules active at low femtomolar concentrations. Based on these results the ascarosides appear as a modular library of small molecule signals, integrating building blocks from three major metabolic pathways; carbohydrate metabolism, peroxisomal β-oxidation of fatty acids, and amino acid catabolism. Our screen further demonstrates that ascaroside biosynthesis is directly affected by nutritional status and that excretion of the final products is highly selective.

Small molecule metabolites play important roles in Caenorhabditis elegans biology, but effective approaches for identifying their chemical structures are lacking. Recent studies revealed that a family of glycosides, the ascarosides, differentially regulate C. elegans development and behavior. Low concentrations of ascarosides attract males and thus appear to be part of the C. elegans sex pheromone, whereas higher concentrations induce developmental arrest at the dauer stage, an alternative, nonaging larval stage. The ascarosides act synergistically, which presented challenges for their identification via traditional activity-guided fractionation. As a result the chemical characterization of the dauer and male attracting pheromones remained incomplete. Here, we describe the identification of several additional pheromone components by using a recently developed NMR-spectroscopic approach, differential analysis by 2D NMR spectroscopy (DANS), which simplifies linking small molecule metabolites with their biological function. DANS-based comparison of wild-type C. elegans and a signaling-deficient mutant, daf-22, enabled identification of 3 known and 4 previously undescribed ascarosides, including a compound that features a p-aminobenzoic acid subunit. Biological testing of synthetic samples of these compounds revealed additional evidence for synergy and provided insights into structure-activity relationships. Using a combination of the three most active ascarosides allowed full reconstitution of the maleattracting activity of wild-type pheromone extract. Our results highlight the efficacy of DANS as a method for identifying smallmolecule metabolites and placing them within a specific genetic context. This study further supports the hypothesis that ascarosides represent a structurally diverse set of nematode signaling molecules regulating major life history traits.dauer formation ͉ differential analysis ͉ metabolomics ͉ NMR spectroscopy ͉ sex pheromone