Introduction
Most organisms use external small-molecule signals to evaluate their environment and communicate with each other. Some well-studied examples include the use of acylhomoserine lactones by Gram-negative bacteria to determine population density (quorum sensing) and the use of long-chain alcohols and acetates by moths as sex pheromones to attract mates1, 2. C. elegans uses a small-molecule signal, the dauer pheromone, to monitor its population density and modulate its development accordingly. Under favorable growth conditions, worms progress from the egg through four larval stages (L1 to L4) to adulthood. However, if worms encounter poor growth conditions (high population density, low food availability, high temperature) as an L1 or early L2, they progress to the dauer stage3. Dauers, which are adapted for survival under harsh conditions, have thickened cuticles, often remain motionless, do not feed, and survive off fat stores4. Dauers are also considered 'non-aging' because C. elegans can survive as a dauer for several months, and then, once conditions improve, progress to the adult stage with a normal life span of about two weeks5. The dauer response has attracted special interest as it is controlled by signaling pathways that control metabolism, aging and development (such as the insulin and TGF-
pathways) in higher eukaryotes and in C. elegans6, 7, 8, 9, 10, 11, 12, 13. Though the biology and genetics of dauer formation have been well studied, the identity of the dauer pheromone has not been fully established.
Over 20 years ago, researchers showed that dauer formation is induced by a family of small molecules, which they named the dauer pheromone to indicate that it is an intraspecies signal that promotes dauer formation3, 14, 15. Early studies suggested that the responsible molecules most likely belong to a class of hydroxylated fatty acids. More recently, investigators identified a glycoside of the dideoxysugar ascarylose (Compound 3) in the crude dauer pheromone extract and showed that this ascaroside can be used to induce dauer formation16. However, this study contained two puzzling features: more than 88% of the dauer-inducing activity of the crude pheromone extract was lost during the chromatographic steps of the purification, and the concentration of ascaroside Compound 3 needed to induce dauer formation was about 1,400-fold greater than the confirmed concentration of Compound 3 in the culture medium. It is possible that these confusing results were due to a substantial loss in ascaroside Compound 3 during purification or to solubility problems with the purified material16. However, it seems more likely that these features arose because more active compounds were overlooked or because multiple compounds are necessary for a biologically relevant signal. Although pheromones sometimes consist of single compounds, they more often consist of mixtures of several compounds such that their full activity can only be reconstituted by including all active components17, 18. Observable activity levels can sometimes be attained using nonphysiologically high concentrations of a single component of the pheromone mixture or structurally related nonpheromone molecules17.
In light of these observations, we performed a detailed analysis of the dauer-promoting constituents of the crude dauer pheromone to isolate significantly active components. As a first step, we isolated a single dauer-inducing ascaroside (Compound 1) using activity-guided fractionation. We then sought to identify any additional members of this family of compounds that were present in the crude dauer pheromone at significant concentrations. In this manner, we were able to identify two ascarosides (Compound 1 and Compound 2) that are two orders of magnitude more potent than Compound 3 at inducing dauer formation.
To isolate active components of the worm-conditioned medium, we cultured C. elegans in liquid medium for two weeks, and the conditioned medium was then collected, freeze dried and extracted with ethanol. The dauer recovery assay, in which fractions are tested for their ability to inhibit recovery of dauers, was used to guide fractionation (Supplementary Methods online). Fractionation included three steps: (i) C18 reverse-phase chromatography, pooling the most active fractions, (ii) normal-phase chromatography on silica gel, pooling the most active fractions, and (iii) C30 reverse-phase HPLC to afford a single active compound, Compound 1 (Fig. 1). Compound Compound 1 has a molecular formula of C12H22O5, as established by NMR spectroscopy (Supplementary Fig. 1 and Supplementary Table 1 online) and mass spectrometry data. Interpretation of the COSY, HMQC and NOESY spectra led to three substructures—a 3,6-dideoxyhexose, a hydroxybutyl unit, and the methyl ketone—and these substructures were assembled on the basis of HMBC correlations (Supplementary Fig. 2 online). Cross peaks from H1, H3 and H4 to C2 linked the hydroxybutyl and methyl ketone units into 5-hydroxy-2-hexanone, and cross peaks from H5 to C1' and H1' to C5 established the glycosidic linkage and the gross structure of Compound 1. The absolute configuration of Compound 1 was established (Supplementary Fig. 3 online), thus defining Compound 1 as 5-O-ascarylosyl-5R-hydroxy-2-hexanone.
Figure 1: Structures of ascarosides 1, 2 and 3.
All are derivatives of the 3,6-dideoxyhexose ascarylose; they differ only in the identity of the fatty acid–like moiety.
Full size image (13 KB)During the fractionation scheme used to isolate Compound 1, it became apparent that Compound 1 was accompanied by several other ascarosides that seemed to contribute to the biological activity of the crude conditioned medium extract. To identify these additional ascaroside derivatives, we analyzed by NMR spectroscopy the combined remaining fractions from the silica gel column (that is, those not containing Compound 1), as well as the entire unfractionated conditioned medium extract. This analysis established that several ascarosides were present in the crude dauer pheromone. Ascaroside Compound 1 was the most abundant, with a concentration of roughly 210–380 nM in the conditioned medium extract (see Supplementary Methods for concentration determination). In addition, we identified ascaroside Compound 2, a derivative of 8R-hydroxy-2E-nonenoic acid (see Supplementary Fig. 4 and Supplementary Table 2 online for NMR data) that was present at a concentration of roughly 80–130 nM in the conditioned medium extract. We also detected Compound 3, which had been previously described16. The concentrations of Compound 3 in our media samples varied considerably but never exceeded one-tenth the amount of Compound 1 (that is, at most 36 nM). A number of additional ascarosides whose structures seemed to be closely related to those of Compound 1, Compound 2 and Compound 3 were present at even lower concentrations, but they were not characterized in detail (unpublished data).
In order to characterize the relative biological activity of the ascarosides, we chemically synthesized enantiomerically pure samples of ascarosides Compound 1, Compound 2 and Compound 3 (see Fig. 1 and Supplementary Methods). We then tested their relative activities in the dauer formation assay, in which eggs were laid on plates containing various concentrations of ascarosides and a small amount of food. We found that at both 20 °C and 25 °C, ascarosides Compound 1 and Compound 2 are roughly two orders of magnitude more potent than the previously identified Compound 3 (Fig. 2). We also tested the relative activities of ascarosides Compound 1, Compound 2 and Compound 3 in the dauer formation assay in several mutant strains, but we did not note a significant difference in the relative activities in the mutant strains as compared with wild-type worms (Supplementary Fig. 5 online). In order to determine how the different ascarosides may work together to induce dauer formation, we also compared the activity of the individual ascarosides to that of different combinations of ascarosides. In wild-type worms, the activities of the different combinations were roughly additive (Supplementary Fig. 6a online and unpublished data). However, natural pheromone production by the worms in the assay could affect the potency of the individual synthetic ascarosides. Thus, we also tested the activities of combinations of ascarosides in the daf-22 strain, in which the dauer pheromone is either not produced or not exported into the environment19. In this strain, the activities of Compound 1 and Compound 2 were mildly synergistic, but only at low concentrations (Supplementary Fig. 6b).
Figure 2: Comparison of the activities of 1, 2 and 3 in the dauer formation assay.
(a) Percent dauer formation induced by Compound 1, Compound 2 and Compound 3 at 25 °C. Data represent the average of three experiments, and error bars represent 
one s.d. The EC50 of Compound 1 is approximately 120 nM, the EC50 of Compound 2 is approximately 370 nM, and the EC50 of Compound 3 is approximately 18,000 nM. (b) Percent dauer formation induced by Compound 1, Compound 2 and Compound 3 at 20 °C. Data represent the average of three experiments, and error bars represent one s.d. The EC50 of Compound 1 is approximately 460 nM, the EC50 of Compound 2 is approximately 1,100 nM, and the EC50 of Compound 3 is approximately 88,000 nM.
The relative activities of ascarosides Compound 1, Compound 2 and Compound 3 are not predicted by their relative structural similarities; that is, terminal-acid ascarosides Compound 2 and Compound 3 are structurally the most similar of the three ascarosides, but Compound 2 and the methyl ketone ascaroside Compound 1 have similar potencies and are much more potent than Compound 3 in the dauer formation assay. Thus, it is unlikely that the potencies of the ascarosides are due to trivial factors, such as solubility or stability profiles. Instead, their differing potencies must be due to specific interactions with their targets and, if they have different targets, the differing roles of those targets in the dauer formation process.
Our understanding of small-molecule signaling has progressed from early studies on one molecule/one response systems to a much more nuanced view in which combinations of molecules orchestrate a biological response. Here, we show that a blend of two structurally related molecules (Compound 1 and Compound 2) induces dauer formation at physiologically relevant concentrations. Given that our activity-based fractionation used the dauer recovery assay rather than the dauer formation assay to follow activity, it is possible that additional molecules that contribute to dauer formation may have been missed. Further analysis of the crude conditioned medium extract may thus identify additional components that contribute to the dauer signal18. The dauer pheromone most likely interacts with a G protein–coupled receptor or receptors in specific ciliated chemosensory neurons that are directly exposed to the environment20, 21, 22, 23, 24. The identification of pheromone components that are active at physiologically relevant concentrations will help to identify appropriate receptors and downstream signaling proteins.
Methods
General procedures.
NMR spectra were recorded on a Varian INOVA 500 NMR spectrometer (500 MHz for 1H and 126 MHz for 13C) or a Varian INOVA 600 NMR (600 MHz for 1H, 151 MHz for 13C). UV spectra were recorded on a DU 640 spectrophotometer (Beckman) in methanol, and optical rotations were measured on a Dip-370 digital polarimeter (Jasco). High-resolution mass spectrometry was performed on a Q-TOF-2 spectrometer (Micromass) equipped with an Alliance 2690/2695 HPLC system (Waters).
Strains and general culture methods.
C. elegans variety Bristol, strain N2 (wild type), and mutant worms were grown at room temperature (20–23 °C) on NG agar plates, which were made with Noble agar (BD Biosciences) and seeded with OP50 bacteria.
Preparation of conditioned medium extract.
Crude dauer pheromone extract was prepared essentially according to a previously described method15. Worms were cultured in 50 liters of S medium25 for 14–16 d at 22.5 °C on a rotary shaker. 25 liters of OP50 was resuspended in a small volume of S medium and added as a food source at day 1 and then again at day 7 or 8. Conditioned medium was centrifuged, filtered through Celite, and freeze-dried. The solids were extracted with 95% aqueous ethanol (5 liters 
3 times) to afford crude dauer pheromone.
Characterization of Compound 1 and Compound 2.
Conditioned medium extract was fractionated by C18 column chromatography with a stepwise gradient of aqueous methanol (0% to 100%). The most active fractions, which eluted with 10% to 50% methanol, were combined and further fractionated on a silica gel column with chloroform and methanol solvent mixtures (20:1 to 4:1). The most active fraction was identified using the dauer recovery assay and showed a clear pink spot on a silica gel TLC plate stained with anisaldehyde (Rf value of 0.35 developed with CH2Cl2:MeOH, 10:1). This fraction was further purified by reverse-phase HPLC on C30 (Develosil) using an aqueous acetonitrile gradient (0% to 40%) to give approximately 5 mg of pure Compound 1 as a colorless oil; [
]D = -
65.5 (8.1 mM in methanol); UV (methanol): 
max 229 nm (
2.83 102); for 1H and 13C NMR data, see Supplementary Table 1; HR-ESIMS (m/z): [M+Na]+ calcd. for C12H22O5Na, 269.1365; found, 269.1347. The remaining fractions from the silica gel column were combined, evaporated and resuspended in methanol-d4 for characterization by dqf-COSY, HMBC and HMQC experiments. Compound 1 and Compound 2 were synthesized (see Supplementary Methods), and the NMR data of synthetic Compound 1 and Compound 2 were identical to those of the natural samples. The concentrations of Compound 1, Compound 2 and Compound 3 in crude conditioned medium extract were determined as described in Supplementary Methods.
Data analysis.
EC50 values were determined using Prism software. Each titration curve was fit with a sigmoidal curve, in which the lower limit was set at 0 and the upper limit was not defined. The EC50 was defined as the concentration at which each ascaroside reached half its maximal activity (as calculated by Prism).
Note: Supplementary information and chemical compound information is available on the Nature Chemical Biology website.
Author Contributions
R.A.B. designed experiments, performed biological experiments and wrote the manuscript; M.F. performed structure elucidation and biological experiments; F.C.S. performed structure elucidation and chemical synthesis; J.C. designed experiments and wrote the manuscript.
