A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans

Abstract

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

Genetic mapping of variation in dauer larvae development in growing populations of Caenorhabditis elegans

“…QTLs defined only by sequential IL mapping are shown in bold. Butcher et al, 2007;Pungaliya et al, 2009;Park et al, 2012), on food availability and temperature (Golden and Riddle, 1984) and potentially on an assessment of individual quality (Harvey and Orbidans, 2011). Within growing populations, variation between genotypes in the number of dauer larvae could therefore be a consequence of variation in traits that affect the likelihood of dauer larvae development, the perception of the environment, or the way in which the population grows.…”

“…Dauer pheromone, a complex mix of structurally related ascarosides (Jeong et al, 2005;Butcher et al, 2007;Pungaliya et al, 2009;Park et al, 2012), is used by C. elegans to assess population density, with these ascarosides also acting to regulate aggregation, mate recognition and dispersal (Srinivasan et al, 2008;Harvey, 2009;Pungaliya et al, 2009;Izrayelit et al, 2012;Jang et al, 2012). Although there is an extensive and detailed understanding of the genetic pathways that specify dauer and non-dauer larval development in C. elegans (see Hu (2007) for a review), the ecology of the species is still poorly understood.…”

DANSing with Caenorhabditis elegans

“…In a groundbreaking report in this issue of PNAS, Pungaliya et al use differential analysis by 2-dimensional NMR spectroscopy (DANS) to study smallmolecule signals that regulate larval development and mating in the nematode Caenorhabditis elegans (4). In response to crowding, starvation, and increased temperature, C. elegans larvae arrest as an alternative larval stage called dauer (5).…”

“…Furthermore, activity-based purification schemes may preclude identification of labile molecules with biological activity. In this context, the article by Pungaliya et al represents a significant advance (4). They use DANS to compare smallmolecule profiles of wild-type C. elegans and a daf-22 mutant, which is deficient in dauer pheromone biosynthesis (11,12).…”

2D NMR-spectroscopic screening reveals polyketides in ladybugs

“…However, detection and characterization of unique or unexpected small molecules in complex mixtures require specific adaptations of the NMR-spectroscopic approach. In previous studies, we introduced high-resolution dqfCOSY spectra of small-molecule mixtures as a means for detecting chemically labile compounds or compounds associated with a specific genetic background (10)(11)(12). Here, we employ this strategy to survey the structure space of unfractionated extracts derived from the above set of insect samples.…”