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Untargeted metabolomics via high-resolution mass spectrometry can reveal more than 100,000 molecular features in a single sample, many of which may represent unidentified metabolites, posing significant challenges to data analysis. We here introduce Metaboseek, an open-source analysis platform designed for untargeted comparative metabolomics and demonstrate its utility by uncovering biosynthetic functions of a conserved fat metabolism pathway, α-oxidation, using C. elegans as a model. Metaboseek integrates modules for molecular feature detection, statistics, molecular formula prediction, and fragmentation analysis, which uncovers more than 200 previously uncharacterized α-oxidation-dependent metabolites in an untargeted comparison of wildtype and α-oxidation-defective hacl-1 mutants. The identified metabolites support the predicted enzymatic function of HACL-1 and reveal that α-oxidation participates in metabolism of endogenous β-methyl-branched fatty acids and food-derived cyclopropane lipids. Our results showcase compound discovery and feature annotation at scale via untargeted comparative metabolomics applied to a conserved primary metabolic pathway and suggest a model for the metabolism of cyclopropane lipids.

Excreted small-molecule signals can bias developmental trajectories and physiology in diverse animal species. However, the chemical identity of these signals remains largely obscure. Here we report identification of an unusual N-acylated glutamine derivative, nacq#1, that accelerates reproductive development and shortens lifespan in Caenorhabditis elegans. Produced predominantly by C. elegans males, nacq#1 hastens onset of sexual maturity in hermaphrodites by promoting exit from the larval dauer diapause and by accelerating late larval development. Even at picomolar concentrations, nacq#1 shortens hermaphrodite lifespan, suggesting a trade-off between reproductive investment and longevity. Acceleration of development by nacq#1 requires chemosensation and is dependent on three homologs of vertebrate steroid hormone receptors. Unlike ascaroside pheromones, which are restricted to nematodes, fatty acylated amino acid derivatives similar to nacq#1 have been reported from humans and invertebrates, suggesting that related compounds may serve signaling functions throughout metazoa.

Many bacterivorous and parasitic nematodes secrete signaling molecules called ascarosides that play a central role regulating their behavior and development. Combining stable-isotope labeling and mass spectrometry-based comparative metabolomics, here we show that ascarosides are taken up from the environment and metabolized by a wide range of phyla, including plants, fungi, bacteria, and mammals, as well as nematodes. In most tested eukaryotes and some bacteria, ascarosides are metabolized into derivatives with shortened fatty acid side chains, analogous to ascaroside biosynthesis in nematodes. In plants and C. elegans, labeled ascarosides were additionally integrated into larger, modular metabolites, and use of different ascaroside stereoisomers revealed the stereospecificity of their biosynthesis. The finding that nematodes extensively metabolize ascarosides taken up from the environment suggests that pheromone editing may play a role in conspecific and interspecific interactions. Moreover, our results indicate that plants, animals, and microorganisms may interact with associated nematodes via manipulation of ascaroside signaling.

Few nucleoside-derived natural products have been identified from animals, despite the ubiquity of nucleosides in living organisms. Here, we use a combination of synthesis and the emerging electron microscopy technique microcrystal electron diffraction to determine the structures of several N3-(β-glucopyranosyl)uric acid derivatives in Caenorhabditis elegans. These noncanonical gluconucleosides further integrate an ascaroside moiety, for which we present a shortened synthetic route. The production of a phosphorylated gluconucleoside is influenced by evolutionarily conserved insulin signaling.

Few nucleoside-derived natural products have been identified from animals, despite the ubiquity of nucleosides in living organisms. Here, we use a combination of synthesis and the emerging electron microscopy technique microcrystal electron diffraction to determine the structures of several N(3)-(β-glucopyranosyl)uric acid derivatives in Caenorhabditis elegans. These noncanonical gluconucleosides further integrate an ascaroside moiety, for which we present a shortened synthetic route. The production of a phosphorylated gluconucleoside is influenced by evolutionarily conserved insulin signaling.

We describe a mathematical model for the aggregation of starved first-stage C elegans larvae (L1s). We propose that starved L1s produce and respond chemotactically to two labile diffusible chemical signals, a short-range attractant and a longer range repellent. This model takes the mathematical form of three coupled partial differential equations, one that describes the movement of the worms and one for each of the chemical signals. Numerical solution of these equations produced a pattern of aggregates that resembled that of worm aggregates observed in experiments. We also describe the identification of a sensory receptor gene, srh–2, whose expression is induced under conditions that promote L1 aggregation. Worms whose srh–2 gene has been knocked out form irregularly shaped aggregates. Our model suggests this phenotype may be explained by the mutant worms slowing their movement more quickly than the wild type., Author summary Among the most complex of animal behaviors are collective behaviors, in which animals interact with each other so as to produce large-scale organization. Starved first-stage larvae of the nematode Caenorhabditis elegans exhibit such a behavior: they come together to form aggregates of several hundred worms. How and why they do this are unknown. To address these questions, we developed a mathematical model of starved L1 aggregation. This model reproduced the main features of the behavior.

Peroxisomal β-oxidation (pβo) is a highly conserved fat metabolism pathway involved in the biosynthesis of diverse signaling molecules in animals and plants. In Caenorhabditis elegans, pβo is required for the biosynthesis of the ascarosides, signaling molecules that control development, lifespan, and behavior in this model organism. Via comparative mass spectrometric analysis of pβo mutants and wildtype, we show that pβo in C. elegans and the satellite model P. pacificus contributes to life stage-specific biosynthesis of several hundred previously unknown metabolites. The pβo-dependent portion of the metabolome is unexpectedly diverse, e.g., intersecting with nucleoside and neurotransmitter metabolism. Cell type-specific restoration of pβo in pβo-defective mutants further revealed that pβo-dependent submetabolomes differ between tissues. These results suggest that interactions of fat, nucleoside, and other primary metabolism pathways can generate structural diversity reminiscent of that arising from combinatorial strategies in microbial natural product biosynthesis.

Summary In the nematodes Caenorhabditis elegans and Pristionchus pacificus, a modular library of small molecules control behavior, lifespan, and development. However, little is known about the final steps of their biosynthesis, in which diverse building blocks from primary metabolism are attached to glycosides of the dideoxysugar ascarylose, the ascarosides. We combine metabolomic analysis of natural isolates of P. pacificus with genome-wide association mapping to identify a putative carboxylesterase, Ppa-uar-1, that is required for attachment of a pyrimidine-derived moiety in the biosynthesis of ubas#1, a major dauer pheromone component. Comparative metabolomic analysis of wild-type and Ppa-uar-1 mutants showed that Ppa-uar-1 is required specifically for the biosynthesis of ubas#1 and related metabolites. Heterologous expression of Ppa-UAR-1 in C. elegans yielded a non-endogenous ascaroside, whose structure confirmed that Ppa-uar-1 is involved in modification of a specific position in ascarosides. Our study demonstrates the utility of natural variation-based approaches for uncovering biosynthetic pathways.

The nematode Caenorhabditis elegans uses simple building blocks from primary metabolism and a strategy of modular assembly to build a great diversity of signaling molecules, the ascarosides, which function as a chemical language in this model organism. In the ascarosides, the dideoxysugar ascarylose serves as a scaffold to which diverse moieties from lipid, amino acid, neurotransmitter, and nucleoside metabolism are attached. However, the mechanisms that underlie the highly specific assembly of ascarosides are not understood. We show that the acyl-CoA synthetase ACS-7, which localizes to lysosome-related organelles, is specifically required for the attachment of different building blocks to the 4′-position of ascr#9. We further show that mutants lacking lysosome-related organelles are defective in the production of all 4′-modified ascarosides, thus identifying the waste disposal system of the cell as a hotspot for ascaroside biosynthesis.

Small-molecule signaling in nematode dauer formation has emerged as a major model to study chemical communication in development and evolution. Developmental arrest as nonfeeding and stress-resistant dauer larvae represents the major survival and dispersal strategy. Detailed studies in Caenorhabditis elegans and Pristionchus pacificus revealed that small-molecule communication changes rapidly in evolution resulting in extreme structural diversity of small-molecule compounds. In C. elegans, a blend of ascarosides constitutes the dauer pheromone, whereas the P. pacificus dauer pheromone includes additional paratosides and integrates building blocks from diverse primary metabolic pathways. Despite this complexity of small-molecule structures and functions, little is known about the biosynthesis of small molecules in nematodes outside C. elegans. Here, we show that the genes encoding enzymes of the peroxisomal β-oxidation pathway involved in small-molecule biosynthesis evolve rapidly, including gene duplications and domain switching. The thiolase daf-22, the most downstream factor in C. elegans peroxisomal β-oxidation, has duplicated in P. pacificus, resulting in Ppa-daf-22.1, which still contains the sterol-carrier-protein (SCP) domain that was lost in C. elegans daf-22, and Ppa-daf-22.2. Using the CRISPR/Cas9 system, we induced mutations in both P. pacificus daf-22 genes and identified an unexpected complexity of functional conservation and divergence. Under well-fed conditions, ascaroside biosynthesis proceeds exclusively via Ppa-daf-22.1. In contrast, starvation conditions induce Ppa-daf-22.2 activity, resulting in the production of a specific subset of ascarosides. Gene expression studies indicate a reciprocal up-regulation of both Ppa-daf-22 genes, which is, however, independent of starvation. Thus, our study reveals an unexpected functional complexity of dauer development and evolution.

We describe a new type of collective behavior in C. elegans nematodes, aggregation of starved L1 larvae. Shortly after hatching in the absence of food, L1 larvae arrest their development and disperse in search for food. In contrast, after two or more days without food, the worms change their behavior—they start to aggregate. The aggregation requires a small amount of ethanol or acetate in the environment. In the case of ethanol, it has to be metabolized, which requires functional alcohol dehydrogenase sodh-1. The resulting acetate is used in de novo fatty acid synthesis and some of the newly made fatty acids are then derivatized to glycerophosphoethanolamides and released into the surrounding medium. We examined several other Caenorhabditis species and found an apparent correlation between propensity of starved L1s to aggregate and density dependence of their survival in starvation. Aggregation locally concentrates worms and may help the larvae to survive long starvation. This work demonstrates how presence of ethanol or acetate, relatively abundant small molecules in the environment, induces collective behavior in C. elegans associated with different survival strategies.

Neuropeptides are essential for the regulation of appetite. Here we show that neuropeptides could regulate feeding in mutants that lack neurotransmission from the motor neurons that stimulate feeding muscles. We identified nlp-24 by an RNAi screen of 115 neuropeptide genes, testing whether they affected growth. NLP-24 peptides have a conserved YGGXX sequence, similar to mammalian opioid neuropeptides. In addition, morphine and naloxone respectively stimulated and inhibited feeding in starved worms, but not in worms lacking NPR-17, which encodes a protein with sequence similarity to opioid receptors. Opioid agonists activated heterologously expressed NPR-17, as did at least one NLP-24 peptide. Worms lacking the ASI neurons, which express npr-17, did not response to naloxone. Thus, we suggest that Caenorhabditis elegans has an endogenous opioid system that acts through NPR-17, and that opioids regulate feeding via ASI neurons. Together, these results suggest C. elegans may be the first genetically tractable invertebrate opioid model. DOI: http://dx.doi.org/10.7554/eLife.06683.001, eLife digest When and how much an animal eats is controlled by a complex web of signals that are produced by the animal's body and brain. Molecules called opioid neuropeptides are among these signals, and act to control eating in mammals by binding to receptors in the brain and body. These receptors can also bind to similar molecules called opiates (such as morphine); opiates are amongst the oldest drugs used by humans and have diverse effects ranging from pain relief to addiction. While the activities of opiates and opioid neuropeptides have been studied in mammals, relatively little is known about opioid signaling in simpler animals. The mechanisms behind many biological processes have been investigated using a worm called C. elegans as a model system because it has a simple body plan and its genes can be altered easily. The feeding behavior of C. elegans is no exception. This worm feeds by contracting and relaxing its pharyngeal muscle to move food into its gut. When the worms sense that food is available, this ‘pharyngeal pumping’ is regulated by one type of nerve cell. Slow pharyngeal pumping also continues in starved worms when food is not available, possibly to encourage them to eat new potential sources of food. However, this slow pumping does not require the same type of nerve cell. Cheong et al. hypothesized that the slow pumping in starved worms might depend on neuropeptide signaling instead, and have now tested this idea using engineered worms that made lower levels of a number of these molecules. The experiments uncovered a molecule called NLP-24 that promotes the slow pharyngeal pumping. This molecule is similar to opioid neuropeptides found in mammals. Worms that made less NLP-24 than normal grew more slowly; this suggests that they had problems feeding. Moreover, the levels of NLP-24 were found to increase in normal worms soon after they were deprived of food. Further experiments revealed the identity of the receptor for this molecule, which is also similar to mammalian opioid receptors. The discovery that opioid signaling is involved in C. elegans' feeding behavior may well, in future, also help to identify new molecular players involved in opioid signaling. Further studies might also help the search for ways to reduce the problematic side-effects that limit the usefulness of opiate drugs as medicines. DOI: http://dx.doi.org/10.7554/eLife.06683.002

The ascarosides, small-molecule signals derived from combinatorial assembly of primary metabolism-derived building blocks, play a central role in Caenorhabditis elegans biology and regulate many aspects of development and behavior in this model organism as well as in other nematodes. Using HPLCMS/ MS-based targeted metabolomics, we identified novel ascarosides incorporating a side chain derived from succinylation of the neurotransmitter octopamine. These compounds, named osas#2, osas#9, and osas#10, are produced predominantly by L1 larvae, where they serve as part of a dispersal signal, whereas these ascarosides are largely absent from the metabolomes of other life stages. Investigating the biogenesis of these octopamine- derived ascarosides, we found that succinylation represents a previously unrecognized pathway of biogenic amine metabolism. At physiological concentrations, the neurotransmitters serotonin, dopamine, and octopamine are converted to a large extent into the corresponding succinates, in addition to the previously described acetates. Chemically, bimodal deactivation of biogenic amines via acetylation and succinylation parallels posttranslational modification of proteins via acetylation and succinylation of L-lysine. Our results reveal a small-molecule connection between neurotransmitter signaling and interorganismal regulation of behavior and suggest that ascaroside biosynthesis is based in part on co-option of degradative biochemical pathways.