Ascarosides, modular secondary metabolites common to many nematode species, are implicated in many aspects of worm behavior and development as well as in interaction of worms with other organisms. Over 150 ascarosides have been described in C elegans, but only a quarter of them are routinely detected in wildtype cultures, while others are long chain shunt metabolites that accumulate in specific mutants. DAF-22, thiolase that catalyzes the final step in peroxisomal beta -oxidation, is required for biosynthesis of short chain ascarosides. Hence,
daf-22 has been traditionally used as a control in various bioassays where ascaroside activity may be suspected. We set out to map the effect of
daf-22 deletion on the entire worm exo-metabolome in an unbiased way. Two specific questions that we had were (1) are there still undiscovered ascarosides or ascaroside classes and if so, how many; (2) are ascarosides the only molecules affected in
daf-22 mutant and if not, what are the other
daf-22-dependent metabolites? We found that about 10% of detectable C elegans metabolites disappear in
daf-22. After excluding known ascarosides from the analysis, we identified about 80 of the remaining
daf-22-dependent molecules as new ascarosides, which range from small modifications of known structures to new large modular assemblies over 900 Da. Surprisingly, we also discovered many more non-ascaroside compounds that were absent in
daf-22 metabolome, which suggests that ascarosides are not the only compound class affected by peroxisomal beta -oxidation. Its disruption has profound direct and indirect effects on numerous metabolic pathways and leads to rewiring of a large portion of the C elegans metabolome. We repeated the untargeted metabolomic comparison of wildtype and
daf-22 in another worm species, P pacificus and again, in addition to numerous new ascarosides, found a large number of unexpected structures. Overall, we demonstrate that disruption of a single gene can cause global metabolic perturbations and by using an untargeted metabolomics pipeline we can harness this information to extend our knowledge of the worm chemical language.