Cholesterol is one of the hallmarks of animals. In vertebrates, the cholesterol synthesis pathway (CSP) is the primary source of cholesterol that has numerous structural and regulative roles [1]. Nevertheless, the few invertebrates tested for cholesterol synthesis show complete sterol auxotrophy [2-6], raising questions about how animals thrive without cholesterol synthesis and about the prevalence of sterol auxotrophy in animals. In the nematode Caenorhabditis elegans (C. elegans), sterols are the precursors of the steroid hormone dafachronic acid that coordinates development to adulthood [7,8]; thus, sterol-deprived C. elegans arrest at the diapause ''dauer'' larval stage [9]. Using this system, we have identified a pathway that converts plant and fungal sterols into cholesterol through the activity of enzymes with sequence similarity to specific human CSP enzymes. Based on this finding, we propose that two critical steps shaped the evolution of animal sterol auxotrophy: (1) the loss of the orthologs of the first three enzymes of the CSP and (2) the co-opting of other downstream enzymes of the CSP for the utilization of dietary sterols. Using this mechanistic signature, we studied the evolution of cholesterol auxotrophy across the animal kingdom. Complete sets of CSP enzymes in basal animals suggest that the loss of cholesterol synthesis occurred during animal evolution. A sterol auxothropy signature in the genomes of many invertebrates, including nematodes and most arthropods, suggests widespread cholesterol auxotrophy in animals. Thus, we propose that this co-opted pathway supports widespread cholesterol auxotrophy by interkingdom interactions between cholesterol-auxotrophic animals and sterol-producing fungi and plants.

Summary This protocol describes the culturing of the nematode Caenorhabditis elegans (C. elegans) in a sterol-defined experimental system and the subsequent quantitative analysis of C. elegans sterols through gas chromatography-mass spectrometry. Although studied primarily in mammals, sterols are essential biomolecules for most eukaryotes. C. elegans cannot synthesize sterols and thus relies on the uptake of dietary sterols. Therefore, C. elegans is a powerful system to study metabolism in sterol-defined conditions that are described in our protocol. For complete details on the use and execution of this protocol, please refer to Shamsuzzama et al. (2020) .