Sterol profiles (Michellod et al. 2023)

We analyzed the sterol content of seven species of gutless oligochaetes, belonging to the Olavius and Inanidrilus genera, as well as three species of gut-bearing annelids: <em>Capitella teleta</em>, <em>Tubifex tubifex</em> and <em>Eisenia andrei.</em> <strong>Metabolite extraction. </strong>Metabolites were extracted from the worms using the following method for metabolite profiling: Tissues from MeOH fixed worms were transferred to 2 mL screwcap tubes containing a mix of silica beads (Sigmund Linder). The residual methanol was added to the screw cap tube. The tubes were spiked with 100 µL 5α-cholestane (1 mM) and 40 µL ribitol (0.2 mg mL-1). 0.5 mL pre-cooled extraction solution (ACN:MeOH:water (v:v:v) 2:2:1) was added to each tube. Tissues were disrupted by bead beating for 2 cycles of 40 sec (4 m sec-1). The tissues were pelleted by centrifugation (9,600 x g, 2 min), and the supernatants transferred to new tubes. The pellets were extracted one more time with 1.5 mL of extraction solution. The supernatants were combined and evaporated to dryness in a vacuum concentrator without heating (approximately 1.5 h). The obtained aliquots were stored at -20°C until metabolite derivatization. <strong>Derivatization. </strong>Metabolite derivatization was performed by adding 80 µL methoxyamine hydrochloride (MeOX) dissolved in pyridine (20 mg mL-1) to the dried pellet and incubating for 90 min at 37°C using a thermomixer (BioShake iQ, Analytik Jena) under constant rotation at 1350 rpm. Following the addition of 100 µL N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA), each extract was vortexed, and incubated for another 30 min at 37°C on a thermomixer under constant rotation at 1350 rpm. After a short centrifugation, 100 µL of the supernatant was transferred to a GC-MS vial (Insert G27, sping S27 and Mikor-KH-Vial G1; Chromatographic Service) for GC-MS data acquisition. <strong>Data acquisition.</strong> The analysis of all metabolomic samples was conducted on a 7890B GC system (Agilent Technologies) coupled to a 5977A single quadrupole mass selective detector (Agilent Technologies). The gas chromatograph was equipped with a DB-5 ms column (30 m × 0.25 mm, film thickness 0.25 µm; including 10 m DuraGuard column, Agilent Technologies) and a GC inlet liner (ultra inert, splitless, single taper, glass wool, Agilent). Helium was used as gas carrier at a constant flow (0.8 mL min-1). An Agilent 7693 autosampler injected 1 µL of derivatized sample in splitless mode. The injector temperature was set at 290°C. The temperature program started at 60°C for 2 min, then increased to 300°C at 10°C min-1, and held at 325°C for 7 min. Mass spectra were acquired in electron ionization mode at 70 eV across the mass range of 50–600 <em>m/z</em> and a scan rate of 2 scans sec-1. The retention time was locked using a standard mixture of fatty acid methyl esters (Sigma-Aldrich). For more details please refer to Michellod et al. 2023.