Lipid extraction and FAME preparation

Approximately 100 mg of sponge powder of each individual sponge was used per extraction. Sponge lipids were extracted with a modified Bligh and Dyer protocol [30], which was developed at NIOZ Yerseke [31–33]. We adjusted this protocol by replacing chloroform with dichloromethane (DCM), because of lower toxicity. The whole protocol is available online: dx.doi.org/10.17504/protocols.io.bhnpj5dn. In short, sponge tissue samples were extracted in a solvent mixture (15 mL methanol, 7.5 mL DCM and 6 mL phosphate (P)-buffer (pH 7–8)) on a roller table for at least 3 hours. Layer separation was achieved by adding 7.5 mL DCM and 7.5 mL P-buffer. The DCM layer was collected, and the remaining solution was washed a second time with DCM. The combined DCM fraction was evaporated to obtain the total lipid extract (TLE), which was subsequently weighed. An aliquot of the TLE was separated into different polarity classes over an activated silica column. The TLE was first eluted with 7 mL DCM (neutral lipids), followed by 7 mL acetone (glycolipids) and 15 mL methanol (phospholipids). The phospholipid (PL) fraction, which was used for further analysis, was converted into fatty acid methyl esters (FAMEs) using alkaline methylation (using sodium methoxide in methanol with known δ¹³C). Alkaline methylation is recommended for complex lipid mixtures [34]. After methylation, FAMEs were collected in hexane and concentrated to ~100 μL hexane for gas chromatography (GC) analysis.

For this study, two individual sponge samples per species were selected for detailed analysis. Aliquots of the FAME samples were used for double bond identification using dimethyl disulfide (DMDS) derivatization [35]. Samples reacted overnight at 40°C in 50 μL hexane, 50 μL DMDS and 10 μL 60 mg/mL I₂. The reaction was stopped by adding 200 μL hexane and 200 μL Na₂S₂O₃. The hexane layer was collected, and the aqueous phase was washed twice with hexane. The combined hexane fraction was dried, subsequently eluted over a small Na₂SO₄ column using in DCM: methanol (9:1) to remove any water and re-dissolved in hexane in a GC-vial for GC-analysis. Another aliquot of FAME sample was used for methyl-branching identification using catalytic hydrogenation with Adams catalyst (PtO₂) and hydrogen. Each FAME sample, dissolved in ~3 mL ethyl acetate with 10–30 mg PtO₂ and a drop of acetic acid, was bubbled with hydrogen gas for at least 1 h, after which the reaction vial was closed, and stirred overnight at room temperature. Subsequently, each sample was purified over a small column consisting of MgSO₄ (bottom) and Na₂CO₃ (top) using DCM and analyzed after re-dissolving it in ethyl acetate.