7890 gc 5975c ms

To measure fatty acid composition, ~1000 young adult stage worms (containing 0–8 embryos) were washed from feeding plates with water on ice and washed once to remove residual bacteria. After settling on ice again, as much water as possible was removed (~90%). Fatty acids were converted to methyl esters for analysis as previously described^{75 (link)}. The worm suspensions were incubated for 1 h at 70 °C in 2 ml of 2.5% sulfuric acid in methanol. Following incubation, the reactions were stopped by adding 1 ml of water and then mixed thoroughly with 200 μl of hexane to extract the resulting fatty acid methyl esters. We measured relative amount of fatty acid methyl esters by injecting 2 μl of the hexane layer onto an Agilent 7890 GC/5975C MS in scanning ion mode.

A GC–MS system (7890 GC & 5975C MS, Agilent Technologies, Santa Clara, CA, USA), fitted with a DBWax (30 m × 0.25 mm i.d. × 0.25 μm, Agilent Technologies) fused silica capillary column, was applied to analyze the HS-SPME isolates basing on our previous work [15 (link)]. High-purity helium (99.995%, Qianxi Jingcheng Gas Co., Ltd., Beijing, China), with a constant flow rate of 1.0 mL/min, was used as the carrier gas. The oven temperature was programmed as follows: the initial temperature was set at 40 °C; temperature increased to 60 °C at 2 °C/min; temperature kept at 60 °C for 2 min; temperature rose from 60 °C to 140 °C at 4 °C/min; temperature maintained at 140 °C for 2 min; temperature increased to 250 °C at 10 °C/min; temperature held at 250 °C for 2 min. MS conditions were as follows: ion source, 230 °C; electron energy, 70 eV; scanning mode from m/z 30 to 500.

Fatty acid composition was analyzed by gas chromatography/mass spectrometry (GC/MS) using the fatty acid methyl ester (FAME) method [51 (link)]. For each sample, roughly 400 worms were collected in water, allowed to settle, and most of the water removed before freezing the worm pellet. 2.5% sulfuric acid in methanol was added to frozen pellets and then incubated at 70°C for one hour in a glass tube. FAMEs were extracted in hexane and separated using an Agilent 7890 GC/5975C MS in scanning ion mode equipped with a SP-1380 column. Relative amounts of fatty acid methyl esters are reported.