Thermally assisted hydrolysis and methylation (THM) analysis: A new perspective for biochemical investigation of fatty acid composition in enchytraeid tissues

• An effective Py–GC–MS based protocol for the analysis of fatty acids in Enchytraeus tissues is reported.
• Two species were evaluated: Enchytraeus crypticus and Enchytraeus sp. nov.
• Fatty acid content of the enchytraeids was diet dependent.
• The method chosen shows potential for use in chemosystematic and ecological studies.

Fatty acids are ubiquitous components in all organisms, and their applications in taxonomic and ecological studies of enchytraeids are scarce, and their small size (ca. 133 μg fresh weight) may be the main reason. Thermally assisted hydrolysis and methylation (THM) reaction with pyrolysis–gas chromatography (Py–GC) allow determination of fatty acid composition for small size samples. Thus, our objective was to test this methodology with soil enchytraeids cultured in laboratory. We used THM on-line Py–GC using trimethylsulfonium hydroxide (TMSH) reagent to investigate fatty acid composition in tissues of two enchytraeid species: Enchytraeus crypticus and Enchytraeus n. sp. cultured on soil and agar. A total of 12 fatty acids were consistently identified, ranging from C10 to C16. The major fatty acids were C14:0 (myristic acid) and an unsaturated C14:1. Fatty acid distribution was dependent on species and culturing method, suggesting the need of standardization of the culturing substrate and diet in chemosystematics studies. THM using TMSH provided insight on the fatty acid composition of Enchytraeus tissues and may be promising for application in taxonomic and ecological studies of this group of neglected soil animals.