Compound-specific stable isotope analysis of soil mesofauna using thermally assisted hydrolysis and methylation for ecological investigations

Stable isotope mass spectrometric approaches are proving to be valuable tools in unravelling biotic interactions in complex ecosystems, yielding information on trophic preferences and functional roles of individual species. Gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) provides considerable opportunities to assist in studies concerned with ecosystem processes mediated by soil invertebrates and microorganisms by determination of delta(13)C values of individual compounds, for example, lipids, amino acids etc. However, techniques conventionally adopted for "wet" chemical extractions and derivatizations necessary for compound-specific stable isotope determinations restrict the size of soil organism that can be studied and can limit investigations of individuals or even parts of individuals. We demonstrate here that individual soil mesofauna can be probed directly for their fatty acid stable isotope signatures by pyrolysis-GC/C/IRMS. A thermally assisted hydrolysis and methylation (THM) reaction is described for the determination of delta(13)C fatty acid values using trimethylsulfonium hydroxide (TMSH). Authentic fatty acids, acyl lipids, and individual Collembola (Folsomia candida) raised on C(3) and C(4) isotopically labeled yeast were analyzed initially by py-GC/MS with TMSH and then by py-GC/C/IRMS. A kinetic isotope effect (KIE) observed with the THM reaction prevents direct calculation of the fatty acid delta(13)C values by simple mass balance equations. However, the KIE is shown to be both reproducible and robust and can therefore be accounted for by the use of correction factors. The fatty acid methyl ester compositions of individual F. candida and their respective delta(13)C values were determined and shown to agree with those obtained by conventional "wet" chemical procedures applied to much larger numbers of Collembola, thus enhancing the scope to which stable isotopes can be applied to the study of invertebrates in complex food webs in any environment.