Stable isotope 15N and 13C labelling of different functional groups of earthworms and their casts: A tool for studying trophic links

Earthworms (Oligochaeta: Lumbricidae) have substantial effects on the structure and fertility of soils with consequences for the diversity of plant communities and associated ecosystem functions. However, we still lack a clear understanding of the functional role earthworms play in terrestrial ecosystems, partly because easy-to-use methods to quantify their activities are missing. In this study, we tested whether earthworms and their casts can be dual-labelled with 15N and 13C stable isotopes by cultivating them in soil substrate amended with 15N ammonium nitrate and 13C-glucose. Additionally, we also wanted to know whether (i) earthworms from different functional groups (soil-feeders vs. litter-feeders) and their casts would differ in their incorporation of stable isotopes, (ii) if enrichment levels are higher if the same amount of isotopes is applied in one dose or in staggered doses, and (iii) if isotopic enrichment in casts changes when they are stored in a conditioning cabinet or in a pot filled with soil placed in a greenhouse. Our findings show the feasibility of dual-labelling tissues and casts of both litter-feeding (Lumbricus terrestris) and soil-feeding (Aporrectodea caliginosa) earthworms using the same method. The advantage of this method is that earthworms and their casts can be labelled under realistic conditions by cultivating them for only four days in soil that received a one-time addition of commercially available stable isotopes instead of offering labelled plant material. In earthworms, the isotopic enrichment remained at a stable level for at least 21 days; labelled casts could be stored for at least 105 days without significantly decreasing their isotopic signals. This simple and efficient method opens new avenues for studying the role of these important ecosystem engineers in nutrient cycling and their functional relationships with other organisms.