Stable isotope labelling of earthworms can help deciphering belowground–aboveground interactions involving earthworms, mycorrhizal fungi, plants and aphids

• Stable isotope tracing reveals a functional relationship between belowground detrivores, symbiotic mycorrhizal fungi, primary producers and herbivores.
• Mycorrhizal fungi alters plant uptake of 15N from earthworms.
• 15N but not 13C labelling of earthworms appears to be a promising tool to study multitrophic interactions.

Functional relationships between belowground detritivores and/or symbionts and aboveground primary producers and their herbivores are not well studied. In a factorial greenhouse experiment we studied interactions between earthworms (addition/no addition of Lumbricus terrestris; Clitellata: Lumbricidae) and arbuscular-mycorrhizal fungi (AMF; with/without inoculation of Glomus mosseae; Glomerales: Glomeraceae) on the leguminous herb Trifolium repens (Fabales: Fabaceae) and associated plant aphids (Aphis gossypii, A. craccivora; Hemiptera: Aphidoidea). In order to be able to trace organismic interactions, earthworms were dual-labelled with stable isotopes (15N-ammonium nitrate and 13C-glucose). We specifically wanted to investigate whether (i) isotopic signals can be traced from the labelled earthworms via surface castings, plant roots and leaves to plant aphids and (ii) these compartments differ in their incorporation of stable isotopes. Our results show that the tested organismic compartments differed significantly in their 15N isotope enrichments measured seven days after the introduction of earthworms. 15N isotope incorporation was highest in casts followed by earthworm tissue, roots and leaves, with lowest 15N signature in aphids. The 13C signal in roots, leaves and aphids was similar across all treatments and is for this reason not recommendable for tracing short-term interactions over multitrophic levels. AMF symbiosis affected stable isotope incorporation differently in different subsystems: the 15N isotope signature was higher below ground (in roots) but lower above ground (leaves and aphids) in AMF-inoculated mesocosms compared to AMF-free mesocosms (significant subsystem × AMF interaction). Aphid infestation was unaffected by AMF and/or earthworms. Generally, these results demonstrate that plants utilize nutrients excreted by earthworms and incorporate these nutrients into their roots, leaf tissue and phloem sap from where aphids suck. Hence, these results show that earthworms and plant aphids are functionally interlinked. Further, 15N-labelling earthworms may represent a promising tool to investigate nutrient uptake by plants and consequences for belowground-aboveground multitrophic interactions.