Following Dyckmans et al. (2005) we used 13C6H12O6 (99 at.% 13C6-glucose; Sigma–Aldrich, Vienna, Austria) and 15NH4NO3 (95 at.% 15N-ammonium nitrate; Chemotrade, Leipzig, Germany) in

order to dual-label earthworm species, with several modifications as follows ( Fig. 1): first, we looked at soil containing 15NH4NO3 that was incubated for seven days and soil that was not incubated. Secondly, we either applied 100 mg of 13C6H12O6 and 100 mg of 15NH4NO3 once or split it into four applications of Selleckchem GKT137831 25 mg 13C6H12O6 and 25 mg 15NH4NO3 over four days. Thirdly, we established treatments with ground oat flakes addition (as an additional food source) and those with no addition. These treatments were combined resulting in five experiments as shown in Fig. 1; one unlabelled control was set up for each experiment. Treatments with a seven day soil

incubation were prepared by filling 200 g sieved and sterilized soil into polypropylene bags, adding (i) 100 mg 15NH4NO3 and 400 mg unlabelled glucose dissolved in 4 ml selleck chemicals llc deionized water (treatment “once + incub”), or (ii) 100 mg 15NH4NO3 and 400 mg unlabelled glucose dissolved in 4 ml deionized water and 20 g ground oat flakes (particle size <1 mm; treatment “once + incub + oats”), or (iii) 25 mg 15NH4NO3 and 400 mg unlabelled glucose dissolved in 4 ml deionized water (treatment “staggered + incub”). These mixtures were incubated in the dark at 15 °C for seven days. To ensure aerobic conditions and a homogeneous 15N distribution, soil was stirred daily. Treatments that did not include soil incubation were prepared seven days later (Fig. 1). Here, soil was enriched with (iv) 100 mg 15NH4NO3 and 400 mg eltoprazine unlabelled glucose dissolved in 4 ml deionized water (treatment “once + no incub”) or (v) 25 mg 15NH4NO3 and 400 mg unlabelled glucose

dissolved in 4 ml deionized water (treatment “staggered + no incub”). Afterwards, the 15N labelled soil was transferred into polypropylene boxes (volume 500 ml) and 100 mg 13C-glucose dissolved in 2.5 ml deionized water were added to the treatments “once + incub”, “once + incub + oats” and “once no incub”. In treatments “staggered + incub” and “staggered no incub”, 25 mg 13C-glucose dissolved in 2.5 ml deionized water were added. On days 2, 3 and 4 of the labelling period (see next section), 25 mg 15NH4NO3, 400 mg unlabelled glucose and 25 mg 13C-glucose dissolved in 2.5 ml deionized water were added to treatment with staggered isotope labelling (Fig. 1). Overall, all treatments received the same total amount of ammonium nitrate (equals 183 mg N kg−1 soil), glucose (equals 200 mg C kg−1 soil), and water (6.5 ml) during the experiment. To label the earthworms, five individuals of L. terrestris or A. caliginosa, respectively, were held in polypropylene boxes (volume 500 ml) each containing 200 g soil treated and labelled as described above.