Soil invertebrates move vertically to forage and avoid environmental stress. However, how invertebrates change their diet with depth is poorly understood, limiting our understanding of the trophic plasticity of soil invertebrates. Trophic consistency may be due to the existence of similar trophic niches at the microscale across soil layers (‘micro-scale feeding hypothesis’), but also due to feeding at certain depth despite moving between layers. To examine these alternatives, we conducted a microcosm experiment incubating springtails (Ceratophysella denticulata) in six separate forest soil layers (L, F/H, 0–3, 3–6, 6–9 and 9–12 cm depth of the mineral soil) and analyzed changes in Collembola stable isotope ratios (¹³C/¹²C, ¹⁵N/¹⁴N). As expected, ¹³C/¹²C and ¹⁵N/¹⁴N ratios in litter and soil organic matter increased with depth, whereas ¹³C/¹²C ratios of Collembola did not significantly differ across layers suggesting consistent basal resource use supporting the micro-scale feeding hypothesis. By contrast, ¹⁵N/¹⁴N ratios of Collembola increased with depth, following the trend of organic matter from litter to 0–3 cm soil, but not beyond. These results suggest that carbon and nitrogen nutrition of springtails is decoupled, and that the use of litter to calibrate ¹⁵N/¹⁴N values for estimating trophic positions of soil animals requires careful interpretation. Our results highlight the importance of soil depth as determinant of trophic positions of soil animals and point to principal differences in nitrogen resource acquisition between litter and soil in soil animal decomposers. Overall, the vertical structure of soils and microscale view of trophic interactions need closer attention to better understand niche differentiation and resource acquisition of soil animals.

Soil cores (ø 5 cm) were taken in a beech forest in Dassel on October 18, 2022. Soils were separated into six depths: L-layer (litter; depth ~ 3 cm), F/H-layer (3 cm, fragmented litter and humus material), 0–3 cm, 3–6 cm, 6–9 cm, and 9–12 cm soil depth (A_h-layer). The soil texture was silt loam (clay 21%, silt 53%, and sand 26%). Samples were stored at -20°C for two weeks, and then freeze-dried for defaunation. Water was added to adjust moisture content to the level at sampling. Soil animals were inoculated by using 1-6-day old juveniles of Ceratophysella denticulata. After incubating for four weeks in 20°C for 4 weeks, bulk stable isotopes of litter and soil were measured after drying at 60°C and grinding samples in a ball mill (Retsch, MM200). Collembolans were extracted from the litter/soil using heat, kept in 50% diethylene glycol, and then transferred into 70% ethanol for storage. As the available dry weight of collembolans was smaller than 100 µg, the bulk stable isotope ratios of ¹³C/¹²C and ¹⁵N/¹⁴N were measured using a modified setup adopted for small sample size (Langel and Dyckmans, 2014). Atmospheric nitrogen and Vienna PeeDee belemnite were used as primary standards. Acetanilide (C₈H₉NO, Merck, Darmstadt) was used as an internal standard. Natural variation in stable isotope ratios of carbon and nitrogen was expressed as δX (‰) = (R_sample – R_standard) / (R_standard) x 1000, with R the ratio between the heavy and light isotopes (¹³C/¹²C and ¹⁵N/¹⁴N).