Bulk stable isotope and fatty acids analysis
We used bulk tissue analyses of δ13C fin clips and
potential prey to estimate diet composition of experimental individuals.
Freeze-dried and homogenized samples were analysed in WasserCluster
Lunz, Austria. Isotope ratios are reported relative to the international
Vienna PeeDee Belemnite carbonate standard. δ13C
values were mathematically corrected for lipid content following methods
as described by Post et al. (2007). The baseline 13C
value was further corrected to account for variability in basal
resources across the sampling sites (Olsson et al. 2009; Musseau
et al. 2020) using the following equation:
\begin{equation}
\delta^{13}C_{\text{corr}i}=\ \frac{\delta^{13}C_{i}-\delta^{13}C_{\text{inv}}}{\text{CR}_{\text{inv}}}\nonumber \\
\end{equation}where δ13Ccorri is the
corrected carbon isotopic ratio for individual i ,
δ13Ci is the carbon isotopic ratio for
individual i ,
δ13 Cinv is the average
carbon isotope ratio of macroinvertebrates and CRinv is
the carbon range (δ13Cmax −
δ13Cmin ) of macroinvertebrates.
δ13Ccorri is hereafter used as
an indicator of reliance on terrestrial prey (i.e., increasing
value suggests increasing reliance of an individual on terrestrial
prey).
Fatty acids were extracted and analysed from freeze-dried samples (3–10
mg dry mass) that were homogenized, sonicated and vortexed (4 times) in
a chloroform-methanol (2:1) mixture, following Böhm et al. (2014). Total
lipid mass ratios were determined via gravimetry. Fatty acids were
derivatized to obtain fatty acid methyl esters (FAME) using toluene and
sulfuric acid-methanol-solution (incubated at 16 h at 50 C). FAME were
identified using a gas chromatograph (Thermo Scientific TRACE GC Ultra)
equipped with a flame ionization detector (FID) and a Supelco SP-2560
column (100 m, 25 mm i.d., 0.2 µm film thickness). Quantification of
fatty acids were performed by comparison with a known concentration of
the internal standard using Excalibur 1.4 (Thermo Electron Corporation).