Excalibur 1.4

Muscle tissue was harvested and analysed from 15 diploid and 15 triploid Atlantic salmon. Total lipids from homogenized and freeze-dried muscle (25–35 mg) and dietary samples (electronic supplementary material, table S2) were analysed as by Heissenberger et al. [49 (link)]. In brief, samples were sonicated and vortexed in chloroform/methanol (2 : 1 by volume). Organic layers were removed and transferred into solvent-rinsed vials. For the gravimetric determination of total lipid contents (i.e. mg lipids/g dry weight (dw)), duplicate subsamples (100 µl) of the extracts were evaporated and weighed. Fatty acids were derivatized to obtain fatty acid methyl esters (FAMEs) using toluene and sulfuric acid–methanol solution (1% (v/v), 16 h at 50°C). In contrast to Heissenberger et al. [49 (link)], hexane without butylated hydroxytoluene (BHT) was used for each washing step after methylation to avoid BHT-related peak interference in chromatograms. FAMEs were identified by comparison with known standards (Supelco 37 FAME Mix) 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 FAs was performed by comparison with a known concentration of the internal standard using Excalibur 1.4™ (Thermo Electron Corporation).

For the determination of the FA composition, seston from epilimnion and metalimnion lake water (3 L triplicates) was filtered (<30 μm) and retained on precombusted and preweighted filters (GF/C Whatman™ filters; 1.2‐μm pore size, 47‐mm diameter), cryogenically frozen (−80 °C), and subsequently freeze‐dried for 48 hr. Lipids and their FA were extracted, derivatized, and analyzed as described in Heissenberger et al. (2010) using a gas chromatograph (Thermo Scientific TRACE GC Ultra) equipped with a flame ionization detector and separated using a Supelco™ SP‐2560 column (100 m, 25 mm i.d., 0.2‐μm film thickness). Excalibur 1.4™ (Thermo Electron Corporation) was used for calculation and, if necessary, manual resetting of the chromatograms. Fatty acid concentrations were calculated using calibration curves based on known standard concentrations (Supelco™ 37 FAME Mix). In this study, we focused on PUFA that are physiologically required by consumers (LIN = linoleic acid, ALA = α‐linolenic acid, EPA = eicosapentaenoic acid, DHA = docosahexaenoic acid) and hence considered a fundamental dietary source supplied by phytoplankton.