Fatty Acids
Analysis of 41 dietary fatty acids was carried out using procedures described by Chavarie et al. (2016b) (Table 1). Muscle samples were freeze-dried and subsequently homogenized with a mortar and pestle. Lipids were extracted overnight from 1 g of the homogenate in a 2:1 chloroform-methanol solution containing 0.01% BHT (v/v/w) at −20ºC (Folch et al., 1957). After extraction, samples were filtered through Whatman Grade 1 Qualitative filter paper and the filter paper/sample was rinsed twice with 2 ml of the 2:1 chloroform:methanol. Sample extract was collected in a test tube and 7 ml of 0.88 N NaCl solution was added to encourage fatty acids to move into the organic (chloroform) layer. The aqueous layer was discarded after which the chloroform was dried with sodium sulfate prior to total lipid measurement. The extracted lipid was used to prepare fatty acid methyl esters (FAME) by transesterification with Hilditch reagent (0.5 N H2SO4 in methanol) (Morrison et al., 1964). Samples were heated for 1 h at 100 °C. Gas chromatographic (GC) analysis was performed on an Agilent Technologies 7890N GC equipped with a 30 m J&W DB-23 column (0.25 mm I.D; 0.15 μm film thickness). The GC was coupled to a Flame Ionization Detector operating at 350oC. Hydrogen was used as carrier gas flowing at 1.25 ml/min for 14 minutes, and increased to 2.5 ml/min for 5 min. The split/splitless injector was heated to 260 oC and run in splitless mode. The oven program was as follows: 60oC for 0.66 min, increasing by 22.82oC/min to 165 oC with a 1.97 min hold; increasing by 4.56 oC/min to 174oC and by 7.61 oC/min to 200oC with a six min hold. Peak areas were quantified using Agilent Technologies ChemStation software. Fatty acids standards were obtained from Supelco (37 component FAME mix) and Nuchek (54 component mix GLC-463).
All fatty acid values were converted to a mass percentage of the total array, and were named according the IUPAC nomenclature as X:Y n-z, where X is the number of carbon atoms in the fatty acids, Y is the number of methylene-interrupted double bonds in the chain, and n-z denotes the position of the last double bond relative to the methyl terminus (Ronconi et al., 2010). Fatty acids suggested by Iverson et al. (2004) as important dietary fatty acids, which transfer from prey to predator, were used in our analyses. Fatty acids profiles (% of fatty acids) were transformed using arcsin square-root function. Fatty acids groups were identified using a multivariate analysis R Package (Team, 2017), FactoMineR, using a hierarchical clustering analysis based on principal components (Husson et al., 2012). To reduce the number of variables used, A SIMPER (similarity percentage routine) was used to assess which fatty acids were primarily responsible for observed differences among groups (King et al., 1999). A principal components analysis (PCA) was performed on the fatty acid profiles with PC-ORD version 6 (McCune et al., 2011) among piscivorous groups to provide inferences about patterns of resource use as defined by Chavarie et al. (2016b). Two-way Permutational Multivariate Analysis of Variance (PERMANOVA), a non-parametric analog of Multivariate analysis of variance (MANOVA), was used to test for differences in fatty acid composition among the groups identified by FactoMineR and among arms (i.e., to investigate any spatial variations within the piscivorous ecotype). Two-way PERMANOVA were performed in PAST 3 (Hammer et al., 2001) using 9999 permutations. Pairwise post-hoc comparison (Bonferroni corrected) followed to test differences among groups defined by FactoMineR and among arms. Pairwise post-hoc comparison (Bonferroni corrected) also followed to test differences among arms (i.e., spatial variation). Finally, the fatty acid groups determined by FactoMineR were tested for differences in depth of capture using one-way analysis of similarities (ANOSIM) with 9999 permutations using PAST 3.