Advancing Amino Acid Isotope-Inferred Trophic Ecology in an Apex Predator: Insights From a Data-Rich Killer Whale...

Amino acid-specific nitrogen isotope analysis (δ¹⁵N-AA) is increasingly used to infer trophic ecology, yet remains poorly validated in marine predators. We analyzed bulk tissue δ¹⁵N (¹⁵N/¹⁴N) and δ¹³C (¹³C/¹²C), and δ¹⁵N-AA in 105 skin samples collected from individually identified Norwegian killer whales between 2017 and 2023. Forty-two individuals with best documented diets in 2010-2023, based on field observations and corroborated by mercury concentrations and bulk tissue δ¹⁵N values in skin, provided reference diet groups against which δ¹⁵N-AA-derived hypotheses were tested: Fish-only (100% fish prey; n = 15), Intermediate mixed diet (~10% mammalian prey in addition to fish; n = 14), and Top mixed diet (~50% mammals, 50% fish prey; n = 13). Univariate and multivariate approaches identified diet as the primary driver of δ¹⁵N variation across individual amino acids and multivariate isotopic space, whereas sex and life stage had little effect. However, substantial inter-individual variation remained unexplained and appeared to reflect metabolic processes. Metrics δ¹⁵N_{ΣTrophic-ΣSource} (δ¹⁵N difference between the sum of trophic amino acids and the sum of source amino acids) and isotopic difference between glutamic acid/glutamine (Glu) and phenylalanine (Phe) (δ¹⁵N_Glu-Phe) successfully identified Top mixed diet whales but lacked the sensitivity to identify Intermediate mixed diet individuals, whilst δ¹⁵N values of Threonine showed no relationship with trophic position. Proline (Pro) and Phe used as trophic-source pair provided the first plausible δ¹⁵N-AA-derived trophic position estimates for killer whales and performed better than bulk isotope-derived trophic position when a priori knowledge was not available. Multivariate clustering of δ¹⁵N-AA data complemented trophic position estimates by resolving finer-scale dietary variation occurring within trophic levels.