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

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 δ15NΣTrophic-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) (δ15NGlu-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.