Accurate diet estimation has long been a challenging issue for researchers investigating predators because of constraints associated with stomach content analyses. Fatty acid signature analysis offers an alternative avenue to study long-term diet trends in consumers. Despite the wealth of experiments involving fatty acids of fish and their diets, few have evaluated quantitative fatty acid signature analysis (QFASA) with fish consumers. To this end, we fed juvenile lake trout (Salvelinus namaycush), round goby (Neogobius melanostomus), and yellow perch (Perca flavescens) various invertebrate species and back-classified each predator to its respective prey using only fatty acids. Estimates were highly accurate when metabolism of diets was natively accounted for by using fatty acid profiles of predators fed known diets as the “prey library”. While highly accurate results were obtained, accounting for each predator–prey relationship limits the use of QFASA to predators that consume a limited number of species. We call for specific knowledge as to how fatty acid profiles reflect each predator–prey interaction before attempting to use fatty acids to quantify a consumer’s diet. Only after incorporating such data will QFASA provide an accurate view of individual’s diets when stomach content data are not available or are invalid.

Summary It is relatively well‐known that fatty‐acid profiles of consumers reflect their diets. However, with fish, controlled studies that trace fatty‐acid profiles of natural prey into consumers are lacking. We asked whether lake trout (Salmonidae: Salvelinus namaycush) fatty‐acid profiles reflect diets at 4, 8 or 14 weeks after feeding began. We also evaluated if calibration coefficients were similar for each diet, a key assumption of quantitative fatty‐acid signature analysis (QFASA). In this study, juvenile lake trout were fed commercially available frozen diets of chironomids (Chironomidae: Chironomus spp.), copepods (Cyclopoida spp.), or Mysis (Mysidae: Mysis relicta) over a 14‐week period. Accurate classification of lake trout into a priori diet groups was attained after 8 weeks of feeding. Calibration coefficients were significantly different among diet groups, especially for lake trout that were fed chironomids, suggesting that diet‐specific modifications to fatty acids occurred. Chironomid‐fed lake trout grew significantly larger than others despite consuming prey that lacked long‐chain essential fatty acids. Furthermore, chironomid‐fed lake trout provide evidence for the conversion of 18:3n‐3 into longer chain n‐3 fatty acids. Our results call for additional studies to better understand how fatty acids reflect dietary origins prior to employing QFASA on wild freshwater fishes. QFASA could provide accurate diet estimates for freshwater fishes with low‐diversity diet compositions, if calibration coefficients for each predator–prey relationship are incorporated.

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