Use of amino acid isotope analysis to investigate capital versus income breeding strategies in migratory avian species

Income and capital breeding represent opposing ends of a continuum of reproductive strategies. Quantifying nutrient allocation to reproduction is challenging, but recent advances in compound-specific stable isotope analysis hold promise for tracing the source of individual compounds allocated to reproduction. Here, we describe a novel approach of using measured carbon (δ13C) and nitrogen (δ15N) isotope values of individual amino acids (AAs) in pectoral muscle of egg-laying females and egg yolk as a useful tool to quantify the reliance on income versus capital breeding in migrating species. We used white-fronted (Anser albifrons frontalis), lesser snow (A. caerulescens caerulescens), and black brant (Branta bernicla nigricans) geese breeding in tundra ecosystems of northern Alaska as model organisms. All three species relied on mixed capital-income breeding strategies, but models based on AA isotope data estimated higher proportional contributions of endogenous resources to yolk synthesis compared to results based on bulk tissue isotope analyses. Tracing income versus capital nutrient allocation in migratory species at the compound level is a major advance from the current 'elemental' perspective obtained from bulk tissue stable isotope analyses. Our framework is applicable to all taxonomic groups, as long as there is a sufficient spatial or temporal isotopic gradient between resources obtained during the breeding and non-breeding periods.Our study was based on tissues provided by a long-term study of geese breeding at the Colville River Delta in northern Alaska (70.4°N, 150.6°W; see Hupp et al. 2018; U. S. Fish and Wildlife Service migratory bird permit MB789758‐1). In spring and summer of 2012, pectoralis muscle was collected from early arriving female WFGO, SNGO, and BLBR. Unpaired egg tissues collected from co-occurring individuals of each species were obtained following clutch completion. We included muscle and yolk samples from 24 and 25 individuals, respectively. Our study was based on tissues provided by a long-term study of geese breeding at the Colville River Delta in northern Alaska (70.4°N, 150.6°W; see Hupp et al. 2018; U. S. Fish and Wildlife Service migratory bird permit MB789758‐1). In spring and summer 2012, pectoralis muscle was collected from early arriving female WFGO, SNGO, and BLBR. Unpaired egg tissues collected from co-occurring individuals of each species were obtained following clutch completion. We included muscle and yolk samples from 24 and 25 individuals, respectively. Muscle and egg yolk were freeze-dried and ground to a fine powder. Lipids are depleted in 13C and were extracted using a 2:1 chloroform: methanol solution (Folch, Lees, and Stanley, 1957) during three sequential 24-hour soaks (~72 hours total). Samples were then triple-rinsed with deionized water and oven dried at 60ºC for 48 hours. Amino Acid δ13C and δ15N Analysis An 8–12 mg aliquot of lipid-extracted muscle and yolk was hydrolyzed in 1 ml of 6N HCL at 110ºC for 20 hours and then dried at 110ºC under a gentle stream of N2. Amino acid films were then subjected to a two-step derivatization (Silfer et al. 1991) with 4:1 2-isopropanol: acetyl chloride (1 hour at 110°C) and 1:1 dichloromethane: trifluoroacetic anhydride (10 minutes at 110°C). During hydrolysis, asparagine and aspartate are converted to aspartic acid (Asx) and glutamate and glutamine are converted to glutamic acid (Glx). We measured the δ13C and δ15N values of seven essential AAs (phenylalanine (Phe), lysine (Lys), valine (Val), threonine (Thr), leucine (Leu), tyrosine (Tyr), and isoleucine (Ile)) and six non-essential AAs (alanine (Ala), glycine (Gly), proline (Pro), serine (Ser), Asx, and Glx. Amino acids were separated using a Thermo Scientific Trace 1310 gas chromatograph containing a BPx5 60m column (ID 0.32 mm, film thickness 1.0 μm), reduced to N2 at 1000°C in a Thermo Scientific IsoLink II combustion interface and Thermo Scientific Delta V Plus isotope ratio mass spectrometer (IRMS). Samples were analyzed as duplicate injections, bracketed by either a single or duplicate injection of an in-house reference standard comprising a mixture of powdered AAs (Sigma Aldrich) of known isotopic composition measured via EA-IRMS