DOI: 10.5061/dryad.xsj3tx9mg
title: Use of amino acid isotope analysis to investigate capital versus income breeding strategies in migratory avian species

description: 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.
description: 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
 <sup>13</sup>C 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 δ<sup>13</sup>C and
 δ<sup>15</sup>N 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 δ<sup>13</sup>C and
 δ<sup>15</sup>N 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.