Females and eggs collection

Pre-breeding, pre-laying, and laying adult eider females (see below for definitions) were shot at foraging and resting sites located within 5 km from the nesting colon y (from June 11 to July 11; Total N = 15 in 2002, 15 in 2003, and 14 in 2004). Post-Iaying females (i.e., sitting on a full clutch for 24 to 48h) and a few (:::;2 per year) laying females were captured on their nest using nest traps (from June 18 to July 22; Total N = 15 in 2002, 17 in 2003, and 19 in 2004). Females were euthanized using halothane and their eggs were collected. and the. Birds were immediately dissected in the field and endogenous reserves were estimated by weighing wet breast muscle (pectoralis and supra-coracoid from the left side), abdominal fat, total leg mass (left side only, including aIl muscles that originate or insert in the femur or tibiotarsus bones) and body mass (following Jamieson et al., 2006).

Breeding stages of females were confirmed by inspecting follicles: females without developing or post-ovulatory foUicles were considered in the pre-breeding stage (which could also include non-breeding birds); the presence of only developing follicles was associated with the pre-laying stage; females with both developing and post-ovulatory foUicles were considered laying; while post-Iaying females had post-ovulatory foUicles only. Prior to nest-trapping, we visited the nest every one or two days during laying to mark the eggs and determine the laying sequence. First and last laid eggs were coUected at the same time as the female (only the first egg was coUected for laying females). Frequently, the number of post-ovulatory foUicles was higher than the number of eggs found in nests (33 cases out of 52). In those cases, we assumed that missing eggs were the first ones, given the low nest attendance of females at the beginning of laying and the high predation rate of first eggs (Andersson and Waldeck, 2006). In only two cases, the number of eggs found in nest was higher than the number of post-ovulatory foUicles, which indicated nest parasitism. Eggs (boiled), breast muscle, abdominal fat and liver samples were kept frozen at -20°C for subsequent laboratory analyses.

Prey collection

Prey SpeCleS consumed by pre-laying and laying eiders were identified by examination of feces and gizzards, as weIl as behavioral observations (see also Abraham and Ankney, 1986). These included the bivalves Hiatella arctica, Serripes spp., Acmea testudinalis, and amphipods (Gammarus spp.). We collected prey items by scuba-diving at 56 locations within East Bay (l-30m deep) in mid July 2007 (0 to 20 km from the nesting colony). Diving sites were associated with heavily used eider feeding areas identified by previous telemetry surveys (Authors, unpublished data). As prey species and female eiders were not collected the same year, and prey were not coUected during the eider pre-Iaying period, we investigated potential annual and seasonal variations in prey isotopic ratios by collecting amphipods throughout 2003, 2007 and 2008 breeding seasons. We found no evidence of such variation and are th us confident that our sampling design did not generate bias in our analyses (see Appendix 1). AlI prey samples were kept frozen at -20°C except for few small organisms that were preserved in 70% ethanol before laboratory analyses. Laboratory analyses Breast muscle and liver samples, as weIl as egg components (albumen and yolk) and prey species (without exoskeleton for bivalves) were oven dried (600C for 48 hours) and ground to powder.

Abdominal fat samples were soaked in 2: 1 chloroform:methanol for 24 hours and dissolved lipids were collected. Since endogenous and exogenous lipids and proteins can be allocated differently to egg formation, we separated our samples into lipid and lipid-free components. Lipids were extracted from prey samples, yolk, liver and breast muscles samples with successive rinses of the 2: 1chloroform:methanol. Lipid extracts were conserved for every tissue except for breast muscle. Solvent was evaporated completely using a fume hood and the remaining lipid residue stored frozen .. Carbonates were also extracted from lipid-free marine organism samples by treating them with drops of O.lN HCL without rinsing (Carabe! et al., 2006). Those samples were then oyen dried (600C for 24 hours) and powdered with a mortar and pestle. We loaded 1 ± 0.01 mg of each sample in a tin cup and combusted them in a Robo- Prep elemental analyzer (Europa Scientific, Crewe, UK). Resultant gases were delivered, using continuous-flow isotope ratio mass spectrometry (CFIRMS), to a Europa 20:20 mass spectrometer (Euro pa Scientific, Crewe, UK) for stable-carbon and nitrogen isotope ratio determination. Stable isotope ratios are expressed in delta (8) notation relative to the Pee Dee Belemnite or AIR standards for carbon and nitrogen, respectively (see Hobson, 1995). Based on replicate within-run analyses of a keratin (BWB II) and egg albumen lab standards, Aanalytical error was estimated to be ±O.3%0 for 815N and 0.1 %0 for 813C.

Isotope mixing model calculations

We calculated relati ve endogenous and exogenous protein contributions to egg components using the Bayesian-based MixSIR model (version 1.0.4; Moore and Semmens, 2008; and Semmens et al., 2009) based upon 813C and 815N values of prey items, egg components and eider tissues. Six protein sources were included in these models: breast muscles (endogenous source) and five prey types (exogenous sources), aU lipid-free. Protein models were generated for both lipid-free yolk and albumen samples. The relative contribution of endogenous and exogenous lipids to yolk-lipid was ca1culated only from S13C measurements, given the low nitrogen levels in adipose tissues. However, as endogenous lipid S13C values overlapped those of prey items, we could not adequately quantify the specifie contribution of aU potential sources to yolk-lipid. We therefore included only two sources in lipid models (abdominal fat and liver lipids) to estimate the relative contributions of endogenous and exogenous lipids, respectively, and we used IsoError linear mixing model (Phillips, 2001). Carbon isotope values of li ver lipids can be used as an integrative indicator of exogenous lipid ratios, as they provide short-terrn dietary information (Hobson and Clark, 1992). However, liver is also an organ where lipid catabolism occurs and where yolk precursors are synthesized.

Y olk lipids and abdominal fat S13C values were expected to differ if exogenous resources contribute to egg lipids (assuming no fractionation between abdominal fat and yolk lipids; see Gauthier et al., 2003). In such case, the greater the difference between S13C values in fat reserves and those in yolk lipids would indicate a stronger contribution of prey items consumed during egg formation (see also Discussion section). Through the process of isotopie discrimination, stable isotope values in egg components are expected to differ from those in contributing (endogenous or exogenous) nutrient pools. Discrimination factors between food and egg components have been determined in few bird species (Hobson, 1995; Ogden et al., 2004) .

Like Gauthier et al (2003), we used a carnivore mode} of Hobson (1995) to estimate discrimination between endogenous reserves and egg components. This approach was necessary since isotopie discrimination factors between endogenous reserves and egg components have not been determined experimentally (Gauthier et al. (2003). We consequently assumed that lipidfree sources discriminate 1) from lipid-free yolk by +3 .5 ± 0.35 %0 for 815N and 0 ± 0.5 %0 for 813C, and 2) from albumen by +3.1 ± 0.35 %0 for 815N and +0.9 ± 0.5 %0 for 813C. Given that yolk lipids are likely to be derived without discrimination either from the diet or from lipid stores (Hobson, 1995), and that lipids from every source have to pass through the liver before their transport to yolk precursors (Stevens, 2000), we assumed no 813C isotopie discrimination between lipids of sources and the yolk. A number of Bayesian-based isotope models are now available to calculate relative contributions of n+ 1 sources when measuring n isotopes. We thus compared our results obtained with the Bayesian MixSIR model to the outcomes generated by the non-Bayesian IsoSource (Phillips and Gregg, 2003) and the Bayesian SIAR model (Jackson et al., 2009 ; see Appendix 3).

Seasonal variations of source contributions

Distributions of possible endogenous protein contributions to the first egg of early and mid breeders were similar and very low (the most likely endogenous contribution in lipid-free yolk and albumen ranged from 0.9% to 4.3%; Figure 4). However, late breeders apparently mobilized a higher proportion of endogenous proteins to form each egg (most likely contribution of 23.3% and 9.6% for lipid-free yolk and albumen respectively; Figure 4). Given that Ô13C values of abdominal fat overlapped those of exogenous resources (see Figure 3), it was not possible to detect seasonal differences in endogenous lipid investment between early, mid and late breeders. Nevertheless, the difference between abdominal fat and yolk lipid Ô13C for a given female did not vary with relative lay date (after controlling for year: FI,31 = 0.82, P = 0.37), suggesting that endogenous lipid investment in eggs was similar among early and late nesters. The difference between albumen ô13C and ôl5N values of first- and last-Iaid eggs collected within a clutch did not vary with timing of breeding (P 2′: 0.66). However, within a clutch, ôl5N values tended to be systematically slightly higher in albumen of last-laid eggs compared to first-Iaid eggs (average of 0.58; P = 0.08), which may suggest a higher contribution of endogenous reserves at the end of the laying sequence (i.e., last egg closer to breast muscle ôl5N values: Figure 2). On the other hand, we found evidence for a seasonal variation in the differences of lipid-free yolk isotope values between last and first eggs (Figure 5; Û13C: Fl,45 = 3.12, P = 0.08; û15N: Fl,45 = 8.16, P = 0.007). This likely reflects seasonal changes in the accessibility of prey in the vicinity of the eider colon y and suggests that early and late nesters feed on different prey items and partly change their diet during clutch formation (see Discussion).