2.2 Sample collection and preparation
Western sandpipers (n = 97) were captured with mistnets during mid-winter (23 January, 22 February, and 24 February) and at the onset of the breeding migration in spring (19-23 April) of 2012 (Table A1). Each bird was aged (after-hatch-year = adult, second-year = juvenile) and sexed based on plumage characteristics and morphometric measurements so that it could be assigned to one of four demographic groups (adult female, adult male, juvenile female, juvenile male; Page & Fearis, 1971; Pyle, 2008). Approximately 250 µl of blood was collected from the brachial vein in the wing and transferred to a heparinized microcentrifuge tube. Blood was centrifuged in the field for five minutes at 5000 rpm to separate fractions of plasma and red blood cells, which were transported on ice and stored at -20 °C until they were freeze-dried.
Benthic invertebrates (n = 83), biofilm (n = 18), and microphytobenthos (n = 18) were collected on the mudflat in January and April of 2012 (Table A1). Biofilm and microphytobenthos samples were collected at nine locations along a 450-m transect that was perpendicular to shore. At each location, one biofilm and one microphytobenthos sample were collected from the sediment surface by scraping the top 2 mm from 10-cm diameter circular plots into plastic bags. Invertebrates visible to the naked eye were removed. Samples were transported on ice. Microphytobenthos samples were refrigerated (4 °C) overnight, and the microphytobenthos was extracted the next day following the protocol of Kuwae et al. (2008, 2012). Biofilm was stored at -20 °C and was later thawed and dried for 3-4 days in a dark room. A 4-g sample of biofilm and microphytobenthos were each sent for stable isotope analysis. To collect benthic invertebrates, surface sediments (≤ 5 cm deep) were collected into plastic bags, transported on ice, and refrigerated (4 °C). Within two days, sediments were rinsed through a 500 µm sieve. Five classes of invertebrates (Bivalvia, Clitellata, Gastropoda, Malcostraca, and Polychaeta) were retained for stable isotope analysis based on their availability in our study area and previous studies of western sandpiper diet (Mathot et al., 2007; Sutherland, Shepherd, & Elner, 2000). Invertebrates were dried to constant mass, and individuals from each class were pooled to achieve a sample mass of approximately 4 g.