2.3. Sample collection

We collected 42 brant (15 adults, 27 juveniles) from 15 to 21 July 2014 on the YKD, and 41 brant (13 adults, 28 juveniles) and 58 white-fronted geese (16 adults, 42 juveniles) from 31 July to 10 August 2014 on the ACP. We collected geese in conjunction with ongoing mark-recapture studies led by the University of Nevada, Reno, USA and the U.S. Geological Survey. Broods and flightless molting adults were herded into large net pens, and a random sample was euthanized via cervical dislocation. We aged birds as adults or juveniles (i.e., young of the year) via plumage examination and weighed birds (±1 g) using a digital scale. Juveniles were approximately 30 d old at capture (YKD peak hatch date ∼ 17 June, ACP peak hatch date ∼ 7 July; T. Riecke, University of Nevada, Reno, USA and D. Ward and J. Hupp, U.S. Geological Survey, unpublished data). Upon death, each bird was eviscerated from the base of the head to the cloaca, and the heart, lungs, liver, gonads, and kidneys removed for parasite examination. We removed heads just below the lower mandible to examine eyes and nictitating membranes for helminths. Collected tissue was immediately flash frozen in the field using a mixture of 99% ethanol and frozen carbon dioxide (i.e., dry ice) and kept frozen until necropsy (Glass et al., 2002). All collections were authorized by the U.S. Geological Survey Alaska Science Center Institutional Animal Care and Use Committee (Permit # 2014-13) and with permits from the U.S. Fish and Wildlife Service (#MB789758) and the Alaska Department of Fish and Game (#14–092).

Before necropsy, samples were thawed at room temperature and divided into functional microhabitats (i.e., anatomical localities within a host) for examination. The following microhabitats were examined for helminths: head; eye surface, nictitating membrane, and tongue and nasal passages; viscera were divided into small intestine, duodenal loop, ceca, proventriculus esophagus, trachea, lungs, heart, and liver. All microhabitats were separated into 1000-ml beakers and flooded with tap water to prevent desiccation of tissues. For gastrointestinal microhabitats, a longitudinal cut exposed the lumen and its contents. The contents were scraped to dislodge attached helminths, and all contents were diluted with additional tap water. The contents of the beaker settled for 5–10 min, and excess (cleared) fluid was drained. The process was repeated several times (depending on the viscosity of luminal contents) until a concentration of helminths remained in clear fluid. Small aliquots of decant (containing helminths) were transferred to petri dishes and the fluid was examined under a dissecting microscope. Non-luminal microhabitats were macerated to dislodge parasites and rinsed into 500-ml beakers and treated as above. Solid residue remaining from each cleared microhabitat was inspected for helminths under a dissecting microscope.

Nematodes were fixed in glacial acetic acid and permanently stored in 7% glycerol. Trematodes and cestodes were fixed in AFA (70% ethanol, formalin, and acidic acid). A sample of 1–2 cestodes and trematodes per species per host were stained in Harris Hematoxylin and counterstained in eosin. Helminth diagnoses were verified through original taxonomic descriptions; Dr. M. Kinsella (HelmWest laboratory, Missoula, Montana, USA) aided in rare species identification. Voucher specimens including those suitable for molecular analyses are available from the Sam Houston State University Natural History Parasite Collection, Huntsville, Texas, USA (see Supplementary Material A).