Bird capture, husbandry and tissue collection:
We captured adult house sparrows using mist nets from sunrise until
measured wing chord (to the nearest 1 mm), tarsus length (to the nearest
1 mm), and body mass (to the nearest 0.1 g). We also collected
approximately 50 µl of blood from the brachial vein of each bird, which
was stored in 300 µl of DNA/RNA Shield (Zymo R1100-50). Immediately
after, each bird was injected subcutaneously with 100 µl of 1 mg ml⁻¹
LPS (from E. coli 055; Fisher L4005) in sterile saline over the
breast muscle. Birds were housed individually in wire songbird cages
(approx. 35.6 x 40.6 x 44.5 cm) with food and water provided ad
libitum , while maintaining visual and vocal contact. Forty-eight hours
post-injection, between 0700 and 1000, birds were euthanized via
isoflurane overdose followed by rapid decapitation. We then collected
liver, spleen, and gut samples in 500 µl of DNA/RNA Shield, and all
samples were stored at -80°C until further analysis. We chose these
tissues because the goal of the larger project for which we collected
sparrows involves epigenetic regulation of immune gene expression; these
tissues are among the most active lymphoid tissues in the body. All
animal procedures complied with local ethical guidelines, approved by
the USF IACUC (IS00011653) and relevant authorities in the countries of
capture. Export and import of animal tissues followed all relevant U.S.
regulations, including USDA-APHIS permits.