Sample Collection and Simulated Infection
House sparrows were collected from four locations in their native range: Israel (n = 6), Norway (n = 6), Spain (n = 6), and Vietnam (n = 6), and three locations in their introduced range: Australia (n = 3), Canada (n = 6), Senegal (n = 6; Table 1). We classified each introduced site by their date of first introduction: Australia 1860s (Sheldon et al. 2018), Canada early 1900s (Grinnell 1919; Anderson 2006), and Senegal 1970s (Hanson et al. 2020a; Table 1). We captured adult house sparrows via mist netting from sunrise to 11.00 during the non-breeding seasons of 2020-2023. Upon capture, we took a 50 µl blood sample from the brachial vein of each bird and stored it in a cryovial with 300 µl of DNA/RNA shield (Zymo). Immediately after this, we injected each bird with 100 µl of 1 mg/ml-1 LPS (from E. coli 055:B5; Fisher L4005) in sterile saline subcutaneously over the breast muscle. Post injection, we housed birds individually in wire songbird cages (35.6 x 40.6 x 44.5) with food and water ad libitum . Although individually housed, the birds could hear and see one another. Eight hours post-injection, we took an additional 10 µl of blood from the brachial vein. All animal research procedures adhered to local animal research guidelines and were approved in advance by both the USF IACUC (IS00011653) and the relevant authorities in the country of origin. We extracted DNA samples using the DNeasy Kit (Qiagen, Valencia CA USA). Thus, we had paired 0- and 8-hour samples for each individual to screen changes in DNA methylation.