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.