Endangered wild salmon infected by newly discovered viruses

Mordecai, Gideon J; Miller, Kristina M; Di Cicco, Emiliano; Schulze, Angela D; Kaukinen, Karia H; Ming, Tobi J; Li, Shaorong; Tabata, Amy; Teffer, Amy; Patterson, David A; Ferguson, Hugh W; Suttle, Curtis A

Samples were provided by the Fisheries and Oceans, Canada Aquaculture Management Division and Salmon Enhancement Program. Additional samples were collected by the Hakai Institute Juvenile Salmon Program. Hatchery samples are identified by fin clipping, and in this study, wild fish could also encompass unmarked hatchery fish. DNA is extracted for detection of DNA viruses, bacteria and parasites from the same tissues from which we extract RNA to target RNA viruses. Nucleic acid extractions on the audit samples (eight tissues-gill, atrium, ventricle, liver, pyloric caeca, spleen, head kidney and posterior kidney) were as previously described (Laurin et al., 2019) . For the wild Chinook and sockeye samples, homogenization using Tri-reagent was performed in a Mixer Mill (Qiagen, Maryland) on each tissue independently (five tissues-gill, liver, heart, head kidney and brain). Tri-reagent homogenates were organically separated using bromochloropropane, with the RNA-containing aqueous layer removed for RNA extraction and the lower DNA-containing organic layer separated from the organics using a TNES-Urea Buffer (Asahida et al., 1996) . For the DNA extractions, a pool of 250 ml (5 tissues contributing 50 ml each) from each of the tissue TNES aqueous layers was processed for DNA using the BioSprint 96 DNA Blood kit (Qiagen, Maryland) and the BioSprint 96 instrument (Qiagen, Maryland) both based on manufacturer's instructions. DNA was quantified using spectrophotometer readings performed on the Infinite M200Pro spectrophotometer (Tecan Group Ltd., Switzerland) and normalised to 62.5 ng/ml using the Freedom Evo (Tecan Group Ltd., Switzerland) liquid handling unit, based on manufacturer's instructions.