Sample collection
At the time of sample collection, fish were removed one at a time from
the tank and quickly euthanized [28]. The caudal peduncle was
severed and blood collected in non-heparinized capillary tubes and
transferred to 0.5mL centrifuge tubes. Tubes were kept at room
temperature (RT) for 30 minutes to allow for blood clotting and then
centrifuged for 10 minutes at 1000g. Serum was removed to labelled 0.5mL
centrifuge tubes and stored at -80°C. Fish were then weighed and total
length recorded. Gill epithelium was scraped off with a slide coverslip,
placed in aluminum foil, snap-frozen in liquid nitrogen, and placed in
-80°C storage. Blood serum osmolality measurements were made using
undiluted serum when possible, and diluted with pure water (Optima LC/MS
Grade, Fisher Chemical) when necessary due to low volume, with
osmolality readings multiplied by the dilution ratio. Osmolality was
determined using a freezing-point micro-osmometer (Advanced Instruments
model 3300).