Sample collection
At the time of sample collection, fish were removed one at a time from
the tank and quickly euthanized first by manually applied blunt force
trauma and then by pithing and exsanguination (Leary et al., 2020).
Following pithing, the caudal peduncle was severed and blood was
collected in non-heparinized capillary tubes and transferred to a 0.5mL
centrifuge tube. 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 a labelled 0.5mL centrifuge tube and stored
at -80°C until further processing. Following blood collection, fish were
weighed and total length recorded. Gill arches were removed and soft
tissue separated from filament skeleton by scraping with a slide
coverslip and placed on a labelled piece of aluminum foil. Gill tissue
was snap-frozen in liquid nitrogen and placed in -80°C storage until
further processing. Blood serum osmolality measurements were made using
undiluted serum when possible, and diluted with pure water (Optima LC/MS
Grade, Fisher Chemical) when necessary, with osmolality readings
multiplied by the dilution ratio. Osmolality was determined using a
freezing-point micro-osmometer (Advanced Instruments model 3300).