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).