Physiological condition infers habitat choice in juvenile sockeye salmon

Salinity preference experiments were conducted on sockeye salmon smolts at lake-exit, estuary-entry and ocean-entry. Smolt condition factor and energy density were positively correlated with salinity preference in the estuary and ocean outmigration stages. Our results suggest that smolt physiological condition influences migratory behaviour and habitat selection.


Supplementary Material
Physiological condition infers habitat choice in juvenile sockeye salmon

Measures of Stored Somatic Energy
To estimate the energetic status of individual smolts, lipids, water and ash were isolated and measured as proximate constituents following the Bligh and Dyer chloroform extraction method (Bligh & Dyer, 1959).Sample percent lipid was calculated by taking the ratio of isolated lipid extracted from the homogenized tissue (g) to the weight of the sample used (g) and multiplying it by the ratio of the volume of chloroform + lipid used to dilute the homogenate (ml) to the volume of chloroform + lipid extracted to isolate the lipid (ml).Sample percent water was calculated by first taking the ratio of the dehydrated sample weight (g) to the homogenized sample weight (g) to get the percent moisture loss, and then taking the difference in percent moisture loss from 100.Sample percent carbon was calculated by the ratio of fully combusted ash sample weight (g) to the homogenized sample weight (g).Percent protein was estimated as the percent difference from the sum of percent lipid, water and carbon (Trudel et al., 2005).
Energy data from four out of the 263 smolts analyzed were removed from further analysis due to high variance (CV>20%) among replicates for either lipid, carbon or water estimates.The energy density (ED) of each smolt was calculated from lipid (f) and protein (p) measurements using the following equation (Breck, 2008): where ED is expressed in units kJ/g wet weight, f is the fraction of lipid measured per smolt (g/g wet weight), Df is the energy density of lipids reported for coho salmon (O.kisutch, 39.54 kJ g -1 ) (Brett & Groves, 1979;Crossin et al., 2004;Higgs, 1979), p is the fraction of protein estimated per smolt (g g -1 wet weight), and Dp is the energy density of protein reported for coho salmon (23.64 kJ g -1 ) (Crossin et al., 2004;Higgs, 1979).
Triglycerides (TAG) are the major energy storage form of lipids in fish and have high ecological and physiological relevance as indicators of growth potential (Hakanson et al., 1994;Lochmann et al., 1995).A colorimetric method was used to determine percent triglycerides for each smolt (McGowan et al., 1983;Weber et al., 2003).A sample of lipid extract was taken from the chloroform-lipid extraction, the chloroform was evaporated from the sample, and then a colorimetric assay was used to determine the concentration of TAG in each sample.The percent ratio of TAG to lipid was determined by multiplying TAG concentration (g mL -1 ) of the sample by the total volume of chloroform used for lipid extraction and dividing by the mass of lipid extracted from the sample.The percent ratio of TAG to lipid represents the density of TAG within lipid stores in individual fish.Absorbance was read at 540 nm in a FLUOStar Omega © optic fluorescence spectrometer.The percent ratio of TAG to lipid represents the density of TAG within lipid stores in individual fish.All replicate estimates of TAG density were within 20% CV.Measurements of TAG density above 100% were removed from analysis (n = 2).

Osmoregulatory Preparedness: Gill NKA Activity
Gill NKA activity is a common metric to estimate saltwater preparedness in juvenile salmonids, where higher gill NKA activity indicates higher saltwater preparedness (Bassett et al., 2018;Elsner & Shrimpton, 2018;Stich et al., 2016).Gill NKA activity was measured following the enzymatic assay protocol from McCormick (1993).A FLUOStar Omega © optic fluorescence spectrometer was used to measure the rate of oxidation of NADH to NAD+, a proxy for ATP hydrolysis.Reagent quality was checked prior to each assay by running a standard curve of ADP consumption in the absence of NKA, in which the acceptable range was -0.17 to -0.2 mOD nmol - 1 .Each gill sample was measured in triplicate and was coupled with a sample that was inhibited by 0.5 mM ouabain.Assay mixtures of homogenized gill tissue samples and reagents were measured for change in absorbance at 340 nm and 25˚C.The linear slope of ATP hydrolysis was calculated over 10 minutes for each sample in Omega BMG Labtech Software © 5.10 R2.The slope of ATP hydrolysis was then corrected by dividing by the measured ADP extinction coefficient standard curve (x̄ = 20.754mOD nmol -1 NADH, SE = 0.140, n = 3).The enzyme activity of each well was then scaled by the corresponding concentration of protein from each tissue sample using the BCA Protein Assay Kit © (Product No. 23225 from Pierce P.O.Box 117, Rockford, Illinois 61105).Protein assays were measured in triplicate, with bovine serum albumin as the standard, read at 25˚C and 550 nm absorbance.The final metric of NKA activity was determined as the difference between uninhibited and ouabain-inhibited rates of ATP hydrolysis in units of micromoles ADP per milligrams of protein per hour.Eight gill samples out of 263 produced high variation among replicates (CV>20%) and were removed from analysis.

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TABLES
Suppl.Table1.Proportional log odds test for explanatory variables of ordinal logistic regression models followingBilder & Loughin (2014).Suppl.Table2.Model selection for predicting smolt salinity preference by physical and physiological condition across all outmigration stages (lake-exit, estuary-entry, and ocean-entry).Models were selected by the second-order bias correction of Akaike's Information Criterion (AICc).Residuals of Fulton's condition factor (resid.K), triglyceride density (resid.TAG), lipid density (resid.lipid),proteindensity(resid.protein),andenergeticdensity(resid.ED) were used as explanatory variables to account for daily decline during the 4 week non-feeding holding period.Fork length was included in all iterations of the full model to account for an observed size-bias.Suppl.Table3.Model selection for predicting smolt salinity preference by physical and physiological condition in the estuary-entry outmigration stage.Models were selected by the second-order bias correction of Akaike's Information Criterion (AICc).Suppl.Table4.Model selection for predicting smolt salinity preference by physical and physiological condition in the ocean-entry outmigration stage.Models were selected by the second-order bias correction of Akaike's Information Criterion (AICc).Suppl.Table5.Model selection for predicting smolt salinity preference by physical and physiological condition in the lake-exit outmigration stage.Models were selected by the secondorder bias correction of Akaike's Information Criterion (AICc).