DOI: 10.5061/dryad.dv41ns25p
title: Physiological condition and salinity preference in freshwater migrating juvenile sockeye salmon

description: The amount of time that juvenile salmon remain in an estuary varies among
 and within populations, with some individuals passing through their
 estuary in hours while others remain in the estuary for several months.
 Underlying differences in individual physiological conditions, such as
 body size, stored energy, and osmoregulatory function, could drive
 individual variation in the selection of estuary habitat. Here we
 investigated the role of variation in physiological conditions on the
 selection of estuarine and ocean habitat by sockeye salmon (Oncorhynchus
 nerka) smolts intercepted at the initiation of their 650 km downstream
 migration from Chilko Lake, Fraser River, British Columbia (B.C.).
 Behavioural salinity preference experiments were conducted on unfed smolts
 held in fresh water at three time intervals during their downstream
 migration period, representing the stage of migration at the lake exit,
 and the expected timing for estuary entry, and ocean entry (0, 1, and 3
 weeks after lake exit, respectively).
description: The focal population is sockeye salmon from Chilko Lake located
 650 river km north of the Fraser River estuary at an elevation of 1174 m,
 in the interior of British Columbia, Canada (Figure 1). Out-migrating
 yearling sockeye salmon smolts were collected from Chilko Lake between
 22:00 and 4:00 April 30 – May 1, 2019, during the peak outmigration period
 (Macdonald <em>et al.</em>, 2020). Smolts were collected by
 dip net at a smolt fence and transferred to a riverside transport tank
 with continual flow-through of aerated river water. In total, 263 smolts
 were collected for salinity preference experiments. All fish collection,
 holding, and experimentation protocols were reviewed and approved by the
 Animal Care Committee at Simon Fraser University (Protocol number
 1238B-17). Behavioural experiments were conducted on
 smolts at three time intervals during their downstream migration period,
 representing their lake-exit, estuary-entry, and ocean-entry outmigration
 stages (0, 1, and 3 weeks after lake exit, respectively; see Clark
 <em>et al.</em>, 2016; Stevenson <em>et al.
 </em>2019). Ocean-entry here is defined as near full-strength sea
 water that is beyond the freshwater influence of the Fraser River. To
 visualize the salinity gradient that migrating smolts are exposed to in
 Figure 1, monthly average sea surface (0.5m depth) salinity values were
 obtained from the SalishSeaCast NEMO model (Soontiens <em>et
 al</em>, 2016; Soontiens and Allen, 2017) for May
 2019<em>.</em>  A random subset of smolts
 (<em>n </em>= 45) was selected to undergo lakeside salinity
 preference experiments to measure salinity preference, energetic reserves,
 and physiology immediately following outmigration (hereafter, lake-exit).
 The remaining smolts (<em>n</em> = 218) were transported to
 the ALCAN research facility at Simon Fraser University, Burnaby, B.C., and
 housed in freshwater and natural photoperiod for the duration of the
 experimental outmigration period (4 weeks following lake-exit, 2 – 29 May
 2019). Smolts were randomly placed in one of four 80-gallon holding tanks,
 each with a maximum density of approximately 15 g
 m<sup>-3</sup>. Water quality in holding tanks was maintained
 at 8.4˚C (CI<sub>95</sub>[8.25, 8.48]), 98.6 % dissolved
 oxygen (CI<sub>95</sub>[98.04, 99.10]), and &lt;0.25 ppm
 ammonia. Water temperatures were within the range of downstream migration
 conditions (Macdonald <em>et al.</em>, 2020) and were low
 enough to prevent desmoltification (&lt;10 ˚C, Stefansson et al.,
 1998). To reflect observations of wild migrating
 smolts, as well as to maximize the contrast of energetic conditions
 throughout migration, wild-caught smolts were not fed during the 4-week
 holding period in fresh water. Smolts were also above the critical energy
 threshold that limits swim performance (Hvas <em>et al.</em>,
 2021; Wilson <em>et al.</em> 2021), suggesting the ability to
 actively search for different salinities was not impaired. The decision to
 not feed is supported by the observation that the majority of migrating
 sockeye smolts caught near the mouth of the Fraser River in Mission, BC
 have empty stomachs (Patterson, unpublished data, personal communication),
 indicating that feeding is limited during the freshwater migration stage.
 A variable proportion (20-40% from 2004 – 2009, 29% in 2014) of juvenile
 sockeye collected in marine trawls in the Strait of Georgia have empty
 stomachs (Beamish <em>et al.</em>, 2012; Neville <em>et
 al.</em>, 2016) and show gene expression patterns that suggest
 recent fasting (Houde <em>et al.</em>, 2019).
 Experimental Design We performed salinity
 preference experiments to infer habitat choice among migrating smolts. The
 design of the experimental preference tank was modified from previous
 studies to test the preference for three salinities simultaneously within
 each migratory stage (Baggerman, 1960; McInerney, 1963; Price &
 Schreck, 2003b). Three 20 L glass aquaria were placed side-by-side within
 a 120 L aquarium (Figure 2), dividing the larger aquarium into three
 chambers of equal size, while allowing for a 2-inch gap above the dividing
 walls for fish to cross between chambers. The outside and bottom of the
 large aquarium were darkened with black plastic to minimize disturbance to
 fish during the experiment. The walls between chambers were left
 transparent to allow smolts to view conspecifics through the dividing
 walls. Smolts were tested in groups of six to allow for more natural
 schooling behavior of sockeye salmon along the horizontal plane
 (Katzman <em>et al.</em>, 2010, Tierney <em>et
 al.</em>, 2009; Webster & Dill, 2007), regardless of
 individual salinity choice. To distinguish individual smolts during the
 preference test, smolts were anesthetized and marked with coloured
 elastomer (Northwest Marine Technology ©). Tagging occurred the day before
 a trial to allow for a minimum of 18 hours for recovery. The tank was
 illuminated uniformly from above using an LED light and surrounded by
 shade cloth to prevent visual disturbance. A video camera (GoPro Hero 3 ©)
 was positioned facing the long side of the aquarium to record fish
 position within all three chambers throughout the experiment. In addition,
 an experimenter recorded fish position (chamber and depth) and behaviour
 every ten minutes by viewing slots in the shade cloth.
 To start the salinity preference trial, all three chambers were
 filled with fresh, aerated water (0.0 – 0.2 ppm, 8.0 – 9.7 ˚C, 97.2 –
 101.6 % DO, <em>n</em><sub>trials</sub> = 42,
 Figure 2A). Six smolts were randomly selected from holding tanks and
 transferred to the experimental aquarium, ensuring equally dispersed,
 randomized placement throughout the three chambers. The smolts were
 allowed to acclimate to the aquarium and explore the three chambers in
 fresh water for 1 hour. After the 1-hour acclimation period, a horizontal
 salinity gradient was imposed in the experimental tank by displacing the
 water of each chamber with either fresh (&lt;2.0 ppt), brackish (14–16
 ppt), or salt water (30–32 ppt) (Figure 2B shows one of six possible
 orientations of the salinity gradient). The order of salinity of each
 chamber was randomized between each trial to control for any effect of
 chamber location or order on fish behaviour or movement. Each chamber was
 filled by separate water reservoirs that fed into the bottom of each
 chamber over approximately 5 minutes. Instant Ocean (Instant Ocean ©,
 Spectrum Brands 3001 Commerce St. Blacksburg, VA 24060-6671) was added to
 filtered, aerated water to make brackish and salt water, and each was
 added to the respective reservoirs. The freshwater reservoir was filled
 with filtered, aerated water. The higher-density salt and brackish water
 displaced the lower-density freshwater upwards, forming a halocline; the
 upper freshwater layer served as a freshwater bridge in which the fish
 could move freely between chambers. Displaced fresh water was
 simultaneously drained from the aquarium using a gravity filtration
 system. Preliminary trials showed that the halocline was stable for over 2
 hours. Salinity probes (Marine Salinity Waterproof Tester ©, HI98319,
 HANNA Instruments) were fixed within each chamber to monitor salinity and
 temperature throughout the duration of the experiments. Once the salinity
 gradient was imposed, fish position (chamber and depth) was recorded every
 ten minutes for one hour. If a fish was in the freshwater layer above the
 halocline of a chamber, it was recorded as being in fresh water. In total,
 44 experimental preference trials were undertaken. Eleven out of the 263
 smolts tested were removed from behavioural analysis due to disturbances
 during experimental trials (e.g. technical failure of salinity
 gradient). Physiological Sampling
 Following preference trials, smolts were euthanized in tricaine
 methanesulfonate at a concentration of 400 mg l<sup>−1</sup>
 (buffered with 800 mg l<sup>−1
 </sup>NaHCO<sub>3</sub>) until fish operculum beating
 ceased and the reflex response was absent (Neiffer & Stamper,
 2009). Immediately after euthanization, gill tissue samples were clipped
 from the right gill arch and frozen on liquid nitrogen in SEI buffer (250
 mM sucrose, 20 mM Na<sub>2</sub>EDTA, 50 mM Imidazole, pH 7.3)
 to preserve enzyme structure and function. Smolt carcasses were weighed
 and fork lengths measured before they were stored in whirlpacks and frozen
 on dry ice. Gill samples and carcasses were transported frozen on dry ice
 to the laboratory and stored at -80˚C until analysis.
 Gill NKA activity is a common metric to estimate saltwater
 preparedness in juvenile salmonids, where higher gill NKA activity
 indicates higher saltwater preparedness (McCormick, 2013). Gill NKA
 activity was measured following the enzymatic assay protocol from
 McCormick (1993; see Suppl. Mat.). Eight gill samples out of 263 produced
 high variation among replicates (CV &gt; 20%) and were removed from
 the analysis. 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; Trudel <em>et al.</em>, 2005;
 Wilson<em> et al.</em>, 2021; see Suppl. Mat.). The energy
 density (ED) of each smolt was calculated from lipid
 (<em>f</em>) and protein (<em>p</em>) measurements
 using the following equation (Breck, 2008): where
 <em>ED</em> is expressed in units kJ/g wet weight,
 <em>f</em> is the fraction of lipid measured per smolt (g/g
 wet weight), <em>D<sub>f</sub> </em>is the energy
 density of lipids reported for coho salmon (<em>O.
 kisutch</em>, 39.54 kJ g<sup>-1</sup>) (Brett &
 Groves, 1979; Crossin <em>et al.</em>, 2004; Higgs, 1979),
 <em>p </em>is the fraction of protein estimated per smolt (g
 g<sup>-1</sup> wet weight), and
 <em>D<sub>p</sub> </em>is the energy density of
 protein reported for coho<em> </em>salmon (23.64 kJ
 g<sup>-1</sup>) (Crossin <em>et al.</em>, 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 <em>et
 al.</em>, 1994; Lochmann <em>et al.</em>, 1995). A
 colorimetric method was used to determine percent triglycerides for each
 smolt (McGowan <em>et al.</em>, 1983; Weber <em>et
 al.</em>, 2003; see Suppl. Mat.). 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 (<em>n</em> =
 2).
description: # Physiological condition and salinity preference in freshwater migrating
 juvenile sockeye salmon 
 This dataset provides physiological and behavioural data on migrating
 Chilko sockeye yearling smolts. Fish tested in the river treatment group
 were tested lake-side 1 day after lake-exit. Fish tested in the estuary
 and ocean treatment group were transported to a holding facility and held
 in freshwater for 2-4 weeks. Salinity preference experiments were
 conducted over to infer habitat choice among migrating smolts. Fish with
 that spent the majority of the experiment time in a chamber were
 determined to prefer the salinity of that chamber. Immediately after the
 experiment, fish were euthanized and preserved for physiological analysis.
 Size metrics, energetic density, and gill NKA activity were measured for
 each fish and used to predict fish behaviour during migration. ##
 Description of the data and file structure | variable name | variable type
 | unit | level values if appl. | description | | :------------ |
 :------------ | :------------------------------- |
 :---------------------------- |
 :------------------------------------------------------------------------------------------------------------------- | | fish.id | character | | | unique identification number for individual juvenile salmon | | outmig.wk | factor | | | number of weeks since onset outmigration | | treatment | factor | | | treatment group associated with stage of migration | | treatment | | | 1.river | salinity preference experiments conducted 1 day following lake outmigration | | treatment | | | 2.estuary | salinity preference experiments conducted 1 week post lake outmigration | | treatment | | | 3.ocean | salinity preference experiments conducted 4 weeks post lake outmigration | | outmig.day | factor | | | number of days since onset of outmigration | | test.doy | factor | | | day of year that fish was tested for salinity preference | | active | factor | | | | | active | | | 0 | non-active fish; non-swim fish; did not leave initial chamber during acclimation or salinity preference experiment | | active | | | 1 | active-fish; fish explored experimental tank during the salinity preference experiment | | final.chamber | | | | the last chamber the fish was in at the end of the salinity preference experiment | | final.chamber | | | Freshwater | the fish was in the freshwater chamber at the end of the 1 hour salinity preference experiment | | final.chamber | | | Brackish  | the fish was in the brackish water chamber at the end of the 1 hour salinity preference experiment | | final.chamber | | | Saltwater | the fish was in the saltwater chamber at the end of the 1 hour salinity preference experiment | | pref | | | | the preferred salinity chamber | | pref | | | Freshwater | the fish spent the highest proportion of time in freshwater throughout the 1 hour salinity preference experiment | | pref | | | Brackish  | the fish spent the highest proportion of time in brackish water throughout the 1 hour salinity preference experiment | | pref | | | Saltwater | the fish spent the highest proportion of time in saltwater throughout the 1 hour salinity preference experiment | | pref.sd | numeric | | | the standard deviation of preferred chamber | | fl | numeric | mm | | fork length | | mass | numeric | g | | wet weight | | k | numeric | NA | | Fulton's condition factor | | lipid | numeric | % g lipid per g wet weight | whole body percent lipid | | | lipid.cv | numeric | NA | | coefficient of variation of percent lipid | | water | numeric | % g water per g wet weight | whole body percent water | | | water.cv | numeric | NA | | coefficient of variation of percent water | | carbon.cv | numeric | NA | | coefficient of variation of percent carbon | | prot | numeric | % g protein per g wet weight | whole body percent protein | | | tag | numeric | % g TAG per g lipid | percent triglyceride of lipid | | | tag.cv | numeric | NA | | coefficient of variation of percent triglyceride | | ed | numeric | kJ/g | | energetic density | | nka | numeric | umol ADP per mg protein per hour | gill NKA activity | | | nka.cv | numeric | NA | | coefficient of variation of NKA activity | | resid\_k | numeric | NA | | residuals of the linear regression predicting k by day of year | | resid\_mass | numeric | NA | | residuals of the linear regression predicting mass by day of year | | resid\_fl | numeric | NA | | residuals of the linear regression predicting fork length by day of year | | resid\_lipid | numeric | NA | | residuals of the linear regression predicting lipid by day of year | | resid\_prot | numeric | NA | | residuals of the linear regression predicting protein by day of year | | resid\_ed | numeric | NA | | residuals of the linear regression predicting energetic density by day of year | | resid\_edlog | numeric | NA | | residuals of the linear regression predicting the log of energetic density by day of year | | resid\_tag | numeric | NA | | residuals of the linear regression predicting TAG by day of year | | resid\_taglog | numeric | NA | | residuals of the linear regression predicting the log of TAG by day of year | | resid\_nka | numeric | NA | | residuals of the linear regression predicting gill NKA activity by day of year |