Journal of Fish Biology (2014) doi:10.1111/jfb.12475, available online at wileyonlinelibrary.com

The effects of sustained aerobic swimming on osmoregulatory pathways in Atlantic salmon Salmo salar smolts A. J. Esbaugh*†, T. Kristensen‡, H. Takle§ and M. Grosell‖ *University of Texas at Austin, Marine Science Institute, Austin, TX 78373, U.S.A., ‡Norwegian Institute of Water Research, P. O. Box 6215, N-7486 Trondheim, Norway, §Nofima, P. O. Box 5010, 1430 As, Norway and ‖Rosenstiel School of Marine Science, University of Miami, Miami, FL 33149, U.S.A. (Received 12 December 2013, Accepted 18 June 2014) Atlantic salmon Salmo salar smolts were exposed to one of the four different aerobic exercise regimens for 10 weeks followed by a 1 week final smoltification period in fresh water and a subsequent eight-day seawater transfer period. Samples of gill and intestinal tissue were taken at each time point and gene expression was used to assess the effects of exercise training on both branchial and intestinal osmoregulatory pathways. Real-time polymerase chain reaction (PCR) analysis revealed that exercise training up-regulated the expression of seawater relevant genes in the gills of S. salar smolts, including Na+ , K+ ATPase (nka) subunit 𝛼1b, the Na+ , K+ , 2 Cl− co-transporter (nkcc1) and cftr channel. These findings suggest that aerobic exercise stimulates expression of seawater ion transport pathways that may act to shift the seawater transfer window for S. salar smolts. Aerobic exercise also appeared to stimulate freshwater ion uptake mechanisms probably associated with an osmorespiratory compromise related to increased exercise. No differences were observed in plasma Na+ and Cl− concentrations as a consequence of exercise treatment, but plasma Na+ was lower during the final smoltification period in all treatments. No effects of exercise were observed for intestinal nkcc2, nor the Mg2+ transporters slc41a2 and transient receptor protein M7 (trpm7); however, expression of both Mg2+ transporters was affected by salinity transfer suggesting a dynamic role in Mg2+ homeostasis in fishes. © 2014 The Fisheries Society of the British Isles

Key words: anadromous; intestine; ion balance; ionocytes; magnesium homeostasis; water balance.

INTRODUCTION A primary long-term goal for the Atlantic salmon Salmo salar L. 1758 farming industry is to improve the strength and robustness of produced fish. To this end, a number of studies have found sustained aerobic swimming, or exercise training, to be beneficial during fish production, where it can increase growth and reduce stress related to aggressive behaviour and dominance hierarchies (Boesgaard et al., 1993; Jorgensen & Jobling, 1993; Davison, 1997; Castro et al., 2011). One of the most crucial rearing stages, however, is when fish are transferred from fresh water to seawater; a stage that represents a complete reversal in osmoregulatory strategy. At present, little is known †Author to whom correspondence should be addressed. Tel.: +1 361 749 6835; email: a.esbaugh@ austin.utexas.edu

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about the effects of sustained aerobic swimming on the osmoregulatory transition in seawater transfer. The physiological changes in S. salar that allow for migration from freshwater habitats to the marine environment are collectively termed smoltification, or parr–smolt transformation. This endocrine-driven process is associated with a suite of branchial modifications that are necessary to maintain osmotic balance (McCormick et al., 1989, 2009; Tipsmark & Madsen, 2001; Tipsmark et al., 2002, 2008a; Bystriansky et al., 2006; Nilsen et al., 2007; Madsen et al., 2009; Bjornsson et al., 2011; Bystriansky & Schulte, 2011). Freshwater fishes actively take up Na+ and Cl− through specialized ionocytes in the gill. In contrast, Na+ and Cl− are actively excreted in the marine fish gill through the combined action of Na+ , K+ ATPase (NKA), the secretory Na+ , K+ , 2 Cl− co-transporter 1 (nkcc1) and the cftr chloride channel. This is also associated with increased paracellular gill permeability, which allows for paracellular Na+ excretion along the established electrochemical gradient (Marshall, 2002; Evans et al., 2005; Marshall & Grosell, 2006). During smoltification, there is a progressive increase in branchial NKA activity (Tipsmark & Madsen, 2001; Tipsmark et al., 2002) and a change in the dynamics of NKA subunit isoform expression (Bystriansky et al., 2006; Madsen et al., 2009; McCormick et al., 2013). In particular, the freshwater nka𝛼1a subunit is down-regulated while the seawater nka𝛼1b subunit is up-regulated. Both branchial nkcc1 and cftr are also up-regulated during smoltification (Tipsmark et al., 2002; Nilsen et al., 2007). Interestingly, all of these changes are initiated while in fresh water as a preparatory mechanism for seawater transfer. To counteract osmotic water loss to the marine environment, seawater fishes have a highly adapted gastro-intestinal tract that can desalinate imbibed seawater to allow for solute and osmotically driven water absorption (Grosell, 2006, 2011; Marshall & Grosell, 2006; Grosell et al., 2011). The Na+ and Cl− ions absorbed in the intestine are subsequently excreted at the gill resulting in overall net water uptake. Intestinal ion absorption occurs primarily through nkcc2, Na+ , Cl− co-transport (NCC) and various anion exchange pathways (Grosell et al., 2011). Little is known about the response of these transporters during S. salar smoltification; however, seawater transfer has been shown to increase HCO3 − secretion pathways, NKA expression and activity, as well as nkcc2 expression in the intestine of the anadromous rainbow trout Oncorhynchus mykiss (Walbaum 1792) (Grosell et al., 2007, 2011; Gilmour et al., 2012). While increased transport of Na+ and Cl− is crucial for intestinal water uptake, the reduced intestinal transport of Mg2+ is also important for marine water balance. In a freshwater environment, Mg2+ homeostasis is governed largely by renal mechanisms (Marshall, 2002; Marshall & Grosell, 2006; Grosell et al., 2011); however, in a marine environment, such mechanisms would compromise osmotic balance by sacrificing water for urine production. To reduce water loss associated with Mg2+ homeostasis, marine fish intestines are practically impermeable to the Mg2+ in imbibed seawater despite large inward gradients. In fact, Mg2+ concentrations in the distal portions of the intestine can exceed the plasma by over 200-fold (Grosell et al., 2011), and are responsible for ultimately limiting water absorption during hypersalinity exposure (Genz et al., 2011a). Very little is currently known about the mechanisms of Mg2+ transport or exclusion by the intestine, but it appears likely that impermeability in seawater stems from a combination of limited Mg2+ influx and high efflux. In mammals,

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12475

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Mg2+ influx occurs largely through the transient receptor protein metastatin (TRPM) channels 6 and 7 (Holzer, 2011a, b; Yogi et al., 2011; Paravicini et al., 2012), as well as through the slc41a1 and slc41a2 channels (Moomaw & Maguire, 2008). Efflux is thought to occur through an electroneutral Na+ /Mg2+ exchange protein (Handy et al., 1996). On this background, the primary objective of this study was to examine the effects of aerobic endurance training on osmoregulatory pathways during smoltification in S. salar. More specifically, this study examined the effects of different exercise training regimens on the transcription profiles of genes integral to the osmoregulatory pathway related to seawater transfer at the gill (nka𝛼1a, nka𝛼1b, cftr and nkcc1) and anterior intestine (nkcc2). A secondary objective was to examine the role that magnesium channels may play in seawater transfer, specifically trpm7 and slc41a2. It is hypothesized that exercise training will further induce expression of the seawater phenotype during smoltification and that Mg2+ transporters will undergo transcriptional regulation in response to salinity transfer.

MATERIALS AND METHODS E X P E R I M E N TA L D E S I G N Juvenile S. salar belonging to the Salmobreed strain were produced and reared at the Nofima Sunndalsøra Research Station (Sunndalsøra, Norway). All experimental facilities and procedures were approved by Norwegian Animal Research Authority (NARA). A total of 1355 fish were individually tagged and measured [mean ± s.d. = 40⋅7 ± 0⋅2 g and fork lengths (LF ) = 15⋅0 ± 0⋅3 cm]. The fish were randomly allocated among 16 cylindro-conical experimental tanks (500 l; 77–86 fish per tank) and allowed to acclimate for 1 week. The centre of each tank was fitted with a 31⋅5 cm diameter plastic pipe, which formed a swim channel. A frequency-controlled pump (www.hanning-hew.de) directed the water current and a wire mesh fence, attached between the pipe and the edge of the tank, prevented the fish from drifting backwards. The water speeds were calibrated using the average speed measured at 12 points in the tank (four horizontal locations and three depths at each location; www.hoentzsch.com, Hontzsch). Four different sustained exercise-training regimes were tested; the control regime in quadruplicate tanks and the other regimes in triplicate tanks (Fig. 1). Three of the training regimes were continuous velocity: the control low intensity (5⋅7 cm s−1 ), medium intensity (11⋅5 cm s−1 ) and high intensity (23 cm s−1 ) regimes. These mean speeds equate to 0⋅38, 0⋅77 and 1⋅53 BL s−1 , respectively, based on the mean starting body size. The fourth regime consisted of a 16 h low velocity (0⋅32 BL s−1 ) interval followed by an 8 h high velocity interval (1⋅31 BL s−1 ) daily. At no point were fish observed to be obviously resting within an exercise treatment group. The 10 weeks of training were followed by a one-week recovery at control speed prior to transferring the fish to seawater for 8 days after which the experiment was terminated. To stimulate smoltification, fish were exposed to a regime of 12L:12D for the first 6 weeks of the experiment, followed by continuous light for the remaining 5 weeks. Water temperature was measured daily (10⋅5∘ C, range ±0⋅8∘ C) while oxygen saturation was measured weekly and maintained at over 85% with oxygen supplementation. Dead fish were removed with daily inspections and weighed. For all sampling, fish were swiftly netted from the experimental tanks and killed by a sharp blow to the head. Blood for plasma ion analysis was collected from the caudal vein via caudal puncture using 1 ml heparinized syringes. The second gill arch and anterior intestine were excised and immediately frozen in liquid nitrogen and stored and −80∘ C until analysis.

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24 h light

12 h:12 h 1·5 BL s–1 1·3 BL s–1

24 h

0·8 BL s–1

SW 0·4 BL s–1

6 weeks

4 weeks

Exercise training

7 days

8 days

No training

Fig. 1. Diagrammatic representation of the experimental design. Salmo salar were exposed to one of the four different aerobic swim training regimens over a 10 week period (denoted by different line types) followed by a 1 week final smoltification period at low speed (0⋅4 BL s−1 ). After recovery, fish were transferred to seawater (SW) for 8 days prior to sampling. To mimic natural conditions, fish were exposed to a 12L:12D cycle for the first 6 weeks of training, followed by 24 h light exposure for the duration of the experiment.

G E N E E X P R E S S I O N A N A LY S I S Total RNA was extracted from S. salar gill and intestinal tissues using the RNeasy mini kit (Qiagen; www.qiagen.com), according to manufacturer’s guidelines. The extraction included DNAse treatment with the RNAse-free DNAse kit (Qiagen) to remove potential genomic DNA contamination. Tissue lysates was obtained by homogenizing one gill arch or gut section in 500 μl of lysis buffer for 3 × 10 s at 2300g (room temperature) using a Precellys orbital shaker bead mill with Precellys CK14 beads (www.precellys.com). Cell debris was removed by centrifugation at 8000 g for 1 min, before transferring the supernatant to RNeasy columns. Total RNA was quantified using a NanoDrop 1000 (www.thermofisher.com) at a wavelength of 260 nm, and all samples had 260/280 > 1⋅9 and 260/230 > 1⋅5. RNA integrity was assessed using a Bioanalyser 2100 (www.home.agilent.com) for a sub-set of randomly selected samples. Subsequent complementary (c)DNA synthesis was performed using qScript cDNA synthesis master mix (www.quantabio.com) and a 50:50 combination of random hexamers and oligo dT primers. Real-time polymerase chain reaction (rt-PCR) was performed on an Mx3000P rt-PCR system (www.home.agilent.com) using the Brilliant SYBR green master mix kit (www.home.agilent.com; 12⋅5 μl reactions). The gene-specific primers are listed in Table I. Primer sets for nka𝛼1a, nka𝛼1b, nkcc1, putative nkcc2 and slc41a2 were designed against known S. salar sequences, while primer sets from the closely related O. mykiss were used for trpm7 and 18S. Note that NKA primer sets were designed to prevent possible cross amplification. In all cases, standard rt-PCR and gel electrophoresis were used to verify the presence of only a single amplicon. Both the thermocycler set-up and reaction composition were performed according to the manufacturer’s guidelines. The annealing temperature for all reactions was 58∘ C. Disassociation curves were used to assess the primer specificity and the presence of primer dimers within each reaction. The PCR efficiency of each primer pair was calculated using a cDNA standard curve. All PCR efficiencies were above 90% with an r2 ≥ 0⋅98. Relative mRNA expression was calculated using the delta–delta cycle-threshold (ct) method using 18s as an internal control and the low exercise 10 week training time point as the basis for relative comparisons (Pfaffl, 2001). A N A LY T I C A L M E A S U R E M E N T S A portable i-STAT clinical analyser and EC8+ cartridge (www.abbottpointofcare.com) were used to determine plasma Na+ and Cl− . Approximately 0⋅1 ml of blood was used for analysis. Measurement of branchial NKA activity was conducted using a standard kinetic

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12475

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Table I. Real-time polymerase chain reaction (PCR) primer pairs used for gene expression analysis Gene

Accession number

Orientation

Sequence

nkcc1

AJ417890

nkcc2

AGKD01008560.1*

trpm7

EZ770859

F R F R F R F R F R F R F R F R

GAT GAT CTG CGG CCA TGT TC TCT GGT CAT TGG ACA GCT CTT TG ACT GCA GGA GAA CTC CCG AGC AGG ACG CTC TTG TGG TTG CCT TAG GTG AGC CAG TAA CAG TGT A ATA CTC GGA CCA CGT ACA CAG CAG CCA TCT TCG GAC TGC AC GTG CTG ATC TTG TCC AGG ACC AAG AGG AGA GTC AGA GGC CC ATT GAC CGC GGT GGA TAG TC GGC CGG CGA GTC CAA TCA T GAG CAG CTG TCC AGG ATC CT CCC ATG GAT TTG CTG GGT ATG C CTG TGT GGC AGG TGA ACT CCA C CTC AAT CTC GTG TGG CTG A TCT CGA TTC TGT GGG TGG T

cftr

AF155237.1

slc41a2

NM_001173587

nka𝛼1a

AY692142

nka𝛼1b

CK879688

18S

FJ710873

*Salmo salar genome database.

assay (McCormick, 1993) and performed by a commercial laboratory (www.pharmaqanalytiq.com).

S TAT I S T I C A L A N A LY S I S All gene expression and ion concentration data were analysed using a two-way analysis of variance (ANOVA) with exercise training and sampling time point as the two factors. In cases where no significant interactions were observed between the two variables, the four exercise treatments were combined to examine the effects of salinity transfer alone. Sample sizes for gene expression analysis ranged from six to 12 per treatment, and nine to 15 for plasma ion analysis. A sample size of 15 was used for NKA analysis. All statistical tests were performed using SigmaPlot version 12.5 (www.sigmaplot.com) at a level of significance of P ≤ 0⋅05.

RESULTS Branchial and intestinal gene expression was examined after 10 weeks of exercise training, after a 1 week final smoltification period and finally 8 days after transfer to seawater (Fig. 1). Different training regimens had no significant effect on any of the branchial genes that were examined (Figs 2 and 3). The most dynamic expression changes were observed during the final smoltification period with all four genes showing up-regulation with low intensity training. Conversely, a relative down-regulation was observed during the final smoltification period in high intensity and interval training in nka𝛼1b and nkcc1 when compared to the post-10 week training time point. Interestingly, increased expression of nkcc1 and cftr after seawater transfer was only observed with low intensity training, while nka𝛼1b expression was only increased in

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12475

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(a)

*

Relative nka1a mRNA

7 6 5 4 3 2 1

***

0

*

6

Relative nka1b mRNA

(b)

* *

4

*

2

** r ns fe SW

tra

m ls na Fi

Tr

ai

ni

ol t

ng

0

Fig. 2. Branchial gene expression of (a) nka𝛼1a and (b) nka𝛼1b as a consequence of exercise training regime and experimental time point in Salmo salar ( , low; , medium; , high; , interval). All data are normalized to the 18S gene and gene expression data are calculated relative to the low intensity training data point (mean ± s.e.). A significant difference from the training time point within an exercise regime (*) and a significant difference from the low training regimen (†) within a time point are denoted [two-way analysis of variance (ANOVA); n = 12 for training, n = 11–12 for de-training, n = 7–9 for seawater transfer].

the low and medium exercise treatments. All four exercise treatments showed the characteristic drop in nka𝛼1a expression upon transfer to seawater. Mean ± s.e. branchial NKA activity of the respective treatments during the final smoltification period ranged from 14⋅4 ± 0⋅7 to 15⋅5 ± 0⋅8 with no significant differences (P ≥ 0⋅05; n = 10–15) and no significant change in intestinal nkcc2 expression was observed in any exercise treatment or at any time point (Fig. 4). Plasma ion analysis revealed a decrease in Na+ during the final smoltification period in all exercise treatments, as well as a decrease in Cl− in the interval exercise regimen (Table II). There was little difference in plasma ions as a consequence of exercise regimen. The expression of two magnesium channel genes, trpm7 and slc41a2, was examined in the intestine of S. salar smolts (Fig. 5). There was no effect of exercise on the expression of either gene at any time point; however, seawater transfer did result in a general trend towards a change in expression of both genes. A trend of down-regulation was observed after seawater transfer for slc41a2 expression, but only the medium exercise regimen was significantly different from the 10 week time point. The trpm7 gene showed an increasing trend in expression, but only the high intensity treatment was statistically significant. To more fully elucidate the effects of seawater transfer on gene expression, a final analysis that combined the four exercise treatments at each time point was performed

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12475

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(a)

7

Relative cftr mRNA

6

* 4

*

2

0

(b)

*

4 3

*

2

*

1

*

0

fe

ol

ns tra SW

Fi

na

ls

m

ni ai Tr

r

t

ng

Relative nkcc1 mRNA

5

Fig. 3. Branchial gene expression of (a) cftr and (b) nkcc1 as a consequence of exercise training regime and experimental time point in Salmo salar ( , low; , medium; , high; , interval). All data are normalized to the 18S gene and gene expression data are calculated relative to the low intensity training data point (mean ± s.e.). A significant difference from the training time point within an exercise regime (*) and a significant difference from the low training regime within a time point (†) are denoted [two-way analysis of variance (ANOVA); n = 12 for training, n = 11–12 for de-training, n = 7–9 for seawater transfer].

(Fig. 6). Branchial nkcc1 was omitted from this analysis because it showed a significant interaction between exercise treatment and sampling time point (P < 0⋅05; two-way ANOVA). This analysis revealed characteristic up-regulation of branchial cftr and nka𝛼1b as a consequence of seawater transfer, as well as the commonly observed down-regulation of nka𝛼1a. Intestinal slc41a2 was significantly down-regulated as a consequence of seawater transfer, while intestinal trpm7 was up-regulated.

DISCUSSION The results of this study suggest that there was a preparatory effect of swim training on branchial ion excretion pathways, although this did not coincide with a down-regulation of freshwater ion uptake mechanisms. During smoltification, freshwater S. salar undergo gill re-modelling to prepare for the osmoregulatory and ionoregulatory challenges of the marine environment. Most notably, salmonids undergo NKA isoform switching whereby the 𝛼1a isoform is down-regulated while the 𝛼1b is up-regulated (Richards et al., 2003; Bystriansky et al., 2006, 2007; Madsen et al., 2009; Bystriansky & Schulte, 2011; Gilmour et al.,

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12475

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Relative nkcc2 mRNA

6

4

2

ns fe r

ol t SW

tra

m ls Fi na

Tr ai ni ng

0

Fig. 4. Intestinal gene expression of nkcc2 as a consequence of exercise training regime and experimental time point in Salmo salar ( , low; , medium; , high; , interval). All data are normalized to the 18S gene and gene expression data are calculated relative to the low intensity training data point (mean ± s.e.). No significant differences were detected [two-way analysis of variance (ANOVA); n = 11–12 for training, n = 8–10 for de-training, n = 6–9 for seawater transfer].

2012; McCormick et al., 2013). This is central to the change from a high affinity freshwater NKA necessary for Na+ uptake from a hypo-ionic environment to a high capacity NKA necessary for Na+ excretion (Jorgensen, 2008). These results provide further evidence for isoform switching in S. salar and also suggest that high intensity and interval training stimulate seawater level expression of nka𝛼1b during smoltification. This is indicated by a preemptive rise in nka𝛼1b expression in high and interval exercise treatments after transfer to seawater, which eliminated the typical up-regulatory response observed in low and medium exercise treatments (Fig. 2). Similar results were also observed for cftr and nkcc1 expression both of which are crucial for chloride excretion and typically up-regulated in response to seawater transfer (Tipsmark et al., 2002; Hiroi & McCormick, 2007; Mackie et al., 2007; Nilsen et al., 2007). Interestingly, expression of these genes was also elevated under medium exercise levels. Table II. Sodium and chloride concentrations in the plasma of Salmo salar exposed to one of the four aerobic swim training regimens (mean ± s.e.). Significant differences within an exercise treatment (*) and between exercise treatments within a time point (†) are denoted [two-way analysis of variance (ANOVA); n = 9–15] Exercise treatment

Na+ (mM) Cl− (mM)

Time point

Low

Medium

High

Interval

Pre-training Training Final smolt Pre-training Training Final smolt

143 ± 1 138 ± 1 126 ± 5* 135 131 ± 1 135 ± 1

144 ± 1 138 ± 1 135 ± 2*† 133 131 ± 1 131 ± 1

144 ± 1 140 ± 1 126 ± 4* 134 131 ± 1 135 ± 1

143 ± 1 140 ± 1 122 ± 4* 133 131 126 ± 4*†

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12475

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(a)

9

Relative slc41a2 mRNA

5 4 3 2

*

1 0

(b) Relative trpm7 mRNA

10

*

8 6 4 2

r

t

fe

ol SW

tra

ns

m ls na Fi

Tr ai ni ng

0

Fig. 5. Intestinal gene expression of the magnesium channels (a) slc41a2 and (b) trpm7 as a consequence of exercise training regime and experimental time point in Salmo salar ( , low; , medium; , high; , interval). All data are normalized to the 18S gene and gene expression data are calculated relative to the low intensity training data point (mean ± s.e.). Significant differences from the training time point within an exercise regime (*) are denoted. No significant differences within a time point were detected [two-way analysis of variance (ANOVA); n = 11–12 for training, n = 8–10 for de-training, n = 6–9 for seawater transfer].

Recent work has shown that S. salar smolts contain at least two different populations of ionocytes in their gills (McCormick et al., 2013). One cell type contains nka𝛼1a and is predominantly expressed during the freshwater residency period. The second cell type contains nka𝛼1b and is hypothesized to co-localize with nkcc1 and cftr. This second cell type is up-regulated during smoltification as fish prepare to move to seawater, with both types being present in about equal amounts during the pre-migration period. The seawater cell type is exclusively expressed post-migration. It is likely that increased exercise intensity, both continuous and interval, increases the distribution of seawater cell types. Because no pre-smolt fish were analysed in this study, it is unclear if the described expressional changes are the result of pre-emptive generation of the seawater cell type, or if exercise simply enhances the natural smoltification process already underway. It is also important to remember the osmorespiratory compromise of gill breathers and it can be safely assumed that increased seawater cell distribution is a preparatory response to the increased osmotic burden associated with exercise. In practise, it is common for S. salar smolts reared under an exercise training regimen to be exposed to a period of reduced activity prior to seawater transfer, here referred to as the final non-swimming smoltification period, stemming from the logistical difficulties of transferring large numbers of fish. One interpretation of these results is that an

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7

*

Relative mRNA

6 5

*

4 3

*

2 1

*

*

7 trp m

c2

41 a2 slc

nk c

tr cf

1 b nk a

nk a 1

a

0

Fig. 6. The effect of experimental time point on the relative gene expression of nka𝛼1a, nka𝛼1b, cftr, nkcc2, slc41a2 and trpm7 within the combined aerobic exercise training treatments (mean ± s.e.) ( , training; , final smolt; , seawater transfer). A significant difference from the 10 week training time point is indicated (*) [two-way analysis of variance (ANOVA); n = 34–48].

absence of training during this period eliminates any preparatory benefit of high intensity training, because seawater gene expression levels are reduced relative to the post-10 week training time point. It is important to consider the temporal influence on gene expression levels as it is possible that the expression levels of seawater genes simply returned to baseline levels once a suitable branchial phenotype had been produced. In fact, the similarity in branchial NKA activity levels between exercise treatments during the final smoltification period suggests similar branchial phenotypes. Subsequent seawater transfer would then act to stimulate renewed transcriptional regulation of the relevant genes. Interestingly, the final smoltification period is also coincident with a drop in plasma Na+ levels (Table II). This probably explains the increased nka𝛼1a expression in low intensity treatments during this period. In total, the results of this study suggest that aerobic swim training may alter the potential seawater transfer window of S. salar smolts, a finding that could have major implications for the S. salar aquaculture industry. The gastrointestinal tract of marine teleosts is a highly adapted tissue that is crucial for water balance in the marine environment (Grosell et al., 2011); however, little is known about the preparatory re-modelling of this tissue during smoltification. Contrary to expectation, nkcc2 in the anterior intestine showed no transcriptional changes in response to salinity change or in the period leading up to salinity change (Figs 4 and 6). Similarly, there were no effects of exercise training on expression levels in the anterior intestine. Previous work has demonstrated that seawater transfer of the closely related O. mykiss results in up-regulation of the intestinal HCO3 − secretion pathway, most notably in carbonic anhydrase, V-type ATPase and sodium bicarbonate co-transporter expression (Grosell et al., 2007). It should be noted that these responses are variable as other studies on O. mykiss have failed to illicit such trends in intestinal gene expression (Genz et al., 2011b; Gilmour et al., 2012). In S. salar, there is evidence that intestinal proteins involved in transcellular and paracellular water movement, such as aquaporin (Aqp)-8b, tricellulin and occludin, are up-regulated during smoltification (Tipsmark et al., 2008a), while seawater transfer caused up-regulation of intestinal claudin-15 and claudin-25b, as well as of Aqp-1a, Aqp-1b and Aqp-10 (Tipsmark et al., 2008b,

© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12475

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2010a, b). Given these data, it is somewhat surprising that nkcc2 did not respond to salinity transfer. It may be that nkcc2 represents a constitutively expressed baseline ion uptake pathway in fish anterior intestine while the HCO3 − secretion pathway represents a seawater-induced pathway. In contrast to nkcc2, both the Mg2+ transporters showed transcriptional regulation in response to seawater transfer (Fig. 6). There were no apparent effects of exercise training or any indication of preparatory re-modelling in advance of seawater transfer. Interestingly, the response of the two transporters was relatively opposite, with trpm7 showing an approximately five-fold increase in expression while slc41a2 decreased by half. The net result of this expression change was that both transporters were in about equal abundance in seawater, while slc41a2 was about four times more abundant in fresh water. Little is known about the relative importance of these transporters to Mg2+ homeostasis in fishes; however, the available evidence from mammals suggests that slc41a2 is a passive channel involved in Mg2+ influx (Goytain & Quamme, 2005; Moomaw & Maguire, 2008). In a freshwater environment, intestinal Mg2+ uptake is of obvious benefit. In a marine environment, Mg2+ uptake at the intestine would compromise water balance because whole body Mg2+ homeostasis is maintained by the kidney (Hickman, 1968a, b; Bijvelds et al., 1998; McDonald & Grosell, 2006). To effectively excrete excess Mg2+ , the kidney would have to sacrifice water for urine formation and therefore down-regulation of this transporter in seawater would clearly benefit water balance. The up-regulated trpm7 transporter is also involved in Mg2+ uptake; however, this channel has a kinase domain and is negatively gated by intracellular Mg2+ concentration. It appears possible that increasing the levels of this channel allows greater control over apical Mg2+ influx, which would aid in making Mg2+ relatively impermeable without fully sacrificing uptake pathways. It appears clear that the mechanisms related to Mg2+ transport in marine fishes are in need of future study to more fully elucidate how marine fishes are able to maintain Mg2+ balance. In conclusion, this study has provided evidence that sustained aerobic swimming can induce gene expression changes in the gills of S. salar smolts. This osmoregulatory plasticity provides additional evidence that sustained aerobic swimming has implications for aquaculture; however, further research is needed to fully demonstrate the degree to which sustained aerobic swimming ultimately benefits animal robustness. In addition, this study has provided insight into the transporters involved in Mg2+ homeostasis in the anterior intestine of fishes and how these transporters respond to seawater transfer. The experimental study conducted by Nofima was funded by the Fishery and Aquaculture Industry Research Fund (FHF) and the Research Council of Norway (grant number: 190067). M.G. is a Maytag professor of ichthyology and is supported by NSF (IOS 1146695) and A.J.E. is supported by NSF (EF 1315290).

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© 2014 The Fisheries Society of the British Isles, Journal of Fish Biology 2014, doi:10.1111/jfb.12475