Fish Physiology and Biochemistry

, Volume 36, Issue 4, pp 845–853

Seawater tolerance in Atlantic salmon, Salmo salar L., brown trout, Salmo trutta L., and S. salar × S. trutta hybrids smolt


    • Norwegian Institute of Water Research
  • J. Koksvik
    • Section of Natural HistoryNorwegian University of Science and Technology
  • J. V. Arnekleiv
    • Section of Natural HistoryNorwegian University of Science and Technology
  • K. Hindar
    • Norwegian Institute of Nature Research
  • F. Kroglund
    • Norwegian Institute of Water Research
  • T. Kristensen
    • Norwegian Institute of Water Research

DOI: 10.1007/s10695-009-9359-x

Cite this article as:
Urke, H.A., Koksvik, J., Arnekleiv, J.V. et al. Fish Physiol Biochem (2010) 36: 845. doi:10.1007/s10695-009-9359-x


High levels of hybridization between Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) have been reported in the Gyrodactylus salaris infected Rivers Vefsna and Driva in Norway. The survival and behaviour during the sea phase of such hybrids is unknown. The reported work documents ionoregulatory status after 24 h seawater challenge tests (24hSW) and gill Na+/K+-ATPase (NKA) activity of migrating wild smolts of Atlantic salmon, brown trout and hybrids at two sampling dates during the 2006 smolt run in River Driva. Salmon, trout and hybrids contributed to 27, 52 and 21% of the catches, respectively. The large contribution of hybrids suggests both a high hybridization rate and a high survival rate from fry to smolt. Both salmon and hybrids had a well-developed seawater tolerance at the time of downstream migration, revealed by small ionoregulatory effects and no or low mortality rates during the 24hSW tests. The trout were not fully adapted to seawater, and high mortality rates were observed (71 and 92%) during the 24hSW tests. The NKA activity was not significantly different between salmon and hybrids. Most of the hybrids were physiologically capable of direct entry to full strength seawater. The incomplete seawater tolerance in trout compared to salmon corresponds well with differences in life-history patterns between these two species. The life history strategy of the hybrids during the sea phase is not known, and further investigations on the marine behaviour and survival is needed to evaluate the role of hybrids in the risk of spreading G. salaris to nearby river systems.


SmoltificationSeawater toleranceSalmo salarSalmo truttaHybrids


Hybridization between Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.) is well known and is reported both from Europe and North-America (Jansson and Ost 1997; Gephard et al. 2000). The frequency of hybrids in natural populations varies, and an occurrence of 13% has been considered as high (Jansson and Ost 1997) although higher contributions have been reported (Jordan and Verspoor 1993). In Norway, Hindar and Balstad (1994) found an average frequency of 0.24 and 0.87% hybrids within healthy juvenile fish populations for the time-periods 1980–1986 and 1986–1992, respectively. In natural environments, the occurrence of hybridisation between Atlantic salmon and brown trout is thought to be a result of sneak mating (Gephard et al. 2000) or increased occurrence of escaped farmed salmon (Youngson et al. 1993; Hindar and Balstad 1994).

Typically, the occurrence of hybrids is stronger in weak, vulnerable or threatened populations of salmon than in strong and viable populations (Hindar and Balstad 1994). In Norway, infection by Gyrodactylus salaris has led to serious negative consequences for 46 salmon stocks (Johnsen and Jensen 1991; Johnsen et al. 2005). In River Driva, production and catches of Atlantic salmon have been dramatically reduced since the infection of G. salaris in the early 1980s. While salmon contributed to 75 ± 13% of the total catch through sport fishing in 1969–1975, the contribution was reduced to 54 ± 19% between 1976 and 1981. After 1981 salmon have only contributed to 28 ± 15% of the catch.

High levels (29–60%) of hybridization between Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) has been reported in the long-term G. salaris infected Rivers Driva and Vefsna, Norway (Arnekleiv et al. 2006; Johnsen 2007). The susceptibility of hybrids to G. salaris infection seems to be intermediate to that of trout, which is almost non-susceptive, and to salmon, where prevalence normally reaches 100% in affected rivers (Soleng et al. 1999).

In addition to spread through human activities, spread by migration of infected individuals into new non-contaminated neighbouring rivers could be one of the main causes behind the dispersal of G. salaris (Soleng et al. 1998; Høgåsen and Brun 2003; Jansen et al. 2007). Seawater (33–35‰ salinity) is found to be lethal to G. salaris within hours. Survival and temperature dependent population growth occurs at a salinity of 5‰ (Soleng and Bakke 1997).

A model of G. salaris dispersal, with primary introduction by stocking of infected fish and secondary regional dispersal depending on salinity in the fjord systems, has been developed (Jansen et al. 2007). As a carrier of the parasite, hybrids could represent a possible threat of further spread to nearby non-contaminated river systems (Klemetsen et al. 2003), although the migration of hybrid smolts in the fjord system are unknown.

The migration behaviour of sea-phase hybrid smolts and the probability of returning to or seeking out new nearby freshwater systems are thought to be closely dependent on the ion regulatory capacity (smoltification status) of the fish. While the general features of the smoltification processes are similar in all anadromous salmonids, species differ in the timing and degree of development of complete seawater ion regulatory capacity (Hoar 1988; Tanguy et al. 1994; McCormick et al. 1998). Atlantic salmon smolts quickly leave estuarine and coastal areas and enter full strength seawater, while brown trout and other salmonid smolts [e.g. Arctic char (Salvelinus alpinus L.)] may reside in low salinity areas for much of the marine life stage (Lacroix and McCurdy 1996; Bystriansky et al. 2006; Thorstad et al. 2007). Thus, they have a reduced need for complete pre-adaptation of ion regulatory capacity to full strength seawater (Finstad et al. 1989; Bystriansky et al. 2006).

If hybrids, which are known carriers of G. salaris, show the same ion regulatory pattern and migrating behaviour as trout, these fish could be a major, but so far undocumented, contributor to the spread of the parasite. The aim of this study was to determine the smoltification status of migrating hybrids and compare with that of trout and salmon as a first step in elucidating the marine behaviour and potential risk of hybrids as an agent in spreading G. salaris to nearby river systems. Smoltification status was therefore investigated by the use of 24 h seawater challenge tests (24hSW) and registration of gill Na+/K+-ATPase activity (NKA).

Given the differences in ion regulatory capacity between Atlantic salmon and brown trout (Finstad et al. 1989; Bystriansky et al. 2006), we hypothesized that the ion regulatory capacity of hybrids would be somewhere between.

Materials and methods

Study site, sampling procedure

The experimental studies were conducted in from April to June 2006 with wild fish collected from the River Driva, central Norway (Fig. 1). The river is the largest in the region with a total length of 150 km and a mean annual discharge of 68 m3 s−1. About 85 km of the main river is available for anadromous salmonids.
Fig. 1

The River Driva in Norway with placement of the smolt-trap in year 2006

Fish were collected in a rotary screw trap. The trap was in operation every night from 25th April to 20th June 2006 with the exception for short periods with extreme water discharge. Fish collected on May 7th and 22nd were used in the study. After emptying the trap, all fish were examined for wounds and other abnormalities, and fish to be used in the experiment were stored in several perforated plastic cages with a volume of 60 l in the river. To ensure as natural physical conditions as possible, the fish cages were kept in the river until exposure started 8 h later.

Seawater exposure tests

Hypo-osmoregulatory capacity was tested using 24 h seawater tolerance tests (24hSW) (Finstad et al. 1989). The fish were dip netted directly from the storage cages in the river into an experimental tank (75 l) containing water at a salinity of 35.0‰. The oxygen level was maintained by use of aerators, and the tanks were partially covered with plastic lids and kept in shady areas. Dead fish were recorded every 6 h. After 24 h of seawater exposure, blood was collected from surviving individuals. Fish held in a similar experimental tank (75 l) with freshwater served as a control group. This group was sampled after 24 h with the same protocol as the experimental group.

Water temperature in the experimental tanks varied during exposure from 7.1 to 11.2°C and 7.2 to 9.8°C on the two sampling dates, respectively.

Blood sampling, plasma and gill analyses

Fish were netted from the tanks, anaesthetised (5 mg l−1 metomidate, Marinil™, Wildlife Labs., Inc., Fort Collins, CO, USA) (Olsen et al. 1995) and killed by a blow to the head. Length and weight were measured, and blood was sampled from the caudal vessel using heparinised (Heparin Leo 5000 I.E. m l−1) syringes. Blood was stored on ice and centrifuged within <3 min. After centrifugation, plasma was stored in airtight cryo-vials at −20°C until analyzed. Total handling time was around 0.5 min per fish and 6 min for the group.

Blood plasma osmolality and chloride concentrations were measured using a Wescor 5500 vaporpressure-osmometer and a radiometer CMT chloride titrator, respectively.

The second gill arch on the left hand side was dissected out and frozen in 2-ml eppendorf tubes in a SEI buffer solution for measurements of Na+/K+-ATPase (NKA) activity. The samples for NKA were stored at −28°C before analysis was performed according to McCormick (1993). Briefly, gill tissue was homogenized in 150 μl SEID (SEI buffer containing 0.1% deoxycholic acid) and centrifuged at 5000g for 60 s. About 10 μl of supernatant was added in duplicate wells of a 96-well microplate containing 200 μl assay medium, with and without 0.5 mM ouabain, and read at 340 nM for 10 min at 25°C. NKA activity was determined as the ouabain sensitive fraction of the enzymatic coupling of ATP dephosphorylation to NADH oxidation, expressed as μmol ADP mg prot h−1.

Genetic analyses

Interspecific hybrids were identified using species-specific diagnostic markers 5S rDNA (Pendas et al. 1995) and GnRH (Gross et al. 1996), and in some cases a microsatellite locus showing non-overlapping allele sizes between Atlantic salmon and brown trout (SsOSL 438). Different restriction patterns of the mitochondrial DNA ND1 segment digested with HaeIII were used to identify the maternal origin of the hybrids (Cronin et al. 1993).

Water quality

Water samples from River Driva were obtained on two dates (May 5th and 23rd) and analyzed. Accredited analytical methods were used for all parameters. The River Driva had a high pH (>6.9), high acid neutralizing capacity (ANC) (>170 μeqv l−1l), high alkalinity (Alk) (>179 mmol l−1), high Ca (>3.5 mg l−1) and low levels of aluminium (Al) (total Al < 70 μg l−1), where the labile fraction (LAl) was < 5 μg l−1. These values indicate that River Driva is not affected by acidification or levels of labile Al that cause negative effects on smoltification (Kroglund et al. 2008). Based on the above chemistry and the lack of point discharge sources, we assume that the water quality in Driva is satisfactory and not the cause for any interspecies variation in smoltification.

Statistical analyses

All statistical tests were performed using the JMP v 6.0.3 software. The data were initially fitted a full factorial model with species, exposure (freshwater controls or 24hSW) and date of sampling (8th or 23rd of May) as model effects. No significant effects or interactions of date were found, so a simpler model, using species and exposure as model predictors, was fitted to pooled data from the two sampling dates. Significant effects of species or exposure, as well as significant species*exposure interactions were found for all three response variables (Table 1, Fig. 4). For further exploration of among-treatment level effects post-hoc Tukey–Kramer HSD tests were applied.
Table 1

Statistical analysis summary

Response variable

Model-fit statistics

Parameter estimates







Std error

t ratio

Prob > |t|

Gill Na+,K+-ATPase (μmol ADP mg protein h−1)















Species [Hybrid]





Species [Salmon]





Exposure × Species [Hybrid]





Exposure × Species [Salmon]





Plasma chloride (mmol l−1)















Species [Hybrid]





Species [Salmon]





Exposure × Species [Hybrid]





Exposure × Species [Salmon]





Plasma osmolality (mOsm l−1)















Species [Hybrid]





Species [Salmon]





Exposure × Species [Hybrid]





Exposure × Species [Salmon]





The data were fitted in a LSM model with species (Atlantic salmon, Atlantic salmon × brown trout hybrids, and brown trout), exposure (freshwater controls or 24 h SWT) and species × exposure interactions


No fish died during handling or holding in the storage cages. All fish used in the experiment were sexually immature. The tests were performed on salmon (146.5 ± 8.5 mm, 22.4 ± 4.1 g; N = 50), trout (170.4 ± 16.4 mm, 38.8 ± 11.8 g; N = 32) and hybrids (153.0 ± 8.9 mm, 25.9 ± 4.3 g; N = 25). The trout were significantly larger regarding weight and length than both salmon and hybrids

Sampling time and downstream migration

A total number of 320 salmon, 250 hybrids and 620 trout were registered in the trap, and the percentage proportion was 27, 21 and 52%, respectively (Fig. 2). The first 24hSW test was conducted when 55.7, 51.4 and 29.9% of the total catch of salmon, hybrids and trout were registered in the trap. For the second 24hSW test, these values were 73.2, 73.7 and 76.5%, respectively (Fig. 2).
Fig. 2

Cumulative frequencies (%) of the downstream migration of wild Atlantic salmon (——), brown trout (.......) and salmon × trout hybrids (– –) in the River Driva during the 2006 smolt-run. Asterisks (*) shows the time of sampling for fish used in the seawater tolerance test conducted on May 8th and 23rd. The trap was in operation every night from 25th April to 20th June; indicated by #

Mortality rate

During the 24hSW tests conducted on May 8th and 23rd 71 and 92% of the trout died, respectively. All salmon survived the tests, while mortality rate for the hybrids were 29 and 10%, respectively (Fig. 3). No fish died in the freshwater control group (Fig. 3).
Fig. 3

Mortality as cumulative frequencies (%) during 24 h seawater tolerance test (35‰) conducted on May 8th and 23rd in the 2006 season for wild Atlantic salmon (——), wild trout (......) and hybrids of these (- - - -) in the River Driva. Mortality was registered for every sixth hour. For fish kept as control in freshwater, no mortality occured

Na+, K+-ATPase activity (NKA)

There were no differences in NKA levels between hybrids and salmon, or between hybrids and trout in freshwater (Fig. 4). The freshwater levels of NKA were significantly higher for salmon compared to trout.
Fig. 4

Least square mean values +1 SE of Atlantic salmon, hybrids and brown trout smolts from freshwater control groups (FW) or 24 h, 35‰, seawater tolerance test (SWT). a Gill Na+,K+-ATPase (μmol ADP mg protein h−1) activity, b plasma chloride (mmol l−1) and c plasma osmolality (mOsm l−1). Bars not connected by the same letter are significantly different (Tukey–Kramer HSD, P < 0.05)

After the 24hSW test, the hybrids had significant higher NKA levels compared to hybrids in freshwater. For salmon and trout, this was not detected (Fig. 4).

The hybrids had significant higher NKA values compared to trout after the 24hSW test (Fig. 4). This was not the case between hybrids and salmon.

Plasma values

After the 24hSW test, both salmon and hybrids had significant lower plasma osmolality and plasma chloride compared to trout (Fig. 4). There were no significant differences in plasma chloride and plasma osmolality between hybrids and salmon (Fig. 4).

After the 24hSW test, the trout had a significantly higher plasma osmolality and plasma chloride compared to trout in freshwater (Fig. 4). For salmon and hybrids, this was not detected (Fig. 4).

Within the control groups held in freshwater only minor fluctuations in plasma chloride and plasma osmolality were observed, and there were no significant differences between species (Fig. 4).


Based on performance in 24hSW tests, most of the downstream migrating hybrid smolts from river Driva were physiologically adapted to entry into full strength seawater. The hypo-osmoregulatory capacity of Atlantic salmon was also fully established at the time of downstream migration, while the downstream migrating trout performed poorly in the 24hSW test and had a lower survival rate compared to salmon and hybrids.

The levels of NKA both for the salmon and hybrids were indicative of complete smoltification. The salmon NKA levels are in agreement with earlier studies on migrating wild salmon smolts conducted in other rivers in Norway (Nilsen et al. 2003).

The low survival rate and registered plasma values and NKA values indicated that the trout were not adapted to full seawater at the time of downstream migration. The incomplete seawater tolerance for trout compared to salmon corresponds well with differences in life-history patterns between these two species. Anadromity is a more characteristic life-history event of salmon than of trout. While salmon quickly leave estuarine and coastal areas and enter full strength seawater (Finstad et al. 1989; Thorstad et al. 2007), brown trout/sea trout and other salmonids (e.g. Arctic charr) may reside in or close to the estuaries for most of the marine life stage, thus reducing the need for complete preadaptation of ion regulatory capacity to full strength seawater (Bystriansky et al. 2006). In the Sunndalsfjord where River Driva drains, earlier investigation has showed a widespread freshwater layer (Molvær 1990). The trout in this system may not have the need for a well-adapted seawater tolerance during migration to the sea, since brackish water may be their major marine environment.

The infection rate of G. salaris on fish used in the 24hSW test were not registered, as the procedure for determining infection rates would involve anesthetizing and handling of the fish that might influence their subsequent performance in the test.

However, other samples of Atlantic salmon, brown trout and hybrids from the smolt trap in 2006 were investigated for infections of Gyrodactylus spp. The prevalence were 100% in salmon (N = 27), 73% in hybrids (N = 26) and 35% in trout (N = 17), while mean abundance (min–max) was 1296 (7–9450), 270 (2–1800) and 19 (2–43), respectively (Johnsen 2007).

The susceptibility of hybrids to G. salaris infection seems to be intermediate to that of trout, which is almost unsusceptible, and to salmon, where prevalence normally reaches 100% in affected rivers (Soleng et al. 1998). Possible G. salaris infection (abundance/prevalence) did not seem to impair the seawater tolerance during 24 h of seawater exposure for either the salmon or hybrids. The capacity for hybrids to survive in seawater was also indicated by the insignificant differences in levels of NKA between salmon and hybrids.

A positive relationship between seawater tolerance and body size seems to be a general phenomenon in salmonids (McCormick and Saunders 1987; Hoar 1988). In this study, the trout was significantly larger than both hybrids and salmon, revealing that the hypo-osmoregularity capacity for the salmon were not size dependent but a result of the smoltification process. The observed difference in seawater tolerance between salmon and trout reflects commonly observed species traits that are not due to sampling, handling or water quality. The stable and normal values for plasma osmolality and plasma chloride and NKA in the control groups indicate that the experimental conditions were not causing stressful reactions that may influence upon the results (Brown 1993; Nonnotte and Boeuf 1995; Wedemeyer 1996).

Downstream migrating smolts of Atlantic salmon/trout hybrids exhibited a seawater tolerance indicating that these fish were physiologically capable of direct transfer to full strength seawater. A low mortality rate in the hybrids suggests that some individual fish have incomplete seawater adaptation at the time of migration. The reported work also documents that the hybrids are more “salmon like” than “trout like” in their capacity of seawater tolerance. Further investigations are needed on the marine behaviour of the hybrids to reveal the potential risk of spreading G. salaris to other river systems.


We are grateful for the field assistance given by Lars Rønning and Gaute Kjærstad at the Norwegian University of Science and Technology, Prof. Sigurd Stefansson and Dr. Tom Ole Nilsen University of Bergen, Department of Biology for conducting the NKA analyses and Dr. Carolyn Knight and Dr. Thrond Oddvar Haugen, Norwegian Institute of Water Research for improving the manuscript. The reported work was financed by the county of Møre and Romsdal through project no (43/2006). This study was conducted as a part of a long-term study in salmonid smolts financed by the TrønderEnergi power company. Funding of manuscript preparation was kindly provided by Norwegian Institute of Water Research and Norwegian University of Science and Technology.

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© Springer Science+Business Media B.V. 2009