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Intestinal transport mechanisms and plasma cortisol
levels during normal and out-of-season parr–smolt
transformation of Atlantic salmon, Salmo salar
Kristina Sundella,*, Fredrik Jutfelta, Thorleifur Agustssona,b,Rolf-Erik Olsenc, Erik Sandbloma, Tom Hansenc,
Bjorn Thrandur Bjornssona
aFish Endocrinology Laboratory, Department of Zoology/Zoophysiology, Goteborg University,
Box 463, S-405 30 Goteborg, SwedenbdeCODE Genetics, Inc., Sturlugata 8, IS 101 Reykjavık, Iceland
c Institute of Marine Research, Matre Aquaculture Station, N-5984 Matredal, Norway
Received 31 October 2002; accepted 18 December 2002
Abstract
The intestine is one of the major osmoregulatory organs in fish. During the salmon parr–smolt
transformation, the intestine must change its functions from the freshwater (FW) role of preventing
water inflow, to the seawater (SW) role of actively absorbing ions and water.
This development can be assessed as an increased intestinal fluid transport (Jv) during the parr–
smolt transformation. The developmental changes taking place during parr–smolt transformation are
governed by a number of endocrine systems, of which cortisol is the main stimulator of Jv. In order
to further elucidate the mechanisms behind the elevation of Jv during parr–smolt transformation,
juvenile Atlantic salmon were followed during natural (1 + age) as well as photoperiod-induced
(0 + age) smoltification. Plasma cortisol levels, gill and intestinal Na+,K+-ATPase activity, Jv (only
during natural smoltification) and intestinal paracellular permeability were measured. In natural
smolting as well as in photoperiod-induced smolting, normal patterns of plasma cortisol levels and
gill Na+,K+-ATPase activity, with clearly defined, transient peaks were obtained. When fish were
transferred to SW, a second elevation in plasma cortisol levels and gill Na+,K+-ATPase activity
occurred, whereas Jv remained at similar levels as in FW fish. As to the mechanisms behind the
increased Jv during parr–smolt transformation, the intestinal Na+,K+-ATPase activity increases in
the anterior intestine and the paracellular permeability, as judged by transepithelial resistance (TER),
appears to decrease in the posterior intestine. These events correspond with the increase in Jv seen
0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved.
doi:10.1016/S0044-8486(03)00127-3
* Corresponding author. Tel.: +46-31-7733671; fax: +46-31-7733807.
E-mail address: [email protected] (K. Sundell).
www.elsevier.com/locate/aqua-online
Aquaculture 222 (2003) 265–285
during this developmental stage. Furthermore, the increase in the physiological parameters follows
the changes in plasma cortisol levels, shifted by a couple of weeks. When the fish were transferred to
SW, a further increase in Na+,K+-ATPase activity was apparent in both anterior and posterior
intestine and the paracellular permeability decreases. To summarize, the increased Jv seen during the
parr–smolt transformation of Atlantic salmon may be due to an increase in the paracellular water
flow of the posterior intestine. When the fish enter SW, the water flow appears to be directed from
the paracellular pathway towards a more transcellular route with increased intestinal Na+,K+-ATPase
activity as the main driving force.
D 2003 Elsevier Science B.V. All rights reserved.
Keywords: Osmoregulation; Parr; Smolt; In vitro; Ussing chambers; Intestinal permeability; Transepithelial
electrical resistance; Mannitol; Na+,K+-ATPase activity; Cortisol plasma levels; Salmon; Salmo salar
1. Introduction
In addition to being an organ of nutrient uptake, the intestine is a major organ for
maintenance of ion and water balance in fish. Already in 1930, Smith demonstrated that
fish in seawater (SW) had high drinking rates and high rates of intestinal ion and water
absorption. This ion-driven water uptake compensates for water that is osmotically lost to
the environment. Fish in freshwater (FW), on the other hand, have low drinking rates
(Perrott et al., 1992) and absorb Na+ and Cl� mainly from dietary sources, in order to
replace salts lost by diffusion to the external medium (Baldisserotto and Mimura, 1994).
In anadromous salmonids, complex changes in physiology, morphology, biochemistry
and behaviour take place in FW, during the parr–smolt transformation, preparing the fish
for marine life (McCormick and Saunders, 1987). During smoltification, the intestinal
function must thus change from its FW role of preventing water inflow, to that of actively
absorbing ions and water.
The developmental changes during the parr–smolt transformation are governed by a
number of endocrine systems, of which cortisol is a major component together with
growth hormone and thyroid hormones. Interrenal activity increases (Specker, 1982;
Young, 1986) and cortisol levels in plasma show a distinct, transient peak during
smoltification in spring (Specker and Schreck, 1982; Virtanen and Soivo, 1985; Lan-
ghorne and Simpson, 1986; Young et al., 1989; Shrimpton et al., 1994; Shrimpton and
McCormick, 1998). The peak in plasma cortisol levels is coincident with the development
of physiological smolt indicators such as increased gill Na+,K+-ATPase activity (McCor-
mick et al., 1991, 1995) and increased hypoosmoregulatory ability (Langhorne and
Simpson, 1986; Young et al., 1989; Bisbal and Specker, 1991; Seidelin and Madsen,
1997). Furthermore, the developmental elevation in intestinal fluid transport (Jv) seen
during parr–smolt transformation of Atlantic salmon (Veillette et al., 1993) is mediated by
cortisol (Veillette et al., 1995).
The major driving force of intestinal fluid transport is considered to be basolaterally
located Na+,K+-ATPases (Loretz, 1995; Movileanu et al., 1998). Thus, the preadaptive
increase in Jv during parr–smolt transformation of Atlantic salmon should coincide with
an increased Na+,K+-ATase activity in the basolateral membranes of enterocytes. However,
K. Sundell et al. / Aquaculture 222 (2003) 265–285266
available data present conflicting results. Some studies have failed to demonstrate
induction of intestinal Na+,K+-ATPase activity either with cortisol treatment or during
seasonal (smoltification) changes (Bisbal and Specker, 1991; Nielsen et al., 1999; Seidelin
et al., 1999), whereas other studies have succeeded (Madsen, 1990; Rey et al., 1991;
Sundell and Bjornsson, unpublished data).
In addition to the transcellular transport driven mainly by the Na+,K+-ATPase, the
paracellular pathway is also a possible route for movement of ions and water via the tight
junctions (TJ). As the tight junctions are dynamic structures that are physiologically re-
gulated (Anderson and Van Itallie, 1995), modulation of TJ permeability may also provide a
means of regulating intestinal ion and water transport (Madara and Pappenheimer, 1987).
The aim of the present study was to investigate the mechanisms behind the devel-
opmental increase in intestinal Jv during the parr–smolt transformation and subsequent
seawater transfer of Atlantic salmon and the role of cortisol in these processes. The
mechanisms were studied in yearling fish undergoing spring smoltification under natural
photoperiod and temperature, as well as in underyearling fish undergoing photoperiod-
induced smoltification. In the second model, the fish were larger at onset of the experiment
and the smolting events were more synchronised in time.
2. Materials and methods
2.1. Spring smoltification of yearling Atlantic salmon (experiment 1)
2.1.1. Fish and holding conditions
Juvenile Atlantic salmon, Salmo salar, were raised and kept at a local hatchery,
Fiskeman i Laxforsen, Anneberg, Sweden. On January 4th 1998 (4 weeks prior to the
first sampling), 250 salmon were transferred to each of two replicate outdoor tanks (1�1
m, water depth 50 cm), under natural photoperiod. The tanks were supplied with water
from a nearby stream at ambient temperature, gradually rising from 2 to 10 jC, duringthe experimental period. On May 25th, 1998 (4 weeks before the end of the experiment),
50 fish from each tank were transported to the fish facility at Department of Zoology,
Goteborg University, and transferred to duplicate 1-m3 indoor tanks containing filtered
and recirculating SW (30x). These fish were kept at simulated natural photoperiod and
at a constant temperature of 10 jC. All fish were fed commercial dry pellets, according to
a feeding schedule used by the hatchery (EWOS, aquaculture feeding tables).
2.1.2. Experimental design
Sampling of fish in FW was conducted on 12 occasions from February 4th to June 30th,
1998, approximately every second week. Sampling in SW was carried out after 1 and 4
weeks, 1 day after the corresponding sampling in FW. On each sampling date, 15 fish from
each of the two replicate tanks were sampled (thus n = 30 for each sampling date; some of
the fish were sampled for purposes other than reported in this study). The fish were
randomly netted; three times four fish and one time three fish from each tank and the fish
from the first two nettings were used in the present study. The fish were immediately
sacrificed by an overdose of anaesthesia (0.05% 2-phenoxyethanol l� 1, Sigma).
K. Sundell et al. / Aquaculture 222 (2003) 265–285 267
2.2. Photoperiod-induced smoltification of underyearling Atlantic salmon (experiment 2)
2.2.1. Fish and holding conditions
The experiment was carried out at Matre Aquaculture Station, Norway (61jN), usingjuvenile Atlantic salmon of the NLA strain. The salmon were hatched in mid-January 2000
and reared under continuous light from first feeding in late February. Two weeks prior to
the initiation of the experiment, 120 salmon with an average weight of 35 g were
transferred to each of two identical indoor tanks (1�1 m, water depth 30 cm), and kept
under continuous light. The tanks were supplied with FW from the Matre hydroelectrical
power plant, with temperatures gradually declining from 13.1 to 10.3 jC. At the start of
the experiment (August 21st, 2000), the fish were subjected to a transient, square-wave
change in photoperiod, from continuous light (24L) to short day (12L:12D) for 6 weeks,
followed by a return to 24L for 6 more weeks. This protocol has been shown effective in
inducing out-of-season smoltification of 0 + age Atlantic salmon (Hansen, 1998; Bjorns-
son et al., 2000). On November 20th, 2000, the remaining fish were transferred to two
identical tanks supplied with borehole SW. The fish were subjected to continuous light,
with a temperature ranging between 11.7 and 9.9 jC, until sampling on March 3rd, 2001.
All fish were fed commercial dry feed (Biomar LTD, Trondheim, Norway) at 2% of body
weight with pellet sizes adjusted to fish weights.
2.2.2. Experimental design
Sampling in FW was conducted on four occasions: on August 18th, just prior to the
switch from 24L to 12L:12D, on October 4th, just prior to the switch back to 24L, and on
October 25th and November 15th, 3 and 6 weeks after the return to 24L. Fish in SW were
sampled approximately 14 weeks after SW transfer. On each occasion, eight fish from
each replicate tank were randomly netted and immediately sacrificed by an overdose of
anaesthesia (0.05% 2-phenoxyethanol l� 1, Sigma).
2.3. Sampling procedures and analyses
2.3.1. Sampling procedures
All fish were weighed (wet weight) and measured (fork length) and the condition factor
(CF) was calculated (CF = body weight� 100� fork length� 3). After anesthesia, blood
was collected from the caudal vessels using 1-ml heparinized syringes. The blood was
centrifuged at 3000� g for 5 min to obtain plasma, which was aliquoted, frozen on dry ice
and stored at � 80 jC until analyses. The fish were then decapitated and the two first gill
arches on the right side dissected out and placed in ice-cold SEI buffer (150 mM sucrose,
10 mM Na2-EDTA, 50 mM imidazole at pH 7.3). The gill tissue was frozen in liquid
nitrogen directly after sampling and stored at � 80 jC until analyses. In experiment 1, the
body cavity was opened laterally and the intestine, from just posterior to the last pyloric
ceca to the anus, was carefully removed and placed in an ice-cold salmon Ringer solution
(140 mM NaCl, 2.5 mM KCl, 15 mM NaHCO3, 1.5 mM CaCl2, 1 mM KH2PO4, 0.8 mM
MgSO4, 10 mM glucose and 5 mM HEPES buffer (pH 7.8) for the fish in FW, and 150
mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.0 mM MgCl2, 7.0 mM NaHCO3, 0.7 mM
NaH2PO4, 10 mM glucose and 5 mM HEPES buffer (pH 7.8) for the fish in SW). The
K. Sundell et al. / Aquaculture 222 (2003) 265–285268
tissue was then subjected to one of three treatments: (i) Intestines from eight fish were cut
open along the mesenteric border, carefully rinsed in the appropriate Ringer solution and
the mucosa was scraped off using two glass microscope slides. The mucosal scrapings
were placed in ice-cold intestinal buffer (200 mM glycine, 300 mM sucrose, 45 mM Na2-
EDTA, 50 mM EGTA, 50 mM imidazole at pH 7.6) and immediately frozen in liquid
nitrogen for later analyses of intestinal Na+,K+-ATPase activity. (ii) Intestines from eight
fish were carefully flushed with ice-cold Ringer, placed in e-flasks containing Ringer
solution and transported on ice to the laboratory for further analyses of Jv. (iii) Intestines of
six fish were cut open along a mesenteric border, rinsed, placed in salmon Ringer solution
and transported on ice to the laboratory for measurement of paracellular permeability.
In experiment 2, after blood and gill sampling, the body cavity was opened laterally and
the intestine from just posterior to the last pyloric ceca to the anus was carefully removed
and placed in ice-cold salmon Ringer. The intestines were then separated into two parts:
the anterior part between the pyloric ceca and the ileorectal valve, and the posterior part
from the ileorectal valve to the anus. The intestinal segments from two sets of eight fish
were treated as described under (i) and (iii) above, respectively.
2.3.2. Plasma cortisol levels
Plasma cortisol levels were measured in unextracted plasma using a radioimmunoassay
procedure according to Young (1986) and validated by Bisbal and Specker (1991).
Cortisol antibodies were obtained from Endocrine Sciences, CA (lot 345102280).
2.3.3. Gill Na+,K+-ATPase activity
Gill samples were thawed on the day of assay, the storage buffer discarded, and the gill
filaments homogenized in 1 ml of SEI buffer containing 0.1% of sodium deoxycholate,
using a glass/glass tissue grinder (Contes Glass, Vineland, NJ). After centrifugation at
3000� g for 30 s, 10 Al of the supernatant was added in duplicate to 200 Al assay medium,
with and without 0.5 mM ouabain, in 96-well microplates and read at 340 nM for 10 min
at 25 jC, according to the microassay protocol of McCormick (1993). Protein concen-
trations of the samples were assessed according to Lowry et al. (1951). Na+,K+-ATPase
activity was expressed as Amol ADP mg protein� 1 h� 1.
2.3.4. Intestinal Na+,K+-ATPase activity
Intestinal mucosal samples were thawed on the day of assay. For samples from
experiment 1, this corresponds to the day after sampling. For experiment 2, this was 6
days after sampling. The storage buffer was discarded and the mucosal scrapings were
homogenized in intestinal buffer using a glass/glass tissue grinder (Contes Glass), 2� 10
strokes. Following centrifugation (5000� g for 1 min), Na+,K+-ATPase activity was
measured in 10 Al of the supernatant, as described for gill samples above.
2.3.5. Intestinal fluid uptake rate (Jv)
The intestines were prepared, as non-everted sacs for gravimetric determination of fluid
transport, as described by Veillette et al. (1993). Briefly, the intestine were tied at the distal
end, filled with Ringer solution (at 10 jC) and tied at the proximal end. The intestinal sacs
were then weighed and incubated in e-flasks filled with Ringer (as described above) and
K. Sundell et al. / Aquaculture 222 (2003) 265–285 269
partly submerged in a cooling bath (at 10 jC) that was equipped with a turntable that
rotated slowly. The Ringer solution was aerated with a gas mixture of 99.7% air and 0.3%
CO2, and the intestinal sacs were equilibrated for 30 min. Over the next 100 min, the
intestinal sacs were weighed every 10 min and the rate of water loss was determined by
linear regression analysis of the sac weights. The rate of water loss was normalized to the
surface area of the sac to yield a rate of mucosal to serosal net water movement expressed
as Al cm� 2 h� 1.
2.3.6. Paracellular permeability
The paracellular permeability of the intestinal segments was assessed by measurements
of transepithelial resistance (TER) and the apparent permeability of the hydrophilic marker
molecule 14C-mannitol in an Ussing chamber system. Together with TER, continuous
monitoring of transepithelial potential (TEP) and short circuit current (SCC) was used as
control of preparation viability.
The intestinal segments were mounted into modified Ussing chambers (Grass and
Sweetana, 1988). The chambers were filled with the appropriate salmon Ringer solution
and the temperature was kept at 10 jC by a cooling mantle. Mixing and oxygenation was
obtained by gas lift with a gas mixture of 99.7% air and 0.3% CO2. The exposed tissue
surface area was 0.75 cm2 and the half-chamber volume 5 ml.
The chambers were equipped with four electrodes each: one pair of Pt electrodes for
current passage and one pair of Ag/AgCl electrodes (Radiometer, Copenhagen) bathing in
3 M KCl solution for measurement of transepithelial potential (TEP) differences. Electrical
connections between the half-chambers and the voltage recording Ag/AgCl electrodes
were made first by 0.9% NaCl agar bridges from the half-chambers to a container with
0.9% NaCl solution, and then by 3.0 M KCl agar bridges to the container with the
recording electrodes. The tip of the 0.9% NaCl agar bridges was positioned no further than
1 mm from the tissue surface. Pt electrodes and Ag/AgCl electrodes were connected to an
external electronic unit (TEMA Processteknik, Uppsala, Sweden) with a voltage-con-
trolled current source (U/I converter) and an amplifier (� 250). The U/I outputs and
amplifier inputs were connected to six pairs of relays, which allowed a simultaneous
measurement of six chambers. The electrical measurements and data collection were
controlled by a PC via A/D–D/A board (LabPC+, National Instrument, Sweden). The
controlling software was developed in LabView (National Instruments) by Dr. J. Karlsson
and J. Grasjo, Department of Pharmaceutics, Uppsala University. The procedures for
electrical measurements, automatic data analysis and presentation are described in Wik-
man-Larhed and Arthursson (1995). In short, direct current pulses of 15, � 15, 30, � 30
and 0 AA for 100 ms, with a 235-s duration for each pulse, are sent across the epithelium.
The voltage response for each current is after 200 ms measured for 20 ms, to minimize
possible disturbance from the main power supply of 50 Hz. Eight recordings of each
voltage response are sampled at 5-ms interval and averaged. A linear least-squares fit of
the current–voltage pairs is then performed. The slope of this line shows the trans-
epithelial electrical resistance (TER), the intercept with the voltage axis describes the TEP,
and the short-circuit current (SCC) is determined as: SCC=�TEP/TER. The electrical
parameters were measured once every 5 min to avoid increases of the epithelial
capacitance.
K. Sundell et al. / Aquaculture 222 (2003) 265–285270
The potential differences between the Ag/AgCl electrodes and the electrical resistance
originating from the electrode/agar–salt–bridge system and the Ringer solution were
corrected for by determining these parameters in the chambers without intestinal
epithelium mounted.
TEP values are referenced to the apical, i.e. the mucosal side. All tissues were allowed
to equilibrate for 60 min, to stabilize, before the experiments started.
Measurement of apparent permeability of 14C-mannitol (MW: 184; Amersham, St.
Louis, MO, USA) was initiated by changing the Ringer solution in the mucosal compart-
ment to a Ringer containing 14C-mannitol (spec. act. 0.02–0.03 MBq ml� 1) and in the
serosal compartment to normal Ringer solution. Samples of 40 Al were withdrawn from
the serosal compartment every 10 min for 90 min and replaced by the same volume of
fresh Ringer. Four milliliters of Optiphase high safe II (Wallac, Finland) was added to each
sample and the radioactivity assessed in a liquid scintillation counter (Beckman LS 1801,
Sweden).
The apparent permeability coefficient (Papp) was calculated using Eq. (1).
Papp ¼ dQ=dt � 1=AC0 ð1Þ
where dQ/dt is the steady-state appearance rate of the compound on the serosal side, C0 the
initial concentration of the compound on the mucosal side of the membrane, and A is the
exposed tissue surface area.
2.3.7. Statistical analyses
All data are expressed as mean valuesF S.E.M. All data were subjected to Cochran’s test
for equal variances. The data sets not showing homoscedasity were log-transformed which
resulted in equal variances. The log-transformed data were then subjected to appropriate
analysis of variance. Differences between intestinal parameters from experiment 2 were
analyzed using a three-factorial analysis of variance, in a mixed model, with time and region
as fixed factors and tank as random factor. Differences in all other parameters measured
were analyzed using two-factorial analysis of variance. Here, a mixed model was also used
with time as fixed factor and tank as random factor. No tank effects were seen at the
significance level of p>0.25 (Underwood, 1997), and therefore, the fish from the replicate
tanks were pooled for all further analyses. To obtain detailed information about differences
between sampling points in experiment 1, a Student–Neuman–Keuls post hoc procedure
were used when appropriate. In experiment 2, independent t-test (two-tailed) for sequential
time-points was used to explore differences in time. Significance was accepted at p < 0.05.
SPSS statistical software (SPSS, Chicago, IL) was used for all statistical procedures.
3. Results
3.1. Spring smoltification of yearling Atlantic salmon (experiment 1)
For salmon smolting under natural photoperiod, the condition factor decreased
significantly in mid-May ( p < 0.05). This decrease was reversed in late May followed
K. Sundell et al. / Aquaculture 222 (2003) 265–285 271
by another decrease towards the end of the experiment (Fig. 1A). The size of the fish
increased from 36.7F 1.3 to 62.7F 2.5 g in weight and from 14.8F 0.2 to 18.3F 0.2 cm
in length, with most of the growth occurring in May and June (Fig. 1B). The fish exhibited
a gradual loss of parr marks and increased silvering during the experiment (data not
shown).
Plasma cortisol levels and gill Na+,K+-ATPase activity increased gradually from mid-
April to a peak in May, with plasma cortisol levels reaching the peak about 20 days prior
to the peak in gill Na+,K+-ATPase activity. This was followed by a gradual decrease in
both parameters towards the end of June (Fig. 2A and B).
The intestinal fluid transport (Jv), measured in whole intestines, was relatively stable
during the first two-thirds of the study. In early May, the Jv started to increase and reached
a distinct peak in late May, after which the Jv decreased towards the end of the
experimental period (Fig. 3A).
Fig. 1. Condition factor (A) and body weight and fork length (B) of 1 + age Atlantic salmon during parr– smolt
transformation under ambient temperature and photoperiod conditions (filled symbols) and after transfer to SW
(open symbols). Data are shown as meansF S.E.M. (n= 30) and data on condition factor during parr– smolt
transformation were initially analyzed using two-way ANOVA. No tank effects were observed and the fish from
the replicate tanks were pooled for all further analyses, including a SNK post hoc procedure. Different letters
above data points indicate significant differences ( p< 0.05).
K. Sundell et al. / Aquaculture 222 (2003) 265–285272
The small size of the salmon, prior to their growth spurt in May, limited the success rate
of analyses of the paracellular permeability and intestinal Na+,K+-ATPase activity, in
particular during the early part of the study. The lower size limit of fish to enable a
successful preparation for the Ussing chamber setup is approximately 50 g, and this mean
weight was not reached until June. Thus, the number of successful preparations was
therefore low, with n = 2–4 throughout the experiment for Papp, and n = 3–5 for April and
May determination of Na+,K+-ATPase activity, instead of the maximal n = 8. No signifi-
cant changes could be demonstrated in either paracellular permeability or intestinal
Na+,K+-ATPase activity. However, there was a tendency towards an increased intestinal
Na+,K+-ATPase activity from mid-April to the end of the experiment (Fig. 3B), and a
similar tendency towards increased paracellular permeability in mid-April, followed by a
decrease towards the end of June.
Fig. 2. Plasma levels of cortisol (A; n= 30) and gill Na+,K+-ATPase activity (B; n= 16) of 1 + age Atlantic salmon
during parr–smolt transformation under ambient temperature and photoperiod conditions (filled symbols) and
after transfer to SW (open symbols). Data are shown as meansF S.E.M. and data obtained during parr– smolt
transformation were initially analyzed using two-way ANOVA. No tank effects were observed and the fish from
the replicate tanks were pooled for all further analyses, including a SNK post hoc procedure. Within each scatter
plot, different letters above data points indicate significant differences ( p< 0.05).
K. Sundell et al. / Aquaculture 222 (2003) 265–285 273
After 4 weeks in seawater, the Na+,K+-ATPase activity in gill and intestine, as well as
the plasma cortisol levels increased well above the corresponding values in FW fish (Figs.
2A,B and 3B), whereas no increase was seen in Jv (Fig. 3A) or Papp.
3.2. Induced smoltification of underyearling Atlantic salmon (experiment 2)
At the starting point of the experiment, August 18th, 2000, the mean body weight and
length (Fig. 4B) of the fish were 39.8F 2.8 g and 14.2F 0.4 cm, and the condition factor
1.35F 0.02 (Fig. 4A). During the ‘‘winter’’ phase of the experiment (6 weeks on
12L:12D), the CF increased to a value of 1.44F 0.02. Following the return to 24L, the
Fig. 3. Rate of intestinal fluid uptake, Jv (A; n= 8), and intestinal Na+,K+-ATPase activity (B; n= 3–8) of 1 + age
Atlantic salmon during parr– smolt transformation under ambient temperature and photoperiod conditions (filled
symbols) and after transfer to SW (open symbols). Data are shown as meansF S.E.M., and data obtained during
parr– smolt transformation were initially analyzed using two-way ANOVA. No tank effects were observed and
the fish from the replicate tanks were pooled for all further analyses, including a SNK post hoc procedure.
Different letters above data points indicate significant differences ( p< 0.05).
K. Sundell et al. / Aquaculture 222 (2003) 265–285274
CF decreased again to 1.28F 0.02 and then further to 1.19F 0.02, after 3 and 6 weeks,
respectively (Fig. 4A). CF did not change significantly following adaptation to SW for 14
weeks.
At the first sampling point in FW, plasma cortisol levels and gill Na+,K+-ATPase
activity were 4.0F 0.81 ng ml� 1 and 2.82F 0.43 Amol ADP h� 1 mg protein� 1,
respectively (Fig. 5A and B). After 6 weeks on 12L:12D, plasma cortisol levels were at
the same level, but gill Na+,K+-ATPase activity was significantly lower (2.41F 0.52 ng
ml� 1 and 1.48F 0.16 Amol ADP h� 1 mg protein� 1). Plasma cortisol levels increased
Fig. 4. Condition factor (A) and body weight and fork length (B) of 0 + age Atlantic salmon. Sampling was
performed after rearing on continuous light from start of first feeding (i.e. for 6 months; open bars), during
photoperiod-manipulated parr– smolt transformation under ambient temperature conditions, 6 weeks on 12L:12D
light regime and thereafter return to continuous light for 6 more weeks (light grey bars) and after transfer to SW
for 14 weeks (dark grey bars). Data are shown as meansF S.E.M. (n= 16). Data on condition factor from the
photoperiod-manipulated parr– smolt transformation and the subsequent transfer to SW were initially analyzed
using two-way ANOVA. No tank effects were observed and the fish from the replicate tanks were pooled for all
further analyses. * Denotes significant difference ( p< 0.05) compared with the time-point before using a
sequential, independent t-test as post hoc procedure.
K. Sundell et al. / Aquaculture 222 (2003) 265–285 275
significantly after 3 weeks on 24L (23.3F 3.9 ng ml� 1) whereas Na+,K+-ATPase activity
remained in the same range as during the simulated ‘‘winter’’ phase (1.84F 0.25 Amol
ADP h� 1 mg protein� 1). After 6 weeks on 24L, the gill Na+,K+-ATPase activity increased
Fig. 5. Plasma levels of cortisol (A) and gill Na+,K+-ATPase activity (B) of 0 + age Atlantic salmon. Sampling
was performed after rearing on continuous light from start of first feeding (i.e. for 6 months; open bars), during
photoperiod-manipulated parr–smolt transformation under ambient temperature conditions, i.e. 6 weeks on
12L:12D light regime and thereafter return to continuous light for 6 more weeks (light grey bars) and after transfer
to SW for 3 1 2= months (dark grey bars). Data are shown as meansF S.E.M. (n= 16). Data from the photoperiod-
manipulated parr–smolt transformation and the subsequent transfer to SW were initially analyzed using two-way
ANOVA. No tank effects were observed and the fish from the replicate tanks were pooled for all further analysis.
* denotes significant difference ( p< 0.05) compared with the time-point before using a sequential, independent t-
test as post hoc procedure.
K. Sundell et al. / Aquaculture 222 (2003) 265–285276
significantly (3.93F 0.45 Amol ADP h� 1 mg protein� 1, Fig. 5B) and the plasma cortisol
levels decreased again (7.33F 1.35 ng ml� 1, Fig. 5A). Following SW transfer, gill
Na+,K+-ATPase activity increased further (9.9F 0.75 Amol ADP h� 1 mg protein� 1),
whereas the plasma cortisol levels were in the same range (8.8F 1.87 ng ml� 1, Fig. 5A
and B). No mortalities occurred in the SW-transferred groups.
The intestinal Na+,K+-ATPase activity of the anterior intestine (Fig. 6) was lowest after
6 weeks on 12L:12D (0.73F 0.19 Amol ADP h� 1 mg protein� 1) and had increased
significantly after 6 weeks on continuous light (2.15F 0.59 Amol ADP h� 1 mg
protein� 1). On the other hand, the intestinal Na+,K+-ATPase activity of the posterior
intestine did not change (Fig. 6). After SW transfer, the Na+,K+-ATPase activity of both
the anterior and the posterior intestine increased significantly compared with the enzyme
activity of fish in FW (Fig. 6). For all sampling points, the anterior intestine had a higher
Na+,K+-ATPase activity than the posterior intestine (Fig. 6). The TER and Papp are both
mainly estimates of the paracellular permeability of the intestinal epithelium. During
photoperiod manipulation, no significant changes in either of these parameters could be
demonstrated (Fig. 7A,B). However, TER of the posterior intestine was constantly higher
than TER of the anterior intestine, and in the posterior intestine, there was a tendency
towards a decrease in TER from the ‘‘winter’’ phase (6 weeks on 12L:12D; 133.2F 10.6
Fig. 6. Na+,K+-ATPase activity in anterior and posterior intestine of 0 + age Atlantic salmon. Sampling was
performed after rearing on continuous light from start of first feeding (i.e. for 6 months; open bars), during
photoperiod-manipulated parr–smolt transformation under ambient temperature conditions, i.e. 6 weeks on
12L:12D light regime and thereafter return to continuous light for 6 more weeks (light grey bars) and after transfer
to SW for 14 weeks (dark grey bars). Data are shown as meansF S.E.M. (n= 8). Data from the photoperiod-
manipulated parr–smolt transformation and the subsequent transfer to SW were initially analyzed using three-
factorial ANOVA. No tank effects were observed and the fish from the replicate tanks were pooled for all further
analyses. An overall significant difference in Na+,K+-ATPase activity between anterior and posterior intestine was
obtained ( p< 0.05) and * denotes significant difference ( p< 0.05) compared with the time-point before using a
sequential, independent t-test as post hoc procedure.
K. Sundell et al. / Aquaculture 222 (2003) 265–285 277
V cm2) to the two sampling times 3 and 6 weeks after the return to 24L (111.4F 6.4 and
111.4F 6.3 V cm2, respectively). This pattern was reversed in SW, and a significantly
higher TER was measured in both anterior and posterior intestine of SW-adapted fish
compared with fish in FW (Fig. 7A).
Fig. 7. Transepithelial resistance (TER; A) and apparent permeability for the hydrophilic marker molecule
mannitol (Papp; B) in anterior and posterior intestine of 0 + age Atlantic salmon. Sampling was performed after
rearing on continuous light from start of first feeding (i.e. for 6 months; open bars), during photoperiod-
manipulated parr– smolt transformation under ambient temperature conditions, i.e. 6 weeks on 12L:12D light
regime and thereafter return to continuous light for 6 more weeks (light grey bars) and after transfer to SW for 14
weeks (dark grey bars). Data are shown as meansF S.E.M. (n= 8). Data from the photoperiod-manipulated parr–
smolt transformation and the subsequent transfer to SW were initially analyzed using three-factorial ANOVA. No
tank effects were observed and the fish from the replicate tanks were pooled for all further analyses. An overall
significant difference in TER between anterior and posterior intestine was obtained ( p< 0.05) and * denotes
significant difference ( p< 0.05) compared with the time-point before, using a sequential, independent t-test as
post hoc procedure. No significant difference in Papp was obtained.
K. Sundell et al. / Aquaculture 222 (2003) 265–285278
4. Discussion
In the present study, data on the physiology and endocrinology of Atlantic salmon
smoltification from two successful aquaculture strategies can be compared. One is the
established practice of letting 1 + age fish smoltify in spring under natural photoperiod,
and the other is the recent practice of inducing smoltification of large 0 + age fish during
fall through the use of photoperiod manipulation. In terms of elucidating regulatory
mechanisms during parr–smolt transformation, the out-of-season induction of smoltifica-
tion through distinctly timed changes in photoperiod, offers many advantages regarding
starting size of the fish as well as the timing and synchronization of developmental events.
On the other hand, it is essential to establish that the physiological changes that take place
during 0 + age salmon smoltification are comparable to those observed during 1 + age
smoltification of Atlantic salmon. To date, only limited data on Atlantic salmon under-
yearling smoltification exists. However, changes in plasma growth hormone levels
(Bjornsson et al., 2000), gill Na+,K+-ATPase activity, hypoosmoregulatory ability and
seawater tolerance (Berge et al., 1995; Duston and Saunders, 1995; Handeland and
Stefansson, 2001) have been found to be comparable to those occurring during 1 + age
smoltification. The present study strengthens the view that photoperiod-induced smolti-
fication in underyearlings elicits similar endocrine and physiological responses as occur
during normal smoltification (McCormick et al., 1991, 1995). The data on growth,
condition factor, body silvering, gill Na+,K+-ATPase activity and SW survival indicate
that both the yearling and underyearling fish of the present study smoltified during the
experiments. Furthermore, the almost 10-fold, transient increase in plasma cortisol levels
during the photoperiod-induced smoltification of underyearling fish is well in line with
reports for several species of naturally smolting salmonids (Specker and Schreck, 1982;
Virtanen and Soivo, 1985; Langhorne and Simpson, 1986; Young et al., 1989; Shrimpton
et al., 1994; Shrimpton and McCormick, 1998).
Although smoltification-related increases in cortisol levels are well established, only
few studies have so far addressed the question of whether this change is governed by
photoperiod. In Atlantic salmon, a rapid increase in daylength in early spring induced an
increase in plasma cortisol levels (McCormick et al., 2000), and although plasma cortisol
levels increase even when Atlantic salmon are kept on continuous light, this increase is not
as pronounced as in fish kept under natural photoperiod (Stefansson et al., 1989). The
present study further supports a causal relationship between photoperiod and plasma
cortisol levels, as plasma cortisol values were at low and stable levels during the 6-week
‘‘winter’’ phase (12L:12D), and then increased to a distinct and transient peak 3 weeks
after return to continuous light.
An important aspect of smoltification is that a minimum period of short-day exposure is
required for Atlantic salmon to complete the process, following an increase in daylength
(Clarke and Shelbourn, 1986; Bjornsson et al., 1989; Berge et al., 1995). However, the
question why the short-day period is needed has not been addressed. In the present study,
the condition factor increased and the gill Na+,K+-ATPase activity decreased during the
simulated winter in agreement with previous data (Berge et al., 1995). It may be speculated
that these physiological changes are related to changes in plasma growth hormone (GH)
levels. This, as GH levels have been found to decrease during a 6-week exposure of
K. Sundell et al. / Aquaculture 222 (2003) 265–285 279
underyearling Atlantic salmon to short daylength (Bjornsson et al., 2000), and the
hormone is known to stimulate gill Na+,K+-ATPase and decrease condition factor during
the smoltification process (see for references, Bjornsson, 1997). The endocrine and
physiological changes occurring during ‘‘winter’’ (this study, Berge et al., 1995; Bjornsson
et al., 2000) demonstrate that developmental changes are already taking place during this
short-day phase. Therefore, if these changes are necessary for the preceding smoltification
process, this may help explain the importance of a minimum winter period.
In SW living fish, there is an elevated intestinal ion and fluid transport compared with
FW fish, reflecting the need for fish in SW to absorb water (Smith, 1930; Skadhauge,
1969). This ion-coupled water transport is ultimately dependent on the basolaterally
located Na+,K+-ATPases (see Loretz, 1995). The present study suggests that the prea-
daptive elevation in intestinal fluid transport (Jv) seen during parr–smolt transformation of
Atlantic salmon is also, at least partly, due to an increase in intestinal Na+,K+-ATPase
activity. This mechanism has also been suggested, but not directly measured, for coho
salmon, Oncorhynchus kisutch, and Atlantic salmon, where the selective Na+,K+-ATPase
inhibitor, ouabain, was shown to decrease the Jv across intestinal sac preparations by 67–
100%, (Collie and Bern, 1982; Veillette et al., 1993). The lack of increase in Jv after 4
weeks of SW acclimation, despite an increased intestinal Na+,K+-ATPase activity, is
difficult to interpret. Similar patterns has been shown under certain occasions in other
studies (Veillette et al., 1993), whereas most studies have demonstrated a higher Jv for
SW-adapted than FW-adapted salmonids (Collie and Bern, 1982; Veillette et al., 1993).
While the major mechanism of ion transport across the intestine is understood, the main
route for water flow, transcellular or paracellular, has not yet been established (Alves et al.,
1999). The permeability for both these routes can be physiologically controlled by
regulatory mechanisms. The paracellular permeability is mainly controlled by regulation
of the tight junctions (Madara and Pappenheimer, 1987; Daugherty and Mrsny, 1999),
whereas the transcellular permeability to water can be regulated by the composition of the
membrane lipids (Hill et al., 1999) and/or by the incorporation of aquaporins into the
membranes (Ma and Verkman, 1999). Several studies have addressed the question of
regulation of ion conductance of the intestinal tight junctions in fish (Bakker and Groot,
1989; Bakker et al., 1993; Loretz, 1995), but no reports are available on the regulation of
paracellular permeability to water flow.
Fish intestinal epithelia have mostly been reported to have TER between 30 and 200 V
cm2 and can thus be characterized as leaky epithelia (Claude and Goodenough, 1973;
Loretz, 1995; Sundell, unpublished). The TER of such leaky epithelia mainly reflects the
resistance in the paracellular pathway (Loretz, 1995) and is thus considered as a measure of
the paracellular permeability. The water transport across leaky epithelia is generally
considered to be paracellular (Collie, 1985; Ma and Verkman, 1999), but in the SW-
adapted eel, considerable water flow across isolated vesicles of the intestinal brush-border
membrane have been demonstrated (Alves et al., 1999). This clearly suggests a transcellular
route for water flow in fish intestine, in line with recent studies on mammalian water
transport (Lennernas, 1995). The elevated TER of the SW-transferred Atlantic salmon, of
the present study, is well in agreement with a recent study on rainbow trout, where SW-
adapted fish had higher TER and Papp for mannitol than FW-adapted fish (Sundell,
unpublished). Thus, for both rainbow trout and Atlantic salmon, the demonstrated decrease
K. Sundell et al. / Aquaculture 222 (2003) 265–285280
in paracellular permeability in SW suggests that an increased transcellular water uptake,
rather than a paracellular, is responsible for the increased Jv in SW-adapted fish. This is
plausible, as the drinking rate of fish is higher in SW than FW (Perrott et al., 1992), which
results in an increased exposure of the intestinal mucosa to water-borne substances. It
would therefore be beneficial for SW fish to restrict the route for passive passage of
substances, i.e. the paracellular pathway, and instead increase the transcellular water flow.
Regarding the regulation of transcellular flow of water across intestinal epithelia, recent
studies have demonstrated the presence of several aquaporins in the intestine of fish
(Lignot et al., 2002) and mammals (Ma and Verkman, 1999), but the function of these
proteins is still not known. No clear model, as suggested for the kidney collecting duct
(Klussman et al., 2000), has so far been demonstrated for the intestine. It is clear, however,
that the intestinal lipid composition can change after SW adaptation. Transfer of masu
salmon and rainbow trout from FW to SW resulted in an increased level of n-3
polyunsaturated fatty acids (n-3 PUFA) of the intestinal brush-border membrane (Leray
et al., 1984) and total intestinal tissue (Li and Yamada, 1992). This increased proportion of
n-3 PUFA in the brush-border membrane was concomitant with an increased fluidity of the
membrane (Leray et al., 1984), which can be correlated to increased water permeability
(Brasitus et al., 1986; Lande et al., 1995). This is consistent with the observations in the
present study, where intestinal paracellular permeability, as judged by increased intestinal
TER, of Atlantic salmon decreases after the fish has been adapted to SW. Thus, together,
these results suggest a higher resistance for water through the paracellular pathway
concomitant with lower resistance through the transcellular pathway after SW transfer
leading to an increased proportion of water flow through the cells.
Cortisol is the main stimulator of increased intestinal fluid transport during the parr–
smolt transformation of Atlantic salmon (Veillette et al., 1995). In rainbow trout, cortisol
implants increased the paracellular permeability, as judged both by measurements of TER
and Papp for mannitol (Sundell, unpublished results). During the photoperiod-induced
parr–smolt transformation, the TER was about 20% lower after 3 and 6 weeks on
continuous light. While this decrease was not statistically significant, taking other
available data into account, which show cortisol to increase Jv during parr–smolt
transformation in Atlantic salmon (Veillette et al., 1995) and to increase paracellular
permeability in rainbow trout (Sundell. unpublished), the physiological mechanisms can
be speculated upon. Thus, it appears likely that the transient increase in plasma cortisol
that occurs during parr–smolt transformation will increase Jv through an increased
intestinal paracellular permeability while the fish are still in FW.
The salmon intestine consists of several morphologically distinct parts. Distal to the
pyloric ceca, two regions can be distinguished, the anterior and posterior (rectal) intestine,
which are separated by the ileorectal valve. The anterior intestine is mainly responsible for
nutrient uptake (Collie and Ferraris 1995; Loretz 1995), whereas ion and water uptake take
place along the length of the intestine (see Loretz, 1995). Thus, the Na+,K+-ATPase
activity of the anterior intestine has a double role in creating Na+ gradients to propel both
nutrient uptake and osmoregulation. This is supported by the consistently higher Na+,K+-
ATPase activity of the anterior intestine, as demonstrated in the present study as well as in
earlier studies on brown trout (Nielsen et al., 1999) and rainbow trout (Rey et al., 1991).
Furthermore, intestinal Na+,K+-ATPase activity following the return to continuous light
K. Sundell et al. / Aquaculture 222 (2003) 265–285 281
(i.e. during the photoperiod-manipulated smoltification) increased only in the anterior part,
which can be suggested to be due to an increased need for nutrients during this energy-
demanding developmental stage (McCormick et al., 1989). The Jv, on the other hand, is
generally higher in the posterior than the anterior part of the intestine (Collie and Bern,
1982; Veillette et al., 1993) and is mainly elevated in the posterior part during parr–smolt
transformation (Veillette et al., 1993). These results are in agreement with the possible
effect on the paracellular permeability in the posterior intestine, where TER had a tendency
to decrease following return to continuous light. Thus, the increased Jv in the posterior
intestine could be due to an increased paracellular permeability during the parr–smolt
transformation.
To summarize, it is still not fully elucidated through what mechanisms cortisol
increases Jv during the parr–smolt transformation. However, the increased Na+,K+-
ATPase activity and the decreased paracellular permeability following SW entry suggest
that the Jv, during this phase, is mainly driven by the increased ion transport and that the
route of water flow may be directed towards a more transcellular pathway.
Acknowledgements
The authors thank Barbro Egner, Gunilla Eriksson and Ivar Helge Matre for excellent
technical assistance, and Elisabeth Jonsson and Victoria Johansson for their assistance
during sampling. Per Nilsson and Carl Andre are acknowledged for their helpful
discussions regarding the statistical analyses. This study was financed by grants from the
Swedish Council for Agricultural and Forestry Research and the Wallenberg Foundation
VIRTUE project to BThB and KS, as well as by the Royal Society of Arts and Sciences in
Goteborg and C.F. Lundstroms Stiftelse to KS. All experimental and animal care
procedures were approved by the appropriate ethical committees for animal research in
Sweden and Norway.
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