intestinal transport mechanisms and plasma cortisol levels … · parr–smolt transformation of...

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


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


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.


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


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).


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).


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).


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.


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.


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.


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.


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


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


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


(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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