Intestinal transport mechanisms and plasma cortisol levels during normal and out-of-season parr–smolt
transformation of Atlantic salmon, Salmo salar
Kristina Sundell
a,*, Fredrik Jutfelt
a, Thorleifur A ´ gu´stsson
a,b, Rolf-Erik Olsen
c, Erik Sandblom
a, Tom Hansen
c,
Bjo¨rn Thrandur Bjo¨rnsson
aaFish Endocrinology Laboratory, Department of Zoology/Zoophysiology, Go¨teborg University, Box 463, S-405 30 Go¨teborg, Sweden
bdeCODE Genetics, Inc., Sturlugata 8, IS 101 Reykjavı´k, Iceland
cInstitute 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 matterD2003 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:k.sundell@zool.gu.se (K. Sundell).
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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.
D2003 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 Clmainly 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,
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 Bjo¨rnsson, 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 (11 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 10jC, during the 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, Go¨teborg 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 10jC. 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 (thusn= 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 l1, Sigma).
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), using juvenile 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 (11 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 10.3jC. 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; Bjo¨rns- 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 9.9jC, 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 l1, 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 weight100fork length3). After anesthesia, blood was collected from the caudal vessels using 1-ml heparinized syringes. The blood was centrifuged at 3000gfor 5 min to obtain plasma, which was aliquoted, frozen on dry ice and stored at80jC 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 80jC 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 3000gfor 30 s, 10Al of the supernatant was added in duplicate to 200Al 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 toLowry et al. (1951). Na+,K+-ATPase activity was expressed asAmol ADP mg protein1h1.
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 (5000g for 1 min), Na+,K+-ATPase activity was measured in 10Al 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 byVeillette et al. (1993). Briefly, the intestine were tied at the distal end, filled with Ringer solution (at 10jC) 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 asAl cm2h1.
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 10jC 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 cm2and 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. Gra˚sjo¨, Department of Pharmaceutics, Uppsala University. The procedures for electrical measurements, automatic data analysis and presentation are described inWik- man-Larhed and Arthursson (1995). In short, direct current pulses of 15, 15, 30, 30 and 0AA 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 ml1) and in the serosal compartment to normal Ringer solution. Samples of 40Al 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).
Papp¼dQ=dt1=AC0 ð1Þ
where dQ/dtis the steady-state appearance rate of the compound on the serosal side,C0the initial concentration of the compound on the mucosal side of the membrane, andAis the exposed tissue surface area.
2.3.7. Statistical analyses
All data are expressed as mean valuesFS.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 ofp>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, independentt-test (two-tailed) for sequential time-points was used to explore differences in time. Significance was accepted atp< 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 36.7F1.3 to 62.7F2.5 g in weight and from 14.8F0.2 to 18.3F0.2 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 meansFS.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, withn= 2 – 4 throughout the experiment for Papp, andn= 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 meansFS.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 39.8F2.8 g and 14.2F0.4 cm, and the condition factor 1.35F0.02 (Fig. 4A). During the ‘‘winter’’ phase of the experiment (6 weeks on 12L:12D), the CF increased to a value of 1.44F0.02. 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 meansFS.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 1.28F0.02 and then further to 1.19F0.02, 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 4.0F0.81 ng ml1 and 2.82F0.43 Amol ADP h1 mg protein1, 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 (2.41F0.52 ng ml1and 1.48F0.16 Amol ADP h1 mg protein1). 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 meansFS.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, independentt-test as post hoc procedure.
significantly after 3 weeks on 24L (23.3F3.9 ng ml1) whereas Na+,K+-ATPase activity remained in the same range as during the simulated ‘‘winter’’ phase (1.84F0.25Amol ADP h1mg protein1). 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 for3 1 2= months (dark grey bars). Data are shown as meansFS.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, independentt- test as post hoc procedure.
significantly (3.93F0.45Amol ADP h1mg protein1,Fig. 5B) and the plasma cortisol levels decreased again (7.33F1.35 ng ml1, Fig. 5A). Following SW transfer, gill Na+,K+-ATPase activity increased further (9.9F0.75 Amol ADP h1 mg protein1), whereas the plasma cortisol levels were in the same range (8.8F1.87 ng ml1,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 (0.73F0.19 Amol ADP h1 mg protein1) and had increased significantly after 6 weeks on continuous light (2.15F0.59 Amol ADP h1 mg protein1). 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; 133.2F10.6
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 meansFS.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, independentt-test as post hoc procedure.
Vcm2) to the two sampling times 3 and 6 weeks after the return to 24L (111.4F6.4 and 111.4F6.3V 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 meansFS.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, independentt-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 (Bjo¨rnsson 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; Bjo¨rnsson 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 (Bjo¨rnsson 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, Bjo¨rnsson, 1997). The endocrine and physiological changes occurring during ‘‘winter’’ (this study,Berge et al., 1995; Bjo¨rnsson 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 200V 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(Lennerna¨s, 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 Egne´r, Gunilla Eriksson and Ivar Helge Matre for excellent technical assistance, and Elisabeth Jo¨nsson 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 Go¨teborg and C.F. Lundstro¨ms 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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