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First published online October 5, 2006
Journal of Experimental Biology 209, 4040-4050 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02462

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Plasticity of osmoregulatory function in the killifish intestine: drinking rates, salt and water transport, and gene expression after freshwater transfer

Graham R. Scott^1,*, Patricia M. Schulte¹ and Chris M. Wood²

¹ Department of Zoology, University of British Columbia, Vancouver BC, V6T 1Z4, Canada
² Department of Biology, McMaster University, Hamilton ON, L8S 4K1, Canada

^* Author for correspondence (e-mail: scott{at}zoology.ubc.ca)

Accepted 31 July 2006

	Summary

TOP Summary Introduction Materials and methods Results Discussion References

 
We have explored intestinal function in the euryhaline killifish Fundulus heteroclitus after transfer from brackish water (10% seawater) to fresh water. Plasma Na⁺ and Cl^– concentrations fell at 12 h post-transfer, but recovered by 7 days. Drinking rate decreased substantially at 12 h (32% of control value) and remained suppressed after 3 and 7 days in fresh water (34 and 43%). By contrast, there was a transient increase in the capacity for water absorption measured across isolated intestines in vitro (3.3- and 2.6-fold at 12 h and 3 days), which returned to baseline after 7 days. These changes in water absorption could be entirely accounted for by changes in net ion flux: there was an extremely strong correlation (R²=0.960) between water absorption and the sum of net Na⁺ and net Cl^– fluxes (3.42±0.10 µl water µmol^–1 ion). However, enhanced ion transport across the intestine in fresh water would probably not increase water uptake in vivo, because the drinking rate was far less than the capacity for water absorption across the intestine. The increased intestinal ion absorption after freshwater transfer may instead serve to facilitate ion absorption from food when it is present in the gut. Modulation of net ion flux occurred without changes in mRNA levels of many ion transporters (Na⁺/K⁺-ATPase _1a, carbonic anhydrase 2, CFTR Cl^– channel, Na⁺/K⁺/2Cl^– cotransporter 2, and the signalling protein 14-3-3a), and before a measured increase in Na⁺/K⁺-ATPase activity at 3 days, suggesting that there is some other mechanism responsible for increasing ion transport. Interestingly, net Cl^– flux always exceeded net Na⁺ flux, possibly to help maintain Cl^– balance and/or facilitate bicarbonate excretion. Our results suggest that intestinal NaCl absorption from food is important during the period of greatest ionic disturbance after transfer to fresh water, and provide further insight into the mechanisms of euryhalinity in killifish.

Key words: Fundulus heteroclitus, intestine, water absorption, ion flux, drinking rate, cortisol, Na/K-ATPase, NKCC, CFTR, carbonic anhydrase, fish

	Introduction

TOP Summary Introduction Materials and methods Results Discussion References

 
The euryhaline killifish Fundulus heteroclitus lives in brackish water estuaries and salt marshes on the eastern coast of North America. The salinity of these habitats routinely fluctuates, so killifish must dynamically regulate ion balance in their natural environment. This species is exceptionally euryhaline, and can tolerate salinities ranging from fresh water to nearly four times sea water (Griffith, 1974). Killifish have therefore been used extensively in the past to understand the basic physiological mechanisms of osmoregulation in aquatic animals (reviewed by Wood and Marshall, 1994). More recently, killifish have been useful in understanding osmoregulatory plasticity, and are revealing many of the physiological mechanisms of euryhalinity in fish (Marshall, 2003).

For example, killifish rapidly reduce ion loss and activate ion uptake across the gills after freshwater transfer (Wood and Laurent, 2003). Ion loss is minimized by decreasing paracellular permeability (Karnaky, 1992; Scott et al., 2004b) and by suppressing active ion secretion via secretory ion transporters; the latter can occur by protein internalization (Marshall et al., 2002b), protein phosphorylation and inactivation (Marshall et al., 2000; Kültz et al., 2001), or suppression of ion transporter expression (Scott et al., 2004a). Ion uptake across the gills is known to be activated by increasing the expression and activity of absorptive ion transporters (Scott et al., 2004a; Scott et al., 2005a; Scott et al., 2005b), and by increasing cell proliferation (Katoh and Kaneko, 2003; Scott et al., 2005b) and differentiation (Marshall et al., 1999; Daborn et al., 2001; Katoh et al., 2001).

In contrast to gill function, much less is known about how intestinal function is modulated in response to freshwater transfer. Similar to other euryhaline fish, killifish acclimated to fresh water are known to have lower drinking rates than those acclimated to seawater (Potts and Evans, 1967). This response presumably helps minimize potentially confounding water absorption in fresh water; however, little is known about the temporal pattern of the response, or whether water and ion transport across the intestine behave similarly. Regardless, killifish rapidly re-establish osmotic balance after transfer to fresh water (Jacob and Taylor, 1983; Scott et al., 2004a), so this modulation of intestinal function is effective.

Water absorption across the intestine of fish is primarily driven by transepithelial ion transport (Loretz, 1995; Schettino and Lionetto, 2003; Grosell et al., 2005). Na⁺ and Cl^– transport across the apical (luminal) surface of the intestine likely occurs through Na⁺/K⁺/2Cl^– cotransporters (NKCC) and Na⁺/Cl^– cotransporters (but see Howard and Ahearn, 1988). Basolateral (serosal) Na⁺/K⁺-ATPase provides the electrochemical gradient for this process, and transports Na⁺ across the basolateral surface. Apical Cl^– absorption may also occur in exchange for HCO₃^–, which is formed by the hydration of CO₂ by carbonic anhydrase (CA) (Howard and Ahearn, 1988; Grosell et al., 2005). Chloride transport may also involve a CFTR Cl^– channel (Marshall et al., 2002a).

The objective of the present study was to characterize intestinal function in the common killifish Fundulus heteroclitus after transfer to fresh water. Drinking rates, intestinal water and ion transport, Na⁺/K⁺-ATPase activity, plasma cortisol, and expression of genes potentially involved in ion transport were assessed at several times after transfer from brackish water to fresh water. Our initial hypotheses were that both drinking and intestinal ion and water transport would decrease after transfer, and that the latter would be coupled to changes in expression of the measured ion transport genes, as previously seen in the gills and opercular epithelium (Scott et al., 2004a; Scott et al., 2004b; Scott et al., 2005a; Scott et al., 2005b). Our results confirm some of these hypotheses but disprove others, and reveal surprising dissociations between drinking rate, fluid absorption and the molecular components of intestinal osmoregulation in killifish.

	Materials and methods

TOP Summary Introduction Materials and methods Results Discussion References

 
Experimental animals
Adult killifish Fundulus heteroclitus L. (3–8 g body mass) were captured from estuaries near Antigonish, Nova Scotia, Canada. Fish were held in static charcoal-filtered Fiberglass tanks at a salinity of 10% seawater (3.5) made up with dechlorinated Hamilton, Ontario tap water ([Na⁺], 0.6 mmol l^–1; [Cl^–], 0.7 mmol l^–1; [Ca²⁺] 1.0 mmol l^–1, [Mg²⁺] 0.2 mmol l^–1; hardness=120 mg l^–1 as CaCO₃; pH 8.0). Fish were maintained at room temperature (18–22°C) in a 14 h:10 h L:D photoperiod. Fish were fed once daily to satiation with a mix of commercial flakes (Wardley Total Tropical Gourmet Flake Blend, Hartz Mountain Corp., Secaucus, NJ, USA) and frozen brine shrimp (San Francisco Bay Brand, Newark, CA, USA). All animal care and experimentation was conducted according to McMaster University animal care protocol #02-10-61.

Salinity transfer protocol
Fish were acclimated to a salinity of 10% seawater (brackish water) for at least 1 month before salinity transfer. Fish were transferred from brackish water to fresh water because (1) it is more environmentally representative of what killifish would naturally encounter in estuaries, (2) brackish water is the preferred salinity for killifish (Fritz and Garside, 1974), and (3) we sought to compare the results of the present work with previous studies using the same protocol. In experiments 1 and 3, drinking rates (experiment 1) and intestinal water and ion fluxes (experiment 3) were measured in fish before transfer, or 12 h, 3 days, or 7 days after transfer to fresh water. In experiment 2, the Na⁺ and Cl^– concentrations in the fluid phase of the gut contents were measured before and 12 h after transfer to fresh water. In experiment 4, mRNA expression in the whole intestine was measured 12 h, 3 days or 7 days after transfer either to brackish water (i.e. a sham treatment where animals were transferred to a new tank having the same salinity) or to fresh water. In experiment 5, Na⁺/K⁺-ATPase activity in intestinal segments as well as plasma cortisol were measured before and 12 h, 3 days, or 7 days after transfer either to brackish water or to fresh water. All fish transfers were made using a net. As feeding is known to be essential to keep the fish healthy after transfer to fresh water, in all series, feeding was continued until 24 h before the actual experiment, the same protocol as used previously (Wood and Laurent, 2003). The only exception was for one treatment in experiment 2 where an alternate protocol was evaluated (see below).

Experiment 1: drinking rates
Drinking rates were measured in static polyethylene chambers containing 200 ml of the appropriate water. Chambers were fitted with a lid and aeration line and were wrapped in black plastic to minimize disturbance of the fish. The fish were allowed to settle for 2 h prior to measurement. At the start of each measurement period, approximately 8 µCi (0.29 MBq) of radiolabelled polyethylene glycol ([³H]PEG-4000, 57.70 MBq g^–1; NEN Life Science Products Inc., Boston, MA, USA) was added to the chamber. A water sample (5 ml) was taken immediately for radioactivity measurements, as well as at 3 h and 6 h, when the experiment was ended. The fish was then killed with a lethal dose of tricaine methanesulfonate anaesthetic (0.8 g l^–1 MS-222; Syndel Laboratories, Vancouver, BC, Canada) neutralized with NaOH, the exact time was noted, and then the fish was removed from its chamber, rinsed in clean water, and weighed. A terminal blood sample was collected by caudal puncture using a modified 100 µl Hamilton syringe. Blood was transferred to lithium-heparinized capillary tubes, centrifuged at 500 g for 5 min, and the separated plasma used for [³H]PEG-4000 radioactivity measurements. There was never any radioactivity in the plasma samples, indicating that [³H]PEG-4000 was not absorbed but always stayed in the gastrointestinal tract. The gastrointestinal tract was then exposed by a ventral incision and ligated at both ends (anterior oesophagus and rectum) to prevent loss of contents. The entire gastrointestinal tract was removed, weighed, and then digested in 0.8 ml of 2 mol l^–1 HNO₃ at 65°C for 48 h in a sealed vial. These samples were centrifuged, and supernatants (0.7 ml) were removed for radioactivity measurements. Drinking rate was calculated by determining the volume of external water taken into the tract from the radioactivity counts in the total tract digest and the reference water samples, and expressing this volume relative to the mass of the individual fish and the [³H]PEG-4000 exposure time. The actual experimental period (approximately 6 h) was scheduled such that the nominal time (e.g. 12 h post-transfer) would be in the middle.

Experiment 2: Na⁺ and Cl^– concentrations of the gut contents of killifish
In experiment 2, fish were rapidly killed as in experiment 1. The whole intestinal tract was ligated immediately posterior to the oesophagus and at the anus, then removed. The entire gut contents were collected, and centrifuged at 10 000 g for 1 min. The supernatant was collected, and the volume of the supernatant and mass of the solid material were measured gravimetrically. Na⁺ and Cl^– concentrations were measured in the free supernatant. Ion concentrations were determined after either 24 h (when solid material was still present in the gut) or 3 days of starvation (when solid material remaining in the gut was much reduced or absent), to determine if the presence of food impacted the ionic composition of the gut fluids. In some fish, gut contents were too small to be extracted; data for such fish are not reported.

Experiment 3: intestinal water transport and ion flux
Before transfer to fresh water, and 12 h, 3 days and 7 days after transfer, fish were lightly anaesthetized in MS-222 (0.1 g l^–1), killed by a blow to the head, and then the blood was sampled, as described above. The plasma was frozen (–20°C) for later analysis of Na⁺ and Cl^– concentrations. The killifish lacks a distinct stomach (Babkin and Bowie, 1928), so the whole intestinal tract posterior to the oesophagus was removed. Heat-flared PE-50 polyethylene tubing was inserted and tied into the anterior end, and the tract was flushed thoroughly to clear any remaining chyme, using modified Cortland saline (Wolf, 1963). The composition, in mmol l^–1, was NaCl 133, KCl 5, CaCl₂.2H₂O 1, MgSO₄.7H₂O 1.9, NaH₂PO₄.H₂O 2.9, glucose 5.5; pH 7.4. The sac was then filled with a 0.5 ml (exact volume) of this saline, which had been radiolabelled with 0.1 µCi ml^–1 (0.004 MBq ml^–1) ²²Na (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA), the posterior end was tied closed, and the catheter was sealed with a pin. Saline (rather than brackish water or fresh water) was used in the sac to avoid passive water or ion movements due to osmotic gradients. Furthermore, the results from experiment 2, as well as those in previous studies (Shehadeh and Gordon, 1969), indicated that gut fluid in vivo is brought near isosmotic saline regardless of salinity or whether fish are starved or fed. A sample of the filling solution (1 ml) was taken for ²²Na counting, and analysis of total Na⁺ ion concentration ([Na⁺]) and [Cl^–].

The sac was blotted on tissue paper in a standardized manner, weighed to 0.1 mg accuracy on an analytical balance, then suspended in a scintillation vial containing 11 ml of the same saline, but not radiolabelled. The external saline was continually bubbled with a humidified gas mixture containing 99.7% O₂ and 0.3% CO₂ (i.e. P_CO2=2.25 mm Hg). The incubation period was 4 h, with sac mass recorded at 0 h, 2 h and 4 h, and samples of the external (serosal) solution (1 ml) taken at these same times. In addition, final samples were taken of the internal (mucosal) saline at 4 h for ²²Na counting and analysis of total [Na⁺] and [Cl^–]. The sac was cut open, and the gross area of the exposed epithelial surface determined by tracing its outline onto graph paper. Fluid transport was determined from changes in sac mass, and was linear over time. Net ion fluxes were calculated from measured changes in the net Na⁺ and Cl^– contents of the mucosal solution (volume x concentration) over the 4 h period. Unidirectional Na⁺ fluxes were calculated from the appearance of ²²Na counts in the serosal solution, and the specific activity (c.p.m. nmol^–1) for Na⁺ of the mucosal solution. Unidirectional flux rate was calculated over the first 2 h period only to avoid the uncertainties of correcting for significant isotopic recycling in the second 2 h period. An exponential decay function was used to estimate the mean mucosal specific activity during the 2 h period. All flux rates were expressed as a function of surface area. A typical 4 g killifish had a gross intestinal surface area of about 10 cm².

Experiment 4: intestinal gene expression
In experiment 4, fish were killed by a blow to the head, followed by rapid decapitation, and the intestinal tract was removed and then immediately frozen in liquid nitrogen. Tissues were stored at –80°C until analyzed. RNA was extracted and reverse transcribed as previously described (Scott et al., 2004a; Scott et al., 2005a). Briefly, total RNA was extracted from tissues (approximately 20 mg) using Tripure isolation reagent (Roche Diagnostics, Montreal, QC, Canada). RNA concentrations were determined using a spectrophotometer and RNA integrity was verified by electrophoresis. Extracted RNA was stored at –80°C following isolation. First strand cDNA was synthesized by reverse transcribing 3 µg total RNA using 10 pmol oligo(dT₁₈) primer and 20 i.u. RevertAid H Minus M-MuLV reverse transcriptase (MBI Fermentas, Burlington, ON, Canada).

Quantitative real-time PCR (qRT-PCR) analysis of gene expression has also been described previously (Scott et al., 2004a; Scott et al., 2005a; Scott and Schulte, 2005). Primer sequences for killifish cDNA, Na⁺/K⁺-ATPase _1a (accession number AY057072) (Semple et al., 2002), cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel (acc. no. AF000271) (Singer et al., 1998), absorptive (apical) Na⁺/K⁺/2Cl^– cotransporter 2 (NKCC2, acc. no. AY533707) and elongation factor 1 (EF1, expression control; acc. no. AY430091) (Scott et al., 2004a), carbonic anhydrase 2 (CA2, acc. no. AY796057) (Scott et al., 2005a), and the signalling protein 14-3-3a (acc. no. AF302039) (Kültz et al., 2001) have been reported previously (Scott et al., 2004a; Scott et al., 2005a). These genes were chosen because we have previously shown that they respond to salinity transfer in the gills and/or opercular epithelium of killifish. Quantification of gene expression by qRT-PCR was performed on an ABI Prism 7000 sequence analysis system (Applied Biosystems, Foster City, CA, USA). A randomly selected control sample was used to develop a standard curve for each primer set, and all results were expressed relative to these standard curves. Expression of each gene of interest was then standardized to expression of the EF1a gene, which does not change in the intestine following salinity transfer (data not shown), and were expressed relative to the 12 h brackish water control samples. All samples were run in duplicate (coefficients of variation were 10%). Control reactions were conducted with no cDNA template or with non-reverse transcribed RNA to determine the level of background or genomic DNA contamination, respectively. Genomic contamination was below 1:87 starting cDNA copies for all templates.

Experiment 5: Intestinal Na⁺/K⁺-ATPase activity and plasma cortisol
In experiment 5, fish were rapidly killed as in experiment 1, intestines were removed, and blood was sampled and stored as described above. The intestinal tract was cut into anterior, middle, and posterior segments, which were immediately frozen separately in liquid nitrogen and then stored at –80°C until analyzed. Na⁺/K⁺-ATPase activity was determined by coupling ouabain-sensitive ATP hydrolysis to pyruvate kinase- and lactate dehydrogenase-mediated NADH oxidation as outlined by McCormick (McCormick, 1993), as we have previously reported (Scott et al., 2004a; Scott et al., 2005a). Plasma cortisol was determined by radioimmunoassay, as previously described (Scott et al., 2003).

Ion and radioactivity measurements
Sodium and chloride concentrations were determined using flame atomic absorption spectrophotometry (SpectrAA-220FS, Varian, Mulgrave, VC, Australia) and coulometric titration (CMT-10 chloridometer, Radiometer, Copenhagen, Denmark), respectively. The only exception was in the assay of gut fluid samples, where the small volume obtained precluded use of the chloridometer, so chloride was measured by a colorimetric assay (Zall et al., 1956). The same certified NaCl standard (Radiometer) was used for all analyses. ²²Na radioactivities in mucosal and serosal saline samples were determined using a Minaxi Autogamma 5000 counter (Packard Instruments, Downers Grove, IL, USA). For [³H]PEG-4000 radioactivities, 0.7 ml of the 2 mol l^–1 HNO₃ intestinal digest or 20 µl of plasma was added to 10 ml of an acid-compatible scintillation cocktail (Ultima Gold; Packard Bioscience, Meriden, CT, USA), and 5 ml water samples were added to 10 ml of an aqueous compatible cocktail (ACS; Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). Radioactivity was measured by scintillation counting (Rackbeta 1217; LKB Wallac, Turku, Finland). Quench was shown to be uniform across samples, and data were corrected for the slight difference in counting efficiencies between the two scintillation fluors.

Statistical analyses
Data are expressed as means ± s.e.m. All data passed tests of normality and homogeneity of variance, so ANOVA (1-way or 2-way, where appropriate) was used to ascertain overall differences. In experiments 1–3, the effects of freshwater transfer were assessed by comparison with pre-transfer controls using Student–Newman–Keuls (SNK) post-hoc comparisons. Because gene expression, Na⁺/K⁺-ATPase activity and plasma cortisol levels can change as a result of handling the fish alone (Scott et al., 2004a), the effects of freshwater transfer in experiments 4 and 5 were assessed by comparing expression levels with time-matched brackish water controls using SNK post-hoc comparisons. The effects of handling in experiments 4 and 5 were assessed using SNK comparisons with 12 h brackish water controls or pre-transfer controls, respectively. All statistical analyses were conducted using Sigmastat version 3.0 and a significance level of P<0.05 was used throughout.

	Results

TOP Summary Introduction Materials and methods Results Discussion References

 
Drinking rates, plasma ions and cortisol after freshwater transfer
Killifish drank significantly less after transfer to fresh water (Fig. 1). Before transfer from brackish water, the drinking rate of killifish was 1.32±0.22 ml kg^–1 h^–1, but 12 h after freshwater transfer the drinking rate decreased to 32% of the pre-transfer value. The reduced drinking rate persisted at 3 and 7 days after transfer, at 34% and 43% of the brackish water value.

View larger version (5K): [in this window] [in a new window]   Fig. 1. Drinking rate of killifish decreases after transfer from brackish water (BW, 10% sea water; grey bars) to fresh water (FW; black bars). Values are means ± s.e.m. (N6). *Significant difference from pre-transfer (Pre) brackish water control (P<0.05).

View larger version (14K): [in this window] [in a new window]   Fig. 2. (A) Plasma concentrations of Na+ (solid bars) and Cl– (hatched bars) in killifish decrease after transfer from brackish water (BW, 10% seawater; grey bars) to fresh water (FW; black bars) and recover slowly thereafter. (B) Plasma cortisol concentrations increase transiently as a result of transfer to freshwater, compared to both pre-transfer and time-matched BW controls. Values are means ± s.e.m. (N6). *Significant difference from pre-transfer (Pre) brackish water control (P<0.05). Significant difference between time-matched FW and BW groups.

Water and ion transport in the intestine after freshwater transfer
The ion concentrations of gut fluids were high, ranging between 46–72% of the Na⁺ concentration of physiological saline and 64–78% of the Cl^– concentration (Table 1). Although freshwater transfer reduced [Na⁺] of gut fluids (overall effect, determined by 2-way ANOVA), levels were still more than 100-fold higher than [Na⁺] in imbibed freshwater. Furthermore, [Cl^–] of gut fluids did not change as a result of transfer to freshwater. The degree of starvation had no effect on the ion concentration of the gut fluids, though the solid mass of the gut contents was greatly reduced after 3 days of starvation. However, transfer to freshwater and prolonged starvation appeared to reduce the volume of gut fluids (all these comparisons relative to brackish water fish that had only been starved for 1 day; data not shown). Regardless, these results suggest that absorption of water and ions across the gut occurs from a mucosal solution that is closer in composition to extracellular fluids than to ingested water, and that this is not changed by the presence of food.

Water absorption in killifish intestine increased transiently after transfer to freshwater (Fig. 3). Bulk water flow across isolated intestines was 0.00109±0.00031 ml cm^–2 h^–1 in brackish water, and increased by 3.3-fold at 12 h after transfer to freshwater. Bulk flow remained elevated (2.6-fold) 3 days after transfer, but by 7 days after transfer bulk flow was not significantly different from the brackish water value.

View larger version (6K): [in this window] [in a new window]   Fig. 3. Bulk water transport rate across the isolated intestine increases transiently in killifish, after transfer to fresh water (FW; black bars) from brackish water (BW, 10% seawater; grey bars). Net absorption occurs in the positive direction. Values are means ± s.e.m. (N6). *Significant difference from pre-transfer (Pre) brackish water control (P<0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 4. (A) Net Na+ (solid bars) and Cl– (hatched bars) flux across the isolated intestine increases transiently in killifish, after transfer to fresh water (FW; black bars) from brackish water (BW, 10% seawater; grey bars). (B) Unidirectional Na+ influx across the isolated intestine does not change significantly after fresh water transfer, but the small non-significant increase is enough to account for the change of net Na+ flux shown in A. Net absorption occurs in the positive direction. Values are means ± s.e.m. (N6). *Significant difference from pre-transfer (Pre) brackish water control (P<0.05). Net Cl– flux was found to be greater than net Na+ flux by two-way ANOVA.

View larger version (6K): [in this window] [in a new window]   Fig. 5. Bulk water absorption rate correlated to net strong ion absorption rate (sum of net Na+ and Cl– flux rate) across killifish intestine, in brackish water (BW, 10% seawater; grey squares) and at several time intervals after transfer to fresh water (FW; black triangles and diamonds). Bulk water transport was extremely well correlated to net ion transport (r2=0.960, P<0.0001), with a slope of 0.00342±0.00010 ml water per µmol strong ion. Grey broken curves represent 95% confidence limits of the regression.

Gene expression and Na⁺/K⁺-ATPase activity in the intestine after freshwater transfer
Transfer to freshwater did not change the expression levels of any of the mRNAs analyzed in the intestine of killifish (Table 2). Levels of Na⁺/K⁺-ATPase _1a, Na⁺/K⁺/2Cl^– cotransporter 2 (NKCC2), and CFTR Cl^– channel mRNAs all remained constant throughout the experiment, not changing as a result of either transfer to freshwater or time. Expression of carbonic anhydrase 2 (CA2) mRNA and mRNA for the signalling protein 14-3-3a were also unaffected by freshwater transfer (when compared to time-matched brackish water controls), but decreased throughout the experiment, suggesting that there could have been an effect of time (e.g. time after handling) on their expression.

The response of Na⁺/K⁺-ATPase activity to transfer to freshwater was analyzed in anterior, middle and posterior segments of the intestine (Fig. 6 and Table 3). Transfer to freshwater increased activity in the anterior portion of the intestine approximately 1.5-fold at 3 days after freshwater transfer (Fig. 6). By contrast, there were no effects of fresh water per se on activity in the middle or posterior segments (Table 3). There were significant effects of time on activity in these segments, but the pattern of these differences were inconsistent between segments.

View larger version (7K): [in this window] [in a new window]   Fig. 6. Na+/K+-ATPase activity in the anterior segment of the intestine increases transiently in killifish, after transfer to fresh water (FW; black) from brackish water (BW, 10% seawater; grey). Values are means ± s.e.m. (N8). *Significant difference from pre-transfer (`pre') brackish water control (P<0.05). Significant difference between time-matched FW and BW groups.

	Discussion

TOP Summary Introduction Materials and methods Results Discussion References

 
The ability of killifish to regulate water and ion homeostasis during environmental salinity fluctuations is exceptional, and depends on the concerted responses of many tissues, including gills, opercular epithelium, intestine and kidney. The mechanisms of euryhalinity at the gills and opercular epithelium are now reasonably well understood (Marshall, 2003; Scott et al., 2005a), but less is known about the intestine and kidney. In the present study we have enhanced our understanding of how intestinal function contributes to euryhalinity in killifish, and demonstrated that drinking rates and intestinal water and ion transport are dynamically and differentially regulated in response to freshwater transfer.

Plasticity of intestinal function after freshwater transfer
Earlier reports indicated that killifish that are fully acclimated to fresh water drink at rates only 10–35% of those acclimated to seawater, and at rates 42% of those that are fully acclimated to near-isosmotic brackish water (40% seawater) (Potts and Evans, 1967; Malvin et al., 1980). Similar differences have been seen in other euryhaline species (e.g. Mozambique tilapia, Oreochromis mossambicus) (Lin et al., 2000). The results of the present study are in agreement with this pattern and with our original hypothesis: killifish transferred to fresh water reduced their drinking rate by 68% within 12 h, and drank only 32–43% as much as animals fully acclimated to 10% seawater over the next 7 days (Fig. 1). In some anadromous species (e.g. Atlantic salmon Salmo salar and Japanese eel Anguilla japonica), drinking rates are similar to those of killifish in seawater, but unlike in killifish, drinking appears to stop altogether in these species in fresh water (Fuentes and Eddy, 1997; Aoki et al., 2003). In the present study, the initial fall in drinking rate appears to recover slightly over time in fresh water. We are unaware of any previous studies examining the time course of drinking rates after transfer to fresh water, but drinking rates rose continually over time after transfer from fresh water to seawater in Atlantic salmon (Fuentes and Eddy, 1997).

After water was ingested, its composition in the gut was adjusted to levels that are reasonably close to the composition of extracellular fluids (Table 1). These results were expected based on previous studies in other fish species (Shehadeh and Gordon, 1969; Ando et al., 2003), and was true in both brackish water and fresh water, and regardless of whether or not food was present in the gut. When starved fish drink hyposmotic water, salt is presumably secreted into the oesophagus or water is absorbed from it, before isosmotic fluid is reabsorbed in the intestine. Alternatively, when hyperosmotic water (or food) is consumed, water is added or salt is absorbed in the oesophagus (Ando et al., 2003). Therefore, although net absorption of water or ions from the environment depends on the composition of ingested water and food, the intestine absorbs water and ions from the fluid phase of chyme that is near-isosmotic. Clearly, ingested food is a much larger source of ions than ingested water when killifish are in fresh water.

In contrast to our initial hypothesis, and in contrast to the pattern in drinking rate, intestinal water absorption increased greatly after transfer to freshwater (Fig. 3). However, the response was transient, and had virtually ceased by 7 days. At 12 h post-transfer, the reduction in drinking rate to 32% of the brackish water value was coupled with a 3.3-fold increase in water absorption across isolated intestine. This might suggest that water absorption by the intestine in vivo falls more slowly than indicated by the drinking rate data alone, but another interpretation is more likely, and is discussed in a later section. Interestingly, eels acclimated to fresh water have similar intestinal water absorption rates to the killifish in this study, but in seawater-acclimated eel, intestinal water absorption rates are substantially higher (Ando, 1975; Aoki et al., 2003).

Contribution of the intestine to water and ion homeostasis in vivo
Even though the ionic composition of ingested fluid is adjusted once it enters the gut (Table 1) (Shehadeh and Gordon, 1969), water absorption from the environment by the intestinal tract in vivo cannot exceed the drinking rate. An approximate but instructive comparison can be made between the drinking rates recorded in vivo, and the bulk water absorption rates measured in vitro. Assuming that a 4 g killifish has an intestinal surface area of about 10 cm² (see Materials and methods), the estimated absorption rate would be about 2.5 ml kg^–1 h^–1 in brackish water, not too far from the measured drinking rate of 1.32 ml kg^–1 h^–1, indicating that supply and absorptive capacity are approximately matched. However after 12 h in fresh water, the absorptive capacity is about 8.75 ml kg^–1 h^–1, far in excess of the measured drinking rate (supply rate) of 0.42 ml kg^–1 h^–1. This calculation does not take into account additional fluid on the supply side that may be secreted in vivo by the oesophagus, biliary and pancreatic systems, etc.; nevertheless, it suggests that the observed bulk water transport rates recorded in vitro after transfer to fresh water do not occur in vivo.

What is the meaning of this discrepancy? At 12 h post-transfer, plasma [Na⁺] and [Cl^–] (Fig. 2), as well as whole body concentrations of these ions (Wood and Laurent, 2003) have fallen precipitously, but thereafter start to recover. Similar patterns have been seen in many previous studies on killifish transferred to fresh water (Jacob and Taylor, 1983; Wood and Laurent, 2003; Scott et al., 2004a; Scott et al., 2004b). We suggest that the increased net Na⁺ and Cl^– absorption, from chyme that would normally be present in the intestinal tract of animals that are feeding, is critical to ionic homeostasis at this time. In other words, killifish probably increase ion absorption to access the large reservoir of ions in food, not to absorb more water. Concurrent increases in the capacity for water absorption in vitro occur as a consequence of the increased ion absorption, but net water absorption from the environment would be minimal in vivo because of the reduced drinking rate (Fig. 1). Similar calculations to those outlined above indicate that the measured capacity for net ion absorption after 12 h in fresh water would amount to about 100 µmol kg^–1 h^–1 for Na⁺ and 150 µmol kg^–1 h^–1 for Cl^–, when measured unidirectional uptake rates at the gills have dropped to about 700 and 0 µmol kg^–1 h^–1, respectively, and net gill fluxes of both ions remain negative (Wood and Laurent, 2003). Because ion supply rate from drinking is so low in fresh water, this capacity for intestinal ion absorption could only be realized in vivo in animals that are feeding. The role of food in the normal ionic homeostasis of fish is often overlooked, but has been recently highlighted by Marshall and Grosell (Marshall and Grosell, 2005) as an important area for future work. We know that feeding is essential to keep killifish healthy in fresh water (Wood and Laurent, 2003), as it is in salmonids when gill uptake mechanisms are impaired, such as during low pH exposure (D'Cruz and Wood, 1998). Indeed, gut uptake of Cl^– must be critically important in killifish and other species (e.g. eel, Anguilla sp., and bluegill, Lepomis macrochirus) that lack branchial Cl^– uptake in fresh water (reviewed by Tomasso and Grosell, 2005).

Even though drinking rate is reduced in fresh water, if water uptake across the intestinal tract occurs in vivo as a result of heightened ion absorption, the kidney undoubtedly has the capacity to deal with it. Glomerular filtration rate (GFR) measured in fresh water in a previous study (Scott et al., 2004b) is approximately 15-fold higher than the drinking rates measured in the present study, and 4-fold higher than the sum of drinking rate and extrarenal clearance rate (an index of whole-body water permeability) (Scott et al., 2004b). Water excretion by the kidney therefore appears more than capable of maintaining total whole-body water content in fresh water.

Molecular responses to transfer to freshwater
In contrast to our initial hypothesis, the expression levels of a number of genes, inferred from mRNA levels, did not change after transfer from brackish to fresh water (Table 2). Although mRNA expression of two genes (those of carbonic anhydrase 2 and the signalling protein 14-3-3a) tended to decrease over time, this was not due to an effect of transfer to freshwater, but rather an effect of handling itself. Expression of Na⁺/K⁺-ATPase _1a, Na⁺/K⁺/2Cl^– cotransporter 2, and CFTR Cl^– channel remained constant in all fish throughout the experiment. This is markedly different from the patterns we have observed earlier in gills and opercular epithelium from killifish subjected to an identical brackish water to fresh water transfer (Scott et al., 2005a). In this previous study, mRNA expression of CFTR went down and that of 14-3-3a went up in both tissues, while carbonic anhydrase 2 expression changed in opposite directions in the two tissues. Note that mRNA levels of the absorptive Na⁺/K⁺/2Cl^– cotransporter 2 were assayed in the intestine in the present study, so it cannot be compared with the downward mRNA response of the secretory Na⁺/K⁺/2Cl^– cotransporter 1 reported in gills and opercular epithelium (Scott et al., 2005a).

In contrast to Na⁺/K⁺-ATPase _1a expression, Na⁺/K⁺-ATPase activity increased in the anterior portion of the intestine after freshwater transfer (Fig. 6), and the time course of this increase was roughly similar to that of plasma cortisol (Fig. 2B). One plausible conclusion from these data is that the changes observed in intestinal ion transport rates are partly due to post-transcriptional regulation of Na⁺/K⁺-ATPase activity, and that these changes may be partly controlled by plasma cortisol. Post-transcriptional regulation is thought to be important in killifish gills and opercular epithelium (Marshall, 2003; Scott et al., 2005a), and cortisol is known to be important for several aspects of ion transport physiology in the intestine (Veillette et al., 1995; Lin et al., 2000). However, in the present study, the increase in Na⁺/K⁺-ATPase activity in the anterior portion of the intestine occurred later (at 3 days) than the largest rise in ion transport (at 12 h), suggesting that other molecular mechanisms must be important during the early stages of freshwater transition.

The molecular mechanisms responsible for changes in ion transport across killifish intestine in these early stages do not appear to include transcriptional regulation of the CA2, CFTR or NKCC2 isoforms measured in this study. Transcriptional regulation of other isoforms and/or ion transport proteins may instead be important after freshwater transfer. However, mRNA for all genes examined in the present study were expressed at high levels in the intestine (data not shown), which implies that they may still have an important role in the intestine. If these genes are important during freshwater transition, they could be regulated by post-transcriptional mechanisms. Understanding the molecular basis for ion transport in the killifish intestine deserves further study.

Relative Na⁺, Cl^– and water transport rates in vitro
The strong correlation between water transport rate and the net flux rates of Na⁺ and Cl^– (Fig. 5) suggest that changes in water absorption after freshwater transfer are entirely driven by transepithelial ion transport. Other mechanisms for changing the rate of water absorption, such as regulating the abundance or channel properties of aquaporins and thus intestinal water permeability, may therefore be less important in killifish after freshwater transfer. In eel, aquaporins were expressed at higher levels in the intestine of seawater-acclimated fish compared to those in fresh water (Aoki et al., 2003; Martinez et al., 2005), which has been correlated with a higher water absorption rate (Aoki et al., 2003). Unfortunately, the relative contributions of regulated water permeability and osmotic driving force (i.e. ion transport) to transepithelial water absorption are not known in these species.

Our data are in accord with the general view, based mainly on similar in vitro studies with seawater fish, that an isosmotic solution is absorbed across the intestine of fish (Loretz, 1995). The slope of the relationship in Fig. 5 of this study indicates that the strong ion concentration of the absorbed fluid was about 292 mmol l^–1 (see Results), which would make it nearly isosmotic to blood plasma and to the incubation saline (note this neglects the small contribution from the unmeasured cation/anion, as discussed below). A very similar value (272 mmol l^–1) may be estimated from the data of other researchers (Marshall et al., 2002a) for posterior intestine sacs of seawater-adapted killifish under similar `symmetrical' conditions.

It is notable that net Cl^– fluxes were always higher than net Na⁺ fluxes, indicating that additional uptake of cation or efflux of anion must occur. Greater Cl^– absorption across the intestine of killifish would obviously help to compensate for the inability of this species to actively absorb Cl^– in fresh water (Patrick et al., 1997; Patrick and Wood, 1999). This suggestion is supported by the observation that the difference between net Cl^– flux and net Na⁺ flux appeared to increase after freshwater transfer. However, net Cl^– fluxes are also higher than net Na⁺ fluxes across the intestine of fresh water-acclimated flounder Platichthys flesus (Smith et al., 1975) and rainbow trout Oncorhynchus mykiss (Nonnotte et al., 1987), both of which can actively absorb Cl^– at the gills, so this suggestion is uncertain. Furthermore, a recent review (Marshall and Grosell, 2005) has noted that an excess of Cl^– over Na⁺ absorption across the intestine is the normal pattern for a variety of marine species.

Although it may play a role in Cl^– homeostasis per se, the difference between net Cl^– flux and net Na⁺ flux across the intestine may simultaneously be a consequence of other physiological processes. For example, the intestines of seawater fish are known to secrete an appreciable amount of bicarbonate, which may be an important part of seawater ionoregulation (Wilson et al., 2002). Chloride is likely exchanged for bicarbonate at the luminal surface (Grosell et al., 2005), so there are probably two routes for intestinal Cl^– absorption: bicarbonate linked (via Cl^–/HCO₃^– exchange) and Na⁺ linked (via NKCC and/or Na⁺, Cl^– cotransporters). The difference between intestinal Cl^– and Na⁺ absorption may therefore reflect the extra influx pathway for chloride, that is, Cl^–/HCO₃^– exchange. This is normally thought of as a phenomenon peculiar to marine fish, but the present findings, together with a report of a great excess of [Na⁺] over [Cl^–] and very high pH (9.0) in the rectal fluid of fresh water-adapted trout (Shehadeh and Gordon, 1969), suggest otherwise. The possible acid–base consequences of such a strategy for a fish in fresh water deserves future study.

	Acknowledgments

 
This work was funded by the Natural Sciences and Engineering Research Council of Canada through Discovery Grants to C.M.W. and P.M.S., and a Canada Graduate Scholarship to G.R.S. C.M.W. is supported by the Canada Research Chair program. The authors would like to thank W. S. Marshall for kindly supplying killifish, L. Diao for invaluable technical support, and M. Grosell for useful discussions.

	References

TOP Summary Introduction Materials and methods Results Discussion References

 

Ando, M. (1975). Intestinal water transport and chloride pump in relation to sea-water adaptation of the eel, Anguilla japonica. Comp. Biochem. Physiol. 52A,229 -233.[Medline]

Ando, M., Mukuda, T. and Kozaka, T. (2003). Water metabolism in the eel acclimated to sea water: from mouth to intestine. Comp. Biochem. Physiol. 136B,621 -633.[CrossRef][Medline]

Aoki, M., Kaneko, T., Katoh, F., Hasegawa, S., Tsutsui, N. and Aida, K. (2003). Intestinal water absorption through aquaporin 1 expressed in the apical membrane of mucosal epithelial cells in seawater-adapted Japanese eel. J. Exp. Biol. 206,3495 -3505.[Abstract/Free Full Text]

Babkin, B. P. and Bowie, D. J. (1928). The digestive system and its function in Fundulus heteroclitus. Biol. Bull. 54,254 -277.[Abstract/Free Full Text]

D'Cruz, L. M. and Wood, C. M. (1998). The influence of dietary salt and energy on the response to low pH in juvenile rainbow trout. Physiol. Zool. 71,642 -657.[Medline]

Daborn, K., Cozzi, R. R. F. and Marshall, W. S. (2001). Dynamics of pavement cell-chloride cell interactions during abrupt salinity change in Fundulus heteroclitus. J. Exp. Biol. 204,1889 -1899.[Abstract/Free Full Text]

Fritz, E. S. and Garside, E. T. (1974). Salinity preferences of Fundulus heteroclitus and F. diaphanus (Pisces: Cyprinodontidae): their role in geographic distribution. Can. J. Zool. 52,997 -1003.[Medline]

Fuentes, J. and Eddy, F. B. (1997). Drinking in Atlantic salmon presmolts and smolts in response to growth hormone and salinity. Comp. Biochem. Physiol. 117A,487 -491.[CrossRef]

Griffith, R. W. (1974). Environment and salinity tolerance in the genus Fundulus. Copeia 1974,319 -331.[CrossRef]

Grosell, M., Wood, C. A., Wilson, R. W., Bury, N. R., Hogstrand, C., Rankin, C. and Jensen, F. B. (2005). Bicarbonate secretion plays a role in chloride and water absorption of the European flounder intestine. Am. J. Physiol. 288,R936 -R946.

Howard, J. N. and Ahearn, G. A. (1988). Parallel antiport mechanisms for Na⁺ and Cl^– transport in herbivorous teleost intestine. J. Exp. Biol. 135,65 -76.[Abstract/Free Full Text]

Jacob, W. F. and Taylor, M. H. (1983). The time course of seawater acclimation in Fundulus heteroclitus L. J. Exp. Zool. 228,33 -39.[CrossRef]

Karnaky, K. J. (1992). Teleost osmoregulation: changes in the tight junction in response to the salinity of the environment. In Tight Junctions (ed. M. Cereijido), pp.175 -185. Boca Raton: CRC Press.

Katoh, F. and Kaneko, T. (2003). Short-term transformation and long-term replacement of branchial chloride cells in killifish transferred from seawater to freshwater, revealed by morphofunctional observations and a newly established `time-differential double fluorescent staining' technique. J. Exp. Biol. 206,4113 -4123.[Abstract/Free Full Text]

Katoh, F., Hasegawa, S., Kita, J., Takagi, Y. and Kaneko, T. (2001). Distinct seawater and freshwater types of chloride cells in killifish, Fundulus heteroclitus. Can. J. Zool. 79,822 -829.[CrossRef]

Kültz, D., Chakravarty, D. and Adilakshmi, T. (2001). A novel 14-3-3 gene is osmoregulated in gill epithelium of the euryhaline teleost Fundulus heteroclitus. J. Exp. Biol. 204,2975 -2985.[Abstract/Free Full Text]

Lin, L. Y., Weng, C. F. and Hwang, P. P. (2000). Effects of cortisol and salinity challenge on water balance in developing larvae of tilapia (Oreochromis mossambicus). Physiol. Biochem. Zool. 73,283 -289.[CrossRef][Medline]

Loretz, C. A. (1995). Electrophysiology of ion transport in teleost intestinal cells. In Cellular and Molecular Approaches to Fish Ionic Regulation (Fish Physiology, Vol.14) (ed. C. M. Wood and T. J. Shuttleworth), pp.25 -56. San Diego: Academic Press.

Malvin, R. L., Schiff, D. and Eiger, S. (1980). Angiotensin and drinking rates in the euryhaline killifish. Am. J. Physiol. 239,R31 -R34.

Marshall, W. S. (2003). Rapid regulation of NaCl secretion by estuarine teleost fish: coping strategies for short-duration freshwater exposures. Biochim. Biophys. Acta 1618,95 -105.[Medline]

Marshall, W. S. and Grosell, M. (2005). Ion transport, osmoregulation, and acid-base balance. In Physiology of Fishes (ed. D. H. Evans and J. B. Claiborne), pp.177 -230. Boca Raton: CRC Press.

Marshall, W. S., Emberley, T. R., Singer, T. D., Bryson, S. E. and McCormick, S. D. (1999). Time course of salinity adaptation in a strongly euryhaline estuarine teleost, Fundulus heteroclitus: a multivariable approach. J. Exp. Biol. 202,1535 -1544.[Abstract]

Marshall, W. S., Bryson, S. E. and Luby, T. (2000). Control of epithelial Cl^– secretion by basolateral osmolality in the euryhaline teleost Fundulus heteroclitus.J. Exp. Biol. 203,1897 -1905.[Abstract]

Marshall, W. S., Howard, J. A., Cozzi, R. R. F. and Lynch, E. M. (2002a). NaCl and fluid secretion by the intestine of the teleost Fundulus heteroclitus: involvement of CFTR. J. Exp. Biol. 205,745 -758.[Abstract/Free Full Text]

Marshall, W. S., Lynch, E. A. and Cozzi, R. R. F. (2002b). Redistribution of immunofluorescence of CFTR anion channel and NKCC cotransporter in chloride cells during adaptation of the killifish Fundulus heteroclitus to sea water. J. Exp. Biol. 205,1265 -1273.[Abstract/Free Full Text]

Martinez, A. S., Cutler, C. P., Wilson, G. D., Phillips, C., Hazon, N. and Cramb, G. (2005). Regulation of expression of two aquaporin homologs in the intestine of the European eel: effects of seawater acclimation and cortisol treatment. Am. J. Physiol. 288,R1733 -R1743.

McCormick, S. D. (1993). Methods for nonlethal gill biopsy and measurement of Na⁺, K⁺-ATPase activity. Can. J. Fish. Aquat. Sci. 50,656 -658.

Nonnotte, G., Aubree-Pelletier, A. and Colin, D. A. (1987). Transient changes of the intestinal absorption of sodium and chloride in the rainbow trout after abrupt transfer into sea-water. Comp. Biochem. Physiol. 88A,437 -441.[Medline]

Patrick, M. L. and Wood, C. M. (1999). Ion and acid-base regulation in the freshwater mummichog (Fundulus heteroclitus): a departure from the standard model for freshwater teleosts. Comp. Biochem. Physiol. 122A,445 -456.[CrossRef]

Patrick, M. L., Pärt, P., Marshall, W. S. and Wood, C. M. (1997). Characterization of ion and acid-base transport in the fresh water adapted mummichog (Fundulus heteroclitus). J. Exp. Zool. 279,208 -219.[CrossRef]

Potts, W. T. W. and Evans, D. H. (1967). Sodium and chloride balance in the killifish Fundulus heteroclitus. Biol. Bull. 133,411 -425.[Abstract/Free Full Text]

Schettino, T. and Lionetto, M. G. (2003). Cl^– absorption in European eel intestine and its regulation. J. Exp. Zoolog. Part A Comp. Exp. Biol. 300, 63-68.

Scott, G. R. and Schulte, P. M. (2005). Intraspecific variation in gene expression after seawater transfer in gills of the euryhaline killifish Fundulus heteroclitus. Comp. Biochem. Physiol. 141A,176 -182.[CrossRef]

Scott, G. R., Sloman, K. A., Rouleau, C. and Wood, C. M. (2003). Cadmium disrupts behavioural and physiological responses to alarm substance in juvenile rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 206,1779 -1790.[Abstract/Free Full Text]

Scott, G. R., Richards, J. G., Forbush, B., Isenring, P. and Schulte, P. M. (2004a). Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer. Am. J. Physiol. 287,C300 -C309.

Scott, G. R., Rogers, J. T., Richards, J. G., Wood, C. M. and Schulte, P. M. (2004b). Intraspecific divergence of ionoregulatory physiology in the euryhaline teleost Fundulus heteroclitus: possible mechanisms of freshwater adaptation. J. Exp. Biol. 207,3399 -3410.[Abstract/Free Full Text]

Scott, G. R., Claiborne, J. B., Edwards, S. L., Schulte, P. M. and Wood, C. M. (2005a). Gene expression after freshwater transfer in gills and opercular epithelia of killifish: insight into divergent mechanisms of ion transport. J. Exp. Biol. 208,2719 -2729.[Abstract/Free Full Text]

Scott, G. R., Keir, K. R. and Schulte, P. M. (2005b). Effects of spironolactone and RU486 on gene expression and cell proliferation after freshwater transfer in the euryhaline killifish. J. Comp. Physiol. B 175,499 -510.[CrossRef][Medline]

Semple, J. W., Green, H. J. and Schulte, P. M. (2002). Molecular cloning and characterization of two Na/K-ATPase isoforms in Fundulus heteroclitus. Mar. Biotechnol. 4, 512-519.[CrossRef][Medline]

Shehadeh, Z. H. and Gordon, M. S. (1969). The role of the intestine in salinity adaptation of the rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 30,397 -418.[CrossRef]

Singer, T. D., Tucker, S. J., Marshall, W. S. and Higgins, C. F. (1998). A divergent CFTR homologue: highly regulated salt transport in the euryhaline teleost F. heteroclitus. Am. J. Physiol. 43,C715 -C723.

Smith, M. W., Ellory, J. C. and Lahlou, B. (1975). Sodium and chloride transport by the intestine of the European flounder Platichthys flesus adapted to fresh or sea water. Pflügers Arch. 357,303 -312.[CrossRef][Medline]

Tomasso, J. R. and Grosell, M. (2005). Physiological basis for large differences in resistance to nitrite among freshwater and freshwater-acclimated euryhaline fishes. Environ. Sci. Technol. 39,98 -102.[Medline]

Veillette, P. A., Sundell, K. and Specker, J. L. (1995). Cortisol mediates the increase in intestinal fluid absorption in Atlantic salmon during parr-smolt transformation. Gen. Comp. Endocrinol. 97,250 -258.[CrossRef][Medline]

Wilson, R. W., Wilson, J. M. and Grosell, M. (2002). Intestinal bicarbonate secretion by marine teleost fish-why and how? Biochim. Biophys. Acta 1566,182 -193.[Medline]

Wolf, K. (1963). Physiological salines for fresh-water teleosts. Prog. Fish Cult. 25,135 -140.

Wood, C. M. and Laurent, P. (2003). Na⁺ versus Cl^– transport in the intact killifish after rapid salinity transfer. Biochim. Biophys. Acta 1618,106 -119.[Medline]

Wood, C. M. and Marshall, W. S. (1994). Ion balance, acid-base regulation, and chloride cell function in the common killifish, Fundulus heteroclitus–a euryhaline estuarine teleost. Estuaries 17,34 -52.[CrossRef]

Zall, D. M., Fisher, M. D. and Garner, Q. M. (1956). Photometric determination of chlorides in water. Anal. Chem. 28,1665 -1678.[CrossRef]

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