Fish Physiology and Biochemistry vol. 5 no. 4 pp 181-186 (1988) Kugler Publications, Amsterdam/Berkeley

Transepithelial ion exchange in smolting atlantic salmon (Salmo Salar L.) D.R.N. Primmett I, F.B. Eddy 2, M.S. Miles 3, C. Talbot 3 and J.E. Thorpe 3 1Department o f Human Anatomy, University of Oxford, South Parks Road, Oxford OXI 1QX, U.K. 2Department o f Biological Sciences, University of Dundee, Dundee DD1 4HN, U.K. 3Freshwater Fisheries Laboratory, Pitlochry PH16 5LB, U.K. Keywords: Na + exchange, salmon migration, stress

Abstract Whole-body (but predominantly gill) Na § exchange, gill Na § § activity and seawater tolerance were examined in juvenile Atlantic salmon during the smolting period. Transepithelial net Na + gain decreased steadily from late February showing a net loss in April and early May, returning to approximate equilibrium in mid-May. This seasonal net loss of Na § to the environment occurred slightly after maximal gill epithelial Na +/K § activity and preceded maximal seawater tolerance. The results are discussed in relation to changes in gill permeability and salt intake via the diet.

Introduction The physiological mechanisms supporting the annual migration of salmon smolts to the sea is unknown, but subsequent survival in sea water depends on inhibition of freshwater (FW) ionoregulatory mechanisms and activation of appropriate saltwater (SW) ones (Evans 1984). FW-adapted salmon absorb Na + from the hypotonic environment across the gill epithelium at a rate equal to or exceeding diffusive ion loss (Evans 1984). Their gill is a tight epithelium; Na + transport is predominantly transcellular via an electroneutral antiport system associated with respiratory cells with low conductance junctional complexes (see Payan and Girard 1984). SW-adapted salmon excrete Na + to the hypertonic environment across the gills to account for diffusive ion entry across the body surface together with ionic intake by drinking (Evans 1984). Their

gill is one o f the most leaky epithelia known and a sizeable portion of the Na § transport occurs along paracellular pathways associated with chloride cells with high conductance junctional complexes (see Payan and Girard 1984). Chloride ceils are responsible for electrogenic C1- extrusion in SW-adapted fish (see Potts 1984). However, chloride cell recruitment is observed in juvenile FW salmon just prior to seaward migration (Langdon and Thorpe 1984). This appearance of chloride ceils has often been interpreted as modification preparatory for a future seawater existence (Conte and Wagner 1965; Langdon 1985) without questioning the functional effects of such a change in branchial epithelial permeability while the animals are still in freshwater. If a mechanism promoting Na § loss is activated in FW salmon, then one could predict that the fish would undergo an ionoregulatory disturbance. Indeed, during the pre-migratory period (i.e. mid-March to early May)

Correspondence to: Department of Biological Sciences University of Dundee, Dundee DDI 4HN, U.K.

182 juvenile FW salmonids may exhibit lower plasma electrolytes and osmolarity (e.g. Fontaine 1951; Kubo 1953; Koch et al. ,1959; Houston 1960; Houston and Threadgold 1963; Folmer and Dickhoff 1981) and high plasma cortisol levels which are known to stimulate Na +/K + / A T P a s e (Langh o m e and Simpson 1981; Thorpe et al. 1987). At this stage changes in muscle water content and increased urine flow suggested increased water permeability (Foda 1974; Eddy and Talbot 1985). In this study, to test whether FW ionoregulatory ,homeostasis is maintained ivr premigratory Atlantic salmon, we examined: (1) the exchange of Na + between these animals and their FW environment, (2) gill epithelial Na +/K + / A T P a s e activity (since the gill is the major site of such exchange), and (3) the ability of these fish to survive direct transfer to SW.

Materials

and

methods

Three experiments were conducted from February to May on one year old, sibling, juvenile freshwater-adapted Atlantic salmon (Salmo salar L.) which were obtained from the Smolt Rearing Station, Department of Agriculture and Fisheries for Scotland, Almondbank, Scotland.

Salinity tolerance

At monthly intervals, ten fish were selected at random from the main stock tank and subjected to a 36 ppt salinity tolerance test as described by Langdon and Thorpe (1985), but for 120h.

Sodium f l u x e s

Groups of fish were transported at weekly intervals to the aquarium at the University of Dundee where they were kept in stock tanks supplied with running aerated water at 10 ~ +_ 2~ Eight fish which had not been fed during the previous 18h were selected from stock then gently transferred to individual glass chambers each containing 500 ml of constantly aerated freshwater temperature I 0 + 2 ~ salt

content in mmol.l -I (0.25 Na +, 0.1 K +, 0.1 Ca ++, pH 7 - 7 . 5 ) , and seasonal photoperiod. Minimal disturbance o f the animals was achieved by darkening the chamber walls but allowing adequate illumination via an aperture (approximately 10 cm 2) at the top so that during the experiment the animals were not disturbed by the experimenter. Rapid aeration of the water screened the animals from external sounds and vibrations. Each container was equipped with a 60 cm length of plastic tubing running from the medium to the exterior such that the radioisotope (see below) could be added or water samples removed with virtually no disturbance to the fish. For each fish (weight range = 8.4 17.5 g), after at least 7h acclimation time, 7.4 kBq 22Na were added to the water; thorough mixing through strong aeration to the chamber was allowed for l h before experimentation. At the beginning and end of the subsequent 24h period a water sample of 2 ml was removed from each chamber from which unidirectional and net whole-body Na + fluxes were determined by: (1) measuring the disappearance of 22Na from the medium using a Panax Reigate Series gamma well counter and (2) measuring changes in water Na + concentration with a Pye Unicam SP9 atomic absorption spectrophotomer, respectively. Net flux was calculated from changes in Na + content of the medium, influx from changes in medium Na + specific activity and Na + efflux by difference, according to the method of Maetz (1956) and Wood et al. (1984).

Na + / K + / A TPase

At approximately weekly intervals, 2 yr old smohing sibling Atlantic salmon were transported from Almondbank to the Fisheries Laboratory at Pitlochry, where gill extracts for Na +/K + / A T P a s e were prepared according to Zaugg (1982) and activity was determined by the method of Soderling, Lebel, Laikko and Larmas (1981), using a LKB 1250 luminometer.

183

1~176

The fish showed progressively greater ability to survive in 36 ppt seawater with season (Fig. 1). One hundred percent mortality occurred within 24h in February, but by May 80~ of the fish survived for more than 120h.

a net loss in early May, then followed by a small net uptake. In these experiments whole body Na + fluxes were measured of which branchial exchanges are the major component, with urinary and faecal net losses being minor routes maximally 10-15~ of the total, but generally less (Heisler 1984; Salman and Eddy 1987). 1. Late February to early March : Net Na + influx decreased from 59+ 15 to 35 + 15 t ~ m o l . k g - l . h - I , while unidirectional influx and efflux changed from 210+ 17and 150+31 to 202_+ 10and 167+21 t~mol N a + . k g - l . h -+ respectively. Similar values for fluxes for parr and very early smolts were reported by Potts et al. (1970). 2. Mid- to late March : Influx and efflux values increased to 298_+16 and 288+14 ~mol N a + . k g - l . h - I respectively by March 30th; the rise in influx failed to match the rise in efflux, resulting in a significant decrease in net Na + flux of about 25%. 3. Early to mid-April: Unidirectional Na + influx and efflux decreased relative to the previous period, but efflux exceeded influx causing a slight net Na + lossof 15+9to 19_+11~molNa+.kg-l.h-t;this significant net loss of sodium lasted until mid-May. 4. Late April: There was no significant change in the slightly negative Na + net flux compared to the previous period; however, both unidirectional influx and efflux reached their highest levels at 355 _+30 and 370 + 60 ~mol Na +. k g - l.h - 1 respectively. 5. Early May:. Unidirectional Na + efflux declined to early April levels, but influx decreaged even further (to February levels), resulting in the largest negative Na + net flux observed during the experiment ( - 38 _+ 14 p~mol Na + . k g - l . h - i). 6. Mid-May: Unidirectional Na + fluxes increased to late March levels and values for influx exceeded those for efflux restoring net Na + gain. Also, at this time, the experimental animals lost their parr marks and acquired a silver colouration.

Sodium fluxes

Na +/K +/A TPase

Presmolting fish showed a strong net Na + gain in February, decreasing linearly (r = 0.973, Fig. 2) to

Gill epithelial Na + / K + / A T P a s e activity significantly increased from a value of about 1.2 #mol

Accumulated mortality ~176 60-

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Fi,e. I. Accumulated precent mortality of smohing Atlantic salmon over 120h in 36 ppt seawater. 9 = 20-25 February; = 25 March 4April; 9 = 29 A p r i l - 7 May; o = 27 May 10 June.

Blood ions At approximately 3 week intervals fish were rapidly removed from the stock tanks, killed by a blow to the head and blood extracted from the heart or caudal aertery/vein. Blood samples were immediately centrifuged and the plasma frozen at - 2 0 ~ for subsequent Na + analysis, as described earlier.

Results

Salinity tolerance

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A T P . m g protein - ] . m i n - l in early February to a seasonal maximum of about 2.5 t~mol A T P . m g protein- l.min- I by early April and then declined, achieving a value of about 2.0 t~mol A T P . m g protein- ~.min- I by mid-May.

Blood plasma sodium ion level This showed a small but insignificant increase between February and July; values from mid March to the end of March showed the greatest variability, while at the end of May the highest values were noted (Fig. 3).

Discussion

The most notable feature of this study is that smolting Atlantic salmon undergo significant changes in whole-body transepithelial Na + exchange while in freshwater. The net Na + gain seen in February, prior to the onset of seaward migration, steadily decreased, attaining negative values by mid-April and a maximum Na + net loss by early May; this fall in net Na + loss was associated with significant increases in unidirectional Na + influx and efflux. A Na + net gain was reestablished by mid-May, however, values for unidirectional fluxes (in either direction) still significantly exceeded pre-migratory

185

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