Camp. Eiochem. Physiol. Vol. 96A, No. 2, pp. 303-308, 1990 0300-9629/90 $3.00 + 0.00 Printed in Great Britain 0 1990 Pergamon Press plc

STUDY OF TRANSBRANCHIAL Na+ EXCHANGE IN SALMO SALAR SMOLTS AND POST-SMOLTS DIRECTLY

TRANSFERRED TO SEA WATER

JEAN-MICHEL NANCE, MICHEL BORNANCIN*, FRANCOIS SOLA, GILLES BOEUF? and JEAN-DENIS DUTIL$

Laboratoire de Physiologie Cellulaire et Cornparke U.A. CNRS 651, Universitt de Nice, Part Valrose, 06034 Nice Cedex, France. Telephone: 9352-9936

(Received 2 January 1990)

Abstract-l. Branchial ionic transport capabilities were studied in smelt and post-smelt Atlantic salmon. We used the perfused isolated head technique to make instant measurements of transbranchial Na+ exchange during direct transfer to sea water.

2. There was a 30% shift from arterial to venous circulation when the perfused head preparation was exposed to sea water. While smelts excreted Na+ at a low rate (68 rEq/hr/lOO g) in freshwater, they exhibited a net excretion rate of 1265 pEq/hr/lOO g in sea water.

3. Smelts artifically kept in freshwater past the smolting period (post-smolts) behaved as rainbow trout: their net Na+ fluxes were positive in freshwater (37 pEq/hr/lOOg), but also in sea waler (4997 p Eq/hr/ 100 g).

4. Our results suggest that Na+ excretion by the gills is mainly effected through the filaments.

INTRODUCTION

Many studies of salmonid smolting have been under- taken in the last few years: reviews by Hoar (1976), Wedemeyer et al. (1980) and symposia (Bern and Mahnken, 1982; Thorpe et al., 1985). This work has concentrated on the influence of environmental factors (particularly temperature and photoperiod), endocrine control and growth as determinants of migration.

Recent work (Boeuf et al., 1985; Boeuf and Prunet, 1985; Boeuf, 1987) has described the main physio- logical events at smolting in Atlantic salmon S&no s&r. Koch et al. (1959), Parry (1960) and Houston (1964) had already shown that Atlantic salmon had a potential for adapting to sea water that was very dependent on its physiological state (Parr or smolt), and that the latter withstood transfer to sea water without notable transient dehydration. McCartney (I 976), Saunders and Henderson (1978), Johnston (1983) and Boeuf et al. (1985), showed that this species develops activation of the Na,K-ATPase system in the gill membranes during springtime smolting in freshwater, as had been previously shown in Oncorhynchus (Zaugg and McLain, 1970, 1972).

Saunders and Henderson (1978) concluded that Na.K-ATPase activity in the gill is an excellent criterion for the physiological smolt state through its role as a sensitive indicator of the animal’s osmoregulatory capacity, agreeing in this with the observations of Boeuf et al. (1978) in the Coho

*To whom correspondence should be addressed. tIFREMER (Brest) Station Ressources Vivantes, 29263

Plouzane, France. SMinistbre des Pi?ches et des Octans, Division de la

Recherche sur les Peches, 850 Route de la mer, C.P. 1000 Mont-Joli, Quebec G5H 324, Canada.

salmon. During the course of smolting, a period of important development in salmon, hypo-osmoregu- latory capacities develop, particularly in the gills, transforming the only slightly euryhaline freshwater parr into smolt, preadapted to a marine environment.

The model proposed by Smith (1932) and further completed by Krogh (1939) first described the ways by which the fish maintains water and mineral bal- ance. The freshwater fish is hyperosmoregulatory, freshwater enters through the gills, fish drink very little and the kidney excretes dilute urine. Electrolyte (Na+, Cl-) loss is compensated by an active branchial uptake. In sea water fish, water is absorbed by the gut together with salt to compensate water loss along the salt concentration gradient between the fish and the external medium. The ionic uptake is com- pensated by an active branchial excretion. This model is classic and was well described and discussed by Maetz (1971). Numerous authors contributed to describe the precise mechanisms implicated in the ionic branchial fluxes, particularly in the so-called “chloride-cells” (see Foskett et af., 1983). The role of Na,K-ATPase in ionic transport is well documented particularly in sea water fish and in euryhaline fish which adapted to sea water after transfer from fresh- water. In this latter case, development of new chloride cells, of Na,K-ATPase activity and of active ionic (Na+, Cl-) fluxes were parallel (see Maetz and Bornancin, 1975; Sargent et al., 1980; De Renzis and Bomancin, 1984).

This study examined the branchial ionic fluxes in smolts at the time of smolting and those of smolts kept in freshwater beyond the smolting period (post- smolts). Ionic fluxes were measured on the perfused isolated head (Payan and Matty, 1975) in order to differentiate influx and ef?lux, and distinguish the role of the respiratory epithelium and the primary lamella

303

304 JEAN-MICHEL NANCE et al

“e”o”s I I efferent 1//l ’ 3” ” “a aa

Fig. I. Diagram of the blood circulation in salmonids (from Smith and Bell, 1978; sea also Gardaire et al.,

1985). v = ventricle; sv = cardiac venous sinus; va = ventral aorta; aba = afferent branchial artery; eba = efferent branchial artery; bv = branchial vein; jv = jugular vein;

da = dorsal aorta. P = perfusion.

(or filament). The technique has been adapted for fish weighing from 30 to 40 g. Two circulatory systems have been described in trout gills (Laurent and Dunel, 1978, 1980); the arterio-arterial vasculature has an exclusive relationship with the respiratory lamella and the central venous sinus which irrigates the filament and is derived from the efferent vascula- ture of the respiratory lamella (Fig. 1). The filaments are composed of epithelial cells and of classical chloride cells that develop during smelting (Pisam et al. 1988). The genera1 morphology of salmon gills described by these authors is very similar to that of trout gills described by Laurent and Dune1 (1978).

MATERIALS AND METHODS

Animals

The Atlantic salmon were obtained from the IFREMER hatchery of Le Conquet (West Brittany) and were reared in freshwater on a small river (pH 6.5-7, Na+ = 1.48, Cl- = 1.20, HCO; = 0.50, Ca*+ = 0.33 mmol/l. In Septem- ber preceding the smolting year (1986) the upper modal group fish (see Boeuf et al., 1985) were transferred to

2 x 2 m Ewos tanks at a density of about 100/m’. They were fed on dry pellets-%6 IFREMER (Gaignon, 1987) from an automatic feeder. Photoperiod and temperature were natural (48” North; temperature regimes given in Fig. 2a).

Two batches from a single population were separated: -the first batch (mean weight 35.5 g) was studied at the

time of smolting, using the Na,K-ATPase activity in the gill as indicator of the sea water adaptability (Fig. 2b);

-the second batch (post-smolts: mean weight 45.5 g) was studied after having been maintained in freshwater for two months beyond the smolting period.

Measurements qf transbranchial Na+ fluxes

The new perfusion medium was described by Bornancin et al. (1985) with physico-chemical parameters similar to those of the salmon blood. A blood sample was taken from the caudal vein for chemical analysis, the fish was hepar- inized and the head of the salmon was severed 1 cm behind the pectoral fins and the gills immediately irrigated by a current of freshwater (0.6 I/min). Oesophagus was ligatured twice to avoid leakage. Both perfusion delivery (approx. 192 ml/hr) and constant aortic pressure (28 k 4 mmHg) were maintained by a pulsatile adjustable pump (NP 31 Bran and Lubbe) in the ventral aorta. Those conditions (Bornancin et al., 1985) have been proved to prevent any oedema formation, and this was regularly checked by morphological examination of the gills at the end of the experiment. The head was perfused for a 10min period before sample collection. After passing through the branchial circulation the arterial and venous efferent liquids were collected separately over I min and weighed for control estimations of flow rate. Total time of experiments never exceeded 1 hr and the preparation is viable for flux measurements (Avella et al., 1987).

Sodium fluxes were first measured while the head was submerged in tap water containing 1 mmol/l of sodium. For this concentration active Na+ uptake which varies according to Michaelis-Menten law is near its maximal value (Avella and Bornancin, 1989). Then this medium was rapidly drained and instantly replaced by sea water and the fluxes measured under those conditions (Nance et al., 1987) after IO min of sea water contact (see Results); the duration of influx measurement was 6min. Each fish was its own reference. During experimentation the temperature was maintained at 12°C and adrenalin (10e6M) was present throughout.

I-

I-

a M Fresh Water temperature

b H Gill Na ,K-ATPase activity from February 11 to july 11,1988.

january , february 1 march 1 april 1 may 1 julY 1 june

I I I I I I I II I I I I II I I I I I In z 2 ii4 z :: ?i 2 s 2 _ _ _ r -

Time (Number of days)

30

f

!i 20 .

i

m E

10 u * 2

0

Fig. 2 (a). Temperature measured on the 5th day of each month. (b) Gill Na,K-ATPase activity from I I February to II July 1986.

Transbranchial Na+ exchange in Salmo salar 305

Inward rransbranchial Na+ fluxes

The external medium contained 22Na+ (50,000 dpm/ml). Sampling the arterial and venous fluids allows the inward flux to be calculated as follows:

J Ill

SL = Q, x perfusion flow in ml/min x 60 min x loo

q,,,/external [Na+] x weight in g

J ,,=(Qv-Qa) x venous flow in ml/min x 60 min ” q,,,/external [Na+] x weight in g

where

SL = secondary lamella PL = primary lamella Q, = arterial fluid radioactivity dpm/ml Q, = venous fluid radioactivity dpm/ml

Q,. Q,. 9_, were determined on a Packard gamma-counter. [Na+] in pmol/ml was determined by Eppendorf flame photometer.

Outward transbranchial Na+ fluxes

The perfusion medium was marked with 22Na+, sampling of the external medium allows the outflux to be calculated as follows:

Aexternal radioactivity (dpm/ml)/min

Total flux: x external vol. in ml x 60 min x 100

Q,,,/perfusate [Na+] x weight in g

[Q,,, - Q,,,,] x perfusion flow

J,,, SL: in ml/min x 60 min x 100

Q,,,/perfusate [Na+] x weight in g

[Q,,,(,, - Qernv,] x perfusion venous

J,,, PL: flow in ml/min x 60 min x 100

Q,,,,/perfusate [Na+ ] x weight in g

where PI,, . Qe~caJ, QW are respectively perfusate, efferent arterial, efferent venous radioactivities in dpm/ml.

Measurements of Na,K-ATPase acfivily

Gill filaments were taken from fish of the same batches as fishes used for flux studies. The filaments were rinsed with 0.25 M sucrose (pH 7.4) and immediately stored in liquid nitrogen. Measurements of gill Na,K-ATPase were obtained according to the method described by Lassere et a/. (1978).

Table I. Perfusatc distribution in the gill before and after direct transfer to sea water (n = 8)

Freshwater Sea water Collected flow (ml/mn f SD) (ml/mn k SD)

Total flow 3.20 k 0.10 3.18 k 0.12 Arterial flow 2.68 k 0.18 2.01 * 0.15

Total flow rate is the sum of measured arterial flow rate + measured venous flow rate.

RESULTS

(I) At the time of transfer to sea water, perfusate distribution in the gills of smolts or post-smolts was modified (Table 1).

The total (arterial + venous) flow rate was main- tained constant by the pulsatile pump and as indi- cated in Table 1, it did not change at the moment of transfer to sea water. However, a 30% reduction (0.6 ml) in arterial flow rate was observed, and the venous flow was enhanced by 0.6 ml. This shift was reversible since the arterial perfusion recovered its entire flow if the preparation was once again placed in freshwater.

(II) Transbranchial Na+ influxes and effluxes measured in freshwater and in sea water on a per- fused smolt or post-smolt head.

As illustrated in Table 2, the smolts had an instant excretory capacity as soon as they were placed in salt water. Measurements made in freshwater showed that they lost sodium ions through the entire branchial epithelium. In sea water, the epithelium of respiratory lamellae was highly permeable and let sodium penetrate; the filamental epithelium, on the other hand, excreted sodium and, overall, a net sodium excretion was observed.

In post-smolts in freshwater in July, we noted that (Table 3):

(1) Sodium was taken up through the respiratory epithelium in freshwater.

(2) At the time of transfer to sea water, large amounts of sodium penetrated through the respir- atory as well as the primary lamellae. The gills had

Table 2. Transbranchial sodium fluxes in Salmo solar smelts. Net negative fluxes are out-flowing and correspond to ion loss (N = 5)

Freshwater Sea water FIUX

(pWhr/lOOg)

Secondary lamellae Primary lamellae

Influx Outflux Influx- Outflux (X f SD) (X + SD) (X i SD) (X t SD)

6+l 4Oi3 2105 + 320 1190 f 146 I?1 35 + 5 Not detectable 2180 rt 357

Net flux -68 -1265

Measurements in freshwater were made prior to sea water measurements. Sea water influx through the primary lamellae could not be calculated, differences between Q, and Q, (see Materials and Methods) were in the region of experimental error and were not measurable.

Transfer to sea water was performed with a perfused head preparation. Each animal was its own control.

Table 3. Transbranchial sodium flux in Solmo s&r post-smolts. Net oositive fluxes are in-flowing and correspond to.an ion gain

Flux Freshwater

Influx Outflux Sea water

Influx Outflux (X f SD) (X _+ SD) (X k SD) (X k SD)

Secondary lamellae Primary lamellae Net flux

58 i 8 20 + 3 2975 i. 468 286 _+ 34 16*4 17*7 2416+481 108 + 35

+37 + 4997

Transfer to sea water was performed with a perfused head preparation. Each animal was its own control (N = 6)

306 JEAN-MKHEL NANCE et al.

lost their ability to excrete sodium and if directly transferred to sea water showed a Iarge net influx.

(III) Net Na+ fluxes evolved over time in sea water (Fig. 3a, b)

-the arterial net flux across the respiratory la- mellae decreased over time and reached a stable mini- mum within 10 min, in both smolts and post-smolts;

-in smolts, the gill part irrigated by venous blood excreted Na + . This excretion was instant and increased very rapidIy. It also reached a stable maximum, after about 10min. This excretion was such that, even considering expe~mental error, it may have compensated the arterial influx. Consequently, the smolt suffered no dangerous plasma sodium load through the gills.

Unlike smolts, gill fluxes in post-smolts were not balanced during transfer (Table 3). The net influx via the venous effluent, although less pronounced, was added to the arterial flux and thus contributed to a possible plasma Na+ load originating in the gills.

The results shown in Tables 2 and 3 took these developments into account; the fluxes were measured after 10 min.

(IV) Arterial fluxes varied with respect to weight and according to the physiological state of the fish under study.

l Smolts 0 Post-smolts z 6000

g 5000

“, g 4000

‘iii 3000 IL

3 Jg 2000 I,..‘,_..*.

0 1 2 3 4 5 6 7 8 9 1011 Tlnw (mid

0 Smolts

0 Post-smelts

3 c 0 .,.‘..,.‘I* 3234567 8 9 10

E -loo0 Time (mid

f is E

-2000

H -3000 ,t

-4000~ L Fig. 3. Na+ gill flux in Salmo sulur transferred to sea water. (a) Development over time of net arterial fluxes in Saimo salar. (b) Development over time of net venous fluxes in Sulmo salar. These two figures represent sodium fluxes measured on isolated perfused heads kept in 35.0% sea water. Similar results were obtained in four other experiments.

0 Influx In smelts A Influx in post-smoits 1 l outllux In smelts A Outflux In post-smelts

Of.,.,.,.,.,.,.,.,., 34 36 38 40 42 44 46 46 50 62

Weight ( g )

Fig. 4. Na+ gill flux in Sulmo salar transferred to sea water. Development of arterial sodium fluxes according to the weight and physiological state of Salmo s&r. Each point represents a single fish. Least squares regression lines are

superimposed on the data.

Discontinuities in the relationship between size and sodium fluxes in the respiratory lamellae suggest influxes and efftuxes varied according to the weight of the fish under study. This change, represented in Fig. 4 shows the distinction between smolts and post-smolts.

Fluxes measured in the filaments also varied with weight, but the trend is not so clear.

DISCUSSION

In freshwater, Atlantic salmon smolts studied here seem to show a net Nat loss, since losses through the gills are greater than the gains. This phenomenon was described by Chartier-Baraduc (1959) and Houston (1959) but not reported by Folmar and Dickhoff (1980). In the post-smolts, it became ctear that all the physiological characteristics of the gills of smelts had disappeared, i.e. the instant capacity for excreting Na+ in sea water, as well as net losses in freshwater. The post-smelt then appeared truly acclimated to its freshwater environment.

This hydromineral regulation in smolts kept in freshwater would become again very comparable with that of the freshwater Salmo gairdneri with the pumping of sodium ions performed by the secondary lamellar epithelium (Nance et al., 1987; Avella et al., 1987). Smolting therefore leads to the development of a mechanism capable of excreting Na+ in salt water, it is located on the filament, a localization common to marine fishes. This potentiai, highly adaptive if followed by active migration to sah water, is only transient. This clearly differentiates it from those salmonids that do not undergo smelting, like the rainbow trout which dies as a result of direct transfer to sea water (Bath and Eddy, 1979) since it cannot control its hydromineral equilibrium.

Moreover, contact with sea water causes an imme- diate redistribution of the branchial circulatory flow, reducing irrigation of the lamellar section in favour of the filamentous section. This may emphasize the respiratory stress observed during transfer (Eddy, 1982). Increased circulation of the biood near the chloride cells on the filaments should result in a far more effective reguiation of plasma osmolarity. Thus,

Transbranchial Na+ exchange in Salmo salar 307

if the secondary lamellae do participate in the excre- tory phenomenon, the vasoconstriction which affects them can limit passive ionic net influxes at their level.

Pisam ef al. (1988) describe the existence, on Atlantic salmon smolt filaments, of “accessory” cells contiguous with the chloride cells which show significant development in some marine fish, like eels adapted to sea water (Sardet, 1980). These cells disappear in freshwater post-smolts.

The pre-existence of such a multicellular organiz- ation is a criterion for preadaptation to changes in environment. The high level of Na,K-ATPase described in smolting salmon develops in parallel to this structure (Langdon and Thorpe, 1985; Boeuf et al.. 1985; Barron, 1986; Boeuf, 1987). The anatomical and biochemical status of gill filaments explains the excretion of Na+ ions observed very soon after the contact of smolts with sea water. Smolt gills can therefore be considered identical to those of marine fish, since direct or gradual contact with sea water triggers the iono-regulatory mechanisms prepared during smolting.

Values of Na+ fluxes presented here are very high, as under the same conditions, i.e. direct transfer to sea water of 150 g rainbow trout we observed fluxes 3-4 times lower (Nance et al., 1987). A general observation is that flux intensities might be inversely related to the fish weight, and for example Maetz and Pit (1977) observed values of Na+ effluxes as high as 8255 ~mol/hr/l00g for 44Og weight sea water mullet.

In other respects, it was observed a sudden and huge swallow of the ligatured piece of oesophagus kept in the head when the freshwater was instantly replaced by sea water. It was not possible to quantify this swelling which was always present in each exper- iment. In 1972, Kirsch isolated the head region of the eel and observed a considerable ingestion of water at the moment of osmotic shock. Kirsch and Mayer-Gostan (1973) reported that the eel drinks about 680pl/hr/lOO g during the first hour after transfer to sea water and Pit et al. (1974) that sea water adapted mullet has a drinking rate of 1533 f 345 pl/hr/lOO g. Bath and Eddy (1979) reported a value of drinking rate increasing from 500 ul/hr/lOO g in the first hour to 2500 pl/hr/lOO g to the sixth hour after transfer of little (13.3 g) rainbow trout to two-thirds sea water. Salmon could attain comparable drinking rates which correspond to 380- 1250 pmol/Na+ entry/hi-/l00 g body weight, and which is of the same order of magnitude as the net Na + fluxes reported here.

Discontinuities in gill physiology with size and season in smolts maintained in freshwater likely point to the existence of a maximum size for smolting and confirm the existence of a relationship between grow. th and smoking. Thus, we have long been able to link smolting with small size in Sulmo s&r (Elson, 1957: Thorpe et al., 1982; Boeuf et al., 1985) and does not occur in a specific “time slot” (Eriksson and Lundquist, 1982) which thus corresponds to an aver- age weight which may vary with cohorts and years. Moreover, considering one particular population, in spring, smolting affects only the upper mode of the population (Thorpe et al., 1982; Boeuf et al., 1985). It is thus not surprising to find in graphic analyses a

range of weight representing a homogeneous category of the population, within which the animals display a characteristic physiological condition. Beyond this weight, the fish maintained in freshwater continue to grow while desmolting and a different relationship between fish weight and Na+ gill flux is observed.

Moreover, desmolting is accompanied in turn by a physiological condition similar to that described in rainbow trout (Boeuf and Harache, 1982; Langdon and Thorpe, 1985), and is integrated in the scheme by which smolting is a cylic phenomenon inhibited by unknown internal factors (Langdon and Thorpe, 1985; Higgins, 1985). It transforms the Sulmo s&r parr into a fish which have got some characteristics of marine fish and can survive with some difficulties in freshwater as is indicated by mortality observed in smolts maintained in freshwater beyond normal migration period.

Acknowledgements-We thank A. Leroux for technical assistance, C. Ungar for translating the manuscript and E. Floc’h for typing it. The Laboratoire de Physiologie Cellu- laire et Comparee (M. Bornancin) received financial support from IFREMER.

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