J. exp. Biol. (1979). 83, 181-192 l 8 liiVith 4 figures

Printed in Great Britain

IONIC REGULATION IN RAINBOW TROUT(SALMO GAIRDNERI) ADAPTED TO FRESH WATER

AND DILUTE SEA WATER

BY F. B. EDDY AND R. N. BATH

Department of Biological Sciences, University of Dundee, Dundee DDi 4HN

(Received 4 January 1979)

SUMMARY

Small rainbow trout (5-20 g) adapt to salinities of up to at least 22%o butnot to full strength sea water. In adapted fish plasma ions are regulated nearthe fresh water values, but there is an ionic invasion of the tissues, particularlyby Cl~ in muscle cells. Analysis of ionic regulation in adapted fish indicatesthat balance is maintained mainly by expending energy on Cl~ regulation.Fish in full strength sea water can no longer regulate Na+ or Cl~ in plasma ortissues, which results in high tissue concentrations of these ions and eventualdeath.

INTRODUCTION

Like other salmonids, mature rainbow trout are euryhaline and readily adapt to seawater. The physiology of sea water adaptation has been discussed by Hoar (1976) andHolmes & Donaldson (1969) while the earlier literature has been reviewed by Parry(1966). Three important points emerge for rainbow trout: (i) Larger individuals adaptmore easily to sea water than smaller ones, (ii) Survival is greatly improved if the fish arefirst preadapted to dilute sea water, (iii) Adaptation to sea water is accompanied by aninitial adjustment period lasting 3-4 days when the fish has to tolerate increased plasmaand body ion concentrations (Houston, 1959).

Much of the work on sea water adaptation has been upon various salmon -species,many of which, unlike rainbow trout, metamorphose from parr to smolt before enter-ing sea water. Atlantic salmon parr and rainbow trout of similar size (10-20 g) areunable to withstand direct transfer to sea water, while smolts survive the transition(Parry, 1958). Ionic regulation in Atlantic salmon parr and smolts was studied in somedetail by Potts, Foster & Stather (1970). Smolts in fresh water had low Na+ and Cl~turnover rates which increased tenfold after 1 day in sea water. Parr and early smoltsshowed smaller increases and, as was shown earlier by Parry (i960), they were lesstolerant of full strength sea water.

In the present work ionic regulation of small rainbow trout adapted to variousdilutions of sea water was studied by measuring a variety of parameters, including saltturnover, drinking rate, and ion concentrations.

182 F. B. EDDY AND R. N. BATH

MATERIALS AND METHODS

Rainbow trout (average weight 10-3 g) were obtained from a local hatchery and keptin dechlorinated water at 10 ± i°C. The were allowed to settle for several weeks andwere fed commercial trout pellets except during experiments.

Sea water (SW) of 23%, salinity was collected from the North Sea and diluted withdechlorinated water to give J SW (n%o) and § SW (22%o). Fish were adapted to•J SW for 10 days, then transferred to $ SW for a further 10 days and finally to fullstrength SW. Weight; length; haematocrit; plasma Na+, K+ and Cl~ concentrations;body and muscle water; and Na+, K+ and Cl~ contents of body and muscle weremeasured for fish in fresh water (FW), ^ SW, § SW and SW.

Fish were killed by a blow to the head and the tail amputated so that blood could becollected from the caudal artery into heparinized capillary tubes which were centri-fuged to determine haematocrit and then deep frozen for subsequent analysis of plasmaions. A muscle sample was taken from the dorsal block anterior to the dorsal fin,scraped clean of skin and weighed to the nearest milligram, as was the remainder ofthe fish. At this stage the fish were divided into two groups; the first was dried in anoven at 108 °C to constant weight for determination of water content while the secondwas homogenized and disrupted ultrasonically in a known volume of distilled waterto liberate readily exchangeable ions. These were eluted for 1 week at 4 °C, when thesample was centrifuged for ionic analysis of the supernatant.

Plasma and supernatant were diluted with distilled water to measure Na+ and K+concentrations using an Eel 100 flame photometer and Cl~ was determined witha Buchler-Cotlove ampiometric titrator.

Potentials were determined as described by Potts & Eddy (1973) and Bath & Eddy(1979). After inserting the peritoneal cannula the fish were allowed to recover for atleast 20 h before continuous recordings were made using a Bryans-Southern 28000pen recorder.

Na+ and Cl~ effluxes. 22Na and 38C1 were obtained from the Radiochemical Centre,Amersham. The radio-isotopes were mixed with trout saline to give a solution with aspecific activity of approximately 20/iCi ml"1 of each. Fifty/il was injected parenterallyinto each fish which was then placed in 750 ml of appropriate constantly aeratedmedium. Two ml samples were removed at known intervals. MNa activity was deter-mined using a Packard 5250 automatic y spectrometer and total ft activity with a Tri-carb Corumatic liquid scintillation counter. MC1 activity was obtained by subtractingthe MNa y9 activity previously derived by y counting. A sample of the injection mixturewas similarly assayed to determine the exact activity injected.

The rate constant, Kgtf, was determined graphically using the equation

where t is time, Ceq counts in the medium at time * (Motais, 1967). Km is the slope ofthe line, here fitted by regression analysis, obtained when (Ceq — CtICe(i) is plottedagainst t.

Drinking rate was determined by the method of Evans (1968) using 1MI-labellewPVP.

Inulin space. This was measured by injecting 14C-labelled inulin into the peritoneal

Ionic regulation in rainbow trout 183

cavity, allowing 24 h for equilibration throughout the body and then sampling plasma,muscle and whole body (as described earlier). The MC activity of the samples wasdetermined by liquid scintillation counting. Inulin space, /nflp, can be calculated by theequation

T 14C per ml sample water

i»Ln — „ _ : ; X IOO.p 14C per ml plasma water

Ion spaces. Na+,, Cl^,, and chloride-potassium space (Cl-K^p) were calculatedusing the mean values shown in Table 1 via equations given by Holmes & Donaldson(1969) and Conway (1957).

Intracellular water and ions. /CH,o and IClon respectively, were calculated from themean values in Table 1 using the equations:

(i) ICHl0 » total tissue — inulin space water.

.... T-, total tissue ion — extracellular ion content111) . /C|n n •= T7—: .w 1 — extracellular water content

Extracellular ion concentration was taken as:

/rjgp x plasma ion x Donnan* equilibrium concentration factor.

RESULTS

Rainbow trout used in the experiments survived indefinitely in FW, ^ SW, and f SWbut survival in SW averaged 5 days. Ionic composition of trout adapted to varioussalinities is shown in Table 1; SW values refer to fish which had been in this mediumfor 4 days. Full adaptation to J and § SW is indicated by constant plasma ion con-centrations and by well regulated body and muscle water content. Fish in SW showbreakdown of osmotic and ionic regulation; there being large increases in plasma ionconcentrations and significant dehydration of both body and muscle.

Whole body ions showed a modest increase in concentration in salinities up to § SW,while muscle ions, especially Cl~, showed rather larger increases. Further evidence offailure to maintain ionic balance in SW fish is shown by massive increases of all ions inboth whole body and muscle.

A similar situation was observed for body and muscle ion spaces, where the greatestincreases were observed in muscle Cl~ and (Cl-K8p), especially at the highest salinity.In contrast inulin space showed a slight decrease with salinity.

Ion contents calculated for the intracellular compartment are also shown in Table 1.With increasing salinity the values for muscle increase rather more than those for totalbody and the greatest increase was observed for Cl~ content of muscle cells.

Blood haematocrit was variable and there was no significant difference between thevalues recorded in the various salinities.

Drinking rates are shown in Fig. 1. FW fish drank approximately 0-5 ml kg"1 h-1,rather lower than 1-3 ml kg"1 h""1 recorded for salmon smolts (Potts et al. 1970), but incontrast to the results of Shehedah & Gordon (1969) where no drinking was observedn FW rainbow trout. Drinking rate increased to 2-5 ml kg"1 h - 1 in \ SW and 4 ml

-1 h-1 in f SW which are similar to the values reported by Shehedah & Gordon• As indicated by Manery (1954).

Tab

le I

Par

amet

er

Un

its

FW a

dapt

ed

+ SW

ada

pted

4

SW

ada

pted

Pla

sma

Na

+

CI-

K

+

Wat

er

Tot

al bod

y M

uscl

e P

lasm

a

g/

~w

g

wet

wt

g/~

oo

g

wet

wt

g/-

l3

PI- Io

n8

Tot

al bod

y N

a+

Tot

al b

ody

Cl-

T

otal

bod

y K

+

Md

e Na+

Mu

sde Cl-

Mus

cle

K+

m-m

ole/

kg bo

dy H

.0

m-m

ole/

kg bod

y H

I0

m-m

olef

kg h

dy

H.0

m

-mol

e/kg

mu

sde

HrO

m

-mol

e/kg

mus

cle -0

m

-mol

e/kg

mus

cle

H.0

(i)~

ota

l body

In

ulin

spa

ce

ml/

xoo

ml m

usd

e H

rO

Na

spa

a

25.9

4 f 1

.035

(5)

m

l/~

oo

ml m

uscl

e H

rO

Cl

spa

a

37'2

3 m

l/~

oo

ml

mu

sde

HrO

(

Cy

p)

32

.78

ml/

~o

o ml m

uscl

e H

rO

28.0

1

Ionic regulation in rainbow trout

186 F. B. EDDY AND R. N. BATH

7

6

- 500

i£ 4

Goo

1 3Q

2

1

1

\\

-

-o-

i i i i

t.

>

• i

0 20 40 60 80 100% seawater

Fig. i. Drinking rates for rainbow trout adapted to fresh water, J SW, i SW and | SW. The SWvalue is an estimate based on the values given by Shehedah & Gordon (1969). Mean values ± 1s.E. At least six fish were used at each salinity.

0-20

016

7 012

008

004

00 20 80 10040 60

% seawater

Fig. 2. Na+ ( • ) and Cl" (O) efflux constants (K h"1) for fish adapted to FW, J SW, J SW, | SWand SW. Mean and standard error are indicated. For each salinity at least six fish were used.

Ionic regulation in rainbow trout 187

Table 2. 5a// balance in rainbow trout adapted to various salinities

It has been assumed that branchial efflux is balanced by branchial influx plus drinking, andfaecal and urinary losses are negligible. Values in m-mole kg"1 h"1.

Na+ Cl-

Medium Efflux Drinking Influx Efflux Drinking Influx

FW 0-6910 00001 06909 0-2135 coooi 0-2134iSW I'SS69 0264 1-2929 1-004 0-337 0-667ISW 7-619 0-95 667 7-566 i-ai 6-353SW 12-73 2'3' i°'42 I4'01 a'95 n-oo

Salt absorption by the gut i« based on values given by Shehedah & Gordon (1969), 80 % in FW, 66 %in i SW, 80 % in f SW and 80 % inSW.

(1969). It is of interest to note that drinking increases in proportion to salinity andtherefore the osmotic gradient between body fluids and the external medium.

In FW fish the potential was slightly negative at 0-4 mV (Table 1) (the polarity refersto the inside of the fish). In ^ and § SW potentials remained steady at around + 2 mVduring the measurement period which in some cases continued for up to 24 h.

Fig. 2 shows Na+ and Cl~ efflux constants. In FW the rate for Cl~ is o-68% h - 1

while that for Na+ is larger at 17% h-1. The rates increase slightly in £ SW and thenrapidly up to § SW reaching 14% h"1 for Na+ and 18% h"1 for Cl~. In SW the ratesincreased little above the § SW values indicating a possible saturation of the effluxmechanism. These data indicate that entry to hyperosmotic media stimulates the effluxmechanisms.

Ionic balance of trout adapted to various salinities is shown in Table 2. In thesecalculations it was assumed that:branchial efflux = branchial influx + drinking, and that urinary and faecal salt lossesare negligible.

DISCUSSION

The results show that small (5-20 g) rainbow trout are well able to adapt to dilutesea waters, up to at least 22%,, but survive only a few days in full strength SW evenafter lengthy adaptation to § SW. Failure to survive SW appears to be associated withan inability to control plasma ion concentrations and with an invasion of the tissue cellsby inorganic ions, especially Cl~. This point is discussed in a subsequent section.

Tissue and body ion contents

The results indicate that adaptation to salinity is associated with increased cellularsalt concentration, particularly in muscle (Table 1). Body and muscle ion spacesincreased with salinity, particularly the Cl~ and (Cl-K8p) spaces which are sometimesused to give an estimate of extracellular space. However, it is unlikely that they reflectan increase in extracellular space where even in dilute SW the values become very high,e-g- 45 % f°r Cl~ space in § SW and also the inulin space remained relatively constant

t various salinities (Table 1). The Cl~ space of FW trout (3278 ml/100 ml bodyater) agrees well with the values reported for various FW fish (Lutz, 1972).Thus adaptation to dilute sea waters involves a number of mechanisms. First, the

salt load is not distributed evenly throughout the body but is concentrated mainly

i88 F. B. EDDY AND R. N. BATH

l-3r

1-2

Z 10

0-9

oo 0-8

0-7

0-6

20 40 60 80: seawater

100

Fig. 3. Relative Na+ and Cl~ permeability of Whole body, muscle and plasma in rainbow trout.Values calculated from mean values in Table 1. A, Plasma; O> whole body; • , muscle.

in the cellular compartment. However, to obtain a complete picture of the salt loaddistribution, it would be necessary to analyse the principal body tissues. The resultsindicate that muscle is relatively more permeable to Cl~ (Fig. 3). Since the whole bodyNa+:C1~ ratio remains steady with increasing salinity, this suggests that other tissuesmay be relatively more permeable to Na+ as has been indicated in liver cells of perchadapting to $ SW (Lutz, 1972). Secondly, the uneven distribution of the salt loadbetween extracellular and intracellular compartments allows the former to remainrelatively constant, thus protecting delicate tissues such as the brain from osmoticstress. In SW these mechanisms are insufficient and the tissues are exposed to osmoticstress.

Salt and water balance

The basic principles of salt and water balance in marine fish were originally outlinedby Smith (1930) and later by Maetz (1971). Trout entering a hyperosmotic environ-ment suffer an osmotic water loss which is replaced by drinking the medium. Theimbibed salt is excreted via the gills and there is strong evidence to suggest that ineuryhaline and marine species salt output is associated with branchial 'chloride' ormitochondrion rich cells (Maetz & Bornancin, 1975, Thomson & Sargent, 1977). Inaddition to the active salt output there are also bidirectional passive movements ofboth Na+ and Cl~ across the gills whose exact nature is unknown, but may containdiffusional and exchange diffusional components (Potts et al. 1970, MaetzBomanum, 1975).

While the data in Table 1 give steady-state information on ionic regulation inrainbow trout, they give less guidance as to how this state is achieved than do drinking

Tab

le 3

. S&

m

and

chlo

ride

bal

ance

in

rain

bow

trou

t aa

kpte

d to

4

and 4

SW a

nd in f

ull

stre

ngth

SW

Na

+

Cl-

A

r t

-,

Equ

ilib

rium

M

easu

red

Pot

enti

al

To

tal

Pas

sive

C

alcu

late

d

Med

ium

po

tent

ial

Na+

C

I-

efA

ux

infl

ux

pass

ivee

fflu

x

Fw

-0.3

6 -1

57

+I*

0.

6g10

-

-

4 S

W

+ 2'4

+

za

-1

3.4

1.55

69

1.a9

29

1.30

06

Pas

sive

C

alcu

late

d in

flux

ps

asiv

e ef

flux

-

-

0.66

7 0.W

6.35

3 14

397

11

.~6

3

37

3

(Equ

iliir

iurn

pot

enti

als

wer

e ca

lad

ated

using t

he

Ne

mt

equa

tion

whe

re R

is the u

nive

rsal

gas

constant, F the F

arad

ay, s th

e io

nic

charg

e, C

, th

e in

tern

al c

once

ntra

tion

, C. the a

tcrn

nl c

once

ntra

tion

and

E is

th

e po

tent

ial.

Pas

sive

&lux

waa

cal

cula

ted

from

th

e eq

uati

on

Pm

. fiu

x - C

c E

F

pass

. id

ux

- E

UP

RT.

Flu

xes

are

in m

-mol

e/kg

-I

h-l

. T

he

'act

ive'

efflux

is a

h e

xp

d

as a

per

anta

ge

of t

he

tota

l efflux. P

ota

rial

s arc

give

n in

mV,

pola

rity

referring t

o i

nsid

e of

fish. I

n f

d

wat

er t

he

pass

ive

inth

u ia

neg

ligi

ble.

)

i9° F. B. EDDY AND R. N. BATH

rates and fluxes (Figs, i and 2, and Table 2). For a fish to be in ionic balanceuptake must equal salt output:

branchial efflux + urine + faeces = drinking + branchial influx.

In SW fish salt losses via urine and faeces are negligible. The branchial efflux may beconsidered to consist of two components, the active and the passive effluxes:

branchial influx = active efflux + passive efflux - drinking.

Assuming that the branchial influx is entirely passive, then using the potentials datathe passive efflux can be calculated (Table 3), i.e. exchange diffusion and active influxare equal to zero.

In $ SW Na+ concentrations in blood and water are almost equal and the equilibriumpotential for Na+, calculated from the Nernst equation (Table 3) is close to the mea-sured potential of + 2-3 mV. Thus little energy is needed to maintain Na+ balance andthe active component of the efflux is relatively small. However the external Cl~ con-centration (174 mM) is higher than the plasma value (109 mM), the Nernst potentialis about — 12 mV, and thus Cl~ tends to enter the fish down both concentration andelectrical gradients. This necessitates energy expenditure for Cl~ regulation and the'active' component is correspondingly high at about 58% of the total Cl~ efflux.

Similar arguments can be applied to fish adapted to § SW; in this case both Na+ andCl~ enter down concentration and electrical gradients and energy must be expended tomaintain ionic balance, hence the large 'active* components of the Na+ and Cl~effluxes (Table 3), Cl~ being the greater. This analysis has also been extended to SWfish; in this case the 'active' components are similar to those for § SW adapted fish,suggesting an inability to increase this portion of the efflux. However, in these fishionic balance cannot be assumed since they continually gain salt up to the time ofdeath.

It is probably incorrect however to assume that the influx is entirely diffusional(potential dependent), i.e. there may well be an exchange diffusion component. Anexchange diffusion component for Na+ efflux for the intertidal fish Xiphister in 100%SW was proposed by Evans (1967) while a similar component of the passive Na+

fluxes in the SW smolt was suggested by Potts et al. (1970). Part of the passive Na+

and Cl~ fluxes in some marine fish such as the mullet (Maetz & Pic, 1975) may beexchange diffusion.

From the present data there is no way of assessing this component but if an exchangediffusion component is included in the calculations for passive efflux (Fig. 4) one seesthe following effects. As this component is increased the active efflux is steadily de-creased, the values for Na+ being approximately 10% lower than those for Cl~throughout, except for fish adapted to \ SW where the 'active' component for Na+remains almost constant. Thus it appears that as the hypothetical exchange diffusioncomponent is increased the energy required for ionic balance would be reduced.

In conclusion small rainbow trout are well able to adapt to salinities up to at leastf SW but not to full strength SW. In adapted fish plasma ions remain near the FWvalues, but there is ionic invasion of the tissue cells, e.g. Cl~ into muscle cells. Analysiof ionic balance in adapted fish shows that ionic equilibrium is maintained mainly

Ionic regulation in rainbow trout 191

ii

uu

80

60

40

20

n

m

I | 1 1 1 1

20 40 60Exchange diffusion

total influx

80 99-99

Fig. 4. Percentage active Na+ and C\~ efflux in rainbow trout adapted to i and 1 SW as a func-tion of increasing percentage exchange diffusion. Values were calculated using the data andequations shown in Table 3. • , Na+, i SW; O, Na+, | SW; A, Cl", i SW; A, Cl," i SW.

greater energy expenditure, especially in Cl~ regulation. Fish in full strength SW canno longer maintain ionic equilibrium and Na+ and Cl~ invade both tissues and plasma,resulting in eventual death.

This work was supported by NERC grant GR3/2942.

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7-a

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