Sodium-potassium balance in the black molly (Poecilia latipinna)

SODIUM-POTASSIUM BALANCE IN THE BLACK MOLLY (POECILIA LA TIPZNNA) EILEENA. BESWICK Department

of Physiology,

University

of Queensland,

(Rrcriwd

4 July

Brisbane,

Queensland

4067. Australia

1977)

Abstract---l.

Black mollies were adapted for 17 days to tapwater. or water containing 154 mmole/l NaCl or 3.4 mmole/l KC1 only, or water containing both 154 mmole/l NaCl + 3.4 mmole/l KCI. 2. After exposure to both the water which contained Na+ + K’ and the water which contained K + alone. the Na/K ratio in skeletal muscle was approx 0.5: 1. by Na’. 3. During exposure to the K’-free water. K+ was lost from the muscle and replaced The Na/K ratio increased to 1.5: 1 in tapwater and to 4: 1 in 154 mmole/l NaCl. Bone and skin changed in a similar manner. 4. It is concluded that accumulation of Na’ was due to the absence of K’ rather than to the presence of Na + in the tankwater.

IYTRODL’CTION Fish adapted to freshwater take up Na’ from the water while seawater-adapted fish surrounding extrude Na’ (Homer Smith, 1930). It is now generally accepted that the site of Na+ exchange is the gill (Maetz. 1969: Evans et al., 1973). The rate of Na’ exchange can be influenced by many factors including internal acid-base balance, and the external concentrations of Ca2’ and K+ (Evans, 1973. 1975). Maetz (1969) demonstrated the presence of Na’ fluxes in both directions of the order of 2600 ~mole/hr/lOO g in seawater adapted flounder. with a net outflux of about l00~mole/hr/lOOg. The outflux was balanced by a K + influx of Na’ 120 ~mole/hr/lOO g. If the external K+ was removed there was a consistent reduction of the Na outflux of 80~mole/hr/lOOg, resulting in an increase in both plasma Na’ and Na+ space. Conversely, K’ added to the external medium stimulated Na outflux. In seawater-adapted Dormatator maculatus, Evans et a/. (1973) demonstrated that both K+ linked Na’ extrusion and Na+-Kf activated ATPase could be described by the Michaelis-Menten equation and had similar K, values of 2mmolejl Kf. They concluded that Na+ extrusion is coupled to K’ uptake and is mediated by Na’-K’ ATPase. External K’ is essential for the functioning of the Cl- pump also. Seawater-adapted eels transformed to freshwater showed a 100% increase in Cl- efflux when 10 mmole K2S04 was added (Epstein et al., 1973). A well-established treatment for certain fish diseases and parasites is to place the fish in a salt solution for several days (Emmens. 1953; Van Duijn, 1973; Cohen. undated). To determine the effect of salt adaptation on the internal Na’ and K+ balance. black mollies (P. latipinna) were adapted over 9 days to water containing added Na’. Kf or Na’ + K’. The Na’ and K’ contents of plasma. muscle, bone. skin. gut and liver were then monitored during the adaptation period and the following week.

MATERIALS AND METHODS

Black mollies (P. latipinna) weighing approximately 1g were obtained from Pisces Enterprises, Brisbane. and allowed to adapt for 1 week to laboratory conditions (24”C, 15 hr fluorescent light. 9 hr darkness). Twenty-four fish were placed in each tank containing 8 I. of Brisbane tapwater (average composition Na’ 1.5 mmole/l. K’ 0. I mmole/l. Cl- 2 mmole/l. hardness, 70 ppm CaCO,, 70ppm MgCO,). The water was filtered through a plastic filter containing activated charcoal and glass wool and was bubbled with compressed air. Fish were fed with Litrabon Staple Food. Fish were then adapted to 1 of 4 salt treatments. Na’. Kt. Na’ + K+, or no added salts. One ninth of the total Na’ or K’ was added to each tank from day 0 to day 8 inclusive. From day 8 on, the Na’ concentration in those tanks containing Na+ was 154 mmole/l and the K’ concentration in those tanks containing K’ was 3.4 mmole/l. which is approximately one third the Na’ or K+ in seawater. Four fish from each treatment group were sampled on days I, 5, 9. 13 and 17. The fish remaining after each sampling period were immediately transferred to clean water containing the appropriate salt content. The fish were starved for 24 hr, anaesthetired with MS222 (ethyl-m-aminobenzoate methanesulphonate) and washed in distilled water for 15 sec. Blood samples were taken by placing the fish on its back; the body wall was ruptured above the heart, the pericardial cavity blotted with filter paper to remove pericardial fluid. and a heparinized microhaematocrit tube drawn to a fine point was plunged into the ventricle. The tube was sealed with wax. centrifuged for 2 min at 15,000 9 and the haematocrit read: a plasma sample varying between 3 and 10~1 was drawn off with a lO$ syringe and transferred to a 2 ml cup. The sample syringe was flushed with deionized water and the washings transferred to the cup to give a final volume of 20~1. The fluid was evaporated, 0.2 ml of deionized water was added to each cup. and Na’ and K’ concentrations read by feeding the entire 0.2 ml of the sample into a 2-channel flame photometer attached to a 4.channel recorder which integrated the output. Integrated values were used to estimate plasma Na’ and K _ concentrations from standard curves. Five tissues. skin. skeletal muscle. bone. gut and liver were sampled for Na’ and K’ levels. The tail of the fish

226

EILF:E.N

A Br Swlch 30 I

No

w . 0’

’

!

1

5

.-.

_.__i-_l___ 9

13

n

1

5

D

9

17

D%

Days

Na/K

Na+K

Fig. I. Changes in plasma Na-. K’ and Na’ + K’ contents. and Na’:K’ ratio of controls ( A addition (N) of 154 mmole Na-11 (-- O--). 3.4 mmole K + :I ( -A-~ ). or I54 mmole Na’ + 3.4 mmole K-11 (--e) to the tankwater and durmg the succeeding week

or during

Table

I. Summary

Source of variation Na’ treatment Ka* treatment time Na’ x K’ Na+ x time K’ x time Na’ x K’ x time Error variance

of analyses

d.f. I

1 4

I 4

4 4 51

* P < 0.05. ** P < 0.01

of variance of changes trated in Fig. 1.

Na’ 6.X8* 1.16 6.35’*

1.a 0.8 1 I .6X 0.33 635.70

m Ion content

Variance ratios K’ Na’ + K’ 1.15

4.29s 5.x7** I .46 0.36 0 92 0.39 I2.X7

7.s5** 0.56 6.71** 1.99 0.80 I .90 0.32 632.28

of plasma.

Na’

IIIUS-

‘K*

0.5h 1.49 5.30** 0.28 0.33 0.68 0.10 66.03

J

Sodium-potassium

balance

in the black

227

molly

K

Na

2m

Na/K

Na+K

M

c

t Q

‘5 L

P

Y .

z

z” 03

0

: 5

1

Days Fig. 2. Changes

Table

in Na’.

K’

2. Summary

Source of variation Nat treatment K’ treatment time Na+ x K‘ Na+ x time K+ x time Na+ x K’ x time Error variance

n

u

9

Days

and Na’

of analyses

+ K’

contents. and Nat/K’ as in Fig. 1.

of variance of changes trated in Fig. 2

d.f.

Na’

1 1 4

5.67* 12.54** 0.22 2.60 0.57 3.81* 0.88 61 1.35

I 4 4 4 60

* P < 0.05. ** P < 0.01

ratio

of skeletal

in ion content

Variance ratios K’ Na’ + K’ 5.41* 31.29** 1.25 0.2 1 0.97 4.73** 1.25 281.88

1.91 0.20 0.90 5.02* 1.70 2.53 1.77 207. I9

muscle.

of muscle

illus-

Na’/K+ 7.59** 13.39** 0.81 4.17’ 1.19 3.18* 1.61 1.86

Legend

K

Na/K

Days

Days Fig. 3. Changes

in Nav.

K’

Table 3. Summary

and

N’a-

+ K

contents, Fig. I.

and

Na + K.

ratio

of bone.

Legend

of analyses of variance of changes in ion content of bone. illustrated in Fig. 3

Source of variation Na + treatment K ’ treatment time Na’ x KNa. x time K ’ x time Nax K’ x time Error variance

* P < 0.05. ** P <

d.f.

Na-

I

5.95*

I 3

5.61

I 3 4 4 60 0.01

7.77*+ 022 1.50 2.06 1.12 328.57

Variance ratios Na’ + K’ K’ 3.87 26.64:’ 7.22** 0.09 0.57 3.03* 0.80 9x.1 1

4.16* 0.20 3x70** 0.09 1.72 0.72 2.86* 169.22

Na’.!K

X.3?** 15.99** 0.20 2.46 1.50 431** 1.10 0.81

as in

Sodium-potassium

balance

in the black

229

molly

No+K

Fig. 4. Changes

to Na+.

K’

Table 4. Summary

and

Na/K

Na+ + K’

of analyses

contents. Fig. 1.

of variance of changes in Fig. 4

Source of

variation Na+ treatment K’ treatment time Na+ x K’ Na+ x time K’ x time Na+ x K+ x time Error variance

d.f 1

I 4

I 4 4 4 60

* P < 0.05. ** P < 0.01

and

Nat 0.78 8.08;: 3.12* 2.40 0.80 0.28 0.46 234.30

Na’/K’

ratio

in ion content

Legend

of skin, illustrated

Variance ratios Na+ + K’ K’ 5.70* 3.81 12.73** 0.62 1.35 1.69 0.75 167.57

of skin.

0.66 0.73 6.31** 2.53 1.66 0.66 0.35 453.04

Na’/K’ 9.52** 11.50** 8.04*’ 0.66 0.64 3.33* 1.35 0.16

as in

230

EILELN

A. Btsw~ca M

NCI

202

I

No/K

Na+K

Fig. 5. Changes in Na’.

Table

K

5. Summary

*

and Na’

+ K * contents,

K

’

ratio of gut

of analyses of variance of changes in ion content in Fig. 5

Source of variation

d.f.

Na* treatment K + treatment time Na’ x K’ Na‘ x time K+ x time Na’ x K* x time Error variance

I

5.85’

I

0.I 7

* P < 0.05. **

and Na.

3 I 4 4 3 60

P < 0.01.

Na’

22.92** 1.O’ 0.33 0.32 1.O6 65.55

of gut. lllustratcd

Variance ratios K’ Na’ + K’ 0.01 3.03 7.36** 2.37 1.57 1.55 0.34 73.54

3.44 2.71 1901** 3.70 I .42 0.47 0.20 123.55

Legend as 111Fig.

Na’,K’

I .96 0.35 10.9x** 0.05 0.46 I .50 I.1 I 0.018

I

Sodium-potassium

balance

in the black

molly

231

Na

>

Na/K

Na+K

Fig. 6. Changes

in Na+,

K+

Table 6. Summary

Source of variation Na’ treatment K+ treatment time Na* x K+ Na’ Y time K’ x time Na’ x K’ x time Error variance

and

Na’

of analyses

d.f.

1 I 4 1 4 4 4 59

* P < 0.05. ** P < 0.01.

+ K+

contents. Fig. I.

and

Na+/K’

of variance of changes in Fig. 6

Na+

0.80 0.0003 I8.33** 0.03 1.41 1.58 I.1 1 48.44

ratio

in ion content

of liver.

of liver, illustrated

Variance ratios Na’ + K’ K’ I.14 0.12 22.514~~ 1.73 2.68* 2.80: I.18 61.07

Legend

2.26 0.08 41.21*’ 0.90 2.46 I .45 1.22 93.62

Na’/K+ 0.07 0.01 6.74** 0.15 1.67 1.55 I .07 0.008

as in

HLSWK

rhe cloaca was sevcrcd. \calcd. and the fin raq\ of the la11 removed. The rkln wa\ carefull! peeled otl’. 2 fillets ol’ skeletal muscle cut elf and the rcmalning skeletal muscle dissected away l’rom the spinal column; the gut and hver wcrc removed. All 5 tissues were blotted on filter paper to remove surface moisture. welghed in alu-

h

po~tcr~or to

mmium foil pans. dried In a vacuum oven for a minimum 01‘ 72 hr and welghed again. The drlrd samples were dlssolved in 0.5~ I .O ml of concentrated mtrlc acid. the volume made up lo 5 ml for skin. bone gut and liver. and to IO ml for muscle. Na’ and K’ concentrations were estimated using an EEL flame photometer. Water content was calculated b! subtraction of wet and dry weights. The total Na’ + K ’ content and the Na:K ratios were also calculated. The Na ’ spaceof skeletal muscle was calculated from the formula

Na,,, IS the muscle Na + m mmole.#kg of tissucwatcr. Na,, is the plasma Na + in mmole~l of plasma. H,O,, is plasma water In g;lOOml and 0.942 is the Gibbs Donnan ratio of the Na ’ distribution between plasma and extracellular

Huld (Manery 1954). The Naj. K’ and Na* + K . contents and the Na K ~ratlo of plasma. muscle. bone. \kln. gut and liver, and the Na space of muscle wcrc each tested hy a 3-wa! analysis of varI;mcc for signllicant cfTcct\ 01‘ Na’ and K’ In the lank water. and of tlmt‘.

HESL’I.TS Two deaths were recorded in fish treated with Na * alone on dais I I and I?. No deaths occurred in any other treatment group. K ’ in the tankwater. irrespective of the presence or absence of Na *. had the greatest effect on the Na’ and K’ contents of the tissues. When K + was absent from the tankwater. the K’ content of plasma, skin. muscle and bone decreased and. in skin. muscle and bone. was coupled with an increase in Na’ content of approximately, equal magnitude. so that there was ;I large increase In the Na+!K’ ratio but no significant change in total Na‘ + K- content. The magnitude of chongcs was greatest in muscle followed bg hone. skin and plasma. K * in the tank water interacted with time to decrease the K ’ content of muscle and bone. and to increase the Na’ content of muscle (Figs I 3: Tables l-4). The Na space of muscle after I3 days was about 35 ml IWg when K’ was present in the tankwater. but was 55 ml ‘I00 g in tapwater controls and 80 ml! I00 g when onl! Na + was present 111the tankwater (Fig. 7 and Table 7). Superimposed on the large changes induced by the absence of K + from the tankwater were small changes due to the presence of Na-. There were significant Increases in the Na i content of plasma. muscle. bone and gut and small decreases in the K + content of skm and muscle. The total Na* + Kcontent of plasma and bone increased (Figs I 5: Tables I 5). Apart from the small increase in Na + content in gut due 10 Na- m the tankwater. gut and liver were largely unalrected by the Na‘ or K * treatments (Figs 5 & 6: Tables 5 & 6). In all tissues except muscle. there was a change in the Na’. K + and Na- + K _ contents and the Na* K’ ratio with time. but there was no consistent trend (Figs I 6; Tables 1~6).

The measured concentrations 01’ plasma k all treatment groups ranged front about 7 mmole.1. This is much greater than reported literature for other fish spc’c~cs (Hoar & Randall. lY6Y). Large valutx here possibl! due to leakage OI Kt from massive injur) caused Mhen the plpettc’ MXS inserted into the small heal-t. Despite the high values, the K concen1ratlon m the plasma of fish adapted to water ulthout K ua\ significantl! loher. and ahen Na W;I~ added to the K-free water. there U;IS ;I large drop of up to W’,, in the K* content of muscle and bone. The lost K appeared to be replaced h\ Na _ Fdl from \r;1lcIcontaining Na _ alone became lethargic and .? ti\h died. Since Kuptake IS Inhlhiteci m K-IICC u;llel K-linked Na + extrusion v,ould alw he Inhllxtcd (Evans. 1973. 1975; Maet/. lY6Y1. .A\ K cotltlllwb to be lost pass~velq. plasma K ’ would decrease \< hlle plasma Na ’ would increase. It Mould folloa that. over a period of days. K would be drawn out 01 other tissues to maintain plasma K ’ levels. To maintain intracellular electrical and osmotic balance. K would be replaced b> ;I positive ion. The K ’ content decreased the most in muscle. bone and skin. Indic:lting that these organs must have II large intraccllulal supply of labile K ’ The Ya ’ content incrca5cd 2nd there was no significant change 111 total Na 1 K content. therefore the in~raccllular K ’ uwld h,r~c been replaced largely b> Na ‘. In contrast. NA add4 10 the tankwater ~nducctl onI1 ;I small mcrcasc m the Na content of the Ils;\uc\ added t(, the t:rnk\ralcr The concentration of Na concentration of plasma was ver! similar lo the N:I Fnsor & Ball (1977) ha\c found rhat h!poph>hcctomired P. /atipi,~r~tr could \ur\i\c 111one-thir-d \c;tw;ltcr where the Na + concentration \+;I\ 16Xmmole 1 and thus the Na’ in the tank~~\a~er \\ar unlikel! tt) pro\ide ani stress In itself Na . .space is used as an estimate of e\traccllular space (Manery. 1954: Holmes & Donaldson. lY6Y). and is based on the assumption that all the Ua ih in the extracellular compartment. Chan c’[ rrl. (1967) and Mayer & Nlbelle (1969) both demonstrated the presence of intracellular Ua ‘. the amount of which retlccting the true will var) the Na _ space without value of extracellular space The Na . space of skeletal muscle of fish adapted to K-free \b\atcr contammg IS4 mmole:l NaCl was about X0 g H,O I00 g. and thus under conditions &here a conalderablc quantlt! of Na* had moved into the intracellular compartfrom rxtracellular ment, Na ’ space is \erq different space and suggests that more than half of the Intraccllular K has been lost and replaced Mith Na HOW estimate of the ratio at” ever Na * space is ;I uxful the total Na content to that which one would expect if Na’ wet-c ebenlv distributed throughout Ihe Ir\suc. \%;I5 The Na space 6f fish adapted to Na ’ t K about 35 g I(x) g \+hile tish adapted 10 tapwater had space of about 55 g IOU g. This i\ difl’erent ;I Nu space (>I‘ freah\+ater and from the whole-hod) Na saltwater eels where the !%a- space of saltwater ccl\ exceeded that of freshwater eels (Maer bi Nibclle. IYhY). The Na ’ space01‘mollies adapted lo K ,tlonc

Sodium-potassium

balance

233

in the black molly

have been investigated. It would seem justifiable to speculate that the cause of death in fish adapted to Na’ without K’ may be due to an acid-base disturbance. The role of K+ in acid/base balance bears further investigation. The evidence suggests that at least some species of fish should not be placed in a permanent bath of NaCl without KCl, and that the K’ content of some freshwaters may be sub-optimum for the keeping of some aquarium fish. Acknowlrdgemw-I

for his valuable

technical

wish to thank assistance.

Mr

Stephen

Moss

REFERENCES CHAN D. K. 0..

Fig. 7. Changes

in Na space of skeletal as in Fig. 1.

muscle.

Legend

Table 7. Summary of analyses of variance of changes in Na+ space of skeletal muscle, illustrated in Fig. 7 Source of variation

Na+ treatment K’ treatment time Na+ x K’ Na’ x time K’ x time Na’ x K’ x time Error variance

d.f. 1 I 4 1 4 4 4 51

Variance

ratio

1.05 21.32** 2.44 6.99* 0.87 6.30** 1.20 190.66

* P < 0.05. ** P < 0.01

had a Na’ space similar to that of fish adapted to Na+ + K+. As the Na’ space in fish adapted to tapwater was greater than this, it would appear that these fish have accumulated Na+, even against such a large concentration gradient, due to a sub-optimum Kf concentration in the tankwater. This suggests that there is a small K+-dependent Na+ efflux in fish adapted to freshwater as well as in those adapted to saltwater. The relationship between potassium balance and acid-base regulation in human medicine is well known (Pitts, 1963). In K+ deficiency, intracellular K’ is lost and may be replaced with either Na+ or H’. It is postulated that the increased intracellular H‘ concentration in the renal tubular cells induces an increased excretion of H+ in the urine, and the plasma becomes alkaline. Darrow (1950) has demon-

strated in rats with a primary K+ deficit, the concentration of bicarbonate in serum varied inversely with the intracellular Kf concentration as well as directly with intracellular Na+ concentration. In fish. Na+ uptake in the gills is exchanged for H’ or NH: and Cl- is exchanged for HCO; (Evans. 1975; Kerstetter & Keeler, 1976). but whether K’ uptake influences H’. NH;. or HCO; extrusion does not appear to

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An Inrroductory

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