1. The rate of loss of sodium, chloride and water via the urine and the rate of intake of sodium, chloride and water by ingestion of the medium was determined for the euryhaline teleost, Xiphister atropurpureus.

  2. The urinary losses of sodium and chloride were approximately 0·5 mM/kg. fish/day in both 100% sea water (480 mM-Na/kg.) and 10% sea water. The ingestion of sodium and chloride by drinking the medium amounted to approximately 4 mM/kg. fish/day in 100% sea water and approximately o-i mM/kg. fish/day in 10% sea water.

  3. The low rate of urine flow in 10 % sea water and the low drinking rate in 100 % sea water indicate a relative impermeability to water in both salinities.

Maintenance of relatively stable concentrations of sodium and chloride in varying salinities by euryhaline teleosts involves a complex of physiological functions first described by Smith (1930). In short, marine teleosts balance the net efflux of water and influx of sodium and chloride by drinking the medium, excreting small quantities of urine that contains less sodium and chloride than the blood and excreting the excess sodium and chloride extrarenally, presumably across the gills. In fresh water (or in any medium hypo-osmotic to the blood) the teleost balances the net influx of water and efflux of sodium and chloride by excreting large quantities of urine that contains much less sodium and chloride than the blood and by actively pumping in sodium and chloride from the medium, again, presumably across the gills.

Quantification of the components of this Smith model has progressed slowly but is now receiving increasing interest. Most workers have been concerned chiefly with urine flow rates and concentrations. Black (1957) gives a summary of the early data. Holmes (1961), Sharratt, Chester Jones & Bellamy (1964), Maetz, Bourget & Lahlou (1964), Fleming & Stanley (1965), Holmes & Stanier (1966), Stanley & Fleming (1966a, b) and Motais (1967) have compiled the most recent and complete data on kidney function of teleosts. In most cases these workers utilized open cannulation systems that maintained the experimental fish in a relatively normal state. Earlier workers’ use of divided chambers, ligation of the urinary opening and closed cannulation systems may have had adverse effects on the experimental animal.

Measurements of the intake of ions and water by ingestion of the medium are relatively rare. Smith (1930), Motais & Maetz (1965), Potts & Evans (1967) and Potts et al. (1967) are the only direct measurements. Hickman (1967) has recently reported an ingenious indirect method based on the total loss of sulphate and magnesium ions.

It has been shown (Evans, 1967) that the intertidal blenny, Xiphister atropurpureus is able to regulate the sodium and chloride concentration of its plasma and its intracellular spaces over a range of salinities from 100% sea water (480 mM-Na/kg.; 560 mM-Cl/kg.) to 10% sea water. Further experiments were undertaken to ascertain the roles played by the kidney and the gut in the ionic and osmotic regulation of this species.

The methods for collection, maintenance and anaesthetization and weighing of Xiphister have already been described (Evans, 1967).

The method of urine collection was modified from Stanley & Fleming (1964). Under a dissecting microscope a short (2 cm.) length of polythene tubing (PE 10) was inserted through the urinary sphincter and a short way into the duct leading to the bladder of an anaesthetized fish. The tubing was stitched to the anterior end of the anal fin. The seal between the sphincter and the tubing was tested by gentle pressure on the abdomen; if the tubing filled with urine the cannulation was considered successful. (The seal was again checked by the same method at the termination of the experiment. If leakage occurred at this time the data from that fish were discarded. Leakage was found by this method in only 2 of approximately 100 cannulations. These checks did not preclude the possibility of intermittent leakage during the experiment.) The distal end of the PE 10 tubing was inserted into a long (20−30 cm) length of PE 50 tubing. The fish was returned to the aquarium of the experimental salinity and allowed to recover for at least 12 hr. During this period the cannula filled with urine. At the end of the recovery period the fish was placed in 200−250 ml. of the experimental salinity in a small polythene aquarium. The distal end of the large cannula was threaded through a serum-bottle stopper in the wall of the aquarium and inserted into the end of a 0·2 ml. pipette graduated in microlitres. An air inlet provided sufficient aeration (Fig. 1). Urine flow readings began immediately and lasted from 6 to 12 hr. At the termination of the experiment the animal was removed from the aquarium and anaesthetized. The cannula was removed, the urine was run into a capillary tube and frozen for analysis; the fish was dried and weighed, the blood was drawn, the fish was sexed and then decapitated and discarded.

Fig. 1.

Cannulation system for urine-flow experiments.

Fig. 1.

Cannulation system for urine-flow experiments.

The methods described previously (Evans, 1967) were employed for sodium and chloride analysis of the urine.

14C-inulin was used as an inert tracer to determine the amount of the medium that was ingested by the fish. Fish were placed in 200 ml, of the experimental salinity containing 10−20 μC. of 14C-inulin solution that contained 90% non-radioactive carrier inulin. After 1 hr. the fish was removed from the solution, placed in non-radioactive medium for 30 min., anaesthetized, weighed and the gut removed. The gut was homogenized in 2 ml. 98 % formic acid and this was made up to 10 ml. with distilled water. 1 ml. of the diluted homogenate was added to 10 ml. Bray’s solution (Bray, 1960) and centrifuged. The supernatant was poured into a vial for liquid scintillation counting and labelled ‘A’. The pellet was redissolved in 2 ml. 98% formic acid. 25 % of this solution was added to 1 ml. distilled water and 10 ml. Bray’s solution in a vial for scintillation counting. This vial was labelled ‘B’.

A standard was set up by adding 0-2 ml. of the radioactive drinking solution to a gut that had been removed from a non-radioactive animal and preparing it for liquid scintillation counting in the same way as the gut of the experimental fish. The samples were counted to 1000 counts on a Nuclear Chicago Mark II Liquid Scintillation System. The amount of drinking was calculated from the following relationships:

formula
formula
formula

The experiments were run for only 1 hr. to avoid loss of the ingested inulin from the anus. To test whether inulin was lost by movement across the gut into the blood a few fish were injected with 14C-inulin by means of a polythene tube down the oesophagus. No radioactivity was detected in the blood until after 12 hr. of incubation.The fish were placed in non-radioactive medium for 30 min after the experiment to allow any inulin that had been swallowed to move down the gut, thus lessening the possibility of regurgitation of the ingested inulin. Longer-term (up to 6 hr.) experiments gave approximately the same results as those of 1 hr. This implied that the loss from the mouth was negligible. A few experiments performed on individuals acclimated to 31 % sea water showed that the drinking rate was nearly equivalent to the urine flow; this would be expected at this iso-osmotic salinity. This indirect check supported the validity of the data on drinking rates in other salinities.

The results of the cannulation experiments are presented in Table 1. It must be added that in both salinities five or six individuals displayed urinary losses of sodium and chloride 5 to 10 times that of the mean of the other animals. This could be accounted for by ‘laboratory diuresis’ (Forster & Berglund, 1956); but the experi-mental set-up provided a minimum of stress, and Fleming & Stanley (1965) have shown that in Fundulus kansae an initial and reproducible period of diuresis is found in cannulated individuals acclimated to fresh water but not in individuals acclimated to sea water. It is interesting to note that in the 100% sea water experiments this increase was due primarily to an increase in the sodium and chloride content of the mine (2− 4 times concentrations of sodium and chloride in the plasma), while in the 10% sea water experiments it was due to an increase in the apparent urine flow. In both cases this implied leakage around the cannula (rather than diuresis) and the data from these animals was therefore discarded. Data from both sexes and reproductive and non-reproductive animals were combined because no differences were found.

Table 1.

Losses of Na, Cl and water via the urine[X¯±S.E.(N)]

Losses of Na, Cl and water via the urine[X¯±S.E.(N)]
Losses of Na, Cl and water via the urine[X¯±S.E.(N)]

In calculating the loss of ions in the urine mM/kg. urine was taken as equivalent to mM/l. urine. Urine water content was taken as 100 % by volume to facilitate water loss calculations. Despite the extreme variability of the data one can safely conclude that in both 100 % sea water and 10 % sea water Xiphister has a very low rate of urine flow. The rate in 100% sea water is slightly below that reported for other euryhaline species; Fleming & Stanley, 1965; Sharratt, et al. 1965; Holmes & Stanier, 1966; Motais, 1967). The concentrations of sodium and chloride in the urine are nearly the same as those in the blood. In 10% sea water the urine flow rate does increase, but the losses of sodium and chloride do not because the reabsorption of these ions by the kidney is increased. Comparison of Xiphister’s urine flow rate in 10 % sea water with the flow rates reported for euryhaline teleosts in fresh water can be made if one assumes that there is no change in water permeability when a teleost goes from fresh water to 10 % sea water, and that the urine flow in hypo-osmotic salinities is correlated directly with the osmotic gradient. If an average euryhaline teleost has a urine flow of 75 ml./kg. fish/day in fresh water then it would be expected to have a flow of 50 ml./kg. fish/day in 10 % sea water. Therefore Xiphister’s low rate of urine flow in 10 % sea water indicates that this species is relatively impermeable to water.

Table 2 summarizes the rates of ingestion of the medium by Xiphister. The rates of ingestion of sodium and chloride were calculated by multiplying the drinking rate by the sodium or chloride concentration of the medium. The rate of ingestion of water is taken as being equivalent to the drinking rate because even 100% sea water is over 99 % water by volume. The drinking rate in 100 % sea water is less than 10 % of the rate found for the other teleosts that have been examined (Smith, 1930; Motais & Maetz, 1965; Potts & Evans, 1967; Potts et al. Hickman, 1967). Since the drinking rate in 100 % sea water is thought to be equivalent to the osmotic loss of water it therefore appears that, like the urine flow in 10% sea water, the low drinking rate indicates a relative impermeability to water. A drinking rate of approximately 1 % of the total body water per day (see Evans, 1967) means that if the fish did not drink for 24 hr. it would theoretically be dehydrated by only 1 %. It seems likely that the total body water of an individual fish varies to this degree during the course of 1 day. Ingestion of water is therefore probably intermittent and may merely be a side-effect of eating. If the drinking is indeed intermittent the rates found in these experiments could have been biased by allowing the fish to drink for only one hour. Drinking does occur in 10 % sea water, though it merely aggravates the osmotic problems of the fish. Ingestion of the medium in hypo-osmotic salinities has also been found in Fundulus heteroclitus (Potts & Evans, 1967) and Tilapia mossambica (Potts et al. 1967).

Table 2.

Intake of Na, Cl and H2O by drinking the medium[X¯±S.E.(N)]

Intake of Na, Cl and H2O by drinking the medium[X¯±S.E.(N)]
Intake of Na, Cl and H2O by drinking the medium[X¯±S.E.(N)]

The research described in this publication was submitted to the Department of Biological Sciences, Stanford University in partial fulfilment of the requirements for the degree of Doctor of Philosophy. It was supported in part by a Public Health Service Predoctoral Fellowship from the National Institute of General Medical Sciences. The advice and encouragement of Professors A. C. Giese, D. Kennedy, H. A. Bern and W. T. W. Potts is gratefully acknowledged. Fig. 1 was drawn by Herbert J. McGinnis.

Black
,
V. S.
(
1957
).
Excretion and osmoregulation, pp. 163-205. In M. Brown
.
The Physiology of Fishes
.
New York
:
Academic Press
.
Bray
,
G. A.
(
1960
).
A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter
.
Anal. Biochem
.
1
,
279
327
.
Evans
,
D. H.
(
1967
).
Sodium, chloride and water balance of the intertidal teleost, Xiphister atropurpureus. I. Regulation of plasma concentration and body water content
.
J. exp. Biol
,
(in the Press)
.
Fleming
,
W. R.
&
Stanley
,
J. G.
(
1965
).
Effects of rapid changes in salinity on the renal function of a euryhaline teleost
.
Am. J. Physiol
.
209
,
1025
30
.
Forster
,
R. P.
&
Berglund
,
F.
(
1956
).
Osmotic diuresis and its effect on the total electrolyte distribution in plasma and urine of the aglomerular fish, Lophiius americanus
.
J. gen. Physiol
.
39
,
349
59
.
HICKMAN
C. P.
Jr
. (
1967
).
Sea water ingestion and intestinal absorption and elimination of water and salts in the southern flounder
.
Paralichthys leihostigma. (In preparation
.)
Holmes
,
R. M.
(
1961
).
Kidney function in migrating salmonids
.
Ann. Rep. Challenger Soc
.
3
,
23
.
Holmes
,
W. H.
&
Stainier
,
I. M.
(
1966
).
Studies on the renal excretion of electrolytes by the trout (Salmo gairdnerii)
.
J. exp. Biol
.
44
,
33
46
.
Maetz
,
J.
,
Bourguet
,
J.
&
Lahlouh
,
B.
(
1964
).
Urophyse et osmorégulation chez Carassius auratus
.
Gen. Comp. Endocrinol
.
4
,
401
14
.
Motais
,
R.
(
1967
).
Les mechanismes d’échanges ioniques branchiaux chez les téléostéens
.
Ann. Inst. Oceanogr
.
45
,
1
83
.
Motais
,
R.
&
Maetz
,
J.
(
1965
).
Comparison des échanges de sodium chez un téléostéen euryhalin (le flet) et un téléostéen stenohalin (le serran) en eau de mer. Importance relative du tube digestive et de la branchie dans ces échanges
.
C. r. Acad. Sci`. Paris
261
,
532
5
.
Potts
,
W. T. W.
&
Evans
,
D. H.
(
1967
).
Sodium and chloride balance in the killifish, Fundulus hetero-clitus
.
Biol. Bull mar. biol. Lab. Woods Hole
47
.
461
70
.
Potts
,
W. T. W.
,
Foster
,
M. A.
,
Rudy
,
P. P.
&
Parry Howells
,
G.
(
1967
).
Sodium and water balance in the cichlid teleost Tilapia mossambica
.
J. exp. Biol
.
47
,
461
470
Sharratt
,
B. M.
,
Chester Jones
,
I.
&
Bellamy
,
D.
(
1964
).
Water and electrolyte composition of the body and renal function of the eel
.
(Anguilla anguilla L.). Comp. Biochem. Physiol
.
11
,
9
18
.
Smith
,
H. W.
(
1930
).
The absorption and excretion of water and salts by marine teleosts
.
Am. J. Physiol
.
93
,
480
505
.
Stanley
,
J. G.
&
Fleming
,
W. R.
(
1964
).
Excretion of hypertonic urine by a teleost
.
Science
144
,
63
4
.
STANLEY & FLEMING
, (
1966a
).
The effect of hypophysectomy on the function of the kidney of the euryhaline teleost, Fundulus kansae
.
Biol. Bull. mar. biol. Lab. Woods Hole
130
,
430
41
.
Stanley
,
J. G.
&
Fleming
,
W. R.
,
1966b
.
The effect of hypophysectomy on sodium metabolism in the gill and kidney of Fundulus kansae
.
Biol. Bull. mar. biol. Lab. Woods Hole
131
,
155
65
.