J. exp. Biol. (1976). 65, 5*9-538
With 4 figures
Printed in Great Britain
529
THE ROLE OF ENVIRONMENTAL CALCIUM
IN FRESHWATER SURVIVAL OF THE MARINE TELEOST,
LAGODON RHOMBOIDES
BY JEFFREY C. CARRIER AND DAVID H. EVANS
Department of Biology, University of Miami, Coral Gables,
Florida, 33124, U.S.A.
(Received 20 April 1976)
SUMMARY
(1) The marine teleost fish, Lagodon rhomboides, can only tolerate fresh
water (5 mM Na) if Ca is also present (10 ITIM). Transfer to Ca-free fresh water
is followed by a substantial increase in radioactive Na efflux with little or no
change in the transepithelial potential. Addition of the chelating agent EDTA
(2 mM) further increases Na efflux. Fish left in Ca-free fresh water for 2-5 h
die with a total body Na less than 50 % of that found in animals acclimated
to Ca-supplemented fresh water.
(2) Rates of Na uptake were measured on either sea-water-acclimated or
Ca-supplemented fresh water-acclimated fish transferred to various low Na
media. In both cases Na uptake has a high Km, is saturable, inhibited by
external NH4, H and amiloride, and is not related to changes in the transepithelial potential.
(3) It is suggested that L. rhomboides is dependent upon external Ca to
decrease diffusional Na loss in low salinities so that a relatively inefficient Na
uptake can balance diffusional and urinary Na loss.
INTRODUCTION
The branchial epithelium of teleost fishes is the site of passive and active movement
of NaCl as well as respiratory gas exchange. The mechanisms of ionic movements are
becoming increasingly understood (for recent reviews see, Maetz, 1971, 1974ft;
Maetz & Bornancin, 1975) and it is apparent that in marine teleosts Na efflux consists of
an appreciable passive component superimposed on Na/Na and Na/K exchange
(Potts & Eddy, 1973; House & Maetz, 1974; Evans, Carrier & Bogan, 1974; Kirschner,
Greenwald & Sanders, 1974; Evans & Cooper, 1976), while Cl efflux consists of a
smaller passive component (compared to passive Na efflux), Cl/Cl exchange and active
Cl efflux, which is still relatively unstudied (Epstein, Maetz & de Renzis, 1973;
Maetz & Pic, 1975). The active ionic exchange mechanisms in the marine branchial
epithelium serve to balance the diffusional and oral influx of NaCl. In freshwater
teleosts, the branchial epithelium is apparently much less permeable to both Na and
Cl than in marine teleosts (Potts & Evans, 1967; Evans, 1967, 1969; Maetz, 1971),
and the slight net efflux of both ions (as well as the renal efflux) is balanced by active
ionic uptake mechanisms which function via ionic exchange: Na/H, Na/NH4 and
CI/HCO3 (Maetz, Payan & de Renzis, 1976).
530
J. C. CARRIER AND D. H. EVANS
In recent years it has become apparent that the branchial permeability to NaCl
may be affected directly by external Ca concentrations. Pickford et al. (1966) showed
that hypophysectomized killifish Fundulus kansae can maintain their blood Na at
higher levels in Ca-supplemented, deionized water than in Ca-free, deionized water.
In addition, Potts & Fleming (1971) showed that the Na efflux from F. kansae in
fresh water is reduced by 50 % by the addition of 1 mM-Ca to the external medium.
Cuthbert & Maetz (1972) corroborated these findings by showing that removal of
external Ca (by addition of chelating agents EGTA and EDTA or adaptation to
deionized water) doubles the rate of Na influx into the goldfish Carassius auratus. This
increase can be reversed by the addition of Ca (2-20 miw) to the medium. However,
the Na efflux from C. auratus is apparently unaffected by external Ca. Kerstetter,
Kirschner & Rafuse (1970) and Cuthbert & Maetz (1972) showed that the transepithelial potential across the trout Salmo gairdneri in fresh water is affected greatly
by external Ca, and Maetz (1974a) has recently shown that addition of 1-5 mM-Ca
to deionized water depolarizes the electrical potential across C. auratus from — 44 mV
(blood relative to medium) to + 7 mV due to a greater reduction in Na permeability
than Cl permeability. These data have been corroboiated by Eddy (1975).
It appears that Ca can also affect Na permeability of the branchial epithelium of
fish in sea water. Potts & Fleming (1971) found that the Na efflux from sea-wateracclimated Fundulus kansae increases by 125 % when this species is placed in Ca-free
sea water and Bornancin, Cuthbert & Maetz (1972) found that both the influx and
efflux of Na is doubled when the eel, Anguilla anguilla, is placed in calcium-free sea
water. More recently, Pic & Maetz (1975) have shown that addition of 10 mM-Ca
reduces the efflux of Na and Cl and the blood negative electrical potential across the
mullet, Mugil capito, after transfer from sea water to deionized water. They propose
that the Na permeability is reduced much more than the Cl permeability which leads
to the reduction in the internal negativity.
There is some evidence that the permeability effects of external Ca may be ecologically relevant. Breder (1934) observed that various species of marine teleosts (needlefishes, Strongylura notata and S. timuca; moj arras, Eucinostomis caUforniensis and
E. gula; halfbeak, Ckriodorus atherinoides; horse-eye jack, Caranx latus; gray snapper,
Lutianus griseus; checkered puffer, Spheroides testudineus) were living in a freshwater
lake on Andros Island, The Bahamas. Analysis of the water indicated that the lake
has an unusually high (i-o—1-5 mM) Ca concentration. Breder (1934) also showed that
addition of Ca to New York City tap water prolongs the survival of the queen angelfish
(Holacanthus ciliaris) and the beaugregory (Eupomacentrus leucostitus) in the medium.
More recently, Hulet et al. (1967) have shown that the sergeant major (Abudefduf
saxatiUs) can survive in fresh water only if relatively high (5-15 mM) concentrations
of Ca are present.
Although one might propose that this Ca promotion of freshwater survival by
normally stenohaline marine fish is due to the reduction in epithelial salt permeability,
so that net losses are small, it is obvious that some means for replenishment of salt
must be present in these fish in Ca-supplemented fresh water. Recent evidence
(Evans, 1973, 1975a) indicates that the euryhaline molly, Poecilia latipinna, is carrying
out Na/NH4 and/or Na/H exchange (hereafter referred to as Na/NH 4 -H exchange)
both when acclimated to fresh water and sea water and Evans (1975 A) has suggested
Role of calcium in survival of L. rhomboides
531
that marine teleosts may possess this ' pre-adaptation' for Na balance in fresh water
due to the needs for excretion of unwanted nitrogenous waste and acid.
Preliminary observations indicate that the marine pinfish, Lagodon rhomboides
(family Sparidae), can only tolerate fresh water if the solution is supplemented with
Ca. We have therefore investigated the effect of Ca on Na fluxes and transepithelial
potentials in sea water and Ca-supplemented fresh water and also the kinetics and
inhibition of Na uptake by this species in sea water and Ca-supplemented fresh water.
MATERIALS AND METHODS
Collection and maintenance of animals, experimental solutions
Pinfish (Lagodonrhotnboides)were collected from Biscayne Bay, Miami, Fla. by either
beach seine or trawling. Experimental animals (10-25 g) w e r e maintained in large,
fibreglassed, wooden tanks at a sodium concentration of 175-200 mM. Fish were
maintained at 24+ 10 °C and at a close approximation to local day-length using a
100 Watt tungsten light source connected to a 24 h interval timer. Animals were
fed TetraMin staple food (TetraWerke).
After approximately 2 weeks of acclimation to brackish water, animals were transferred to smaller 80-120 1 aquaria of various salinities. For experiments in fresh water,
animals were transferred to 60-80 mM-Na for 7-10 days. Preliminary experiments
indicated that survival is possible when the external salinity is below 40-45 mM-Na
only when a minimum of 2 mM of Ca, as CaCl2, is present. Therefore, the Ca-supplemented fresh water in the context of this research is defined as water having a NaCl
concentration of 5 mM and a CaCl2 concentration of 10 mM. Animals acclimated to
Ca-supplemented fresh water are referred to as ' freshwater '-acclimated.
Sea water used in these experiments was collected from Biscayne Bay and made up
to 480 mM-Na using artificial sea salts (Instant Ocean, Inc.). Ca-free sea water was
made from the artificial sea-water formulation of Pantin (1962) from which calcium
was omitted.
Analytical procedures
Na concentration of body fluids and experimental baths was measured with an
Instrumentation Laboratories Model 143 Flame Photometer. Total body Na (/JM/g
body wt) was determined by homogenization of whole animals in a Virtis homogenizer
at full speed for one to two minutes. Samples were diluted to 1 1 with double distilled,
deionized water and the Na concentration was determined with the flame photometer.
Ca concentrations were measured by chelation with EDTA in the presence of the
calcium-dependent, photoluminescent protein calcein (Oxford Laboratories Calcium
Titration Kit).
Na efflux
Intraperitoneal injections of 1-2 /tCi MNa in 5-10 /A of aqueous solution (New
England Nuclear or Amersham-Searle) were made under anaesthesia (o-oi % ethylffj-amino-benzoate methane sulphonic acid, MS-222, Sigma Chemical Co.) and the
animals then were placed into an appropriate experimental solution. Samples of the
532
J. C. CARRIER AND D . H . EVANS
experimental baths were removed at various times throughout the experiment and the
radioactivity measured. At the end of each experiment, the radioactivity in the animal
and in the bath was determined and summed to determine the amount of radioactivity
initially injected. Efflux rate constants were then calculated using formula (i):
where K is the rate constant of efflux (fraction of the exchangeable sodium effluxed
per hour), t is the experimental period in hours, Qo is the radioactivity in the animal at
time o, and Qt is the radioactivity in the animal at any time t.
Na influxes
The rate of influx of Na was measured as described previously (Evans, 1973).
The uptake solutions (400 ml) contained various concentrations of NaCl (no Ca) and
the radioactivity in the fish after 5 min was divided by the mean specific radioactivity
of the uptake solution. The animals were weighed at the end of the experiment and
the uptake expressed as /iM-Na. g - 1 . hr -1 . To determine if influx is affected by external
NH 4 or H ions, or amiloride, uptakes were also determined in 1 mM-NaCl solutions
to which either 10 mM-NH4 (pH = 6-8, ammonium added as sulphate salt), o-i mM-H
(pH = 4, maintained by 10 mM Hepes buffer and H2SO4), or 1-5 x io~* M amiloride
had been added. The radioactivity in the fish or samples of the media in both efflux
and influx experiments was measured with a Packard Model 446 Armac Liquid
Scintillation Detector connected to a Packard Model 2001 Tricarb Scintillation
Spectrometer.
Transepithelial potentials (T.E.P.)
T.E.P.S were measured as described previously (Evans et al. 1974). The experimental
fish was placed in a plexiglass-framed, nylon screen box in order to restrict movement
and prevent the withdrawal of the implanted 3 M-KC1, 2 % agar-filled PE 10
bridge.
All data are expressed as mean ± standard error (no. of animals).
RESULTS AND DISCUSSION
Effect of External Ca
(1) Sea-water-acclimated pinfish
Table 1 presents the flux data for Lagodon rhomboides in sea water and calcium-free
sea water. The Na efflux from L. rhomboides is greater than that described for most
other marine teleosts (Maetz, 19746) and is exceeded only by that found in the mullet,
Mugil capito (Maetz & Pic, 1975). Na efflux is profoundly affected by the removal of
Ca from sea water: 2-3 h after transfer to calcium-free sea water the efflux increases
by 35%. The T.E.P. in sea-water-acclimated L. rhomboides ( + 5±2mV (11), blood
relative to bath) is also dependent upon external Ca as indicated by the hyperpolarization to + 3 6 1 4 mV (5) 2-3 h after transfer to Ca-free sea water. If we assume
that the T.E.P. in sea water is predominantly a diffusion potential (Potts & Eddy, 1973;
Role of calcium in survival of L. rhomboides
533
Table 1. Na efflux rate constants for Lagodon rhomboides
in various solutions
Solution
Sea water
Ca-free sea water
Ca-supplemented fresh water
(5 mM-Na+10 mM-Ca)
Fresh water (5 mM-Na)
Freshwater + EDTA (2 rnM EDTA/1)
• X±B.B.
Rate constant (h"1)
0-77±003 (9)*
1-03 ±0-03 (6)t
0-05 ±o-oi (10)
o-i4±o-oi (io)J
o-ao±001 (7)§
(N).
t 2-3 h post transfer from SW to Ca-free SW.
X 1-3 h post transfer from Ca-supplemented FW to 5 mM-NaCl FW.
§ 1-4 h post addition of EDTA.
Kirschner et al. 1974) it appears that removal of external Ca increases the ratio of Na
to Cl permeability. However, our data do not allow a definite statement with regard
to changes in Na permeability per se. The 31 mV hyperpolarization in Ca-free sea
water would lead to a 58% increase in the passive Na efflux (calculated using
Goldman current equation, Evans et al. 1974) but the magnitude of the passive Na
efflux from L. rhomboides is unknown. For instance, if the passive component is 25%
of the efflux (and the Na permeability remains at sea-water levels), the total Na efflux
in Ca-free sea water would have a rate constant of o-88, below that actually found
(Table 1). On the other hand, if the passive component is 75 % of the Na efflux, the
total Na efflux in Ca-free sea water would have a rate constant of I-IO, greater than
that found. In fact, if the passive component is greater than 50 % of the Na efflux,
the change in the T.E.P. can account for the stimulation of Na efflux in Ca-free sea
water. If the passive component is less than 50 % of theNa efflux, one must postulate an
increase in Na permeability plus T.E.P. changes to account for the stimulation of Na
efflux in Ca-free sea water. At present the magnitude of the passive Na efflux is not
known with certainty for any teleost. Potts & Eddy (1973) and Kirschner et al. (1974)
have proposed that most of the Na efflux is diffusive, but Evans & Cooper (1976) have
recently shown in three species of teleosts that most of the Na efflux is via Na/Na and
Na/K exchange and only 10-20 % of the Na efflux is via diffusion.
Thus, we are unable to determine if the increase of Na efflux in Ca-free sea water is
T.E.P. or permeability mediated. Potts & Fleming (1971) and Bornancin et al. (1972)
also found that the Na efflux from sea-water-acclimated F. kansae and A. AnguiUa,
respectively, increased in Ca-free sea water; unfortunately neither the T.E.P. nor the
diffusional efflux were measured, so it is not possible to differentiate between T.E.P.
and permeability effects in these studies.
The effect of Ca-free sea water on the Na efflux from L. rhomboides is time dependent. Seven hours after transfer to Ca-free sea water the rate constant of Na efflux
increases to 1 -76 ± 0-04 h - 1 (6). Whether this further increase is due to a greater
hyperpolarization of the T.E.P. or to an increase in the Na permeability is not known
since T.E.P.S were not measured in these experiments.
534
(2)
J- C. CARRIER AND D. H. EVANS
'Freshwater'-acclimatedpinfisk
While Lagodon rhomboides can survive indefinitely in Ca-supplemented fresh water
(unpublished observations) we found that transfer from calcium-supplemented to
Ca-free fresh water (5 mM-Na) results in rapid death of this species. Within 2-5 h
transfer of 50fishfrom Ca-supplemented fresh water to Ca-free fresh water, 48 animals
were dead. Total body Na content of 5 of these fish was 29-6 ± i-8 fiM Na.g" 1 fish,
a substantial decrease from the total body sodium content of animals maintained in
Ca-supplemented fresh water (65-5 ± 2-8 /iM Na.g" 1 fish, 5 animals). Thus, death
was apparently secondary to a substantial net loss of sodium from the body fluids.
Transfer into Ca-free fresh water also results in a significant increase in the efflux
of Na from L. rhomboides, the rate constant increasing by approximately 280 % after
transfer into Ca-free fresh water (1-3 h after transfer) (Table 1). These data corroborate those of Potts & Fleming (1971) who found that the rate constant for Na efflux
from F. kansae increases by 225 % after transfer of this species to Ca-fresh water.
It appears that even after transfer from Ca-supplemented to Ca-free fresh water
some residual Ca-mediated effects may be present. This is shown by the observation
that addition of 2 mM EDTA leads to a further increase (400 % of Ca-supplemented
freshwater control) in the efflux of Na 1-4 h post treatment (Table 1). Similar
experiments by Cuthbert & Maetz (1972) showed that addition of 2 mM EGTA to
C. auratus in fresh water increases Na efflux nearly fourfold. It therefore appears that
significant amounts of Ca adhere to the epithelium even in Ca-free solutions.
The stimulation of Na efflux in Ca-free fresh water does not appear to result from
changes in the T.E.P. for at low Na concentrations removal of Ca results in either no
change or only a slight increase in internal negativity (Fig. 3), which would result in
a reduction of passive Na loss rather than the stimulation which is observed. Thus
the ability of Ca-supplemented fresh water to promote survival of L. rhomboides
primarily results from a reduction of the passive Na permeability of the permeable
(presumably branchial) epithelial membranes. Hulet et al. (1967) have proposed a
similar idea, based solely upon survival data, for the effect of Ca on the survival of
A. saxatilis in low salinities.
Although it is clear that calcium reduces the permability of L. rhomboides in low
salinities, the effect is not complete and this species still faces a substantial loss of
Na in low Na environments. In fact the rate constant of Na efflux from L. rhomboides
in Ca-supplemented fresh water (Table 1) is some 5 times that normally found in
freshwater-acclimated teleosts (Maetz, 19746). Thus, it appears that although Ca
promotes survival, other factors must be involved which enable L. rhomboides to
maintain Na balance in fresh water. As P. latipinna possesses the freshwater ionic
exchange mechanism for Na/NH 4 -H exchange when acclimated to sea water (Evans,
1973; 1975a, b) it is possible that other species of marine fish may also possess this
system in sea water. Therefore, it is desirable to determine if L. rhomboides carries out
Na/NH 4 -H exchange when acclimated to sea water or calcium-supplemented fresh
water.
Role of calcium in survival of L. rhomboides
a
535
4
4
1 2--
I
«W
10
20
30
External [Na] (mu)
Fig. i
40
50
20
40 60 80 100 120 140
External [Na] (nm)
Fig. a
Fig. I. Rate of sodium uptake vs. external sodium concentration for ' freshwater'-acclimated
L. rhomboidet. Each point is the mean ±standard deviation for from 9—14 animals.
Fig. a. Rate of sodium uptake vs. external sodium concentration for seawater-acclimated
L. rhomboidet. Each point is the mean ± standard deviation for from 0-14 animals.
Sodium uptake
(1) Kinetic analysis
When L. rhomboides is acclimated to sea water or Ca-supplemented fresh water,
Na uptake in low sodium environments is saturable and, therefore, presumably
carrier-mediated (Figs. 1 and 2). The K^ of Na uptake is 22 mM in sea-wateracclimated individuals and 5 mM in 'freshwater'-acclimated individuals. Thus, the
Xjn of Na uptake by L. rhomboides is very similar to that of the sailfin molly Poecilia
latipirma (Evans, 1973) and other brackish water organisms (Maetz, 1974ft).
The rate of Na uptake by 'freshwater'-acclimated L. rhomboides transferred to
1 mM-Na solutions is independent of external Ca. The rate of uptake in 1 mM-Na
is 0-98 ± 0-07 /iM.g-1.hr1 (9) while the rate of uptake in 1 mM-Na +10 mM-Ca is
096 ± 0-04 /iM. g"1. h - 1 (6).
(2) Effect of T.E.P.
The apparent saturation of Na uptake by L. rhomboides might be secondary to
changes in the T.E.P. However, the depolarization occurred with increasing external
sodium, in either 'freshwater'- or sea-water-acclimated L. rhomboides (Figs. 3 and 4).
Such a depolarization would diminish Na uptake (if strictly passive) rather than increase it substantially. It is clear, therefore, that the kinetics of Na uptake in low
Na solutions (by both 'freshwater'- and seawater-acclimated) cannot be ascribed to
changes in the T.E.P.
External Ca has a relatively minor effect on the T.E.P. in low Na solutions (Figs. 3
and 4), for both sea-water and 'freshwater'-acclimated fish. The addition of external
Ca depolarizes the T.E.P. only slightly in 1 mM-Na but has no significant effect on the
T.E.P. at 10, 25 or 50 mM-Na. If the T.E.P. in low solutions is a diffusion potential,
predominantly due to the differential Na and Cl permeability, it appears that Ca
retards Na loss to a greater extent than Cl loss only in 1 mM-Na. In addition, since
'freshwater'-acclimated L. rhomboides show no T.E.P. change, or are more negative,
in Ca-free, 1 mM Na solutions, it is obvious that the observed increase in Na efflux
in calcium-free 5 mM Na solutions (Table 1) does not result secondarily from changes
in the T.E.P.
35
EXB6J
536
J. C. CARRIER AND D. H. EVANS
+ 20
+ 10
+ 10
A
A
I
A
J
0
W -10
H
*e
-10
H
-20
-20
5
10
25
5
50
10
25
External [Na] (ITIM)
External [Na] (ITIM)
Fig. 3
Fig. 4
Fig. 3. Effect of external sodium concentration on the T.E.P. across 'freshwater'-acclimated
L. rhomboides, in calcium-free solutions (•) and calcium-supplemented (10 mM) solutions (A).
Fig. 4. Effect of external sodium concentration on the T E.P. across sea-water-acclimated.
L. rhomboides, in calcium-free solutions (#) and calcium-supplement (10 mM) solutions (A).
Table 2. Inhibition of sodium uptake in Lagodon rhomboides
acclimated to 'freshwater' or sea water by NHit H or amiloride
Rate in ...
Freshwater
acclimated
Decrease (%)
Seawater
acclimated
Decrease (%)
1 mM-Na
oo8±o-o8»
(7)
2-6 ±02
(8)
1 mM-Na +
amiloride
0-30 ±0-04
(6)
69%
1 mM-Na +
pH = 4
10 mM-NH,
1 mM-Na +
O-2O±O-O2
o-ai±o-oa
I-6±O-I
(11)
I-O±O'I
(IO)
(IO)
80%
79%
(9)
61%
38%
• Rates of sodium uptake are expressed in fttt. g" 1 . h - 1 ±8.E. (N).
o-oo±o-o6
(8)
66%
(3) Effect of counter-ions and amiloride
If the Na uptake observed on transfer to low-Na solutions is mediated via the welldocumented Na/NH 4 -H cationic exchange system (Maetz et al. 1976) then it should
be inhibited by addition of high concentrations of the counter ions or amiloride to the
external bath, as has been described for both sea-water-and freshwater-acclimated P.
latipinna (Evans,i975a) and oth.erfreshwaterfish(Krogh, 1939; Maetz & Garcia Romeu,
1964; Kirschner, Greenwald & Kerstetter, 1973). It is clear that addition of either
counterion or amiloride does, in fact, significantly inhibit (38-80% decrease) Na
uptake by either 'freshwater'- or seawater-acclimated L. rhomboides (Table 2).
The findings that Na uptake is saturable, is not affected (at 1 mM-Na) by addition
of external Ca (despite changes in the T.E.P.), and is inhibited by either external NH4,
H or amiloride suggest strongly that Na uptake by L. rhomboides is mediated via
Na/NH 4 -H exchange in either sea water or fresh water.
50
Role of calcium in survival of L. rhomboides
537
CONCLUSIONS
Sea water-acclimated L. rhomboides carry out Na/H and/or Na/NH4 exchange, presumably to rid the body of unwanted metabolic wastes (see Evans (19746) for a review
of this general proposition). Thus, this species of marine teleost possesses a mechanism
for extraction of Na from the low Na environment, one of the pre-requisites for survival in fresh water. However, the K^ of this Na uptake mechanism is relatively high
(indicating a rather low affinity for Na) and the passive Na permeability of L. rhomboides is also high; even in Ca-enriched fresh water the Na efflux is 5 times that found
in most freshwater teleosts. Therefore, in low salinities net loss of Na is so great that
the fish cannot survive unless the passive permeability to sodium is reduced by an
abnormally high level of external calcium. An external concentration of 10 mM Ca
reduces the permeability of the epithelium sufficiently to allow for the relatively low
affinity Na uptake mechanism to balance Na loss. These data support the proposition
that some marine teleosts may possess mechanisms for Na uptake in fresh water but
remain stenohaline due to a relatively high passive permeability to Na (Evans, 1975 b).
This study was supported by NSF Grants GB 36423 and BMS-75-00091 to D. H. E.
and was a portion of the Ph.D. dissertation of J. C. C. The amiloride was kindly supplied by Merck, Sharp and Dohme, Inc.
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