The Role of Environmental Calcium in Freshwater Survival of the Marine Teleost, Lagodon Rhomboides

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, 1974b; 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).

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 mm) 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 trans-epithelial 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 corroborated 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-water-acclimated Fundulus kansae increases by 125 % when this species is placed in Ca-free sea water and Bomancin, 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; mojarras, Eucinostomis califomiensis and E. gula; halfbeak, Chriodorus atherinoides; horse-eye jack, Caranxlatus ; 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 (1·0−1·5 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 saxatilis) 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/NH₄−H exchange) both when acclimated to fresh water and sea water and Evans (1975 b) has suggested 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.

Pinfish (Lagodonrhomboides) were collected from Biscayne Bay, Miami, Fla. by either beach seine or trawling. Experimental animals (10−25 g) were 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 l 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.

Na concentration of body fluids and experimental baths was measured with an Instrumentation Laboratories Model 143 Flame Photometer. Total body Na (μM/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).

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 μM-Na. g⁻¹. hr⁻¹. To determine if influx is affected by external NH₄ 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), 0·1 mm-H (pH = 4, maintained by 10 mm Hepes buffer and H₂SO₄), or 1·5× 10⁻⁴ 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.

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-KCI, 2% agar-filled PE 10 bridge.

All data are expressed as mean ± standard error (no. of animals).

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, 1974b) and is exceeded only by that found in the mullet, Mugil capita (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 ±2 mV (11), blood relative to bath) is also dependent upon external Ca as indicated by the hyperpolarization to +36 ± 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 ;

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 0·88, 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 1·10, 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 the Na 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. Anguilla, 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.

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 50 fish from 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 ± 1·8 μM 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 μM 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. saxatiEs 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, 1974b). 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/NH₄−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/NH₄-H exchange when acclimated to sea water or calcium-supplemented fresh water.

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_m, of Na uptake is 22 mm in sea-water-acclimated individuals and 5 mm in ‘freshwater’-acclimated individuals. Thus, the K_m 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, 1974b).

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 0·9810·07 μM.g⁻¹.h⁻¹ (9) while the rate of uptake in 1 mm-Na +10 mm-Ca is 0·96 ± 0·04 μM. g⁻¹. h⁻¹ (6).

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.

If the Na uptake observed on transfer to low-Na solutions is mediated via the well-documented Na/NH₄-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,1975a) and otherfreshwaterfish(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/NH₄-H exchange in either sea water or fresh water.

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 (1974b) 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_m 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.