NaCl adaptation in Rana ridibunda and a comparison with the euryhaline toad Bufo viridis

Only a few amphibians have been found to tolerate high salinities for extended periods of time (i.e. Rana cancrivora and Bufo viridis)’, most being unable to adapt to salinities higher than 0·7–0·9% NaCl (see Bentley, 1971; and Katz, 1973 a for recent summaries).

The adaptation of the euryhaline toad Bufo viridis to high salinities have been recently described (Katz, 1973 a). This study describes the adaptation of the frog Rana ridibunda to saline environment and compares them with those of B. viridis.

Frogs (Rana ridibunda) were collected in northern Israel. They were kept in running water at 20 ± 2 °C, and were fed on maggots. Bufo viridis were collected at the same location.

The animals were adapted to NaCl in solutions of 3–4 cm in depth. The solutions were changed daily. Weight was followed to the nearest 0·1 g after catheterization of the urine through the cloaca. The animals were not fed during the experiments.

For chemical analyses, the urine was catheterized with pyrex capillaries, the animals were then double pitted and blood was collected from the heart into heparinized capillaries (5000 U/ml). Osmotic pressure of the urine and plasma was determined immediately on a Knauer type osmometer (Berlin), and urea was determined colorimetrically using Sigma reagents (Sigma Bull. no. 14, 1969). Sodium and potassium were determined on stored samples with Beckman model DU flamephotometer. Chloride was titrated according to Schales & Schales (1941).

Whole liver, gastrocnemius and sartorius muscles, the heart and a sample of the skin were taken, weighed to the nearest 0·1 mg, and were dried to constant weight in a 95 °C oven. Water content was calculated from the difference in wet and dry weight. Whole body sodium and chloride content were determined on extracts of dry samples in 0·1 N-HNO₃.

Oxygen consumption was measured using a differential respirometer as previously described (Katz, 1973 a).

Water uptake was determined gravimetrically in whole animals according to Jorgensen (1949), as described by Katz (1973b).

Sodium transport was determined electrically on isolated abdominal skin in ‘Ussing’ type chambers as described by Ussing & Zerahn (1951). Solutions contained, in MM: Na+115·0; K⁺3·5; Ca²⁺ 1·0; Mg²⁺0·5; Cl^- 95·0; SO₄^2-0·5; HCO3^- 2·5 ; H₂PO₄^2- 0·5 ; glucose 1·o ; pH 8·0. The solutions were aerated throughout. Syntocinon was from Sandoz.

Student test was used for statistical analyses.

There was only low mortality (> 10 %) among frogs which were adapted to NaCl solutions of up to 300 mOsM, when transferred gradually from low to high salinity (Fig. 1). Upon immediate transfer to solution of 300 mOsM, however, they lost about 20% of their initial weight and died after 8–10 days (Fig. 2). Table 1 summarizes the chemical analyses of the blood. There was a substantial increase of urea level in adapted frogs (from 10 to about 50 mM). Chloride and sodium increased, but the concentration of potassium did not change appreciably. The osmotic pressure of the blood was always hypertonic to the external solution, but the actual Osmotic gradient across the skin declined rapidly with increasing salinity. Urine was always hypotonic to the plasma.

The effects of adaptation to saline and of dehydration on oxygen consumption of the frogs is summarized in Table 2. It can be seen that there was no change in the oxygen consumption when the frogs were gradually adapted to saline (7–10 days in each salinity before transfer to the next solution) or when they were slowly dehydrated. There was, however, a significant increase in the oxygen consumption during rapid dehydration by either immersion of the animals in 300 mOsM of NaCl or sucrose, or in air.

The water uptake of living frogs did not change significantly during saline adaptation (Table 3). It should be noticed, however, that the osmotic gradient across the skin of ‘tap water’-adapted animals was much larger than in those which were adapted to saline. Hydro-osmotic effect of neurohypophyseal hormones decreased somewhat in salinities higher than 230 mOsM. The effect of adaptation on the rate of water uptake is shown in Fig. 3. It can be seen that ‘23o’-adapted animals did not lose as much water as did ‘tap water’ animals, in NaCl solution of 470 mOsM.

Sodium transport was studied using isolated pieces of abdominal skin in vitro. The results are summarized in Table 4. Sodium transport in skins from saline- adapted frogs (measured by the short-circuit current) decreased, and the resistance of the skin increased. Sodium transport increased following application of neurohypophyseal hormone (syntocinon), and the resistance of the skin declined to normal values.

A remarkable constancy in the water content of the heart and skin, and small variations in that of skeletal muscles and the liver, were observed during adaptation different salinities (Table 5).

The effects of neurohypophyseal hormone (syntocinon) on the water content of internal organs and on chloride concentration in the plasma, are summarized in Table 6. It can be seen that the water in the tissues of the frog is more labile and may change considerably according to the environmental conditions. In the toad, on the other hand, only small fluctuations in the water content of those organs were observed under similar conditions.

Table 7 shows the whole body analyses of sodium and chloride, in animals maintained in distilled water or adapted for over 10 days in NaCl solution of 230 mOsM. The sodium content increased by 20% and that of chloride by 33% in the saline- adapted frogs.

Frogs could not be adapted, under our experimental conditions, to salinities higher than 300 mOsM NaCl. This concentration is hypertonic solution for a frog from tap water and caused death unless the animals were gradually adapted. However, the frogs did not die from dehydration immediately upon transfer, but lost about 20% of their initial body weight, which was then maintained until death (Fig. 2). It is possible that some unidentified factor may accumulate in unadapted animals, but not in gradually-adapted ones. The increase in blood osmolality of frogs under adaptation is rather limited. It seems that the frogs cannot tolerate high sodium concentration, which never exceeds 130 mM, and is unable to accumulate urea, as is the case in Rana cancrivora (Gordon, Schmidt-Nielsen & Kelly, 1961) and Bufo viridis (Katz, 1973 a).

Bentley & Schmidt-Nielsen (1971), found that it was sodium per se rather than the osmotic effect of the solution, which caused death of Rana pipiens when placed in sea water. Sodium accumulated both as a result of drinking and uptake through the skin. Under less acute conditions (i.e. 4iomOsM NaCl), 100% (5 out of 5) of Rana ridibunda died within 3 days, whereas only one individual of Bufo viridis died out of five. Frogs were found to drink under these conditions (Congo red was used as a marker), but not the toads. No drinking was observed in either of these species under adaptation conditions.

The water content of the tissues did not change appreciably during adaptation from tap water to 300 mOsM NaCl. This is not surprising, since the increase in the osmotic concentration of the blood was only 35 % at the highest external salinity, and partly resulted from an increase in the concentration of urea.

No significant change was found in the water uptake of saline adapted frogs (Table 3) ; sodium transport on the other hand, decreased by more than 60% (Table 4). It follows from this, that less sodium is entering with the solution taken up by saline- adapted frogs. The adaptive value of this effect has been stressed, for the toad (Katz, 1975), and is exemplified here by the limited increase in the whole body content of sodium during saline adaptation.

Neurohypophyseal hormone (syntocinon), increased both the water uptake and the sodium transport across the skin of saline-adapted frogs, to levels similar to those measured in ‘tap water’-animals. Only at higher salinities was the effect of hormone decreased, as in Bufo viridis (Katz, 1973 c). The hypophyseal system should certainly adapt to the saline conditions, and natriferic effect should not be stimulated (Bentley, 1969). This would prevent the increase in sodium concentration to toxic levels (Bentley & Schmidt-Nielsen, 1971).

Fig. 4 compares water evaporation from the frog and the toad under similar environmental conditions, and shows no significant difference beween the two species. Thorson (1955), also found similar rates of evaporative water loss from a number of American anurans from various habitats.

Many amphibians have been described from brackish water and saline environment under natural conditions (Neill, 1958; Ruibal, 1959; and others). However, only two species so far, namely Rana cancrivora (Gordon et al. 1961) and Bufo viridis (Gordon, 1962; Tercafs & Schoffeniels, 1962; Katz, 1973 a), could be adapted experimentally to salinities as high as 8o % of sea water or its equivalent. As Rana cancrivora and Bufo viridis (the most adaptable anuran species) belong to two different families, it seems likely that ecological factors were important in determining the development of their adaptability to high salinities. Rana cancrivora is restricted to coastal lowland areas between southern south Viet-Nam and southern Thailand (Gordon et al. 1961), and Bufo viridis is restricted to dry areas and low humidities (Kauri, 1948).

It has already been stressed that amphibians are less homeostatic than other vertebrates (Jörgensen, 1950; Bentley, 1966; Katz, 1973 a; and others). This is true especially for the osmolality and composition of their blood, as well as for the lability of water in their tissues (cf. Smith & Jackson, 1931 ; Katz, 1973 c), but may be true for other parameters. However, it is the ability to accumulate and maintain high urea levels in the blood, which forms the major difference between species which can, and those which cannot, adapt to high salinities. Other differences probably exist, some of which may be of adaptive value (Thesleff & Schmidt-Nielsen, 1962 ; Katz, 1975 ; and others). However, actual mechanisms (e.g. enzyme systems and their control; reabsorption and permeability of urea in the kidney and the skin etc.) which enable the adaptable species to accumulate and maintain urea, remain to be identified. The basic differences in physiological functions of various organs under the extreme situations also remain to be elucidated.

The skilful technical assistance of Miss Judith Weissberg is gratefully acknowledged.