«' Biol. (i97s). 63,

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1 «' Biol. (i97s). 63, figures Printed in Great Britain NaCl ADAPTATION IN RAN A RIDIBUNDA AND A COMPARISON WITH THE EURYHALINE TOAD BUFO VIRIDIS BY U. KATZ Department of Zoology, The Hebrew University of Jerusalem, Israel (Received 12 May 1975) SUMMARY The physiological adaptation of the frog Rana ridibunda to saline environment was studied. It was found that blood was always hypertonic to the external solution, but at the highest salinity tolerated (i.e. 300 mosm) the osmotic gradient across the skin was nearly abolished. Water uptake by the living frog remained unchanged, whereas sodium transport across the skin decreased markedly. Neurohypophyseal hormone increased water uptake and sodium transport to levels similar to those in tap water frogs. Water content of the tissues was not affected by saline adaptation, although it varied appreciably under acute conditions. Oxygen consumption increased in dehydrated frogs, but not in adapted ones. The results are discussed and compared to the euryhaline toad Bufo viridis; the importance of high urea levels for high salt adaptation is stressed. INTRODUCTION 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 % 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. MATERIALS AND METHODS 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 o-i 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 re then double pitted and blood was collected from the heart into heparinized P 49-2

2 764 U. KATZ 85mM-Na + 115mM-Na + 170mM-Na + I Time (days) Fig. 1. The effect of NaCl adaptation on the weight of Rana ridibunda. Animals began to die (t) after transfer to 320 mosm (170 mm-nacl). Average weight of the animals in the beginning was 30 g. t = 2i C. 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 o*i 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 o-i N-HN0 3. 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 (19736). 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; Ca*+ i-o; Mg 2+ o-5; Cl~ 95-0; SO^o^; HCO 8-2-5; HjPO^ 0-5; glucose 1 -o; ph 8-0. The solutions were aerated throughout. Syntocinon was from Sandoz. Student test was used for statistical analyses. RESULTS 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

3 NaCl adaptation in a frog and a euryhaline toad mOsM 100. ridibunda 90 I Time (days) 6 10 Fig. 2. The effect of immediate transfer of Rana ridibunda into solution of 300 mosm-nacl. Average weight of the animals at the beginning was 35 g. All animals died after 9 days, t = 31 C. 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 salineadapted 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 M different salinities (Table 5).

4 Table I. The eflect of NaCl adaptation on the osmolality and composition of the plamuz and urine of Rana ridibunda (Animals were adapted for 7-10 days at each solution. t=zr OC. Mean f S.D. on four separate animals. (Aug. 1970). The t-test compares each group with tap water group (control).) Plasma Urine Experimental I A > I > salinity AOsm K- Urea AOsm C1- Urea (NaCl) (rnos~) Na+ (rnequiv.11) C1- (m~) (~OSM) (mequiv.11) d (111~) p$ Tap water 247 f: f II+I 41f b 165 ~ OSM 255f f 6-74f P < 0.40 > 0.50 < mos~ 300f f z 7f 2 III+~ f f8 P < 0'001 > 0.25 < 0.00 I > 0' ~OSM 337f f 7 8 +_ 2 127f 8 53 k I5 303 f P > 0'001 > > 0'001 > 0'02

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6 768 U. KATZ 100 A 0 a b c d e 110 min' S L Fig. 3. Water uptake of living frogs {Rana ridibunda), in various solutions. A, animals from tap water. B, animals previously adapted to 330 mosm-nacl. Solutions: a, tap water; b, 185 mosm; c, 230 mosm; d, 400 mosm; «, 470 mosm. Average weight of the animals was 32 g. Mean ± s.d. t = 2i C. 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 salineadapted frogs. DISCUSSION 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 rnm, 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 throu

7 Table 4. The effect of NaCl adaptation and of neurohypophyseal hormone (syntocinon) on the electrical properties of frog skin (Rana ridibunda) (Abdominal pieces of skin were equilibrated with chloride ringer in the 'Ussing' chambers for I h (control). Hormone (0.1 U/ml) was then applied to the inside. Mean f S.D. of 12 separate skins in each group. t = 21 OC.) Control (at the end of I h1equilibration) After application of hormone A L r < I Solution of Potential Potential adaptation difference 1, Resistance difference 1, Resistance (~OSAI) (n. ana) (mv) W/cmL) (n. an*) Table 5. The effect of NaCl adaptation on the water content of various organs from Rana ridibunda (Animals were adapted for 7-10 days at each salinity before taken for analyses. t = 21 "C. Mean f S.D. on 4 separate animals at each salinity. t-test compares each group to the tap water (control) group.) HSO 230, NaCl 230, Sucrose Experimental Weight of salinity animals (NaCl) (id Tap water Plasma osmolality (~OSM) Whole body Sartorius Water content of tissues (% of fresh weight) A Heart Liver Skin

8 Table 6. The effect of neurohypophyseal hormone (syntocinon), on the water content of various organs in Rana ridibunda and Bufo viridis (Animals which were adapted to 230 mos~ NaCI, were injected with 2 U per animal of syntocinon and water uptake was followed in either tap water or in the solution of adaptation (230 mos~). After 2 h the animals were killed and water content of various organs was determined. t = 2 I OC. Mean + S.D. (number of animals in parentheses).) Frogs (Ram ridibunda) Toads (Bufo viridis) A \ f L 7 Control 230 ~ OSM NaCl adapted Control ~ OSM adapted (uninjected animals (uninjected animals animals from in animals from in tap water) t A > tap water) Tap water 230 ~ OSM NaCl Tap water 230 ~ OSM NaCl Initial weight (g) (3) 36.3 f 2.3 (7) 28.3 f 4.2 (5) (3) (6) (7) 9 Weight increase after the injection of hormone I.Z+O-5-3' Chloride in the plasma (mequiv./l) f I '6f 41'7 Water content (% of fresh weight) In : (I) Sartorius muscle (2) Liver (3) Skin Table 7. The effect of NaCl adaptation on the whole body content of water, sodium and chloride in Rana ridibunda Experimental salinity (NaCl) Tap water 230 mosm (t = 21 "C. Mean + S.D. of three animals.) Water Weight content (g) (% of fresh wt) Na+ (pequiv./g fresh wt) C1- (pequiv./g fresh wt) \ k? 3

9 NaCl adaptation in a frog and a euryhaune toad If rl B. viridis R. ridibunda 1 70 t Time (h) Fig. 4. Water evaporation from Rana ridibunda and Bufo viridis in the air under acute conditions (in front of a fan). Average initial weight of the frogs was 26-8 ±5-0 g. (Mean±8.D.) ( and they lost aa-2±6-8% of their weight. Average weight of the toads was g and they lost 30-4^:3-9% of their initial weight during the same period of time. t = ai C. 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 salineadapted 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, 162; Tercafs & Schoffeniels, 1962; Katz, 1973 a), could be adapted experimentally

10 772 U. KATZ to salinities as high as 80 % 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 (Jorgensen, 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. REFERENCES BENTLEY, P. J. (1966). Adaptations of amphibia to arid environment. Science, N.Y. 15a, BENTLBY, P. J. (1969). Neurohypophyseal function in amphibia: hormone activity in the plasma. J. Endocr. 43, BENTLEY, P. J. (1971). Endocrine* and Otmoregulation. Berlin, Heidelberg, New York: Springer- Verlag. BENTLEY, P. J. & SCHMIDT-NIELSEN, K. (1971). Acute effects of sea water on frog* (Rana ptpiens) Comp. Biochem. Pkytiol. 4oA, GOHDON, M. S. (1962). Osmotic regulation in the green toad (Bufo viridis). J. exp. Biol. 39, GORDON, M. S., SCHMIDT-NIELSEN, K. & KELLY, H. M. (1961). Osmotic regulation in the crab-eating frog (Rana cancrivora). J. exp. Biol. 38, J0RGENSEN, C. B. (1049). Permeability of the amphibian skin. II. Effect of moulting of the skin of anurans on the permeability to water and electrolytes. Acta phytiol. tcand. 18, J0ROENSEN, C. B. (1950). The amphibian water economy, with special regard to the effect of neurohypophyseal extracts. Acta pkysiol. tcand. 33, Suppl. 78, KATZ, U. (19730). Studies on the adaptation of the euryhaline toad Bufo viridis to high salinities. Oxygen consumption, plasma concentrations and water content of the tissues. J. exp. Biol. 58, KATZ, U. (19736). A simple and accurate method for the study of water movement across amphibian skin. J'. Biol. Education 8, KATZ, U. (1973 c). Cellular adaptations to hypertonic solutions in amphibians previously adapted to high salinities. Ph.D. thesis. The Hebrew University, Jerusalem. In Hebrew, with English abstract. KATZ, U. (1975). Salt induced changes in sodium transport across the skin of the euryhaline toad Bufo viridis. J. Pkysiol., Land. 347, KAURI, H. (1948). Uber die ausbreitung und die ausbretungsumstsnde der wechselkrote (Bufo viridis Lar.) in ostseegebeit. KungL Fyr. Sallsk. Handl. N.F. 59 No. 13, Lund. NEILL, W. T. (1958). The occurrence of amphibians and reptiles in salt water areas, and a bibliography. Bull. mar. Set. Gulf Caribb. 8, RUIBAL, R. (1959). The ecology of a brackish water population of Rana pipiens. Copeia No. 4, SCHALES, O. & SCHALES, S. S. (1941). A simple and accurate method for determination of chloride in biological fluids. J. biol. Chem. 140,

11 NaCl adaptation in a frog and a euryhaline toad 773 SMITH, V. D. E. & JACKSON, C. M. (1931). The changes during desiccation and rehydration in the body and organs of the leopard frog (Rana ptpiens). Biol. Bull. mar. biol. Lab., Woods Hale 60, TERCAFS, R. R. & SCHOFFKNIELS, E. (1962). Adaptation of amphibians to salt water. Life Set. 1, THKSLEFF, S. & SCHMIDT-NIEI^EN, K. (196a). Osmotic tolerance of the muscles of the crab-eating frog (Rana cancrivora). J. cell. comp. Pkytiol. 59, 31-4, THORSON, N. (1955). The relationship of water economy to terrestrialism in amphibians. Ecology 36, USSING, H. H. & ZEHAHN, K. (1951). Active transport of sodium as the source of electrical current in the short-circuited, isolated frog skin. Acta Physiol. Scand. 33,

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