«
'• Biol. (i97S). 63, 763-773
763
4 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 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.
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
764
U. KATZ
85mM-Na +
115mM-Na+
170mM-Na+
I
12
16
20
Time (days)
24
28
32
36
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 - H N 0 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; S O ^ o ^ ;
HCO 8 - 2-5; H j P O ^ 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
NaCl adaptation in a frog and a euryhaline toad
765
300mOsM
. ridibunda
100
90
I
80
70
-4
-2
2
4
6
10
Time (days)
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).
305 ~ O S M
P
P
230 m O s ~
P
Tap water
165 ~ O S M
Experimental
salinity
(NaCl)
12
> 0'001
337f
< 0'001
247 f:12
255f3
< 0.40
300f 9
AOsm
(rnOs~)
I
115+5
111+5
> 0.50
121 f z
> 0.25
131 f 7
> 0.025
Na+
2
8 +_ 2
7f
6f1
7+2
K(rnEquiv.11)
A
Plasma
127f 8
> 0'001
<
I I I + ~
0.00 I
83+6
79 f 6
< 0.70
C1-
12
> 0'02
53 k I5
> 0'02
44+
-
II+I
Urea
(m~)
>
303 f
12
161 f 6 2
41f6
74f 13
AOsm
(~OSM)
I
>
55
32f8
70 + 27
125+ 16
-
1 3 53
(111~)
Urea
4+2
14+ 12
C1(mEquiv.11)
Urine
(Animals were adapted for 7-10 days at each solution. t = z r OC. Mean f S.D. on four separate animals. (Aug. 1970). The t-test compares each
group with tap water group (control).)
Table I. The eflect of NaCl adaptation on the osmolality and composition of the plamuz and urine of Rana ridibunda
b
p$
d
768
U. KATZ
A
100
d
e
10 min'
0
1
S.
a
b
c
-100
-200
-300
-400
-500
-600
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
I
1,
@A/-9
h1equilibration)
Resistance
( n .ana)
Potential
difference
(mV)
<
1,
W/cmL)
L
I
Resistance
( n .an*)
After application of hormone
Table 5 . The effect of NaCl adaptation on the water content of various organs from Rana ridibunda
Potential
difference
(mV)
A
Control (at the end of
Tap water
Experimental
salinity
(NaCl)
Weight of
animals
(id
Plasma
osmolality
(~OSM)
Whole body
Sartorius
Heart
A
Water content of tissues
(% of fresh weight)
Liver
Skin
(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
Solution of
adaptation
(~OSAI)
r
(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.)
Table 4. The effect of NaCl adaptation and of neurohypophyseal hormone (syntocinon) on the electrical properties of frog skin
(Rana ridibunda)
6-9 1.0
85.3 f 4.6
+
+
-
Tap water
36.3 f2.3 (7)
t
\
A
>
+
I.Z+O-5
104-5 10.2
28.3 f 4.2 (5)
230 ~ O S M
NaCl
230 ~ O S NaCl
M adapted
animals
in
+ 5.1 (3)
68.6 4.0
28.1
Control
(uninjected
animals from
tap water)
A
Frogs (Ram ridibunda)
I 16.5
-
+ 6.1
+
32.8 2.3 (3)
Control
(uninjected
animals from
tap water)
f
+ 1.4
100.0 + 7.0
3'5
33.8 4.5 (6)
+
\
+ 6.6 (7)
1.45 0.5
137'6f 41'7
29.9
230 ~ O S NaCl
M
230 ~ O S adapted
M
animals
in
Tap water
L
-
Toads (Bufo viridis)
Tap water
230 mOsM
Weight
(g)
(t =
21
Na+ (pEquiv./g fresh wt)
+ S.D. of three animals.)
Water
content
(% of fresh wt)
"C. Mean
7
C1- (pEquiv./g fresh wt)
The effect of NaCl adaptation on the whole body content of water, sodium and chloride in Rana ridibunda
Experimental
salinity
(NaCl)
Table 7.
Initial weight (g)
Weight increase after the injection
of hormone
Chloride in the plasma (mEquiv./l)
Water content (% of fresh weight)
In :
(I) Sartorius muscle
(2) Liver
(3) Skin
+
(Animals which were adapted to 230 m O s ~
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 m O s ~ )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).)
Table 6. The effect of neurohypophyseal hormone (syntocinon), on the water content of various organs in Rana ridibunda and
Bufo viridis
3
k?
9
NaCl adaptation in a frog and a euryhaUne toad
B. viridis
100 r l
771
R. ridibunda
I f 90
|
80
1 ° 70
t
60
0
1
2
3
4
0
Time (h)
1
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 29-714-1 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
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.
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