Camp. Biochem. Physiol.
Vol. 85A,
No. 4. pp. 703-713. 1986
0300-9629186 $3.00 + 0.00
Pergamon Journals Ltd
Printed in Great Britain
PLASMA OSMOLALITY,
URINE COMPOSITION AND
TISSUE WATER CONTENT OF THE TOAD
BUFO VZZ?ZDZSLAUR. IN NATURE AND UNDER
CONTROLLED
LABORATORY
CONDITIONS
U. KATZ.* D. PAGI, S. HAYAT and G. DEGANI
Department of Biology, TECHNION-Israel Institute of Technology, Haifa 3200, Israel.
Telephone: (04) 293 416
(Received 28 February
1986)
Abstract-l.
The compositions of plasma and urine were studied in toads (Bufo viridis) which were
collected from three locations in Israel, and compared with toads which were kept under constant
laboratory conditions for nearly 2 years.
2. Plasma osmolality was rather constant (over 310 mOsm kg-’ H,O) during the whole year in the active
toads.
3. Urea was the most variable osmolyte in the plasma, and accounted for the higher osmolality in
southern population.
4. Urine osmolality fluctuated in a circannual fashion both in freshly captured and in the toads under
constant laboratory conditions.
5. Water content of the tissues was constant throughout the year, independent of the plasma osmolality.
6. It is concluded that high plasma urea concentration and the excretory system (kidneys and the urinary
bladder) are important in sustaining constant plasma osmolality in active toads. Both mechanisms change
annually and form the basis for the high terrestriality of this species.
INTRODUCTION
The amphibia are a transitory group among vertebrates in the invasion of land. They are characterized
by a wet integument which is rich in glands (Noble,
1931) and which is an avenue for respiration
and consequently is highly permeable to water. All
amphibians are therefore dependent on free water
availability to a greater or lesser degree, though some
of them have invaded areas which are rather dry
(Bentley, 1966). Behavioural, structural and physiological adaptive mechanisms have been identified
in various species, mostly in anuran amphibians
(McClanahan,
1967; Loveridge, 1976; Shoemaker
and Nagy, 1977), which sustain their life under more
arid conditions.
The toad Bufo ciridis, which is a terrestrial species,
has a wide geographical distribution, ranging from
Southern Sweden in the North (Gislen and Kauri,
1959) to North Africa, Israel and the Arab peninsula
in the South (Mendelssohn and Steinitz, 1944). This
species does not hibernate in its southern distribution
regions, which are also without rainfall for a great
part of the year. Quite extensive studies have been
made on this species, under laboratory and experimental (salt) conditions (Gordon, 1965; Katz, 1975,
1980), but the knowledge of its annual life cycle and
osmoregulation
under natural conditions is still
limited (Jorgensen, 1984). It was the purpose of the
present study to investigate the osmoregulation of
this species in nature, and to compare it to constant
laboratory conditions. Our findings show that plasma
*To whom correspondence
should be addressed.
703
osmolality and the tissue water content are kept
relatively constant in active toads throughout the
whole year. Greater variations were found in the
urine composition which seem to be important in
the regulation of constant plasma osmolality.
MATERIALS AND METHODS
Toads (Bufo uiridis) of both sexes were collected (four to
six specimens at each time) during night excursions in three
different locations in Israel. They were transferred to the
laboratory in Haifa or Kfar Giladi (about 2 hr travel), and
analyzed the following morning. A set of some 60 toads
collected in Maale Ephraim was maintained throughout a
whole year under controlled laboratory conditions. They
were kept in 60 x 60cm tanks with soil and free access to
tap water at 16 + 1°C. and 12 hr photoperiod. They were
force fed once a week with calf liver. and their weight was
monitored throughout the whole period.
For analysis, the toads were weighed ( f 0.1 g), urine was
sampled by catheterization through the cloaca, and they
were decapitated. Blood was collected into heparinized
(5000 U/ml) polyethylene tubes, and centrifuged to collect
the plasma. Osmolality was determined on 50~1 samples
(plasma or urine) with a Knauer-Berlin
semimicroosmometer. Chloride was titrated on a Radiometer CMTlO
chloridometer, and sodium and potassium were determined
with a flame photometer. Urea was assayed calorimetrically
at 570 nm using Sigma reagents (Bull, No. 640).
The heart, liver, gastrocnemius muscle and a piece of
abdominal skin were removed, blotted on Whatman No. 2
filter paper and weighed in aluminum foil (+O.l mg). The
whole intestine (from stomach to colon), and the fat bodies
were removed separately and weighed (+ 0.1 g). The ovaries
with the eggs were removed from the females, and the
remaining carcass weighed ( kO.1 g). The tissues and carcass
U.
704
KATZ ef al.
were dried to constant weight (48 hr at 95”C), weighed again
after cooling and the water content calculated from the
difference.
Meterological data were provided by the Israeli
meteorological service. The data were analyzed on the IBM
3081 D main frame computer using SAS library. The
Student’s r-test was used for statistical analyses. and the
results are given as mean k SEM.
RESULTS
Toads from three locations (Kfar Giladi in the
north, Maale Ephraim in the Rift Valley and
Hatzeva/Ein-Gedi
in the south-see
map) were
analyzed. The toads of the control laboratory set
originated from Maale Ephraim. The composition of
the plasma and urine of males and females did not
differ significantly from one another in any of the sets
analyzed. Table 1 compares males and females from
the laboratory set and the means of the toads of all
sets. In the following both sexes are combined for
each set and shown together. The experiments were
carried out during the years 1982-84, and all analyses
were carried out using similar procedures.
Figure 2 shows that the toads in the laboratory set
maintained constant weight throughout the whole
year. Their nutritional state (as judged from their fat
Fig. 1. Map of Israel and the Sinai peninsula showing the
sites where the toads were collected. The broken lines
connect the iso-rainfall points (based on data of the Israeli
meterological service).
Fig. 2. Relative weight and nutritional state of the toads of
the laboratory set during the whole experimental period.
Five toads were analyzed at each time point. Circles show
the first group and triangles the second group, which was
brought into the laboratory 7 months later. Open symbols
represent females, and filled symbols males, except for the
bottom frame where females and males are combined
together.
content and relative weight of the liver) was not
affected significantly by the Iong captivity, and the
mortality was some 15% (nine toads out of 57) during
the whole year. Longer time in captivity (toads which
were kept over 7 months) under laboratory conditions did not show any significant effect on the
composition and osmolahty of the plasma or the
urine (Table 2). The plasma osmolality was relatively
high, but more or less constant throughout the entire
year [Fig. 3(a)]. The average osmoality of the plasma
was 377 +_5 mOsm,kg-’
Hz0 (mean & SEM of 47
toads in seven time determinations,
during a 12
month period). The concentration of urea was relatively high, and contributed between 18 and 40% of
the total plasma osmolahty in the various groups.
The highest urea concentration was found in April
(169 + 10 mmol.lr)
and the lowest in February and
July (69 f 10 mmol.l-‘). In groups Nos 31 and 32 the
concentrations of the solutes did not add up completely to the tota measured osmolality; these
differences, however, are not statistically significant
and could be due to analytical errors.
The osmolality of the urine fluctuated annually
[Fig. 3(b)]: lower osmolalities were measured during
the winter months (February-May),
and higher
osmolalities in the summer (June-December).
The
osmolality of the urine was remarkably high, with the
lowest recorded values (150 mOsm) measured in the
Annual changes in blood and urine in a toad
Table I. Comparison
of plasma
in males and females of Bufo oiridis. 1. The toads from the laboratory
Maale Euhraim and Kfar Giladi). Mean + SEM. Number of toads in Damtheses
and urine composition
toads (laboratorv.
1. Laboratory
Females
Weight
Haematocrit (%)
Plasma osmolality (mOsm)
Plasma urea (mm01 1~ ‘)
Plasma chloride
Plasma sodium
Plasma potassium
Urine osmolality (mOsm)
Urine urea (mm01 I ‘)
Urine chloride
Urine sodium
Urine potassium
705
41.4 +
46.5+
378 +
106i
3.1 (13)
1.5(12)
B(l3)
11 (13)
121 f 5 (13)
116+ lO(l2)
6.Ok 1.2(12)
260 + 22 (9)
101* 14(9)
54k4(9)
69 i 11 (7)
7.4 f 1.7 (8)
set
Males
P*
29.2 f 1.2 (35) 46.7 + 1.2 (21)
376 f 6 (34)
99 * 7 (34)
119k5(31)
115*6(31)
7.7&0.7(31)
244 * 11 (30)
71 f 7 (28)
47 * 5 (30)
61 f7(30)
11.4 * 2.0 (28)
0.001
0.919
0.887
0.589
0.760
0.930
0.253
0.487
0.053
0.48 1
0.595
0.295
set. II. All
II. All toads
Males
Females
57.1 + 2.4 (57)
42.9 f I. 1 (54)
351 _+6 (45)
84?6(51)
111 _+3 (50)
101 f 6(48)
6.4 f 0.5 (46)
219il5(33)
77f6(40)
35 f 5 (37)
52 f 7 (33)
5.7 k 0.8 (35)
P
33.8 f 1.1 (78)
0.001
45.7 * I. 1 (59)
356 f 5 (73)
89 i 5 (76)
I II It 3 (76)
113?4(78)
6.5 + 0.5 (71)
229 f 10 (61)
71 i 6(61)
44 k 4 (62)
57f5(61)
7.7 + 1.1 (59)
0.08 1
0.51 I
0.533
0.853
0.075
0.862
0.585
0.457
0.176
0.514
0.225
Table 2. The effect of long captivity on the plasma and urine composition and on the nutritional state of Eufo uiridis. Two. groups are
compared: the first (I) was kept in the laboratory 7 months longer than the second group (II). Comparison was repeated three times within
4 months. The results were similar and only one comparison (4.10.83) is shown. Mean f SEM of five toads in each group
Urine
Plasma
Osmolality
(mOsm)
Group I
Group II
Comoarison (P)
Jan
370 * 9
352 + 30
0.12
Fob
Urea
cl(mmol~l-‘)
128*7
109+5
0.08
114+6
107+6
0.44
Na+
130+9
137 * 18
0.74
Osmolality
(mOsm)
265 + 12
273 + 3
0.54
Cl(mmol.l-‘)
Urea
137i 10
112*4
0.18
AW
Mar
55 f 6
57 _+6
0.71
SW
Na+
Fat content
W)
85 +7
80f IO
0.66
0.68
0.73
0.95
act
NW
Fig. 3. (a) Osmolality and composition of the plasma of the toads of the laboratory set. Five toads in
the first four columns, and 10 (combining the early and later groups) in the last three columns. Mean + SE.
White columns show the osmolality and the divided columns the various osmolytes (urea, chloride, sodium
and potassium from bottom to top).
Fab
Jun
JUl
Timstwmka)
Fig. 3. (b) Osmolality and composition of the urine of the toads of the laboratory
Explanations are as above.
set. Mean f SE.
706
U. KATZ et
al.
Table 3. Comparison of the osmolality and composition of the plasma in three sets of toads Et&o Grids. 1. Laboratory set
(originated from M. Ephraim). II. Maale Ephraim set. III. Kfar Giladi set. Mean f SEM. Number of toads in parenthesis
Comparison (P)
Weight (g)
Osmolality (mOsm)
Urea (mmol.l-‘)
Cl- (mmol.l-‘)
Na+ (mmol.l-‘)
K+ (mmol.l-‘)
Haematocrit (“/o)
1. Laboratory
set
32.5 + 1.4(48)
376 f 4.8 (47)
101 + 5.6 (47)
ll9*3.8(47)
115*4.6(43)
7.2 + 0.6 (43)
46.7?0.9(4lj
I:11
II. M. Ephraim
II:111
III. K. Giladi
III:1
0.001
0.001
0.55
0.04
0.39
0.12
0.35
46.9 & 2.3 (58)
349 + 5. I (52)
96 +_6. I (52)
109 f 3.3 (50)
109 * 4.4 (50)
5.9 + 0.5 (501
48.1 : 0.8 i4Oj
0.001
0.001
0.001
0.02
0.15
0.55
0.001
52.7 ~fi3.4 (34)
309 * 4. I (20)
48 _t 4.0 (29)
99 + 2.3 (28)
95 + 6.8 (26)
6.5 f 0.5 (25~
37.7 I 1.4 ij?j
0.001
0.001
0.001
0.001
0.043
winter (February).
The volume of urine stored in
the urinary bladder was 3.47 +_ 1.45% (mean + SEM)
of the body weight ranging from 1.4 to 5.5%.
(b) The setsfrom nature
Figure 4(a) shows the osmolality and composition
of the plasma
in nine groups
of toads which
were collected in Maale Ephraim (see map) during
an eighteen month period. The osmolality
of the
plasma
was rather
high throughout
the whole
period
(352 + 4mOsm.kg-’
HZ0 in 52 toads,
mean + SEM), and urea was the most variable solute
ranging from 40 _+ 11 to 159 + 36 mmol .l-‘, with
higher
concentrations
recorded
in the summer
months (significantly different, P = 0.001). The pat-
n.77
0.001
tern of the fluctuations in the osmolality of the urine
[Fig. 4(b)] was similar to the situation in the laboratory set (i.e. lower osmolalities recorded in the winter
months). The volume of urine stored in the urinary
bladder
was 5.32 + 2.19% (mean + SEM) of the
body weight, ranging from 2.5 to 8.8%. Table 3
summarizes the concentration
and composition of the
plasma of the three sets of toads (see map, Fig. 1).
The two sets which originated from Maale Ephraim
resembled each other closely, showing small differences only in the total osmolality
of the plasma.
Larger differences were found in the plasma osmolality and the concentration
of urea when the sets
from Maale Ephraim and Kfar Giladi are compared;
both were lower in the Northern
population
(Kfar
t
Fig. 4. (a) Osmolality and composition of the plasma of the toads from M. Ephraim. Six toads were
analyzed at each time point. The lower panel is from the second year (1983/4). Mean + SE. Explanations
are as in Fig. 3.
Annual
changes
in
blood and urine in a toad
107
Table 4. Comparison of the ratios (R) of the osmolality and concentration of the urine to plasma in the three sets of toads,
Eufo viridis. I. Laboratory set. II. Maale Ephraim set. III. Kfar Giladi set. Mean + SEM. Number of toads in parenthesis
I. Laboratory
Comparison (P)
R-OS (osmolality)
R-w (urea)
R-Cl (Cl-)
R-Na (Na+)
R-K fK+\
0.654 f
0.836 +
0.448 *
0.562 f
1.527 +
0.0264
0.0779
0.0403
0.0508
0.2736
set (35)
I:11
0.038
0.635
0.217
0.150
0.001
Giladi). These two populations differed considerably
in their urine osmolality:
a mean value of
247 f 101 mOsm was found in the population of
Maale Ephraim while in the population of Kfar
Giladi it was 138 f 29 mOsm (mean f SEM).
The osmolality of the plasma and urine at southern
latitude (Hatzeva/Ein-Gedi-see
map) in winter and
summer is shown in Fig. 5. The general picture
conforms with the previous sets: plasma osmolality
was quite similarly constant in winter and summer
(its absolute values, however, were lower than in the
previous sets described above). Urine osmolality in
these toads in summer was nearly four times higher
than in winter.
(c) Relation of urine and plasma concentrations
The urine analyses were made on samples from the
urinary bladder and therefore represent the end product of both the kidneys and the urinry bladder. The
ratios of the urine/blood osmolality and the concen-
II. Maale Ephraim (40)
II:111
0.642
0.857
0.429
0.591
0.973
+
+
+
+
+
0.0353
0.0780
0.0475
0.0623
0.1324
0.524
0.001
0.001
0.358
0.487
III. Kfar Giladi (17)
III:1
0.402 + 0.0828
1.168 kO.0387
0.063 f 0.0184
0.255 f 0.1241
0.591 + 0.2102
0.040
0.001
0.001
0.042
0.016
trations of urea, chloride, sodium and potassium are
summarized in Table 4. The ratio was over 1 for urea
in the Kfar Giladi set, significantly different from the
two other sets. The ratio for potassium on the other
hand was nearly 1 in the laboratory and Maale
Ephraim sets, significantly higher than the ratio in
the Kfar Giladi set. All together the ratios for the
electrolytes and osmolality were lower in the Kfar
Giladi set than in the other two, which did not differ
from each other significantly. All ratios were independent of the plasma osmolality which ranged from 300
to 450 mOsm in the laboratory and Maale Ephraim
sets, and from 260 to 350 mOsm in the Kfar Giladi
set. Figure 6 shows the relation of urinary urea
(U-ur) and sodium (U-Na) to plasma (P-ur) and
sodium (P-Na) respectively, in the three sets of toads.
There is a clear distinction between the laboratory
and the Maale Ephraim sets (I and II) on one hand,
and the Kfar Giladi set (III) on the other hand. It
seems that in the toads from the northern region
Fig. 4. (b) Osmolality and composition of the urine of the toads from Maale Ephraim. Mean + SE.
Explanations are as in Fig. 3.
U. KATZel al
708
1
(Kfar Giladi) sodium retention was stronger than in
the other two sets [Fig. 6(a)], while urea excretion was
related linearly to the plasma urea concentration [Fig.
6(b)]. Comparison of the urinary chloride and sodium
shows also greater sodium retention in the Kfar
Giladi set than in the other two. This is reflected in
the lower concentration of sodium which was found
in the urine of the northern toads (Kfar Giladi),
whereas it was higher and varied widely in the urine
of the toads of Maale Ephraim and those in the
laboratory.
(d) Water content of the tissues
0
Plasma
Jon. 83
Fig. 5. Osmolality and composition of the plasma and urine
of toads from the southernmost area (Hatzeva/Ein-Gedi).
Mean of six toads at each time. Explanations are as in
Fig. 3.
The water content of the various tissues of males
and females (Table 5) did not differ significantly in
the toads from either Maale Ephraim or Kfar Giladi.
In the laboratory set the water content of all the
tissues, except for the liver, differed significantly in
males and females. The water content was lower in
the females and the largest difference was found in the
water content of the skin. The results for both sexes
are pooled.
. .
40
;
U.ur
=42
5t4357&
Fig. 6. Relations of urinary urea (6a) and sodium (6b) concentrations to the respective concentrations in
the plasma. I. Toads of the laboratory set. II. Maale Ephraim set. III. Kfar Giladi set. The equation on
the bottom right of each set of points is the calculated linear regression, and r2 the correlation of the line
to the observed points.
Annual changes in blood and urine in a toad
709
-
/
.
.
_
-.*
+=i
;.
. .
.
40-
..*
/
20 j*
*
**
*
*
.
I .
1.., ,.
al
I
*
44
UJzC1=38.3+0.166U_No
d;OOYi
.
do b
(
(
,
/
“IO?‘2O”~‘N”
160
,
(
180 203
,
,
220
240
a
Fig. 7. Relation of the urinary chloride concentration to
the con~n~ation of sodium. Same sets of toads and
explanations as in Fig. 6.
(e) Relation of tissue water content and plasma
osmolality
The regression equations for the water content in
the three sets of toads show that the water content of
the tissues is independent of the plasma osmolality in
the range of 300400mOsm
(Table 6). Only for the
water content of the skin of the toads from Maale
Ephraim may be a slight dependency be observed on
the plasma osmolahty (a slope of 0.06, significant at
P = 0.04).
Comparison of the water content of the tissues in
toads which were kept in the laboratory and those
from nature shows consistent differences for all the
tissues (Table 7). The water content was higher by
15-50% for the various tissues in the toads which
were kept in the laboratory as compared to the toads
which were analyzed upon coilection. The greatest
difference was found in the water content of the heart.
The water content of the tissues in the toads from
Kfar Giladi (except for the heart) was similar to that
of Maale Ephraim (toads from nature), and lower
than in the laboratory set. Here again the water
content of the tissues seem to be independent of the
U. KATZ et al.
710
Table 6. Linear regression equations (Y = ux -t b) of the water content in the various tissues of Bufo ciridis in relation to the
osmolality of the plasma (Pas). 1. 48 toads in the laboratory set. II. 58 toads in Maale Ephraim set. 111. 20 toads in Kfar Ciladi
set
I. Laboratory
r*
(Pos1
Heart
Liver
Skeletal muscle
Skin
Carcacs
Table 7.
82.9
85.3
83.2
86.5
79.8
0.01
0.03
0.01
0.03
0.01
0.010
0.070
0.027
0.044
0.020
II. Maale
(Pas)
P
0.51
0.09
0.28
0.11
0.35
77.4
68.5
75.3
92. I
80.3
- 0.02
+ 0.003
- 0.002
- 0.06
- 0.02
Ephraim
2
P
fPost
0.43
0.87
0.x9
0.04
0.1 I
0.014
0.0005
0.0004
0.096
0.05
III. Kfar Giladi
r*
65.6 + 0.04
95.1 -0.09
70.6 + 0.01
78.3 - 0.02
0.02
0.06
0.04
0.005
comparison of the mean water content of the various tissues of B&o ciridis which were kept in the laboratory
directly from nature (Maale Ephraim and Kfar Giiadi). Mean rt SEM. Number of toads in parentheses
Water content (%)
of tissues
Heart
Liver
Skeletal
Skin
-
set
(mOsm)
set
(I&,
1 dry
+ 0.03
+ 0.02
+ 0.02
&0.03
0.001
0.001
0,001
0.010
2.44
2.30
2.92
2.70
+ 0.03
& 0.02
f 0.02
2 0.03
2.83 i: 0.01
377 + 5
0.998
0.001
2.80 i 0.01
352+4
0.025
0.001
plasma osmolality in the various sets. Higher water
content was found in the toads of the laboratory set,
which had higher plasma osmolality than the toads
from Maale Ephraim.
DISCUSSION
The present study extends our knowledge and
understanding of the osmoregulation and the general
biology of Bufo viri&-a
terrestrial amphibian-in
nature, and under constant laboratory conditions.
Under laboratory terrestrial conditions the toads
bathe in the water a few times a day, while under
natural conditions they bathe in water mainly during
the breeding season, and only occasionally thereafter,
seeking humid niches, and burrowing under more
severe conditions (Degani et al., 1984; Katz and
Gabbay, 1986). Consequently, the introduction of
irrigation schemes in Israel has enabled Bus0 viridis to
extend its range in recent decades to newly created
humid niches (artifically irrigated), even in the generally more arid areas.
(a) Annual lzj2 cycle of Bufo viridis
The annual life cycle of mature Bufo oiridis in Israel
may be divided into three principal periods. At the
beginning of winter (usually about the second half
of November) upon rainfall, they begin to move
towards the breeding spots (temporary ponds). This
will happen however, only if there is enough rain to
produce ponds which would last for a couple of
weeks or more. Otherwise, the toads keep hiding until
the onset of rain, and only a few emerge before then.
This type of behaviour was greatly emphasized in the
last few winters (i.e. 1983/84, 1984/85, and 1985/6)
which all began rather late and had comparatively
little rainfall. The toads did not emerge until
February; however, fresh ponds which were formed
artificially early in December and January, attracted
a couple of hundred toads which were “fooled” by
these artificial conditions. Mass migration usually
occurs in the principal spawning period upon heavy
rainfall (January and February). Toads continue,
0.82
or brought
weight)
&II)
-..._ ..._
0.00 I
0.122
0.452
-
3.69
2.68
3.52
3.13
muscle
Carcass
Plasma osmolality
1. I.aboratofy
Water content (g.gII. Maale Ephriam
(55)
.-____-
P
0.55 --0.34
0.40
___
III. Kfar Giladt
-.-.-. (30)
__~
(I:%)
3.29 f 0.05
2.13 + 0.02
3.02 t 0.02
0.107
0.001
0.001
2.52 + 0.04
309 jL 5
0.027
0.00 I
however, to spawn sporadically until summer (May,
June or even July). In the second annual period-the
rest of the winter and the spring (February to
April jthe
toads are found quite close to the ponds
and they hide during the day. In the summer
(May-October), they hide out (apparently in more
distant places, which are yet unknown), either beneath stones, or by burrowing in the soil. This type
of annual cycle is predicted only in ordinariIy rainy
years. In bad years with little and more sporadic rain
(i.e. 1983/84, 1984/85, and 1985/86 as mentioned
above) no mass migration was noticed and spawning
spread over the year until midsummer. The toads
seem to be active throughout the whole year, although it is not yet clear for how long they may hide
during summertime, whether they aestivate or how
frequently they emerge to feed. Toads are commonly
active nocturnally throughout the whole year on
moist grass or in plantations.
The annual cycle of the weight of the gonads and
that of the fat bodies conforms to the cycle described
recently by Jorgensen (1984) for this species in
Denmark and in Israel, and also for Bufi bufo
(Jorgensen er al., 1979) in Denmark. It seems that the
principal differences between the toads in the north
(Denmark) and those in Israel, lie in the timing and
length of the spawning period (Jorgensen, 1984). It
begins in Israel earlier-sometimes
in December,
extending to midsummer (June and July), and they do
not hi~rnate during the winter, which is relatively
warm in the Mediterranean
region. Temperature
seems to be more important
in triggering reproduction behaviour in the northern distribution
region (Denmark), while humidity (rain) seems to be
more dominant in the South (Israel).
(b) The osmolali~y and composition of the plasma
Amphibians may be considered rather vulnerable
amongst vertebrates to changing environmental conditions, due to the high water permeability of their
integument. This is reflected in large variations of
their plasma osmolality, both in a given species and
amongst the various species of the group (Bentley,
Annual changes in blood and urine in a toad
1971; Katz, 1975; Balinsky, 1981). Bufo viridis has
characteristically high plasma osmolality, and particularly high and variable concentration of urea (Katz
and Gabbay, 1986).
Merren and Kuchler (1965), and Becht (1969)
reported very small fluctuations in the electrolyte
concentration (sodium and chloride) in frog blood in
nature, whilst Robertson (1978) found some 10%
increase in the sodium concentration of Rana pipiens
plasma in the summer. Jolivet-Jaudet et al. (1984)
observed higher levels of aldosterone-a
major mineral ocorticoid regulatory hormone-in
the plasma of
the terrestrial toad (Bufo juponicus formosus) in the
summer. Our study demonstrates that the plasma
osmolality of active toads Bufo viridis remains remarkably constant throughout the whole year under
constant conditions in the laboratory, It is regulated
within a narrow range, in active toads in nature,
fluctuating less than 15%, with the higher values
measured in the summer months.
The toads which were analyzed in this study represent selectively active toads which seek water. The
osmolality of the plasma of these toads was, however,
rather high but constant (>300mOsm),
while in
most amphibians
it is usually in the range of
230 mOsm (Bentley, 1971). The plasma of the laboratory set, which had free access to tap water throughout the whole year, had the highest osmolality
amongst the various sets, significantly different
(P < 0.001) from the osmolality of the “matched”
toads (Maale Ephraim), which were analyzed upon
collection. These two sets (originating in Maale
Ephraim) however resembled each other closely, and
differed clearly from the toads of Kfar Giladi. The
toads from the southernmost region (Hatzeva/EinGedi), on the other hand, had significantly lower
plasma osmolality. The differences cannot be related
to the geographical or genetic separation only, but
should be related also to the particular ambient
conditions in each case. Further investigation is
needed to resolve this problem.
The relatively high plasma osmolality which was
found in the toads of all the sets, would provide
constantly greater water absorptive potential from
the substrate, and thus would be of great adaptive
value. Urea is the most variable solute in the plasma,
and it accounts for the variations in the plasma
osmolality between summer and winter and amongst
the various sets. The mechanisms which regulate
plasma urea concentration, i.e. increased production
through increased urea cycle enzymes (Balinsky et al.,
1967), and increased uptake across the skin (Katz and
Gabbay, 1986) seems to be influenced by annual
changes more than osmomineral metabolism. This
should form the basis for the observed annual
changes.
(c) Urine osmolulity and its role in osmoregulation
Our measurements do not allow us to differentiate
between the role of the kidneys and the urinary
bladder in osmoregulation, since the urine which was
catheterized was stored for some time in the bladder.
However, the results indicate that the urinary stystem
as a whole is important for the regulation of the
plasma osmolality and its composition. Figure 8
shows that while plasma osmolality fluctuated only a
711
little during the whole year, urine osmolality varied
considerably with lower values measured in the
winter. It is difficult to speculate on the regulatory
mechanisms which are involved in these effects.
Lagerspetz (1977) suggested interaction of season and
temperature in the control of metabolism in amphibia, implicating mainly adrenaline and the thryoid
glands. The neurohypophysis and adrenocorticoides
should be more closely involved in the control of the
osmomineral metabolism, but very little is known yet
about it. It is worth noting though, that the annual
pattern persisted under the constant laboratory conditions [Fig. 8(a)], implying an innate control mechanism. It should be emphasized that only active toads
are presented here. Under more severe conditions of
water scarcity the toads burrow, and their plasma
osmolality is greatly increased, mostly by urea
(Degani et al., 1984; Katz and Gabbay, 1986). The
latter situation
would certainly entail different
control mechanisms and it will be interesting to study
the transition
from the one type to the other.
(d) Tissue water content
Water content of the tissues was maintained constant throughout the whole year in all the sets of
toads investigated. The various tissues had characteristic values of water content for each, and the water
content was independent of the plasma osmolality in
the range of 30&44OmOsm. This was found also in
burrowing toads (Katz and Gabbay, 1986) where
plasma osmolality increased to over 900 mOsm,
mostly by urea. The large variability of the plasma
osmolality in Bufo viridis in nature and under burrowing conditions is accounted for almost entirely, by
urea. This is common to all arid zone (Shoemaker
and Nagy, 1977) and salt adaptable amphibians
(Balinsky, 1981). In sea water elasmobranchii (Smith,
1936), plasma urea is an essential constituent of the
blood osmolality. In amphibious fish (Gordon er al.,
1969) it varies upon aestivation and in the killifish (a
teleost) it was also elevated when the fish was kept in
solutions containing
high external urea concentrations (Griffith et al., 1979). Among other vertebrates, only in one reptile (the turtle diamondback
terrapin) plasma urea concentration increased upon
adaptation to sea water (Gilles-Baillien, 1970).
Increased plasma urea concentration plays a key
role in osmoregulation in the arid invading and salt
adaptable amphibians. It allows increased water absorptive potential across the skin (Katz and Gabbay,
1986) while equilibration with the cell interior does
not impose osmotic stress on the tissues. Only little
is known on the adaptive cellular mechanism and on
the volume regulation under these conditions.
Recent studies on this species (Degani et al., 1984;
Katz and Gabbay, 1986) and studies on other terrestrial species (McClanahan,
1972; Loveridge, 1976)
show that toads burrow during the dry season and
their plasma osmolality is increased considerably
under these conditions-mostly
by urea. The toads
do not void urine under burrowing conditions, rather
they store it in the urinary bladder. The urinary
bladder seems therefore to participate importantly in
water balance, and recycling of water and urea
is anticipated (Katz and Ben-Sasson, 1984). The
differences in the ratios of urine to’ plasma concen-
U. KATZet al.
712
AWl
act
SW
ooc
NW
Jon
Fob
mm
w
MOY
limobllonms)
(bl
---_
/
/
/
/
A
Fig. 8. Annual changes in the total osmolality of the plasma and urine in the laboratory (a) and Maale
Ephraim sets (b). Over lf years are shown in the latter set. Continuous lines show the mean plasma
osmolality, and broken lines the mean osmolality of the urine.
trations
between the
(both in the laboratory
toads
from
Maaie
Ephraim
and in nature) and those from
Kfar Giladi show that the excretory system can
respond differently and selectively under different
environmental conditions.
In summary, it was found that plasma osmolahty
of Bufi r:iridis is kept rather high and mainlined
within a narrow range in active toads throughout the
whole year. In a northern population plasma osmolality was significantly lower due to lower plasma
urea concentration.
Tissue water content is kept
constant throughout the year in all toads. Urine
osmolality shows great fluctuations with lower values
in the winter months. The dist~bution of Bufo ~ir~~~
and its invasion of semi arid zones are discussed in
the light of its inherent capacity for urea accumulating and tolerating ability and is related to the high
efficiency of the excretory system.
Acknou,ledgemenrf-The
technical assistance of Shosh
Gabbay and the secretarial assistance of Jean Kenton are
gratefully acknowledged.
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