6i
CHANGE OF WEIGHT OF MARINE ANIMALS
IN DILUTED MEDIA
BY K. HUKUDA.
(Received 8th June, 1931.)
(With Three Text-figures.)
I. INVERTEBRATES.
L. FREDERICQ (1885) found that the content of soluble salts in the body fluid of
several marine invertebrates is approximately equal to that of the surrounding sea
water, and suggested that salts are able to penetrate the gill membrane like a dialysis
membrane. This theory was tested by several investigators, either by measuring
the amount of osmotic swelling, or by determining (by chemical analysis) the actual
transfer of substances through the membrane.
Margaria (1931) and the author have confirmed that when a typical marine
invertebrate, or an elasmobranch, is transferred to a diluted medium an equalisation
of osmotic pressure between the internal and the external media takes place. If
this equalisation is effected by dilution of the internal medium by the surplus water
entering through the bounding membrane, the weight of the animal as a whole
should increase. If, on the other hand, dilution takes place by the disappearance
of salts and other dissolved substances from the internal medium, the weight should
remain unchanged.
Quinton (1900) observed a progressive increase, or decrease, in the weight of
Aplysia, Sipunculus and Arenicola when kept in diluted or concentrated media.
Bottazzi and Enriques (1901) suggested that this change of weight might be due
to the variable amount of water contained in the gastro-intestinal tube. Osmotic
swelling was noticed by Schiicking (1902) in Sipunculus, by Garrey (1905) in
Asterias, Nereis and Chaetopterus, and by Dakin (1908) in Cancer pagurus, Hyas
araneus, Doris tuberculata, and Arenicola marina. Recently some measurements
were made by Schlieper (1929), who confirmed that the increase of body weight
in diluted media was transitory in Cancer pagurus, reaching a maximum in 3-10
hours, as well as in Nereis diversicolor, beginning to diminish on the second day.
Bethe (1930) found the weight increase in Aplysia also to be transitory, reaching
a maximum on the second day, but failed to observe an increase of body weight in
Carcinus maenas.
Experimental.
The following experiments were performed in Plymouth in September, 1930.
The weights of animals (Portunus puber, P. depurator, Carcinus maenas, Maia
squinado and Cancer pagurus, kept in diluted media, were measured. Moisture
K. HUKUDA
62
adhering to the skin was absorbed by a dry duster, in which the animal was wrapped
and placed on the balance. Weighing was to o-i gm. The greatest possible care was
taken to shake out the water contained in the gill cavities.
The results for the crustaceans used agreed with those of Schlieper (1929),
showing a transitory increase of weight directly after the animals were transferred
to diluted media1.
Table I. Changes of weight of animals in dilute media.
Portunus depurator. Weight in two-thirds sea water.
Time
(hours)
0
4
10.30
26.30
54
82*
No. s
No. 2
No. 6
gm.
0/
/o
gm.
%
gm.
0/
la
II4-3
iiS-9
1147
II5-9
114-6
114-0
IOO
IOI-2
IOO3
IOI-2
IOO-2
92-7
94-7
94-0
93- 2
93-3
93-5
IOO
IO2-2
78-5
78-3
78-2
79o
78-3
79'4
IOO
998
ioi-s
ioo-6
100-7
100-9
998
9-6
100-7
998
IOI-I
• All animals in good condition.
Portunus puber. Weight in two-thirds sea water.
Time
(hours)
0
4
10.30
26.30
47-30
54-3O
82
No. s
gm.
No. 7
°/
/o
IOO
191
194
19-4
192
19-1*
ioi-6
IOI-6
ioo-6
100
* Found dead.
gm.
/o
21-3
22-O
21-5
2I"4
IOO
103-3
IOI-O
100-5
21-4
213+
100-5
ioo-o
f In good condition.
Cancer pagurus. Weight in two-thirds sea water.
Time
(hours)
0
7-3O
25-30
53-3O
68.30
92*
No. 3
No. 2
gm.
3214
325-3
321-5
3217
321-2
3218
0/
/o
IOO
IOI-I
IOO-O
ioo-1
ioo-o
ioo-1
gm.
0/
/o
422-9
4280
424-1
423-2
421-6
418-8
IOO
101-5
100-4
ioo-1
997
99O
• Both in excellent condition.
The increase never exceeded 4 per cent, of the initial weight. The equalisation
of osmotic pressure, however, was almost complete in less than 24 hours, as was
shown by direct measurement (Fig. i,A). The osmotic pressure was determined
1
All dilutions were of tank water with tap water: the animals had been for some time in tank
water (which is slightly more concentrated than sea water) before use: Plymouth tap water may be
taken for the present purpose as equivalent to distilled water. Osmotic pressures are expressed as
a fraction of that of Plymouth sea water.
Change of Weight of Marine Animals in Diluted Media
63
by the vapour pressure method (Hill, 1930 a), (Margaria, 1930). Now if it were
supposed that the equalisation took place by osmosis of external water, it would be
possible to calculate the amount of water necessary to dilute the body fluid to the
degree shown in curve A. For this purpose the water content of the animals was
determined and the following values were found (Table II).
Table II.
Animal...
Water content (%)
Portunus
puber
Portunus
depurator
Cancer
pagurus
Carcinus
mamas
72-2
672
67-0
680
Maia
squinado
8i-5
0
The whole animal was crushed and kept in a thermostat at 95° C. and then at 120 C. until the
weight was constant. The difference between the initial and final weights represented the water
content of the animal.
25
10
15
Time in hours
Fig. 1. Portunus puber. A, osmotic pressure observed (ordinate on left); B, water transfer calculated
by assuming semipermeability (ordinate on right); C, actual increase in body weight observed
(ordinate on right).
If it be assumed, from the analogy of mammalian blood (Hill, 1930 b), that
97 per cent, of the water in crustacean haemolymph is in the "free" state, being
able to participate in osmotic dilution, approximately 70 per cent, of the body weight
of P. puber is free water. The amount of water necessary to increase the dilution
n times, i.e. to decrease the osmotic pressure to i/n of the normal value, is therefore
(n — 1) x 70 per cent, of body weight. The difference between the actual (curve C)
and the calculated (curve B) increase of body weight is so large as to make the
assumption of osmosis, or the existence of a true semipermeable membrane,
impossible. It would have to be assumed that water entered to the extent of onethird of the body weight in 5 hours, and that urine was excreted at the inconceivable rate of 21 per cent, of the body weight in the first hour. For other
crustaceans there are similar determinations, reported by Margaria (1931), of the time
relations of the equalisation of osmotic pressure. It is certain, therefore, that in
these animals the equalisation occurs mainly by the disappearance of salts and
other dissolved substances from the internal medium, not by the transfer of water.
64
K . HUKUDA
The presence, however, of an osmotic swelling, though in a very slight degree,
suggests that the membrane is slightly semipermeable in the sense postulated by
Schiicking (1902), that is to say, it is permeable to water and in a less degree to
salts. The permeability of the bounding membrane thus investigated is really that
in an abnormal condition, the animal being immersed in a medium more dilute
than its natural environment. Whether and how far the normal properties of the
membrane are thereby affected is unknown, although the behaviour of the animals
under investigation was apparently normal {Care, maenas, Portunus depurator and
Cancer pagurus). This applies also to Portunus puber, except on the last day of their
survival, when some of the group showed signs of weakness.
The salts that disappear from the circulation could be accounted for in two ways:
(i) by diffusion through the bounding membrane, or (ii) by being fixed and deposited in the body, either to be stored or excreted. The demobilisation, however,
of salts is not likely, although Bethe (1928) demonstrated the mobilisation of Ca
from a hypothetical Ca reservoir in Carcinus maenas, and Collip (1920) also found
the mobilisation of Ca from the shell of molluscs, but not from the carapace of
crustaceans, into the haemolymph. It is possible that, in the earlier period of
immersion in diluted media, salts and other dissolved substances are excreted,
together with the excess of water. The greater part, however, of the equalisation
of osmotic pressure between the internal and the external media is probably
effected by diffusion of salts through the bounding membrane, leading to the almost
complete disappearance of the concentration gradient. In other words, the bounding
membrane of marine crustaceans is permeable not only to water but also to salts.
The same conclusion has been reached by Quinton (1900) on Carcinus maenas, and
by Bethe (1928, 1930) on Carcinus maenas and Aplysia, by means of chemical
analysis.
II. ELASMOBRANCHS.
L. Fredericq (1904) observed that the osmotic pressure of the blood of Scyllium
kept in diluted sea water decreased to the same level as the external medium. The
new equilibrium, in his opinion, was established by "transport of water from the
external medium into the blood." In animals which had been kept in a diluted
medium containing i-o per cent. NaNO3 no nitrate was found in the blood. Further
support to his view is the remarkable difference between blood and surrounding
medium in the content both of salts and of urea. The urea content of blood, according to v. Schroder (1890), is 2-5-3 P e r cent, and that of soluble salts is i-62-3 per cent. Thus the blood of elasmobranchs and the surrounding sea water are
in equilibrium in osmotic pressure but not in concentration of individual solutes.
The bounding membranes of the dogfish appear to be semipermeable, keeping the
solutes in the internal and external media from diffusing towards each other and
becoming equalised in concentration. If this is so, and if the semipermeability is
maintained even in diluted media, a considerable amount of water may be expected
to pass by osmosis through the membrane.
Change of Weight of Marine Animals in Diluted Media
65
Experimental.
Dogfish (Scyllium) were kept in various dilutions of sea water and weighed at
intervals. A considerable increase of weight (Fig. 2) was invariably observed,
together with marked diffuse oedema, especially on the abdominal surface. The
duration of immersion necessary to increase the body weight by 10 per cent, is
shown below (Table III) (interpolated from the curve of Fig. 2 and similar ones
not shown).
130 •
0N0.3
©No.5
100
20
40
60
Time in hours
Fig. 2. Time-weight curves of dogfishes kept in three-quarters sea water. No. 2, re-transferred into
sea water at the time marked with an arrow, showed a quick decrease of weight toward the original.
Table III.
Three-quarters sea water.
Scyllium...
Initial weight (gm.)
Necessary duration (hours)
1
2
3
4
5
6
I 3 5-8
3280
2
374-7
3*
132-0
1
308-1
3
IO2-2
2*
Mean = 3 hours
Four-fifths sea water.
Scyllium...
Initial weight (gm.)
Necessary duration (hours)
7
8
9
10
11
12
13
225-6
3
240-1
215-5
4
373-8
5
274-6
3*
355-3
5
254-7
5
3i
Mean == 4 hours
Details of the case of dogfish No. 7, which survived in excellent condition for
108 hours until killed, are given below (Table IV), and in Fig. 3.
Table IV.
Time (hours)...
Weight (gm.)
Weight (%)
0
22O-6
IOO
2.3O
242-0
IO94
6.3O
2486
II2-3
22.30
2587
117-0
30
259-5
117-3
59
88
2582
116-8
256-4
116-0
108
2551
115-3
The time-weight curve rose rapidly at first and then less rapidly, reaching a
maximum in 30 hours, after which it decreased gradually, I-I gm. on the first day
JEB-Ixi
5
66
K . HUKUDA
and then 1-5 gm. per day. This slow decrease in weight may be due to loss of water
(or solutes) by excretion of urine, partially compensating the gain by osmosis.
If it be supposed that the bounding membrane is semipermeable, it is possible
to calculate the amount of water necessary to dilute the internal medium to the
same osmotic pressure as the external. The water content of a dogfish, weighing
283-5 gm-> w a s found to be 68-8 per cent, of the total weight. Assume that 97 per
cent, of this water is in the "free " state. Some lower homologues of fatty substances
must have escaped in the procedure of measuring the water content (described
above). The amount of free water therefore is taken as 65 per cent, of the body
weight. At the end of 108 hours the blood of dogfish No. 7 was found to have an
osmotic pressure equal to o-86i of that of sea water. The water, therefore, that had
been transferred into the body was equal to ( - ^ — 1J x 65 per cent. = 16-3 per
cent, of the body weight. 1-075 ^ e r e 1S t n e osmotic pressure, expressed as a fraction
of that of sea water, of the initial external medium, in which the dogfish had been
kept for several days preceding the experiment, so that it corresponds to the initial
100
80
40
60
Time in hours
Fig. 3. Time-weight curve of a dogfish which survived in four-fifths sea water in excellent condition.
20
value of the osmotic pressure of the blood. The increase of weight observed was
15-3 per cent. Considering the complicated nature of the organism, the agreement
between calculated and observed is close. The kidneys of dogfishes are not able to
regulate the water output enough to diminish appreciably the state of hydropsy.
Table V.
Dogfish...
Duration (hours)
Gain in weight (%)
8
9
47
22»
l6l
20-6
•
10
11
12
22
21-4
22
182
22
14-9
22
19-5
Found dead.
In cases which developed more or less serious pathological symptoms, the
increase of body weight was large. This is possibly due to abnormal metabolites
aiding in concentrating the internal medium. Even in cases in which no external
symptoms other than diffuse oedema were noticeable, the unusual environmental
condition must have interfered in some way with the tissue metabolism. At the
end, when the dogfishes were killed and the osmotic pressure of their blood was
Change of Weight of Marine Animals in Diluted Media
67
measured, it was always somewhat higher than the value calculated from the
swelling, except in No. 7, which survived in highly excellent condition for 4 ! days.
The swelling, in fact, generally continued in these cases.
Table VI.
Dogfish...
5
3
i
External medium
(fraction of tank
water)
Duration (hours)
Swelling (%)
Osmotic pressure
calculated*
Osmotic pressure
7
i
7730
t
108
22
43-3O
43'3O
161
187
186
0764
0-828
0822
0861
Killed in
excellent
condition
Found
dead
13
I
IS-3
0867
Died
12
i
22-8
0796
266
Final state
9
* Calculated by the formula 1-075
*
'°
x
0863
0836
0-837
0-900
0864
0-872
Found
dead
Killed
slight
symptoms
Killed
slight
symptoms
^r
r~z~ •
per cent, swelling + 65
A test of semipermeability can be made in another way, i.e. by comparing in
the early stages the degree of swelling with the change of the osmotic pressure of
the blood. The osmotic pressure expected from the degree of swelling, under the
assumption of semipermeability, is calculated by the above formula. Taking the
mean of three similar experiments (No. 4-N0. 6) in three-quarters tank water, the
following results are obtained (Table VII).
Table VII.
Time (hours)...
Weight (%)
Osmotic pressure calculated
(fraction of sea water)
1.30
0
108
100
O-9S9
1-O75
4-30
18.30
US
0-875
121
0-814
Taking the mean of seven experiments (Nos. 7-13) in four-fifths tank water,
the results are (Table VIII):
Table VIII.
Time (hours)...
Weight (%)
Osmotic pressure calculated (fraction of
sea water)
0
2.30
6.30
100
107-6
112-5
1-075
0915
0964
7
"3-5
0-892
22
1180
0843
22.30
30
116-7
0856
119-2
0831
45
119-3
0-830
These calculated values of the osmotic pressure agree closely with those actually
determined by Margaria (1931).
5-2
68
K. HUKUDA
SUMMARY.
1. Several species of marine invertebrates, and an elasmobranch, have been kept
in diluted media. The increase of body weight so caused was compared with the
resulting dilution of the body fluids.
2. The bounding membrane of the invertebrates was permeable to salts when
the animals were immersed in diluted sea water.
3. The bounding membrane of the elasmobranch was semipermeable, i.e.
permeable to water but not to solute. There is a close quantitative agreement
between the osmotic swelling observed and the diminution of the osmotic pressure
of the blood.
It is my pleasant duty to express my sincere gratitude to Prof. A. V. Hill, who
suggested these experiments, and to Dr R. Margaria and Mr J. L. Parkinson for
their co-operation.
REFERENCES.
BETHE, A. (1928). Pflugers Arch. 221, 344.
(1930). Journ. Gen. Physiol. 13, 437.
BOTTAZZI, F. and ENRIQUES, P. (1901). Arch. (Anat) Physiol. Supp. p. 109.
COLLIP, j ; B. (1920). Joum. Biol. Chem. 45, 23.
DAKIN, W. J. (1908). Biochem. Journ. 3, 473.
FREDERICQ, L. (1885). Arch. Zool. exp. ge"n. 3, xxxiv.
(1904). Arch. Biol. 20, 709.
GARREY, W. E. (1905). Biol. Bull. 8, 257.
HILL, A. V. (1930 a). Proc. Roy. Soc. A, 127, 9.
(1930 6). Proc. Roy. Soc. B, 106, 477.
MARGARIA, R. (1930). Journ. Physiol. 70, 417.
(1931). Proc. Roy. Soc. B, 107, 606.
QUINTON, R. (1900). C.R. Acad. Sci. Paris, 131, 905 and 952
SCHLIEPER, C. (1929). Z. vergl. Physiol. 9, 478.
v. SCHRODER, W. (1890). Hoppe-Seylers Z. 14, 576.
SCHOCKING, A. (1902). Arch. (Anat.) Physiol. p. 533.