NITROGEN-EXCRETION IN SELACHIAN
ONTOGENY
BY JOSEPH NEEDHAM, M.A. PH.D.
(Fellow of Caius College, Cambridge)
AND
DOROTHY MOYLE NEEDHAM, M.A., PH.D.
(From the Biochemical Laboratory, Cambridge, and the
Marine Biological Station, Millport, Scotland.)
{Received 14th March 1929.)
(With One Text-figure.)
THE investigations to be described in this paper were originally undertaken in
order to throw light on the utilisation of protein as a source of energy by the
developing selachian embryo. It has been shown that in the case of the chick
(Needham (24), Fiske and Boyden(i4)) and of the frog (Bialascewicz and Mincovna (4)) there exists a peak in the intensity of protein breakdown during embryonic
life, on each side of which the catabolism of these substances is much diminished.
In the case of the mammal (cow and sheep, Lindsay to) and others) the descending
limb of the curve has alone been observed. Our intention was to extend these
investigations to a selachian egg, and we obtained several interesting results, although
special difficulties were encountered, e.g. (a) the lack of any good series of weighings
of selachian embryos, and (6) the remarkably high concentration of urea in elasmobranch tissues, which makes it difficult to regard it wholly as a waste-product.
Experiments by Denis (8) in 1912 showed that in selachians urea is the principal
form in which nitrogen is excreted. Working on Mustelus cants, she found that its
urinary urea formed 84-7 per cent, of the total waste nitrogen, with ammonia
5-1 per cent, and uric acid 0-5 per cent.
We therefore set out to determine the urea and ammonia content of selachian
eggs at all stages during their development. The method used was a modification
of that described by Folin(is) for blood-urea. The contents of the eggs was ground
in a mortar with sand to as uniform a pulp as possible, then diluted, and the proteins
precipitated by boiling with acetic acid at pH just green to brom-cresol-purple.
Owing to the presence of fats and lipoids in the yolk which were not carried down
on the precipitate of protein, centrifuging had often to take the place of filtration at
oint, but we were usually able to prepare a clear or slightly opalescent solution
urea and ammonia estimation. This is accomplished in Folin's method by
8
JOSEPH NEEDHAM and DOROTHY MOYLE NEEDHAM
decomposing the urea with soya-bean urease acting at 500 for half-an-hour ir
presence of a few drops of phosphate buffer at/>H 7*0, and then distilling ove
ammonia rapidly into ice-cold 0-05 N hydrochloric acid after adding a saturated
solution of borax to the urease-urea mixture. The ammonia in the distillate is
estimated colorimetrically by Nesslerisation. Naturally each estimation must be
accompanied by a duplicate without urease in order to know the amount of preformed ammonia present, and this value must be deducted from the other.
The eggs used were those of the "rough-dog," Scyllium canicula, with a few of
Pristiurus melanostoma: the results obtained are given in Table I.
Table I.
Mg. per embryo
Description of stage
of development
Scyllium canicula:
Undeveloped (fertilised ?)
Orange spot only
(mm.)
Diameter of ring: 1
0-023
083
1-24
o-S3
Urea
nitrogen
219
474
615
9
032
080
3 79
480
825
4-25
S-8S
15
0-33
2-16
6-7
8
(cm.)
Length of embryo: o-1
0-2
0-33
0-44
117
0-62
0-4
051
°-5
o-6
o-37.
.08
2'5
30
033
039
°-53
°'44
039
Q.JM
869
11-62
832
9SO
930
35
36
3-8
39
49
53
032
6-90
0-36
79O
61
70
042
0-50
0-36
o-7S
10-63
10-03
15-92
1476
14-44
12-71
Pristiurus melanostoma:
1-5
o-43
981
17
o-s7
o-6i
7-54
2-O
5-6
116
Circular vein i cm. in diameter.
Cephalic plate and medullary tube closed;
blastoderm half over.
4'45
10-60
11-46
10-40
0-30
048
047
Appearance of circular vein.
9-00
8 17
802
871
34
072
Remarks
0-003
0-63
0-97
073
2-3
•
Ammonia
nitrogen
807
12-44
Holes in egg-case still plugged with jelly.
Four holes in egg-case; contents acid to
brom-cresol-purple.
Four holes, from which liquid easily
escaped on slight pressure.
No more "white" or jelly left.
No more'' white'' orj elly left; holes open.
Holes open and bubbles within the egg.
Two holes in the egg-case; contents acid
to phenol red (alk. to brom-cresolpurple).
Nitrogen-excretion in Selachian Ontogeny
9
The determinations were made on one egg only in each case unless two or three
^ f c figures are given beside one length; this indicates that more than one egg of
that embryo-length was analysed. It will be seen that only in very few cases were
we able to obtain more than one embryo of a given age, and the individual estimations necessitated by this fact are the cause of the considerable scattering of the
observations (see Fig. 1). After the embryo had lifted itself off from the yolk and
was simply attached to it by its umbilical cord,
it was not difficult to determine its age by
measuring its length, but in the earliest stages,
while segmentation was proceeding and the blastoderm was enveloping the yolk, the determination
of age presented more difficulty, and we simply
noted the diameter of the orange-coloured blastoderm standing out against the bluish yolk.
As the work proceeded we were surprised to
find comparatively large amounts of urea present
in the earliest stages. The question was unfortunately confused by the fact that the only egg
we were able to obtain which showed absolutely
no trace of development gave an exceedingly low
result. In our opinion, however, a certain amount
of urea is present in the undeveloped egg, before
the embryo has embarked on any appreciable metabolism of its own, and the urea
which it forms and excretes during its development is only a contribution to a stock
already provided by the maternal organism. These considerations are illustrated by
Fig. 1, which shows the data graphically. The points for the stages before the
embryo has a measurable length are all grouped along the ordinate.
As Fig. 1 shows, there is undoubtedly a production of urea by the developing
dogfish embryo, and although owing to the reason already given the scattering of
points is considerable, they seem to fall on each side of a curve very slightly concave
to the abscissa and approaching a straight line during the latter part of its course.
This line begins at a point corresponding to about 4/6 mg. of urea nitrogen in the
undeveloped egg and attains some 20 mg. of urea nitrogen at the time of hatching.
It is probable, then, that each Scyllium egg is supplied by the maternal organism
with this initial amount of urea, and that the catabolism of the embryo adds to it as
development proceeds. The horizontal line on Fig. 1 illustrates diagrammatically
the maternal contribution. As for the ammonia-content of the egg, it seems to remain relatively constant (except for one or two high values) at 0-5 mg. per embryo,
with perhaps a slight falling tendency.
The accessory scale on the right-hand side of Fig 1 measures off in mg. the
contribution of the embryo to the urea of the egg, and the result is shown in Table II.
Unfortunately no good series of weighings exists for Scyllium embryos, but, as is
\«Aknown (Fulton (17), Kearney (20), the length of fish embryos increases at first
faPmore rapidly than the weight. Now this evidently implies that when the points
io
JOSEPH NEEDHAM and DOROTHY MOYLE NEEDHAM
are plotted against weight, the slight concavity towards the abscissa shown in Fie. i
will be greatly accentuated, because the distance between any two points wi^Be
drawn out in the later stages and compressed in the earlier ones. This will happen
because equal increments of length mean much greater increments of weight in the
later stages than in the earlier. Plotted against weight, then, the urea-content curve
would rise sharply to a certain point and then very slowly. Accordingly, when the
urea produced by the embryo is referred to ioo units of embryo weight, a peaked
curve would result.
This expectation we found to be fulfilled as far as possible when we used the
figures of Kearney (21) for Mustelus cants as weight data. We lay no emphasis on the
result, but it will be admitted that the shape of the ascending weight-time curve for
Mustelus probably does not differ much from that of the related Scyllium. As the
last column of Table II shows, over the range covered by Kearney (2*5 to 8'O cm.)
a descending curve is obtained, and the dogfish may therefore be said to be in the
same position as regards its protein utilisation as the mammal, i.e. a curve has been
obtained which is probably the descending limb of a peak.
Table II.
Length in cm.
Scyllium
Urea-nitrogen
mg. per embryo
(embryo's contribution only)
o
0
o-5
i-35
24
i-o
i*5
2-O
43
—
—
—
—
S-8
64
200
34
25
3*0
3'5
4-o
4*5
5-o
55
6-o
6-5
103
70
1 i-o
n
Kearney's
corresponding
weights
(Mustelus), mg.
71
77
84
90
96
n-6
12-2
no
300
400
550
730
920
1150
1450
1720
2050
Mg. ureanitrogen
per 100 mg.
wet weight
—
—
—
—
5-250
3200
2370
1-920
1-550
1-230
1-042
0-899
0-760
0668
O-595
The presence of urea in the eggs of elasmobranchs is not reported here for the
first time. As is well known these fishes have a very special relation to this substance. In 1858 Stadeler and Frerichscw) isolated "kolossale quantitaten von
Harnstoff" from the organs of plagiostomes, obtaining a solid mass of urea nitrate
when they added nitric acid to their final concentrates. One liver of an adult
Scyllium canicula gave them 2 oz. of urea, and similar high figures were reported
for Acanthias vulgaris. Teleostean fishes, however, and the cyclostome, Petromyzon
planeri, yielded practically no urea, at any rate not more than would be present in
mammalian tissues. Stadeler (33) confirmed the selachian results on Raia batismgd
clavata and on Torpedo marmorata and ocellata. In 1861 Schulzedo) repeatedWnd
confirmed Stadeler's work on Torpedo, and in 1888 Krukenberg(«) published an
Nitrogen-excretion in Selachian Ontogeny
II
extensive work on the subject, in which he related his unsuccessful attempts to
^bionstrate urea in the bodies of Teleosts (Lophiusptscatorius, Conger vulgaris, Acipenser sturio), a Cyclostome (Petromyzonfluviatilisand Ammocoetes) and a Cephalochordate (Amphioxus lanceolatus), although he found large amounts of it in the bodies
of Elasmobranch Fishes {Scyllium stellare, Mustelus vulgaris and laevis, Acanthias
vulgaris, Squatina angelus, Torpedo marmorata, Myliobatis aquila) and in the
Holocephali, Chimaera monstrosa. Particularly interesting were his experiments
with eggs—he isolated considerable amounts of urea from a 5 cm. embryo of
Mustelus laevis, and from the yolk of Scyllium stellare and Myliobatis aquila eggs,
but he could find none in their surrounding jelly or "white." An egg of Pristis
antiquorum yielded 3920 mg. per cent, (wet weight) and a Torpedo ocellata egg
1740 mg. per cent. An Acanthias vulgaris embryo, 17 cm. long, had 3360 mg. per
cent, in its muscles, 1800 mg. per cent, in its liver, and 2640 mg. per cent, in its
unused yolk. Other work on urea in selachians was done by Grehant(ig) and by
Rabuteau and Papillon (27).
More light, however, was thrown on the reasons for this richness in urea when
in 1897 Bottazzi(6), working on the osmotic pressure of fish blood, found that
elasmobranchs differed fundamentally from teleosts in being isotonic with the seawater.
Table III.
A
Selachians
Teleosteans
Torpedo marmorata
Trygon violacea
Charax puntazzo
Serranus gigas
-2-26°
-2-44°
-!-O4°
-103°
Bottazzi observed that the selachian osmotic pressure would correspond to some
3-9 per cent. NaCl, but did not emphasise the fact that selachian blood did not
contain anything like so much ash. It was left for Rodier(a8) to show that the
difference was made up almost wholly by urea. DuvaldO has since found that the
salts alone would cover only an osmotic pressure of A — i-o6°. " High blood-urea,"
as Smith(32) says, "is a phyletic character of the orders Selachii and Batoidei," and
its osmotic function was well shown by the reciprocal relation between salts and
urea which Smith found to hold in all selachian tissues and fluids.
Table IV.
Blood-urea
mg. %
Smith (32)
Denis (9)
Selachians
Teleosteans
Dogfish, Mustelus cams
Dogfish, Mustelus cams
Sand-shark, Carcharias littoralis
Skate, Raw erinacea
Mackerel, Scomber scombrus
Goosefish, LopMus piscatorius
Flounder, Paralicthys dentalus
880
800
1000
868
86
40
46
Whence comes all this urea? In 1901 von Schroder (29) extirpated the livers of a
number of fishes {Scyllium canicula) and observed only a small reduction of the
12
JOSEPH NEEDHAM and DOROTHY MOYLE NEEDHAM
urea-content of the muscles, which was 1950 mg. per cent, before and i860 mg. per
cent, afterwards. The liver can therefore not be the main source, and possibly all fl
tissues have the power of forming urea from the amino-groups in the food.
Arginase appears to be found in great quantities in elasmobranchs, thus Hunter and
Dauphineefao) found the following typical figures:
Table V.
Arginase units
Liver
Selachian
Teleostean
Squalus sucklii
Sebastodes maliger
319
29
Kidney
3i
4
The arginase of the dogfish liver was twice as active as the most active teleostean
liver arginase (in the herring, Cliipea pallasii) and forty times as active as the feeblest
(in the tommy-cod, Hexagrammos stelleri). It is probable, however, that the contribution of urea made by arginase to the total urea-content of the elasmobranch cells,
would not be large. Hunter and Dauphinee made the interesting observation that
the undeveloped eggs of Squalus sucklii contained notable amounts of urea, but no
arginase, while both were present in an embryo of 20-5 cm.
It may be added that Baglionio, 2) found that the selachian heart could not beat
properly unless a certain amount of urea was added to the perfusion fluid.
The excretion of urea in selachians does not appear to take place wholly through
the kidneys. Denis (9) found that only 20 to 50 mg. were excreted through the
kidneys of an adult dogfish per kilo per day. The gills have been found by Duval and
Portierdz, 13) to be absolutely impermeable to urea. But van Slyke and White (31)
found that large amounts of urea were contained in the bile (up to 72^3 per cent, of
the total biliary nitrogen), so that the intestinal tract is probably concerned together
with the liver-cells in regulating the urea-content. The kidney does not seem to do
much regulation, as Denis do) found the blood-urea to be uninfluenced by experimental nephritis induced by uranium nitrate or potassium chromate. Another mode
of elimination of urea from the elasmobranch body may be through the peritoneal
pores, which Smith (32) believes to have an excretory function, for the peritoneal
fluid contains 680 mg. per cent.
In the light of all these facts it is not surprising that the dogfish egg should
contain urea before the development of the embryo has begun, nor that the embryo
should produce urea during its development. But before any closer interpretation
of our data can be given it is necessary to consider the question of whether the oviparous dogfish egg (Scyllium canicula) forms a closed system or not, for if it should
not, the urea formed by the embryo could easily escape into the water surrounding
the egg, and the ascending curve might be a measure of the rate of escape as well
as of the rate of formation.
At first sight there is every reason for supposing that the dogfish egg is
closed system. In the earlier stages of development it is completely filled b
yolk and the white, but about half-way through development, slits appear at the
Nitrogen-excretion in Selachian Ontogeny
13
corners of the egg-case, which widen, lose their plugs of jelly and finally gape
n, allowing sea-water to penetrate into the egg-interior. In the case of certain
rays these slits are believed to assist the respiration of the embryo, and Clark(7),
using suspensions of carmine granules, has shown that definite currents pass in and
out of the eggs through them. And we often found bubbles inside the larger eggs
after only slight handling.
We determined to test, therefore, to what extent the eggs of ScyUium retain
urea and ammonia. First of all we made a few experiments with the egg-cases themselves, cutting discs from them about 1-5 or 2 cm. in diameter and fitting them up
as diffusiometers, i.e. held with paraffin wax between two cork rings at one end of a
glass tube. Urea solution was placed above the membrane and water without urea
below, and the apparatus was left for some days, after which time the urea-content
of the lower solution was estimated, and the diffusiometer tested with neutral red
or some other dye to make sure that it had been properly sealed. The results were as
follows:
Exp. 1. 12 c.c. containing 3-12 mg. urea/c.c, i.e. in toto 37-44 mg. urea, were placed
in the upper part of the apparatus, with pure filtered sea-water below. After three days
1-5 mg. were found below the membrane.
Exp. 2. 62*4 mg. urea in sea-water placed above the membrane. After three days
1-3 mg. of urea were found-below the membrane.
Exp. 3. 62-4 mg. urea in sea-water placed above the membrane but with the membrane
reversed, so that the natural outside was on the upper side instead of on the lower. After
three days 0-05 mg. urea was found below.
These facts, which other similar trials confirmed, seem to show that the
horny egg-case membrane lets urea escape. The area through which active diffusion
took place was 1 cm. in diameter, and if about i\ mg. of urea could pass through it
in three days, a whole egg, having a surface of about 20 sq. cm. (Ford (16)) or 25-5
times the area of the piece of capsule taken, could lose about 38 mg. urea in three
days, i.e. enough to drain the egg completely in a short time. The horny egg-case
can therefore not now retain the urea of the egg-interior. More than one line of
argument, therefore, seemed to indicate that the dogfish egg is not a closed system
as regards urea.
We therefore determined to place ScyUium eggs at different stages of development in small vessels of sea-water and to estimate the urea-content of the surrounding
liquid from time to time.
It may be said here that these determinations necessitated a slight change in the
estimation-method. We found that when the phosphate buffer was added to seawater even if diluted, a precipitate was formed, probably of the calcium salt, and that
this precipitate carried down with it or in some way inactivated the urease. If,
however, the buffer was omitted, quite good results were obtained (94 per cent.,
91 per cent, on known amounts). We had also to go into the question of the removal
of urea from sea-water, in order to be sure that any urea lost by the eggs would
. intact for us to estimate. It is usually supposed that the many thousands of
5s of urea which must be added annually to the sea are removed by diatoms and
bacteria. Wishing to avoid the necessity of using a Berkefeld filter, we simply
14
JOSEPH NEEDHAM and D O R O T H Y M O Y L E NEEDHAM
filtered the outside sea-water so that it contained no diatoms or organisms
similar size, and then tested the capacity of urea to remain unchanged in it; 95
cent, was recoverable after ten days.
We then went on to compare the effect of the presence of Scyllium eggs.
Exp. 4. Four eggs were taken (in one the embryo was 3-5 cm., in another i-o cm. and
in the remaining two 2-5 cm.) and placed in 500 c.c. fresh filtered outside sea-water.
After three days the ammonia and urea were estimated. In the 500 c.c. there was 0-48 mg.
of ammonia and 0-18 mg. of urea, or 0-09 mg. per cent, of ammonia, and 0-03 mg. of urea.
In a similar lot of water which had not contained any eggs there was found 0-068 mg.
per cent, of ammonia, and no urea. The urea lost by the eggs was therefore minimal in
quantity, and probably not outside the experimental error. The eggs, moreover, contained
within them between 30 and 50 mg. of urea.
Exp. 5. Five eggs were taken (embryos of 3-6, 3*8, 3'9, 3-0 and 2-5 cm.) and placed in
500 c.c. of sea-water as before. After four days the control showed 0-04 mg. per cent.
ammonia1 and 0-04 mg. per cent, of urea, while the water surrounding the eggs showed
0-08 mg. per cent, of ammonia and 0-08 mg. per cent, of urea. This was especially remarkable as the five eggs between them contained well over 50-0 mg. of urea and the slits
in the egg-cases were all well open, so much so that in three out of the five large bubbles
were to be seen inside. We could only conclude that in spite of a free penetration of seawater into the egg-case, the system embryo-yolk was not giving up more than minimal
amounts of its urea to the exterior.
If then there was no parallel between the horny egg-case of the dogfish and the
strictly closed egg-shell of the chick, was it possible that the embryo and yolk
formed a closed system or something approaching it? Experiments showed that
this was so:
Table V I .
Mg. per fraction
Length of
embryo
in cm.
30
o-5
5'3
Ammonia
Yolk and embryo
White
Yolk and embryo
White
Yolk and embryo
White
039
0-025
0415
o-33
0-04
037
048
0-024
0-504
Ammonia +
urea
871
0-19
11-92
0-07
15-20
0-06
Urea
832
0-165
8485
n-59
0-03
11-62
1472
0-036
14756
Percen tages of the total
30
05
53
Yolk and embryo
White
Yolk and embryo
White
Yolk and embryo
White
9
o
89
98
2
99
11
95
5
1
99
1
It is thus evident that 99 per cent, of the urea and about 92 per cent, of
1
This agrees well with Gad-Andresen's (18) figure of 0-035 mf»- P er cent.
Nitrogen-excretion in Selachian Ontogeny
15
onia of the dogfish egg is retained within the yolk or the embryo and does not
^ ^ in the white or jelly between them and the shell. Unfortunately we did not
make any determinations on embryo and yolk separately, and we are therefore
unable to say whether the urea formed by the embryo during its development is
mainly retained within its body or is excreted into the yolk to be subsequently reabsorbed. An interesting consideration arises from the fact that if the main channel
of elimination of the urea in the adult dogfish is through the bile and perhaps through
the intestinal walls, the urea produced during development would tend to pass backwards into the yolk, for as Balfourfa) says, "the nutriment from the yolk-sac is
brought to the embryo partly through the umbilical canal and so into the intestine,
and partly by means of blood-vessels in the mesoblast of the sack." If then the
vitelline membrane and the blastoderm were impermeable to urea, as the gills are,
the state of affairs experimentally found by us would naturally arise. The cloaca do
not open in ScylMum until a comparatively late stage, and the oesophagus, which
opens for a short time early on, closes again and becomes for a long time a solid
cord of cells.
From the standpoint of general physiology the elasmobranch egg is of great
interest. It has elsewhere been suggested that the nature of the end-products of
protein metabolism arises from the requirements of the embryo (*s). In aquatic eggs,
such as the sea-urchin's or the trout's, the waste-nitrogen can be got rid of as
ammonia or urea, dispersing readily in the surrounding water, but in the terrestrial
egg, on the contrary, the non-diffusible insoluble uric acid has to be brought into
play, as is seen in the eggs of sauropsida and insects. The word " cleidoic " has been
introduced (z6) to designate those eggs which are shut off thus from their surroundings
as closed boxes (/eXetSow, shut up). It is evident that survival value attaches to the
closed-box egg, for the longer an embryo can continue its prenatal existence the
stronger it will be when it at length emerges, and for this end, much more protection
is required than for the minute egg whose development is quickly accomplished.
It is therefore very interesting that the only class of animals lower than the reptiles
which have evolved a structure approaching the cleidoic egg, are just those which
have found out a way to withstand a concentration of urea in their bodies which
would ordinarily be rapidly fatal. If the Scyllium embryo had had to arrange for an
efficient removal of its nitrogenous waste-products, it could perhaps not have
elaborated its egg-case. But the evolution of the egg-case must have taken place in
two stages; first the elasmobranchs, having discovered how to withstand severe
uraemia, were able to enclose their embryos completely, and secondly when it later
became convenient to open the box again, whether for the respiratory watercurrent or other purposes, the selachians, having become adapted to urea-retention,
continued to store it within themselves.
Our absolute values for urea are lower than those of all previous investigators,
no doubt because we used the quite specific urease method instead of extractions
with alcohol and the preparation of urea nitrate as was usually done before. The
c^hents of a Scyllium egg in the early stages weighs about 5 gm. wet and 2 gm.
d^^so that as there is some 10 mg. of urea present then, the egg contains 200
mg. per cent. This is a great deal smaller amount than the figures given by
16
JOSEPH NEEDHAM and DOROTHY MOYLE NEEDHAM
Krukenberg(2z). A Scyllium embryo after hatching, 11-15 c m - long, weighed 3-8^».
and would contain, extrapolating from our curve, about 22 mg. of urea N, or 4 4 ^ 5 .
urea, i.e. 1160 mg. per cent.—more in accord with the older figures. We also made
a few estimations on the eggs of the " spur-dog," Acanthias vulgaris, which are much
larger than those of Scyllium, with the following results:
Table VII.
Mg. per fraction
Length of
embryo
Undeveloped ...
3 cm-.
3-6 cm.
4-2 cm.
Average
Embryo and yolk
Perivitelline liquid
Whole egg-contents ...
Embryo alone (two specimens)
Average
Embryo and yolk
Perivitelline liquid
Whole egg-contents ...
Ammonia N
Urea AT
4-06
192
248
282
420
0-75
4-95
6-70
10-40
8-55
236
1-40
11884
780
136-1
110-9
1298
3795
16775
193-97
20876
201-36
114-04
27-90
141-94
376
There did not seem to be any great increase in the urea of the eggs, much more
being provided by the maternal organism than in Scyllium. As the undeveloped
egg weighs 25-2 gm. approximately, it will contain 888 mg. per cent, of urea, an
interesting value because, as we have already seen, the blood-urea in closely-related
forms has been found by more than one observer to be about 880 or 900 mg. per
cent. The Acanthias egg then has exactly the same percentage of urea as the blood,
unlike that of Scyllium where it is much lower. It is possible that the reason why
the Acanthias egg does not show a steadily rising urea-content during its development is that being ovoviviparous, and held within the maternal uterus, it forms part
of the maternal system, and having all the urea it requires provided at the start,
depends much less than the Scyllium egg on the activity of the embryo itself.
But perhaps the most interesting result which emerges from the analyses of
Acanthias eggs is that just as in Scyllium the major part of the urea is held in the
embryo and yolk.
Table VIII.
Percentage of total N
Length of
embryo
3-0 cm.
4-2 cm.
Yolk and embryo
White
Yolk and embryo
White
Ammonia
Urea
85
77
23
81
19
15
63
37
The partition in Acanthias, however, is not so striking as it is in Scyllium.
Nitrogen-excretion in Selachian Ontogeny
17
CONCLUSIONS.
1. A study has been made of the urea and ammonia production of selachian
embryos, especially in Scyllium canicula.
2. The observation of earlier workers, that the undeveloped dogfish egg contains notable amounts of urea, has been confirmed.
3. During development, the urea-content of the egg increases considerably, the
embryo adding to the original quota supplied by the maternal organism. In the egg
of Scyllium the urea-content is thus increased four or five times.
4. The egg-case of the Scyllium egg does not form a closed system to urea for
its wall is permeable to this substance, and at any rate in the later stages there is a
free penetration of sea-water into the egg-case through the four slits. Nevertheless,
only minimal amounts of urea are found in water surrounding the developing eggs,
and it is probable that the walls of the yolk-sac are impermeable to urea. For
98 per cent, of the urea of the egg is found in the embryo and yolk, so that an
excretion must take place not into the white but into the yolk, as, indeed, might be
expected if the main path of exit of the urea in the adult is through the intestinal
tract.
5. Evidence is adduced which makes it likely that a peak of protein catabolism
exists in the embryonic life of the dogfish, just as in that of the chick and the frog,
and probably of mammals.
6. It is pointed out that the only fishes which have evolved an egg of the cleidoic
type (though not now actually cleidoic) are just those which have evolved the power
of withstanding severe uraemia. This is in accordance with the view that the main
end-product of nitrogen metabolism is causally connected with the manner of life
of the embryo of the form in question.
Our thanks are due to Mr Richard Elmhirst and the staff of the Millport Marine
Biological Station for their cordial help and co-operation during our stay. We also
wish to acknowledge assistance received from the Government Grant Committee
of the Royal Society and from the Thruston Fund of Gonville and Caius College,
and to thank the authorities responsible in each case. During the course of the
work, one of us (D.M.N.) held a Beit Memorial Research Fellowship.
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