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CORNELL UNIVERSITY.
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THE DECENNIAL PUBLICATIONS OF
THE UNIVERSITY OF CHICAGO
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THE DECENNIAL PUBLICATIONS
ISSUED IN COMMEMORATION OF THE COMPLETION OF THE FIRST TEN
YEARS OF THE UNIVERSITY’S EXISTENCE
AUTHORIZED BY THE BOARD OF TRUSTEES ON THE RECOMMENDATION
OF THE PRESIDENT AND SENATE
EDITED BY A COMMITTEE APPOINTED BY THE SENATE
EDWARD CAPPS
STARR WILLARD CUTTING ROLLIN D. SALISBURY
JAMES ROWLAND ANGELL WILLIAM I. THOMAS SHAILER MATHEWS
CARL DARLING BUCK FREDERIC IVES CARPENTER OSKAR BOLZA
JULIUS STIEGLITZ JACQUES LOEB
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THESE VOLUMES ARE DEDICATED
TO THE MEN AND WOMEN
OF OUR TIME AND COUNTRY WHO BY WISE AND GENEROUS GIVING
HAVE ENCOURAGED THE SEARCH AFTER TRUTH
IN ALL DEPARTMENTS OF KNOWLEDGE
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STUDIES IN GENERAL PHYSIOLOGY
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STUDIES IN GENERAL
PHYSIOLOGY
BY
JACQUES LOEB
FORMERLY OF THE DEPARTMENT OF PHYSIOLOGY
NOW PROFESSOR OF PHYSIOLOGY IN THE
UNIVERSITY OF CALIFORNIA
THE DECENNIAL PUBLICATIONS
SECOND SERIES VOLUME XV
PART II
CHICAGO
THE UNIVERSITY OF CHICAGO PRESS
1905
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No 26 4a):
j
Copyright 19s
BY THE UNIVERSITY OF CHICAGO
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XI.
XII.
XIII.
XIV.
XV.
TABLE OF CONTENTS
PART I
The Heliotropism of Animals and its Identity with
the Heliotropism of Plants
Further Investigations on the Heliotropism of Ani-
mals and its Identity with the Heliotropism of
Plants
On Instinct and Will in Animals
Heteromorphosis
Geotropism in Animals
Oreanizalion and Growth -
Experiments on Cleavage -
The Artificial Transformation of Positively Helio-
tropic Animals into Negatively Heliotropic and
vice versa
On the Development of Fish Embryos with Sup-
pressed Circulation
On a Simple Method of Producing from One Egg
Two or More Embryos Which Are Grown
Together
On the Relative Sensitiveness of Fish Embryos in
Various Stages of Development to Lack of Oxygen
and Loss of Water
On the Limits of Divisibility of Living Matter
Remarks on Regeneration
Contributions to the Brain Physiology of Worms
The Physiological Effects of Lack of Oxygen
1X
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265
295
303
809
821
338
345
370
TABLE OF CONTENTS
x
PART II
XVI. The Influence of Light on the Development of
Organs in Animals 425
XVII. Has the Central Nervous System Any Influence
upon the Metamorphosis of Larvae? 436
XVIII. On the Theory of Galvanotropism 440
XIX. The Physiological Effects of Ions. I 450
XX. Onthe Physiological Effects of Electrical Waves 482
XXIJI. The Physiological Problems of Today 497
XXII. The Physiological Effects of Ions. II 501
XXIII. Why Is Regeneration of Protoplasmic Fragments
without a Nucleus Difficult or Impossible ? 505
XXIV. On the Similarity between the Absorption of Water
by Muscles and by Soaps 510
XXV. On Ions Which Are Capable of Calling Forth
Rhythmical Contractions in Skeletal Muscle 518
XXVI. On the Nature of the Process of Fertilization and
the Artificial Production of Normal Larvee
(Plutei) from the Unfertilized Eggs of the Sea-
Urchin 539
XXVII. On Jon-Proteid Compounds and Their Réle in the
Mechanics of Life-Phenomena.—The Poison-
ous Character of a Pure NaCl Solution 544
XXVIII. On the Different Effects of Ions upon Myogenic
and Neurogenic Rhythmical Contractions and
upon Embryonic and Muscular Tissue 559
XXIX. On the Artificial Production of Normal Larve from
the Unfertilized Eggs of the Sea-Urchin
(Arbacia) 576
XXX. On Artificial Parthenogenesis in Sea-Urchins 624
XXXI. Onthe Transformation and Regeneration of Organs 627
XXXII. Further Experiments on Artificial Parthenogenesis
and the Nature of the Process of Fertilization - 638
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TABLE OF CONTENTS
XXXIII. Experiments on Artificial Parthenogenesis in
Annelids (Chzetopterus) and the Nature of the
Process of Fertilization
XXXIV. On an Apparently New Form of Muscular Irrita-
XXXY.
XXXVI.
XXXVII.
XXXVIII.
INDEX
bility (Contact-Irritability?) Produced by Solu-
tions of Salts (Preferably Sodium Salts) Whose
Anions Are Liable to Form Insoluble Calcium
Compounds
The Toxic and the Antitoxic Effects of Ions as a
Function of Their Valency and Possibly Their
Electrical Charge
Maturation, Natural Death, and the Prolongation
of the Life of Unfertilized Starfish Eggs
(Asterias Forbesii) and Their Significance for
the Theory of Fertilization
On the Production and Suppression of Muscular
Twitchings and Hypersensitiveness of the Skin
by Electrolytes
On the Methods and Sources of Error in the
Experiments on Artificial Parthenogenesis
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646
692
708
728
748
766
773
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PART II
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XVI
THE INFLUENCE OF LIGHT ON THE DEVELOPMENT
OF ORGANS IN ANIMALS!
I. EARLIER EXPERIMENTS
Two GREAT series of experiments which Nature herself
has made are at our disposal for answering the question as
to what effect light has on the development of animals;
namely, the intra-uterine development, and the development
of animals living in caves. The fact that intra-uterine
development goes on in complete darkness proves that the
formation of the embryo and its organs, histological differ-
entiation, and considerable growth can occur and continue
for a long time in the absence of light. As far as animals
living in caves are concerned, some of them differ from the
same forms which live in the light in the development of
single organs, such as eyes, antenne, and pigment. It has
not, however, as yet been proved that this peculiarity of the
cave inhabitants is a direct effect of the lack of light upon
their development; but granting that it is the direct result
of lack of light, it follows from a summation of the facts in
hand that where light has any direct effect whatsoever on
development, it evidently makes itself felt only upon the
development of individual organs and not upon the develop-
ment in general.
It is strange that, notwithstanding the definiteness of
these facts, experimental work on the influence of light on
the development of organs in animals has been directed
mostly to the question whether light promotes or inhibits
development and growth of animals in general.
1 Pfliigers Archiv, Vol. LXIITI (1895), p. 273.
425
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426 STUDIES IN GENERAL PHYSIOLOGY
It is not to be wondered at that the various investigators
arrived at diametrically opposite results.
Edwards states that frogs’ eggs do not develop and soon
die when inclosed in a dark box, while the development
takes place in an open box which is exposed to the light.
According to Edwards, even the young larve require a longer
time to develop in the dark than in the light. Edwards’s
statements are based on very few experiments. Dutrochet
repeated the experiments of Edwards, and found that
where the supply of oxygen was sufficient and the tempera-
ture was the same in the two boxes, the eggs of Batrachians
developed as well and as rapidly in the dark as in the
light. These facts indicate that in Edwards’s experiments
the eggs suffered from lack of oxygen and exposure to a low
temperature.
Béclard published a short communication on the influence
of light on the development of the eggs and the larve of
flies. He placed the eggs under colored bell-jars, and found
that after four or five days development was most advanced
under the violet and blue jars, and least under the green. I
cannot understand the experiments of Béclard, as fly larve
hatch in about two days in summer, and up to this time their
size depends on the size of the eggs, since further growth
takes place only when the larve find food in which to bury
themselves. The statements of Béclard regarding the influ-
ence of light on the production of carbon dioxide in animals
are also doubtful.
It is rather strange, though characteristic, that in the
scientific literature of our subject one frequently finds serious
mention of the investigations of General Pleasanton, made
on six pigs. The general put three pigs into a stall with
violet windows, and three into a stall with ordinary windows.
While the three pigs exposed to violet light gained 398
pounds in four months, the others gained during the same
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THE INFLUENCE oF LIGHT ON ORGANS 427
time 386 pounds in weight. From this observation Pleasan-
ton concluded that violet light is favorable to the growth of
pigs. General Pleasanton’s book is printed in blue type, and
gives an explanation of all natural phenomena, from love
down to the activity of a volcano.
Emil Young made a series of experiments on the influ-
ence of colored light on development. He concluded that
violet light hastens to a certain degree the development of
frogs’ eggs, and the growth of embryos, while green light is
fatal to or greatly retards development. Young’s results are
incomprehensible. The larve of frogs develop naturally in
daylight, and the latter contains more green light than light
which has passed through a green screen, and should accord-
ing to Young be fatal. We might, perhaps, assume that
some other light counteracts the effect of the green light
sufficiently to do away with this fatal effect. From Young’s
experiments, however, this does not seem to be the case. It
is quite possible that other conditions (such as the develop-
ment of micro-organisms) affected the results of Young’s
experiments, which lasted through several weeks.
Driesch used monochromatic light and carried out the
same experiments as Young on freshly fertilized eggs of
Rana, Echinus, and Planorbis, and found, in all cases, that
light “has no influence on the segmentation or the forma-
tion of organs; under otherwise similar conditions these
phenomena occur with the same velocity in darkness, in
white, in green, in violet, or in other lights.”
In my experiments on heteromorphosis in Naples, I
observed that the polyps of Eudendrium racemosum are
positively heliotropic, and that the number of polyps which
develop is apparently dependent on the intensity of the light.
It seemed that fewer polyps were developed in weak light
than in strong light. This accidental observation led me to
study the influence of light on the development of organs
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428 STUDIES IN GENERAL PHYSIOLOGY
iu Eudendrium racemosum more closely in Woods Hole.
The results of these observations are briefly reported in the
following pages.
II. NEW EXPERIMENTS
1. The species of Eudendrium studied in Woods Hole
has the same name as that in Naples—namely, Eudendrium
racemosum; it is, however, not certain that the two forms
are identical. The following statements hold for the form
in Woods Hole. When fresh stems of Eudendrium are put
into an aquarium, all the polyps soon fall off, probably due
to unavoidable injury in collecting and handling the mate-
rial. In the course of a few days, however, with a good
supply of oxygen and a sufficiently high temperature, new
polyps are developed. It was the dependence of this new
development on light which was studied.
A large quantity of vigorous colonies was collected each
time. Long stems were picked and put in separate vessels,
ten being distributed into each vessel, all of which contained
an equal quantity of sea-water. Each of the stems usually
formed from ten to twenty polyps. The different vessels
were exposed to various kinds of light. In each experiment
I therefore dealt, not with the development of a single
polyp, but with a large number of them. I thought it
necessary, furthermore, to make another set of control ex-
periments by exposing the same stems successively to differ-
ent kinds of light.
Experiment 1.—On August 8 a number of stems of the
same culture of Eudendrium was divided as equally as pos-
sible between two vessels, in the manner described above.
One of the vessels was exposed to diffuse daylight; the other
was placed in a dark box which was ventilated every even-
ing. The supply of oxygen was the same in the light as in
the dark, and the temperature was always the same in the
two vessels.
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THE INFLUENCE OF LIGHT ON ORGANS 429
On August 14 over fifty polyps had developed in the
vessel which was exposed to light, while in the vessel kept
in the dark not a single polyp had been formed. The
experiment was continued until September 1; the polyps
thrived and increased in number in the light, while in the
dark not a single polyp had yet been formed. The stems
which up to this time had been in the dark were now
exposed to the light. On September 6—that is to say, in
five days—several polyps developed on each stem. The
number of polyps increased from day to day. The same
stems, which in three weeks had been unable to form a
single polyp in the dark, developed a great number of them
in five days when exposed to the light. The control ani-
mals in the light had developed polyps from the first.
Experiment 2.—On August 16 the stems of a new colony
were divided equally among three vessels, two of which were
placed in the dark and one in the light. As usual, numerous
polyps were formed in the light in the course of five days;
no polyps were formed at first in the dark. This experi-
ment was also continued until September 1. By that time
no polyps had yet developed on the stems kept in one of the
darkened vessels; two stems in the other darkened vessel had
developed six polyps. The animals were then exposed to
light; in five days all the stems had produced new polyps.
Experiment 3.—On August 25 one half of a Eudendrium
colony was placed in diffuse light, while the other half was
put in the dark. On September 1 a large number of polyps
had been developed in the light; but in the dark only roots
and no polyps had been developed. Conditions remained
the same until September 5, when the animals which had
been kept in the dark were exposed to the light. On the
following day they were accidentally killed.
These experiments show that light favors the develop-
ment of polyps in Eudendrium; that no polyps, or only very
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430 STUDIES IN GENERAL PHYSIOLOGY
few, are developed in the dark; that darkness does not, how-
ever, interfere with the development of roots.
2. It was of interest now to establish which rays of the
visible spectrum favor the development of polyps. The
effect of light on plants is known to be very strikingly a
function of the wave-length. Assimilation and in part the
formation of chlorophyll are pre-eminently functions of
the long wave-lengths. The heliotropic phenomena are
essentially a function of the blue rays. According to Sachs,
the ultra-violet rays are of special importance in the forma-
tion of blossoms in certain plants. I have shown that the
short light-waves are most effective heliotropically in animals
also. From this, however, no conclusions can be drawn as
to which rays influence most especially the formation of
polyps. I therefore studied this subject experimentally.
Unfortunately, these experiments were hampered because
no other means of obtaining monochromatic light were at
my disposal than the use of blue and red glass. I had
special boxes made for these experiments which were painted
black inside and one wall of which was formed by blue or
red glass. The dark-red glasses which I employed yielded a
light which was fairly monochromatic; the dark-blue glasses
allowed some red to pass through. In the case of the light-
red and light-blue glasses the light was far from monochro-
matic.
Experiment 1.—On August 31 a large number of Euden-
drium were divided between two vessels, one of which was
placed in a box in dark-red light, the other in a box in dark-
blue light. As nearly as I could judge, the red and blue
glasses allowed about equal quantities of light to pass
through. The old polyps perished within three days, but
somewhat sooner in the red than in the blue light. On
September 4 I discovered the first new polyp in the blue
light. The number of new polyps in the blue light steadily
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THe INFLUENCE OF LIGHT ON ORGANS 431
increased, but not a single one was formed in the red light.
On September 8, seventy new polyps with stems 3-10 mm.
long had developed in the blue light. Nota single polyp
had developed in the red light, but a few roots had been
formed. Conditions did not change during the following
days. After nine days not a single polyp had developed in
the red light, while over seventy thriving polyps had been
‘formed in the blue light.
In order to test whether the stems in the red light would
develop polyps if brought into the blue light, I substituted
a blue glass for the red glass on September 9. Two days
later, on September 11, indications of new polyps were
already apparent, and on the following day thirty-two com-
plete polyps had been formed; on the next day the number
had increased to sixty-six. From these experiments it is
seen that in the development of polyps red light acts more
like darkness, while blue light acts like mixed daylight, just
as in heliotropic phenomena.
Experiment 2.—On August 22 a large number of stems
of Eudendrium were equally divided, as in the preceding
experiments, between two vessels, one of which was placed
in blue, the other in red light. After the old polyps had
fallen off, the first new polyps appeared in the blue light on
August 27. At about the same time roots began to develop
in the red light, but no polyps. On August 29 forty vigor-
ously growing polyps had developed in the blue light, while
in the red light no polyps but only roots had developed. On
August 31 the culture in the blue light had formed a forest
of new, well-developed polyps, while the culture in the red
light had only developed several roots. The culture in the
blue light had also developed a few roots. On August 311
replaced the red glass by blue. On September 11 the first
new polyps began to form, whose number from now on
steadily increased.
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432 STUDIES IN GENERAL PHYSIOLOGY
This time I also made the reverse experiment. The
polyps formed in blue light were exposed to red light (the
blue glass was replaced by a red one on September 6). After
five days all the newly developed polyps had perished. By
September 13 only a few diminutive polyps had developed.
Experiment 3.—On August 25 eight stems of Euden-
drium culture were placed behind light-red glass (which
allowed also some blue light to pass through), and nine
stems of the same culture behind blue glass which was not
very dark. On August 30 a number of polyps had devel-
oped, not only behind the light-blue, but also behind the
light-red glass. The light-red screen was then replaced by
a dark-red one; while thé number of polyps constantly
increased in the blue light, development soon ceased behind
the dark-red screen. On September 1 the eight stems in the
red light had only sixteen small polyps, while the nine in the
blue light, which were of the same size and from the same
culture, had eighty polyps. On the following day eighteen
polyps had developed in the red light, while the stems in the
blue light were literally covered with them.
On September 5 the glasses were exchanged; the animals
which up to this time had been behind the red screen were
now exposed to blue light, while those which had been
exposed to blue light were placed behind the dark-red screen.
The number of polyps on the stems in the blue light (which
had formerly been in the red) increased rapidly; on Septem-
ber 9 the number had grown to 27, on the 10th to 40, and
soon. The polyps on the animals in the red light (which
had formerly been in the blue) not only did not increase in
number, but they began to die, and on the 11th only a few
were left which looked sickly.
A fourth experiment corroborated the result that numerous
and vigorous polyps are formed behind a dark-blue screen,
while in red light only roots are formed.
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THe INFLUENCE oF LIGHT ON ORGANS 433
We must therefore conclude that not all the rays of diffuse
daylight influence the formation of polyps equally, but that
only the more refrangible (blue) rays of the visible spectrum
favor the development of polyps, while the less refrangible
(red) rays act as darkness—a fact similar to that established
for heliotropism.
3. We may finally raise the question whether or not light
influences the development of Planula larvee. My studiesin
this direction are not yet completed, but I found that in
isolated cases, in spite of an exposure to darkness for several
weeks, during which time no new polyps were formed, the
development of the larvee progressed as under normal condi-
tions; at least they were normal and not retarded in their
development. I was able to make some experiments with
the larvee themselves. These larve are pear-shaped, and are
able to move forward very slowly by means of cilia. They
are, as I have already shown, energetically positively helio-
tropic. The blue rays are in these experiments more effective
than the red. About forty-eight hours after they begin to
swim, the larvee attach themselves to some solid base, and
within the next twelve hours their pointed ends begin to
grow and to form polyps, while their blunt ends form roots.
When lighted from one side only the whole organism bends
toward the light. The question therefore arose whether the
Planula larva can develop a polyp in the dark. This is the
case when a larva has developed in the presence of light.
When brought into the dark such a larva develops a polyp
within twelve to twenty-four hours. In this, however, we need
not see any contradiction to the other experiments, as in
these the development of the polyps required three to four
days. It would be interesting to determine whether Planula
larvee, if their whole development occurs in the dark, can
develop polyps in the absence of light.
Tn conclusion I wish to mention that the polyp-bearing
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434 STUDIES IN GENERAL PHYSIOLOGY
and growing stems are energetically positively heliotropic.
Only that part of the stem immediately behind the polyp
bends heliotropically. While these curvatures are usually
produced in less than two hours in blue light, no curvature
takes place in dark-red light even after two days; nor do
the heliotropic curvatures appear when the polyps are cut
off. Ishall return later to this and some other facts bearing
on the theory of heliotropism. A stem of Eudendrium which
is illuminated from one side only develops more polyps on the
lighted side than on the shaded side—a thing which explains
itself from the foregoing.
Ill. EXPERIMENTS ON FUNDULUS EMBRYOS
A large number of experiments on Fundulus embryos
show that they develop as completely and as quickly in the
dark as in the light; only the supply of oxygen must be the
same in both cases. In one experiment the eggs were kept
in the dark in a small, tightly closed vessel; those exposed to
the light were kept in a large vessel; in this case the eggs
developed more quickly in the light than in the dark. Ccn-
trol experiments showed very clearly that it was not the
light, but the better supply of oxygen to the vessel exposed
to the light, which caused this difference in the development
of the eggs. Only one constant difference exists between
the eggs cultivated in the light and in the dark, and this
concerns their color. As I have stated repeatedly, a large
number of black and red chromatophores are formed in the
membrane of the yolk-sac, which gradually creep upon the
blood-vessels and surround them like a sheath. Since the
number of these chromatophores progressively increases, the
egg, if developed in the light, finally becomes very dark. In
contrast to this, the eggs kept in the dark are very light and
transparent. This difference may possibly be due to a con-
traction of the chromatophores in the dark, but I am not cer-
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THe INFLUENCE oF LIGHT ON ORGANS 435
tain of this. The other possibility is that in the dark a
smaller number of pigment cells are formed. In embryos
which develop in the light toward the end of development
the pigment cells form a sheath around the blood-vessels.
When the eggs develop in the dark, only isolated chromato-
phores are found upon the blood-vessels; the vessels are for
the most part free from pigment.
I found no noticeable difference in the development of
pigment in the embryo itself. The pigment cells of the
retina, for example, developed apparently as numerously and
contained the same amount of coloring matter in the dark as
in the light. The yolk-sac alone showed the influence of
the light.
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XVII
HAS THE CENTRAL NERVOUS SYSTEM ANY INFLU-
ENCE UPON THE METAMORPHOSIS OF LARVA:??
Gustav TornieR has just published a hypothesis which
is to explain how the acquired characteristics of parents are -
inherited by their offspring. This hypothesis is as follows:
In the more highly organized animals every adaptation of a
functioning peripheral end-organ is accompanied by a corresponding
and equal adaptation in the central nervous system; the central ner-
vous system carries the acquired characteristic to the sexual organ,
which forms with it a functional and nutritive unit, especially to
the sexual cells, in that it compels the latter to undergo similar
transformations. If the sexual cells give rise to new individuals,
the descendants inherit the acquired characteristics of the parents.’
Tornier’s paper is very clear, and even though I cannot
agree with his hypothesis, I consider it important that
Tornier through his precise presentation of his subject has
directed the attention of investigators to the question of the
significance of the central nervous system in the processes
of development.
If Tornier’s idea is correct, then every alteration in the
central nervous system must be accompanied by a similar
change in the end-organs. Before the appearance of Tornier’s
paper I had already made a series of experiments in which
I divided the spinal chord of Amblystoma larvae in order to
determine whether in the change of the larvee to the sexually
mature form the animals with the divided spinal cord would
behave as one or two separate animals; in other words,
whether in an animal with a divided spinal cord the meta-
morphosis of the interior and the posterior portions would
occur simultaneously as in the case of the uninjured animal.
1 Archiv fiir Entwickelungsmechanik der Organismen, Vol. IV (1896), p. 502.
2" Uber Hyperdaktylie,” etc., ibid., Vol. III, p. 180.
436
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THE METAMORPHOSIS OF LARVA 437
Amblystoma is well adapted to an experiment of this kind.
The larva loses an organ at each end in the process of
metamorphosis—the three large external gills at the head
end of the animal, as well as the so-called tail fin at the
tailend. Both disappear simultaneously in normal animals
in a few days; at the same time an alteration occurs in the
pigmentation and marking of the skin. One could not state
a priori how a division of the spinal cord would influence
the processes of development, for it is well known that
immediately after the division of the spinal cord in dogs
severe changes usually occur in the condition of the skin of
the posterior portions of the body, which may lead to the
formation of abscesses, which later again disappear (Goltz).
Division of the spinal cord (close behind the cervical
region) did not have even the slightest effect in a single
instance upon the processes of development; metamorphosis
occurred just as though the animal were uninjured. This
was the more remarkable as in some cases a metamorphosis
occurred immediately after the division of the spinal cord,
while the wound was still open. I believed at first, on the
basis of these experiments, that division of the spinal cord
might accelerate the metamorphosis of the larvee, or cause it
directly. I found, however, that this was not the case. One
animal, for example, was still in the larval stage six weeks
after the operation, while the uninjured control animals had
already completed metamorphosis.
Since the experiments showed without exception that
division of the spinal cord had no effect upon the meta-
morphic processes, we must state in detail what evidence we
have to show that the cord was indeed severed entirely.
First of all the operation was made in such a way that the
separation of the two ends of the spinal cord could be seen
directly in the bottom of the wound. Secondly, the pos-
terior part of the body was paralyzed, that is to say, it was
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438 STUDIES IN GENERAL PHYSIOLOGY
dragged along in the movements of the anterior portion of
the animal as though it was an inanimate mass. 5 | ‘
55 Z: 2.04 2:06
3 | C Sp ¢
2:07 2:09 ac 2:18
one hour and forty minutes. Only the eggs of the last lot
that had been in the solution one hour and forty minutes
showed the beginning of a development. I believe that I
took out the eggs too soon. In some cases such eggs are
able to develop, but in others they are not, and I think it
probable that if the eggs had been left a little longer in so-
lution 1 or Z they would have developed further. I made
some camera drawings of the way in which the eggs were
segmented (Fig. 148). The successive stages of the segmen-
tation of one and the same egg up to the six-cell stage
were drawn. The reader will see from the drawings that the
egg went within twenty minutes from practically an undivided
FIG. 148
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egg into a six-cell stage. It is obvious that these cell-divi-
sions are accompanied by most striking amceboid motions,
which are characteristic of all the eggs without a membrane.
I believe that these amceboid motions exist in the fertilized
eggs just as well, but the membrane prevents them from
becoming so conspicuous as in the unfertilized eggs where
there is no membrane. In the normal eggs these amceboid
motions are more symmetrical, and this is another reason
why they escape our observation. When I made my first
experiments on the effect of more concentrated sea-water
upon the segmentation of fertilized eggs, the idea struck me
that the segmentation by budding (Knospenfurchung) was
the outcome of amceboid motions, and I soon afterward ex-
pressed the idea that the same is true for the process of cell-
division in general.’ The two nuclei of the mother cell are
the centers around which the protoplasm streams and flows.
These amceboid motions are only one episode in the process
of cell-division, for whose full explanation other phenomena
of an entirely different character must be taken into con-
sideration.
Sixth series—The preceding experiment was repeated,
but this time with due consideration of the fact that the
eggs must remain long enough (two hours) in the artificial
solution. The eggs of two females were distributed in three
solutions:
(1) 60 cc. 422 MgCl, + 40 c.c. sea-water
(2) 50 ce. + 50 ce.
(3) Normal sea-water
None of the eggs formed a membrane. Some of those
that had remained in normal sea-water segmented after
twenty hours. They divided into from 2 to 3 cells and not
further. I have already mentioned the fact that the unfer-
tilized eggs of various females differ somewhat in their
1LoEB, Archiv fiir Entwickelungsmechanik, Vol. I (1895), p. 453.
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610 STUDIES IN GENERAL PHYSIOLOGY
tendency to segment in normal sea-water. It may be pos-
sible that these variations enhance or diminish the effects of
artificial solutions upon the development of unfertilized eggs.
The eggs that had been for two hours in solution 1 had the
FIG. 149
next day developed into the characteristic blastules some of
which are represented in Fig. 149. Some of these blastule
originated possibly from the whole mass of one egg, for
instance 1, 3, and 4. But even here the irregular outline
betrays clearly that the blastule originated from eggs with-
out a membrane. As I said in an earlier experiment, the
outlines of the eggs became irregular through the amoeboid
motions of the blastomeres, and in the blastule the outline
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ARTIFICIAL PRopUCTION OF NoRMAL Larve 611
of the irregular morula stage is preserved. This is intel-
ligible if we remember that the blastula originates through
the cleavage cells moving or sticking to the periphery of the
egg. The other blastule represented only smaller pieces of
asingle egg. In some cases one part of the egg disintegrated
and formed débris at- . i.
tached to the other part ~
which reached the blas-
tula stage (5, 6, and 7).
Each one of these blas-
tule was moving and
had to be immobilized
tomakethe cameradraw-
ing. It is impossible to
give a fair idea of the
variety of forms of blas-
tule one sees in such ex-
periments. No egg of
this lot (solution 1)
reached the pluteus
stage. All died the
second day. The eggs
that had been in solu-
tion 2 (equal parts of FIG. 150
22n MgCl, and sea-water) looked very different from the
preceding lot (Fig. 150). After twenty-four hours many
of them had developed into blastule. These blastule left
no doubt that they came from eggs without a membrane, in-
asmuch as in the majority of cases several blastule originated
from one egg. Quadruplets were especially frequent (Fig
150, Iv and v), but twins and triplets were also quite com-
mon. I watched their development, and am thus quite cer-
tain that these multiple embryos sticking together came from
one egg. The feature that distinguished these embryos
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612 STUDIES IN GENERAL PHYSIOLOGY
from those that had been treated with the stronger MgCl,
solution, however, was the fact that the former all had regular
and sharp outlines and were free from débris. The outlines
of the blastule were much more spherical. These blastule
had greater vitality than the others and kept alive during the
next thirty-six hours.
The next morning a
number of them had
(co) reached the pluteus
LS =‘ stage with a perfectly
normal skeleton and
intestine, but they
died the following day
f) (Fig. 151). They had
YS? lived more than two
Oh days. Their develop-
ment was slower than
in the case of fertil-
ized eggs.
All these blastulee
and plutei swam about on the bottom of the dish, not rising
to the surface like the larvee from fertilized eggs.
The control eggs that had been left in the normal sea-
water remained unsegmented, with the exception of a few
which on the second day were found divided into 2 or 3 cells.
The latter, of course, segmented no further. None of these
eggs had a membrane.
Seventh series.—The preceding series had shown that a
mixture of equal parts of 2,on MgCl, and sea-water is more
favorable for the development of the eggs than a mixture
with more MgCl, and less sea-water, for instance 60 e.c.
MgCl, and 40 c.c. sea-water. In the latter mixture the eggs
seemed to suffer more. It must, however, be stated that as
far as the comparative number of eggs is concerned that
FIG, 151
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ARTIFICIAL PRODUCTION OF NorMAL Larva 613
undergo development, the solution with 60 ¢c.c. MgCl, and
40 c.c. sea-water is equally good or even better than the
mixture of equal parts of both. I now tried whether a
mixture with less MgCl, would still be favorable. A mix-
ture of 40 c.c. %2n MgCl, +60 c.c. sea-water was found
ineffective. The eggs remained two hours in this solution,
and a few of them segmented afterwards, but as the number
was comparatively small I did not follow up this experiment.
It is possible that a mixture of 40 c.c. %,9n MgCl, + 60 c.c.
sea-water is too weak to bring about artificial partheno-
genesis of the egg of Arbacia. In one of the preceding
experiments we found that by treating the eggs with a
mixture of 30 c.c. 2,°n MgCl, +60 c.c. sea-water we were
not able to bring about parthenogenesis.
Eighth series.—It was evident that in order to produce
plutei from the unfertilized egg of Arbacia we must confine
ourselves to solutions which contain less than 60 and more
than 40 per cent. of 2.2n MgCl,. In the next experiments
the following four solutions were tried:
(1) 55 cc. 4°” MgCl + 45 e.c. sea-water
(2) 50 = + 50 a
(3) 45 «“ + 55 «“
(4) Normal sea-water
. At three different intervals (two hours, two hours and
ten minutes, two hours and twenty minutes) portions of the
eggs were taken out of these four solutions and put back
into normal sea-water. Two hours later in each of the lots
that had been in the first three solutions about 50 per cent.
of the eggs were segmented into from 2 to 16 cells. None
of them had a membrane. No egg in solution 4 (normal
sea-water) was segmented or had a membrane. The next
morning the eggs that had been in solution 1 were teeming
with blastule. Many of them resembled the blastule of
Fig. 149, but the majority were clean and free from débris.
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614 STuDIES IN GENERAL PHYSIOLOGY
The eggs that had been in solution 2 had a large number of
blastule and gastrule. They were free from débris and
looked very much like those drawn in Fig. 150. The eggs
taken from solution 3 had very few blastule. The latter,
however, were perfect, except that the single egg as a rule
produced more than one embryo. The majority of the eggs
were still in the morula stage. The next morning, forty-
eight hours after the treatment with the MgCl, solution,
each one of the three dishes contained perfect plutei.
Many eggs of solution 3 which the previous day were still
in the morula stage had in the mean time developed into
blastule or plutei. This time the plutei were still alive on
the following day (seventy-two hours after the treatment
with the artificial solution). Their vitality was not much
less than that of the normal plutei which often died just as
early. I mentioned that I had put back the eggs from the
MgCl, solutions into normal sea-water at three different
intervals. Those taken out last gave the best results. It is
very obvious that the unfertilized eggs develop much more
slowly than the fertilized eggs. The latter reach the pluteus
stage at the proper temperature within twenty-four hours or
little more, while the unfertilized eggs reach the pluteus
stage after forty-eight hours at the same temperature. I
had the same experience in all my experiments with unfer-
tilized eggs. The eggs that had been left in normal sea-
water remained undeveloped and not one egg had a mem-
brane. One egg in a hundred was segmented after twenty-
four hours in 2 or 3 cells, but none developed further.
Ninth series.—This time I intended once more to repeat
my experiments ard at the same time make control experi-
ments of an altogether different character. I will first speak
of the repetition of the old experiments. The unfertilized
eggs of one female were put into the following two solu-
tions:
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(1) 50 c.c. 4’n MgCl. +50 c.c. sea-water
(2) Normal sea-water
Two hours later the eggs from solution 1 were put back
into normal sea-water. Three and a half hours later about
50 per cent. of the eggs that had been in solution 1 were
divided into from 2 to 16 cells, but not an egg had a mem-
brane. The control eggs that had been in normal sea-water
all the time were all without membrane and absolutely unseg-
mented. Millions of eggs were examined under the micro-
scope. The next morning the eggs that had been in solution
1 had reached the blastula stage and were swimming about.
A small number were in a gastrula stage and even beginning
to assume a pyramidal form. In the control eggs not one
had developed. Perhaps one in a hundred had amceboid
forms such as precede segmentation in unfertilized eggs, but
not an egg was segmented, and not one had a membrane.
The next day some of the blastule of the other lot had
reached the pluteus stage. The control experiments will be
discussed in the next paragraph.
Possible sources of error and objections.—F rom all these
experiments I draw the conclusion that by putting the unfer-
tilized eggs of Arbacia for two hours into a solution of 60 c.c.
20 n MgCl, + 40 c.c. sea-water the eggs develop into blastule
if brought back into normal sea-water. If we put the unfer-
tilized eggs for about two hours into a solution of equal parts
of 2.n MgCl, and sea-water, the eggs may reach the pluteus
stage. The possible objection might be that the eggs were
fertilized. Such fertilization could only have been caused
by the instruments or hands of the experimenter having
been in contact with spermatozoa, or by the sea-water con-
taining spermatozoa. The first possibility was absolutely
excluded through the above mentioned precautions. The
second possibility was rendered practically impossible, as,
first, the spawning season was practically over, and, second,
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616 STUDIES IN GENERAL PHYSIOLOGY
the spermatozoa lose their power of fertilizing eggs in a very
short time (in about five hours). But that it was absolutely
excluded is proven by the following facts:
1. None of the unfertilized eggs kept in normal sea-water
developed or formed a membrane. J examined millions of
eggs in each experiment. Not one was found that was fer-
tilized. The sea-water used in this case was the same as
that used for the unfertilized eggs that did develop. If the
sea-water had contained spermatozoa, the unfertilized eggs
kept in normal sea-water all the time should have been fer-
tilized.
2. None of the eggs which developed after treatment
with MgCl, solution ever had amembrane. Fertilized eggs
which were put immediately after fertilization into a mixture
of equal parts of %,9 n MgCl, and sea-water and kept there
for two hours did not lose their membrane. In the ninth
series I made the following control experiments: Unfertilized
eggs that had been in the above-mentioned #9 n MgCl, solu-
tion for two hours were put into normal sea-water to which
fresh sperm was added. In this case a number of eggs
formed membranes.
3. No blastula originated from an egg that had been kept
for some time in one of the following solutions:
(1) 100 cc. 4°" MgCl,
(2) 30cec.22n “ -+ 70 c.c. sea-water
(8) 40 “© “ +69 «
(4) 100 c.c. sea-water + 33 gr. (wet) MgCl,
and the solutions mentioned in chap. iii. Yet eggs of the
same female that had been kept for some time in a mixture
of 50 or 60c.c. 2°n MgCl, and 50 or 40¢.c. of sea-water
developed into blastule or plutei. This happened in spite
of the fact that the vitality of the latter eggs had suffered
more than that of those in the above-mentioned solutions
with more sea-water and less MgCl,. Moreover the water
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ARTIFICIAL PRODUCTION OF NoRMAL Larve 617
was always changed in both classes of eggs simultaneously,
and the chances for fertilization of the eggs from sperma-
tozoa contained in the sea-water were equal for both. If the
sea-water had contained any spermatozoa capable of impreg-
nating the eggs, those eggs that had been in solutions with
less MgCl, should have been fertilized first.
4. In almost all the experiments eggs were taken out
of the mixture of 60 to 50 cc. %9 n MgCl, +40 to 50 cc.
sea-water at different periods. In no case did a single egg
develop into a blastula that had been in this solution for less
than one half-hour, and generally only those eggs yielded
blastule that had been in this solution for about two hours.
If the sea-water had contained spermatozoa, the latter should
have fertilized those eggs first which had been a shorter time
in the artificial solution. On the other hand, the eggs that
had been left in the artificial solution more than two and
one-half hours as a rule yielded fewer or no blastule.
5. I stated above that even at the height of the spawning
season eggs are rarely fertilized by spermatozoa contained in
the running sea-water. I do not think one would be likely
to see more than one egg in a thousand undergo develop-
ment under such conditions, provided that no contamination
through the instruments occurred. In our experiments which
were made at the end of the spawning season about 20 to 50
per cent. of the eggs that had been kept in the right solution
developed. It is out of the question to attribute such a
result to spermatozoa contained in the sea-water.
6. As far as I can see, there is only one possible source
of error left. It might be that the sea-water contained
spermatozoa, but that these spermatozoa were not able to
fertilize normal eggs, while a treatment of the egg with
the mixture of 60 c.c. 22 MgCl, + 40 c.c. sea-water in-
creased its susceptibility to impregnation, or a treatment
of the spermatozoon with the same solution increased the
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618 STUDIES IN GENERAL PHYSIOLOGY
fertilizing power of the spermatozoon. Both possibilities
must, however, be discarded. As far as the liability of the
egg to impregnation is concerned, I made the following
experiments in the last series. Unfertilized eggs were put
into a solution of 50 ec. 2°n MgCl, + 50 c.c. sea-water
and left in this solution for two hours. They were then
taken out and fertilized with fresh spermatozoa. At the
same time another lot of the eggs of the same female which
had been kept for two hours in normal sea-water were fer-
tilized with sperm of the same male. Practically every egg
of the latter lot developed into a blastula, while only about
50 per cent. of those eggs that had been in the MgCl, solu-
tion reached the blastula stage. Hence the treatment with
MgCl, diminishes the power of development of eggs, but
does not increase tt. As far as the spermatozoa are con-
cerned, former experiments by Norman, Morgan, and myself
showed that a slight increase in the concentration of the
sea-water destroys the fertilizing power of spermatozoa very
rapidly. In my experiments I added 2 gr. of NaCl to 100
c.c. of sea-water.
The spermatozoa which had been in this solution for only a few
hours, when brought back into normal sea-water, fertilized only
a thousandth part or less of the normal eggs, while the spermatozoa
of the same animal which had remained in normal sea-water
fertilized at the same time almost all the eggs.)
Morgan repeated my experiments, obtaining the same result.’
Norman tried the effects of a slight increase of MgCl, upon
spermatozoa.’ I repeat his statement:
I put sperm at 8:30 into MgCl, solution 24 gr. to 100 ce. of
sea-water. At 8:30 some of the sperm was mixed with normal
unfertilized eggs, and within three minutes the eggs were fertilized.
At 8:42 eggs and sperm were again mixed. In two minutes egg
membranes began to become visible, showing normal fertilization,
1 Logs, Journal of Morphology, Vol. VII (1892), p. 253.
2 MorGAN, Anatomischer Anzeiger, Vol. IX (1894), p. 141.
3 NoRMAN, Archiv fiir Entwickelungsmechanik, Vol. III (1896), p. 106.
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ARTIFICIAL PropvucTIoN oF NorMAL Larva 619
and within another minute all the eggs were fertilized. At 8:52
another test was made, but at this time the egg membrane did not
appear, showing that fertilization did not take place. At 9 o’clock
about one egg in every 100 was fertilized.
Norman repeated these experiments several times with the
same result. They prove that even a small addition of
MgCl, to sea-water, much smaller than in any of our
experiments, suffices to annihilate the power of impregnation
in the spermatozoa in a very short time. In my own experi-
ments the increase in the osmotic pressure of the sea-water
was much greater than in Norman’s experiments. I made
another control experiment in the ninth series which bears
on the same question. Unfertilized eggs were left in a
solution of equal parts of 2,.n MgCl, and sea-water for two
hours. At the end of that time they were put back into
normal sea-water to which sperm was added which had also
been in a solution of equal parts of #,°n MgCl, and sea-
water for two hours. Only very few of the eggs formed a
membrane.
There is, as we saw, a typical difference between the
blastulee and plutei which develop from fertilized and
unfertilized eggs. The former rise to the surface, the latter
swim at the bottom of thedish. If eggs be kept for two hours
in the MgCl, solution and then fertilized with normal sperm,
the blastule rise to the surface. If they be fertilized with
sperm that had been in MgCl, solution for two hours, they
remain at the bottom of the dish like the unfertilized eggs.
It is thus clear, I think, that even this last possible objection
that the treatment with the MgCl, solution increases the
impregnating power of the spermatozoa, or the impregna-
bility of the egg must be discarded. Hence I draw the
conclusion that the unfertilized eggs that had been treated
with equal parts of 2,2n MgCl, and sea-water developed
parthenogenetically.
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620 STUDIES IN GENERAL PHYSIOLOGY
Vv. SOME REMARKS CONCERNING THE NATURE OF THE PROCESS
OF FERTILIZATION
The facts of the preceding section force us to transfer the
problem of fertilization from the realm of morphology into
that of physical chemistry. There is certainly no reason
left for defining the process of fertilization as a morphological
process. The morphology of the spermatozoon itself becomes
of secondary importance as far as the process of fertilization
is concerned.
The spermatozoon not only starts the development of non-
parthenogenetic eggs, but it is also the bearer of the heredi-
tary qualities of the male. From our experiments it becomes
evident that these two functions of the spermatozoon are
not necessarily bound together, for nobody would assume for
an instant that the hereditary qualities that are carried by
the spermatozoon could be imparted to the egg by a change
in the inorganic constituents of the sea-water. We have
learned to attribute the different activities of a cell to
different enzymes. We must in future consider the possible
or probable separation of the fertilizing qualities of the
spermatozoon from the transmission of hereditary qualities
through the same.’
The plutei produced from the unfertilized egg resemble
closely in every regard those produced from the fertilized
egg. The latter in many cases live longer than the former,
but even this was not so in every case, and it is not impos-
sible that in further experiments parthenogenetic plutei with
a greater duration of life will be produced. The only
difference between parthenogenetic and normal blastule is
that the latter rise to the surface of the water, while the
former do not. One might think that this was due to the
influence of the MgCl, solution on the egg. This is, how-
ever, not the case. Eggs that had been in such a solution
1 Logs, Biological Lectures, Woods Hole, 1899, Ginn & Co., Boston.
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ARTIFICIAL PRoDUCTION oF NorMAL Larvae 621
and were fertilized afterward rose to the surface. Even
this difference might be caused to disappear by further
experimentation.
An agency which causes the egg to go through only the
first stages of segmentation, which lead, for instance, to a
division of the egg into 2, 4, or 8 cells, need not necessarily
have much in common with those agencies in the sperma-
tozoon that cause the development of the fertilized egg. But
if the egg can be caused through an artificial influence to
reach the blastula stage and swim about, the artificial cause
must have more in common with the effective element in
the spermatozoon. If however the artificial influences cause
the egg to reach the pluteus stage, or in other words cause
the egg to develop as far as the fertilized egg can be
developed at present in our laboratory, I think the two pro-
cesses of artificial and natural development must be pretty
closely allied.
It is in harmony with our statement that a very large
number of conditions cause an unfertilized egg to reach a
two- or four-cell stage. It suffices to leave the eggs for some
time in sea-water (about twenty-four hours). A slight
increase in the alkalinity of the sea-water causes the begin-
ning of a segmentation much sooner. A short treatment
with sea-water that is faintly acid has the same effect. An
increase in the concentration of the sea-water which probably
causes a loss of water in the egg has the same effect (Morgan).
Morgan found more recently that treatment with a solution
of strychnia salts may lead to a beginning of segmentation.’
Possibly in this case the alkalinity of the sea-water was
modified. But none of these or the other methods mentioned
above has yielded blastule, gastrulz, or plutei.
There is at present only one way known by which the
1MorGAN, Science, Vol. XI (1900), N. S., p.176. R. Hertwig had found this many
years ago.
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unfertilized egg of Arbacia can be caused to develop into a
pluteus.' This consists in treating the unfertilized egg for
two hours with a mixture of about equal parts of a 22n
MgCl, solution and sea-water. It is of theoretical interest
to find how this treatment may possibly affect the egg sub-
stance. The bulk of our protoplasm consists of proteids,
which according to their physical behavior belong to the
colloidal substances. The proteids are characterized by two
qualities which are of the utmost importance in the analysis
of life phenomena. The proteids change their state very
easily, and readily take up or lose water. It is more than
probable that one or both of these qualities may account
for muscular contractility and protoplasmic motion. The
agencies which affect these two variable qualities of the
protoplasm most powerfully are, first of all, certain enzymes
(for instance, plasmase, trypsine, etc.). Almost equally
powerful are ions in certain concentrations. As I have
dwelt upon this point in my three preceding publications,’
it need not be repeated here. But I wish to call attention
to a most interesting paper by Dr. E. Pauli, which has
recently appeared and which throws more light on this sub-
ject... The third agency is temperature.
In our experiments it was evidently the second factor
which affected the condition of the colloids. The transitory
treatment of the unfertilized eggs with a mixture of equal
parts of a %n MgCl, solution and sea-water brings about a
change in the physical conditions of certain colloids which
is not reversed by putting them back into normal sea-water,
and which allows them to develop into normal plutei.
As far as the spermatozoon is concerned, it may bring
about the same change in the condition of the colloids in
1T have not been able to raise the fertilized eggs of Arbacia beyond the pluteus
stage in the laboratory.
2Part IT, pp. 539, 544, and 559.
3 PAULI, Archiv fiir die gesammte Physiologie, Vol. LXXVIII (1899), p. 315.
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ARTIFICIAL PRODUCTION OF NoRMAL Larva 623
the egg, either by its carrying specific ions into the egg or
by carrying enzymes, or in some other way which is as yet
unknown to us. It is certainly remarkable that the sperma-
tozoa contain a large amount of ash (5 per cent., according
to Hammarsten). In the parthenogenetic egg the colloids
are from the beginning in such a condition as to allow the
development to proceed. In other animals it is perhaps
solely the ion constitution of the sea-water or of the blood
which prevents the eggs from developing parthenogenetic-
ally. I shall discuss this point more fully in connection
with further experiments on this subject.
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XXX
ON ARTIFICIAL PARTHENOGENESIS IN SEA-URCHINS'
In the last October number of the American Journal of
Physiology I published a preliminary note on the artificial
production of larve from the unfertilized eggs of the sea-
urchin. I mentioned that unfertilized eggs were able to
develop into normal plutei after having been in a solution of
equal parts of a 2,°n MgCl, solution and sea-water for about
two hours. The control experiments by which the possibility
of the fertilization of these eggs through spermatozoa had
been excluded were briefly mentioned. In the April num-
ber of the same journal a full description of my experiments
was published which I believe puts an end to any doubt con-
cerning the possibility of an error. Nevertheless, I decided
to repeat these same experiments with the additional precau-
tion of using sterilized sea-water. Through the kindness
of the board of trustees of the Elizabeth Thompson Fund I
was enabled to make further experiments on artificial par-
thenogenesis at the Pacific coast. These experiments have
led to a number of new results, which will be published in
the American Journal of Physiology. Here I will confine
myself to a description of the precautions which were taken in
these experiments to exclude the possibility of a fertilization
of the eggs through spermatozoa.
The sea-water used for these experiments was heated the
day before, very slowly, to a temperature of from 50 to 70°
C., and was kept at that temperature for about ten minutes
and allowed to cool very slowly. The control experiments
proved that, as was to be expected, the spermatozoa are
killed by this treatment. During the time the water was
heated no sea-urchin was opened in the laboratory or was even
1Science, Vol. XI (April 20, 1900), p. 612.
624
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ARTIFICIAL PARTHENOGENESIS 625
kept there. The sterilized sea-water was kept in special
flasks and covered jars which were utilized for this purpose
only. Before we started an experiment we disinfected our
hands thoroughly with soap and brush in the same way as is
customary in a surgical operation. Every sea-urchin before
it was opened was exposed for from two to five minutes to a
powerful stream of fresh water, and care was taken to wash
the whole surface of the animal as thoroughly as possible
with fresh water. The mouth of the sea-urchin was then
cut out with scissors that had been sterilized the day before
in the flame and had been kept dry since. Through the
excision of the mouth the sexual glands were exposed, and
their color allowed to decide whether the animal was a male
or a female. If the first animal that was opened was a
female, the intestine was removed with a sterilized forceps,
and care was taken not to bring the forceps in contact with
the ovaries or with the outside surface of the animal. After
the intestine had been removed and nothing left except the
ovaries, the inside of the animal was repeatedly filled with
fresh water and washed out. Then each of the five ovaries
were taken out in foto with a sterilized section lifter, and
special pains were taken that the ovaries did not come in
contact with the surface of the sea-urchin or with the hands
of the experimenter. The ovaries were first put into a dish
of fresh water, were washed off carefully, and then put into
sterilized sea-water.
One portion of the eggs was put into sterilized sea-water
to serve as control material. A second portion was put into
a mixture of equal parts of sterilized sea-water and a *,!n
MgCl, solution. An hour or two later these eggs were
taken out of this mixture and put into sterilized sea-water-
While of the latter eggs as many as 25 per cent. developed
into blastule and swam around the next day, not an egg of
the control material even segmented. We spent hours:
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626 STUDIES IN GENEBAL PHYSIOLOGY
searching the control material for segmented eggs, but were
never able to find a single one.
In addition to these control experiments we made several
others. It was necessary to apply the mixture of equal parts
of the 22» MgCl, solution and sea-water for from one to
two hours to bring about the development of the unfertilized
eggs. We made it a rule to take out one portion of eggs
from this solution much earlier —in some cases after ten min-
utes. In no case did one of these eggs segment or develop.
A third series of control experiments was applied. Solu-
tions with less MgCl, and more sea-water were tried. In
solutions of 30 c.c. 2,°n MgCl,, and 70 c.c. sea-water not an
egg was able to develop.
If the first animal opened in these experiments happened
to be a male, the instruments were at once laid aside for
disinfection, and the next animal was opened by another
experimenter with the same precautions.
In some experiments we used sea-water that had been
filtered through a new Pasteur filter. Although no sperma-
tozoa are able to pass through such a filter, the eggs treated
with a mixture of equal parts of a %,9n MgCl, solution and
filtered sea-water developed, while none of the control eggs
were able to develop.
In one of the former papers I mentioned the fact that
the mixture used for artificial fertilization killed the sperma-
tozoa in a comparatively short time and injured many of
the eggs. Contrary to the common prejudice, it is a fact
that spermatozoa are much more sensitive and are killed
much sooner than the egg.
My experiments at Pacific Grove were carried on with
Strongylocentrotus franciscanus and S. purpuratus. In both
animals artificial parthenogenesis can easily be accomplished.
In the experiments at Pacific Grove I enjoyed the valu-
able assistance of Mr. W. E. Garrey.
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XXXI
ON THE TRANSFORMATION AND REGENERATION OF
ORGANS!
I
SEVERAL of the older scientists, for instance, Bonnet,
Spallanzani, and Dalyell had occasionally observed that in
the place of a head a tail may be regenerated in lower
animals.” These casual observations had been considered as
curiosities or pathological cases, and scientists took no further
notice of them. It occurred to me that it might be possible
to produce the substitution of one organ for another at
desire, and that in this way we might gain an insight into
the physiology of morphological processes. Having tried
in vain to accomplish this result during the year 1888 in
Kiel, I succeeded the following year at Naples. I found
that if the foot of a Tubularian be cut off and the foot
end of the stem surrounded on all sides by sea-water a head
will be produced instead of a foot, while the same end
produces a foot if it is in contact with some solid body,
like the bottom of the aquarium: This arbitrary substitu-
tion of one organ by another I called heteromorphosis in
contradistinction to the case of regeneration in which the
same organ is reproduced. I succeeded in showing that
phenomena of heteromorphosis can easily be produced in all
kinds of Hydroids and in Tunicates.’
Since then a great number of heteromorphoses in various
classes of animals have been obtained. The most brilliant
accomplishment in this field of science is undoutedly
Herbst’s discovery that if in Crustaceans the eye together
1 American Journal of Physiology, Vol. IV (1900), p. 60.
2Part I, p.115. 3 Loc. cit.
627
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628 STUDIES IN GENERAL PHYSIOLOGY
with the optic ganglion be removed, an antenna will be
produced in the place of the eye, while if the eye alone
is cut off an eye is regenerated. The presence or absence of
the optic ganglion decides whether a regeneration or a hetero-
morphosis will follow.’
I found, very early in my experiments, that in certain
Hydroids a heteromorphosis can be produced without any
organ being cut off or any wound being inflicted upon the
animal. In Antennularia—a Hydroid common at Naples—
the arrangement and orientation of the organ as well as
the direction of growth is dominated by gravitation. The
animal consists of a straight vertical stem, which forms
stolons at its lower end and which carries small branches
with limited growth at regular intervals. On the upper
surface of these branches the polyps are found. If such a
stem be suspended horizontally in the water the lateral
branches which are directed downward and which had
finished growing now begin to grow downward very rapidly.
At the same time the polyps on these branches disappear.
The downward-growing parts no longer resemble the old
side-branches but look like roots. A closer examination
reveals the fact that they not only possess the morphological
appearance of roots but also the physiological reactions of the
latter, inasmuch as they are positively geotropic and stereo-
tropic, while the branches do not show these forms of
irritability. In this case the tissue of the polyps which dis-
appeared seems to have been transformed into the tissue of
roots.”
I made a similar observation shortly afterwards at Woods
Hole in another Hydroid, Margelis. When the uninjured
points of a stem of Margelis are brought in contact with a
solid body the point of the stem assumes the form and
1 Hersst, Archiv fiir Entwickelungsmechanik, Vol. IX (1899), p. 215.
2 Part I, p.191.
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TRANSFORMING AND REGENERATING OrGANS 629
reaction of a root. It looks as if the contact with a solid
body brought about a transformation of the stem into root
material which is morphologically and physiologically dif-
ferent from the stem.’ But as neither Antennularia nor
Margelis is sufficiently transparent, it was not possible to
ascertain that a transformation of polyps and stems into
stolons occurs in this case.
Miss Bickford made an observation in my laboratory
which helped in making the assumption of a transformation
of organs more probable. Small pieces were cut from a
stem of a Tubularian. These pieces were smaller in size
than a normal polyp. Miss Bickford found that within
sixteen hours such a piece assumed the form of a polyp.’
Driesch confirmed her observation.’
Last summer I had an opportunity to observe directly
the transformation of organs under the influence of contact.
My observations were made at Woods Hole on a transparent
Hydroid, Campanularia. This Hydroid attaches itself with
stolons to solid bodies. The stem with the polyps grows at
right angles with the solid body to which its stolons are
attached. If these Campanulariz be cultivated on a ver-
tical wall all the stems assume an exactly horizontal position
in the water. The stem of a Campanularia is the mest per-
fect specimen for negative stereotropism I have ever ob-
served. If a stem be cut off and put on the bottom of a
watchglass filled with sea-water, all the polyps that touch
the glass are transformed into the material of the stem.
This material creeps out of the stem, forming stolons wher-
ever it comes in contact with the glass, giving rise to polyps
on its upper surface which is in contact with sea-water.
The polyps continue growing at right angles toward the
1 LoEeB, Woods Hole Biological Lectures, 1893.
2 BICKFORD, Journal of Morphology, Vol. IX (1894), p. 417.
3 Driescu, Vierteljahrschrift der Naturforscher-Gesellschaft, Ziirich, 1896.
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630 STUDIES IN GENERAL PHYSIOLOGY
bottom of the dish. All these processes may occur in less
than a day, and can be observed directly with a lens. I will
try to give a description of these phenomena with the aid of
camera drawings I made while observing them. Fig. 152
shows the condition of a
Campanularia stem that
had been put on the bot-
tom of a watchglass the
FIG, 152 previous day. Originally
it had five perfectly de-
veloped polyps. Only
two of these are left (4 and 5); the three others
(1, 2, and 8) have disappeared. At the lower
t end, a, of the original stem a new stolon, a b,
has grown out. What had become of the three polyps
that had disappeared? I watched them very closely and
found that they were transformed into a shapeless mass
and withdrawn into the stem. I will describe this process
of transformation of polyps into the material of the stem
more minutely with the help of Figs. 153, 154, 155. These
are not taken from the same stem, but as the process
occurs almost always in the same form, this makes no
material difference.
The transformation of a polyp into the less differentiated
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TRANSFORMING AND REGENERATING ORGANS 631
material of the stem begins with a shortening and folding
together of the tentacles (polyp 1 in Fig. 153). This
process is at the beginning the same as that which occurs
upon any stimulation of the polyp and especially in the act
of taking up food. But while in the
latter case the tentacles unfold again,
in the case of the transformation of
the polyptheyremaintogether. Very
soon all the tentacles begin to fuse
into a homogeneous mass. This
process of fusing begins usually at
the peripheral end of the
sa polyp (polyp 2, Fig. 153).
A little later all the ten-
tacles form an undifferen-
tiated mass of protoplasm
(see polyp 1, Fig. 154). ¥
In the next stage (2, Fig. 154) the
2 original differentiation of the crown of
the polyp into tentacles can no longer
be recognized.
At this stage the transformed shape-
less mass of the polyp begins to flow
back into the stem (1, Fig.155). A little later only
a fraction of the original protoplasm of the polyp is
left in the periderm, the rest having crept back into
the stem (2, Fig.155). In polyps 3, 2, and 1 (Fig. 152)
FIG. we see the further stages of this process of the polyp
material flowing back into the stem.
The transformation of polyps and their creeping into the
stem occurs probably in a similar way in an Antennularia
which is put into the water horizontally. The main difference
between an Antennularia and a Campanularia is that in the
latter this transformation is produced by the polyp coming
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632 STUDIES IN GENERAL PHYSIOLOGY
in contact with a solid body, while, in an Antennularia a
change in the position of the polyp toward the vertical
suffices to bring about this result.’
While these processes are going on, the material of the stem
begins to creep or grow out of the original periderm. It
seems to me worth while to
call the attention of the
reader to the fact that in this
case the process of growth is
identical with the process of
progressive motion of a pro-
Ste toplasmic mass. In plants
FIG. 155 Sq growth occurs mostly near the
apex of an organ. If we look
at the increase in size of the
stolon from the point of view of growth we notice that its growing
point is near the apex, just as in plants. But if we look at it
from the point of view of progressiveamceboid motion wenotice
that only the foremost point creeps and that the rest of the pro-
toplasm is pulled out more passively. That the protoplasm
of the stem is under a strain will be seen by a glance at
Figs. 152, 153, 154, and 156. The ccenosare or protoplasm
lies in the periderm in the same way as a stretched rubber
thread would lie. Wherever the periderm is bent the proto-
plasm touches it on the concave side. It follows as nearly
as possible the shortest line in the periderm. It is possible
that the strain under which the ccenosare is kept causes the
protoplasm to flow in the direction of the strain toward the
tip of the stolon. Botanists are inclined toward an ex-
clusively osmotic conception of the process of growth. I
have come more and more to the conclusion that the osmotic
theory of growth is not in harmony with the phenomena
1In former papers I have described the fact that in Eudendrium the polyps
are thrown off when the stems are put into small dishes. Such phenomena may
occur also in Campanularia, but this was not the case here. [1903]
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TRANSFORMING AND REGENERATING ORGANS 633
of absorption. Ido not consider it
impossible that the phenomena of
protoplasmic motion which we can
actually observe in the growth of a
stolon in Campanularia exist also in
the phenomena of growth of other
organisms, plants as well as animals.
I have already called attention to this
possibility in a former paper.
Before we leave this subject I wish
to describe how the nature of the
contact localizes the development of
polyps from stolons and stems. The
piece, bc, Fig. 156, was cut out from
a fresh Campanularia stem and had
been put into a watchglass filled with
sea-water. This piece had a normal
polyp at 7, which was transformed
into a mass of undifferentiated pro-
toplasm and began to flow back into
the stem. Simultaneously a new
stolon began to grow out at c, and
very soon reached the considerable
size,cd. Then anew polyp, h, began
to rise on the upper surface of the
stem. It grewatright angles toward
the watchglass, a point which cannot
be rendered accurately in the draw-
ing. A new stolon, ab, began to
grow or creep out simultaneously at
a. Curiously enough, as soon as this
happened the protoplasm began to
flow back from the old stolon, ¢ d.
At the time the drawing was made
FIG. 156
d
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634 STUDIES IN GENERAL PHYSIOLOGY
it had flowed back to the point e. This was on the third
day of the experiment. I have however noticed that the stem
can send out stolons in different directions simultaneously.
The hereditary arrangement of organs in Hydroids is
unequivocally determined by external circumstances, espe-
cially contact. A germ or larva of a Hydroid will form
roots on one side only, namely the side where it touches
solid bodies: on the opposite side where it touches sea-
water it will produce polyps or stems. The negative stere-
otropism of the latter or their positive heliotropism as in the
case of Eudendrium will cause them to continue growing
away from the solid body into the sea-water. Weismann is
therefore wrong in assuming that the hereditary arrange-
ment of the organs in Hydroids is due to a definite arrange-
ment of the elements in the germ.
II
What is the character of the physical or physiological
processes which underlie the transformation of organs?
Such complicated formations as the polyp in Campanularia
are only possible if certain of the constituents are solid.
The transformation of such a polyp into the more shapeless
flowing or creeping material of the stem can only be due to
a liquefaction of these solid constituents. It is moreover
certain that contact with sea-water favors the formation of
polyps with its more solid elements, while the contact with
solid bodies favors the formation of the more fluid material
of the stem or stolon. Hence it seems as if the nature of
contact in this case determined the state of matter of certain
colloids in the Campanularia.’’ Although I had observed the
influence of the nature of contact upon these phenomena fur
many years I had not been able to form any definite idea of
1I do not need to mention especially that the periderm does not participate in
these liquefactions.
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TRANSFORMING AND REGENERATING OrGAnNs 635
how the nature of the contact could possibly influence these
processes, and I do not think that anyone else has thus far
offered an explanation. While studying the literature on the
coagulation of the blood I came across Duclaux’s account of
this process in his Traité de microbiologic,’ and it seemed
to me that if his notions are correct they might also
be applied to our problem of contact-heteromorphosis.
According to Duclaux it is the character of the contact
applied to the leucocytes which decides whether the enzyme
of coagulation, the plasmase, becomes effective or not. As
long as the leucocyte touches the endothelium of the blood-
vessels the blood remains liquid because the contact of the
leucocytes with the endothelial cells does not allow the fibrin
enzyme to act. If, however, the leucocyte touches a piece of
glass the plasmase becomes active and causes coagulation.
If the glass is covered with a layer of oil coagulation does
not occur. Duclaux assumes that surface tension phenomena
decide the setting free of plasmase on the part of the leuco-
cyte. Whether this latter assumption be correct or not mat-
ters little for our purpose. We only need to carry the
analogy between the influence of contact upon the state of
matter of fibrinogen and the state of matter of certain col-
loids in the Hydroids far enough to assume that both depend
upon definite enzymes becoming active through certain forms
of contact acting upon the cells in which they are formed.
In the case of the blood a solidifying enzyme, in the case of
the polyps a liquefying enzyme is made active if the leuco-
cyte or the polyp come in contact with glass or some other
solid body.
These considerations possibly allow of a wider application
than to the mere case of contact-heteromorphosis. When a
piece of our skin is cut off, the cells of the margin of the
wound begin to multiply and spread out over the gap. We
1Ducnavux, Traité de microbiologie, Vol. II, Paris, 1899.
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636 STUDIES IN GENERAL PHYSIOLOGY
might say the change in the character of the contact causes
an increase in the cell-divisions. This is still more obvious
where whole organs are produced or regenerated. In one of
my former papers I pointed out a very definite chemical dif-
ference between embryonic tissue and muscle tissue.’ The
former is more immune against K ions and more sensitive
toward Ca ions. It has long been noticed, especially by
botanists, that young tissue contains comparatively more K
than old tissue. I am inclined to assume that this accounts
for the fact that young tissue contains more water or has a
greater degree of turgidity than old tissue. An increase in
K allows the protoplasm to take up more water, an increase
in Ca has the opposite effect.’ Ion effects and the effects of
certain enzymes of liquefaction or solidification are often
similar, or may at least support each other. It is not impos-
sible that the increase in cell-divisions among the cells of
the margin of the wound may be due to the different charac-
ter of the contact to which these cells are exposed during or
after the lesion, inasmuch as this different contact sets free
or throws into activity certain enzymes which do not act as
long as these cells are in their natural surroundings, e. g., as
long as they are in contact with other cells.
In returning after this digression to our main subject we
must mention that the nature of the contact is not the only
means by which solid elements in living tissues may be
liquefied. Five years ago I proved that lack of oxygen
liquefies the cell walls in the blastomeres of a teleost egg
(Ctenolabrus),’ and Budgett showed in my laboratory that
lack of oxygen produces the same phenomenon in Infusoria.'
This case may find its explanation through the well-known
experiment of Pasteur on the effect of oxygen on yeast cells.
With plenty of oxygen the yeast cells multiply abundantly,
1 Part II, p. 559. 2 Part IT, p. 510. 3 Part I, p. 370.
4BupeGeErt, American Journal of Physiology, Vol. I (1898), p. 210.
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TRANSFORMING AND REGENERATING ORGANS 637
but produce comparatively little fermentation; with little
oxygen they multiply less but cause a more abundant devel-
opment of alcohol and CO,. In the liquefaction of the cell-
walls of the blastomeres of Cisnclahnis or of Infusoria we may
have the analogue of the increased fermentation in Pasteur’s
experiment. In the latter we have to deal with a special
enzyme, the zymase.
Miescher pointed out that in the salmon a liquefaction of
muscular tissue occurs, and that the liquid products are util-
ized for the formation of sexual cells. Miescher was inclined
to ascribe the liquefaction of the muscle to lack of oxygen.
He noticed that the liquefaction of the muscle was preceded
by a reduction in the blood supply of the muscles.’ My own
and Budgett’s observations agree with Miescher’s views.’
It is possible that the processes of histolysis in the meta-
morphosis of insects are of a similar character, and some
authors have claimed that the histolysis in this case is
brought about by a process of asphyxiation. Metschnikoff
assumes that a phagocytosis plays an important réle in these
phenomena of histolysis. It is certain that in my experi-
ments on Ctenolabrus and in Budgett’s experiments on
Infusoria no phagocytes were present, and it is practically
impossible that they played a réle in the above-mentioned
phenomena in Campanularia. I do not think that the lique-
faction of colloids requires the presence of phagocytes any
more than the liquefaction of crystals.
1 Die histochemischen und physiologischen Arbeiten von F. Miescher, Leipzig, Vol.
I (1897), pp. 94-100.
2It is possible that in the case of Campanularia the histolytic phenomena do not
stop with the liquefaction of certain constituents, but that this process is followed by
hydrolysis. [1903]
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XXXIT
FURTHER EXPERIMENTS ON ARTIFICIAL PARTHENO-
GENESIS AND THE NATURE OF THE PROCESS OF
FERTILIZATION!
1. In my previous communications on the subject of arti-
ficial parthenogenesis’ I had confined myself to the proof of
the fact that the unfertilized eggs of Arbacia and Strongy-
locentrotus franciscanus and purpuratus, are capable of a
development into the pluteus form if kept for from one to
two hours in a mixture of equal parts of a %?n MgCl, solu-
tion and sea-water. The above-mentioned solution, which
brings about the artificial development of the egg, differs in
three directions from the constitution of the normal sea-
water. First, the osmotic pressure of the solution is higher
than that of the normal sea-water; second, one-half of the
salts contained in normal sea-water are removed. It might
be possible that the sea-water contains ions which are in-
jurious to the development, and that the removal of these
ions makes the development of the unfertilized eggs possible.
Third, a considerable amount of MgCl, is brought into solu-
tion, and it might be that the Mg ions have a specific “stimu-
lating” effect upon the development. For the determination
of the nature of the process of fertilization it was necessary
to find out which of the three conditions is essential for the
production of artificial parthenogenesis.
2. I had already mentioned in a previous paper that the
mere change in the constitution of the sea-water, if not
accompanied by an increase in its osmotic pressure, can only
1 American Journal of Physiology. Vol. IV (August 1, 1900), p. 178. These ex-
periments were carried out with the aid of the Elizabeth Thompson Science Fund,
2 Part II, pp. 539, 576, and 624.
638
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ARTIFICIAL PARTHENOGENESIS 639
cause the egg to go through a few segmentations, but cannot
cause the parthenogenetic production of a blastula or a later
stage of development. The increase in the osmotic pressure
of the solution is therefore an essential condition for arti-
ficial parthenogenesis. As the season was at an end, it was
not possible for me to decide last autumn whether the other
two above-mentioned conditions are equally essential.
Through the aid of the Elizabeth Thompson Fund I was
enabled to carry on experiments in co-operation with Dr. W.
E. Garrey at Pacific Grove during the spring,’ and I have
since had a chance to continue this work at Woods Hole.
My new results enable me to give a more definite answer to
the question of the nature of the process of fertilization.
I first tried to ascertain whether the MgCl, plays a specific
role in artificial parthenogenesis, or whether its place may
be taken by some other salt. , I found that the latter is the
case.” A mixture of equal parts of a 1,9 n NaCl solution and
sea-water, or of equal parts of a 42 KCl solution and sea-
water, is just as effective as, if not more so than, a 2,9 n
MgCl, solution. Unfertilized eggs of Strongylocentrotus,
if left for seventy minutes in any of these solutions, devel-
oped, and some of them reached the pluteus stage. Such
eggs remained alive as long as ten days. Even a mixture
of equal parts of a %2n CaCl, solution and _ sea-water
brought about the development of the eggs, but it was
necessary to take the eggs out in about forty to fifty
minutes, as otherwise the solution killed them. None of
the eggs treated with the CaCl, solution developed beyond
the blastula stage, or lived longer than one day.
I noticed that in these experiments with a 19 NaCl or
1 I wish to express my thanks to Professor Jenkins, of Stanford University, for
kindly allowing me the use of the Hopkins Laboratory.
2I had been misled in my original experiments of 1899 through the fact that
the solutions which I considered as isosmotic differed in their concentration. owing
to an error in their preparation. When I resumed the experiments in 1900 I dis-
covered the error and corrected it. [1903]
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640 STUDIES IN GENERAL PHYSIOLOGY
KCl solution only a comparatively small number of eggs
reached the blastula stage, certainly many less than in my
previous experiments with MgCl, on Arbacia. A further
examination revealed the fact that the MgCl, solution
which I had used was, through an error or a misunder-
standing of the assistant who made it, weaker than a 22n
solution. As soon as I found this out, I started experi-
ments with more diluted NaCl and KCl solution. Instead
of using equal parts of a 142n NaCl or’ KCl solution and
sea-water, I used the following mixtures:
20 42n NaCl-+ 30 distilled water + 50 sea-water,
Or— —- 174 42. NaCl + 323 distilled water + 50 sea-water.
In both cases more eggs reached the blastula and pluteus
stage than with the original stronger mixture. In one case
unfertilized eggs developed beautifully after having been
for two hours in a solution of equal parts of 15 21n NaCl
+ 35 distilled water-+50 sea-water. But this was nearly
the lowest limit for artificial parthenogenesis in Arbacia.
Asa rule, 25 per cent. or more of the unfertilized Arbacia
eggs reached the blastula stage.
3. It was thus proved that MgCl, does not play a specific
role in the production of artificial parthenogenesis. It
remained to decide whether it is essential to remove one
part of the normal constituents of the sea-water, or whether
the mere increase of the osmotic pressure suffices. I found
that the increase in the osmotic pressure of the sea-water is
all that is needed. In the experiments in which the maximal
number of unfertilized eggs reached the bastula stage about
1 gram NaCl had been added to the sea-water. We can
produce the same increase in the osmotic pressure of the
sea-water by adding 10 c.c. of the 24n NaCl or 24n KCl
solution* to 90 ¢.c. of sea-water. In this case the mixture
1My 24%4nNaCl solution contained 146.25 g. in a liter. The 2%nKCl solution
contained 186.25 g. in a liter.
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ARTIFICIAL PARTHENOGENESIS 641
contained practically all the constituents of normal sea-water.
Yet if unfertilized eggs of Arbacia are left in such a solution
for from one and one-half to two hours, as many as 50 per cent.
of the eggs may reach the blastula stage when put back into
normal sea-water. Many of these eggs die in the blastula
stage and only a small number reach the gastrula or pluteus
stage. The blastule are like those which I described in one
of my former papers.’ In the majority of cases more than
one blastula develops from one egg. I have seen as many
as six moving blastule arise from one egg. The tendency
to give rise to more than one embryo is greater in the egg
of Arbacia than in the egg of Strongylocentrotus. This
difference is probably due to the fact that even the unferti-
lized egg of Strongylocentrotus often forms a fine membrane
which is much thinner than the one produced through the
entrance of a spermatozoon, but which is sufficient to keep
the blastomeres together. The addition of NaCl or KCl to
sea-water favors the formation of this membrane.
4. In all the experiments mentioned thus far the increase
in the osmotic pressure had been brought about by the
addition of electrolytes. This might be considered as an
indication that the electrically charged ions in the sea-water
played an important rdle in the production of partheno-
genesis. I myself was originally inclined to such an assump-
* tion. I have convinced myself, however, that an increase in
the osmotic pressure of the sea-water through the addition
of cane-sugar or urea can produce parthenogenesis. My
stock solution of cane-sugar (rock candy) was 2n and con-
tained 684.3 g. in a liter, while the stock solution of urea
was 24n and contained 150.31 g. in a liter. I found that
the unfertilized eggs of Arbacia were able to develop after
they had been for from one and one-half to two hours in one
of the following solutions:
1 Part II, p. 576.
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642 STUDIES IN GENERAL PHYSIOLOGY
(1.) 100 sea-water+25 2n cane-sugar
(2.) 824 sea-water-+173 24n urea
Both the sugar solution as well as the urea solution injured
the eggs, the urea solution much more than the sugar solu-
tion. I made an attempt to produce parthenogenesis by
submitting unfertilized eggs to a pure cane-sugar solution
whose osmotic pressure was about equal to that of the sea-
water, to 90 c.c. of which 10 c.c. of a 24n NaCl solution had
been added. When the unfertilized eggs of Arbacia were
put for about two hours into a mixture of 60 2n cane-sugar
+40 distilled water or 55 2n cane-sugar+45 distilled
water, many of them segmented and a few developed into
swimming blastule, but they died within the first twenty-
four hours. This proves conclusively that the development
of the unfertilized egg is produced through an increase in
the concentration of the surrounding solution. As it is
immaterial whether the increase in the osmotic pressure is
brought about by electrolytes or non-conductors, there can
be no doubt that the essential feature in this increase in the
osmotic pressure of the surrounding solution is a loss of
water on the part of the egg.
5. Having reached the conclusion that the loss of water,
or rather the loss of a certain amount of water, causes the
parthenogenetic development of the egg, it seemed possible
to take another step in advance. In all the previous experi-
ments the unfertilized eggs had been submitted to a solution
of higher osmotic pressure for from one to two hours, and
were then put back into normal sea-water to develop. If
the initial loss of water on the part of the egg were all that
is required for the production of artificial parthenogenesis,
it would be possible to find a solution which would not only
take away water from the egg, but which would also allow
development to go on. I remembered from my earlier
experiments on the effects of an increase in the concentration
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ARTIFICIAL PARTHENOGENESIS 643
of sea-water upon development’ that so slight an increase in
the concentration of sea-water as is sufficient to induce par-
thenogenesis allowed the development of the eggs to go on
for at least twenty-four hours. I found that if we put
unfertilized eggs into a mirture of 93 sea-water and 7 24n
NaCl solution, many eggs develop in the solution, and some
of them even reach the blastula stage and swim about. If
we use a mixture of 90 sea-water and 10 24n NaCl solution,
the development stops earlier, for the simple reason that
such a solution is more injurious. Those facts show clearly
that the function of the artificial solution in the production
of parthenogenesis is that it has to deprive the egg of a
certain amount of water. In the majority of cases the
solutions that produce such an effect are at the same time
too injurious to allow the egg to develop or live long enough
to reach the blastula stage. This is the reason why we have
to take the eggs out of this solution and bring them back
into normal sea-water, if we wish them to develop into nor-
mal larvee.
6. A consequence of the loss of water on the part of the
egg is an increase in its osmotic pressure. The osmotic
pressure inside the egg is furnished chiefly or almost ex-
clusively by electrolytes. It is thus not impossible that the
ions in the egg, if their concentration is raised, bring about
that change which causes the egg to develop. If we assume
that the spermatozoon starts the development of the egg in
the same way as in the case of artificial parthenogenesis it fol-
lows that the spermatozoon must possess more salts or a
higher osmotic pressure than the eggs. As I pointed out
in a former paper, this seems to be the case. But there is
no reason why the spermatozoon should not bring about the
same effects that we produce by reducing the amount of
water in the egg in some different way. At present, how-
1Journal of Morphology, Vol. VII (1892), p. 253.
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ever, the only light that can be thrown upon the nature of
the process of fertilization must be expected from an analysis
of the effects of a loss of water upon the egg.
It seems as if the liquefaction of the nuclear membrane
and other constituents of the nucleus were a prerequisite for
cell-division. Norman showed that a certain increase in the
concentration of the sea-water brings about a distribution of
the chromosomes in the egg. Morgan’s observations agree
with this. But as all these observations were made with
solutions whose osmotic pressure was considerably higher
than that of the solutions used in my experiments, new ob-
servations will be required to decide this question. Hoppe-
Seyler, in one of his papers, points out that a loss of water
on the part of the protoplasm brings about a diminution
in the processes of oxidation. We know that lack of
oxygen can bring about the liquefaction of solid con-
stituents. I add these remarks for those who enjoy the
speculative side of biology. But at the best a theory can-
not give us anything more than the facts it includes, and
‘it is therefore clearly our task to supply the lacking ex-
perimental data in this field of biology before we begin
to theorize.
7. I think we should try to discover first of all whether
the process of development can be started by depriving the
egg of water in a few forms only, or whether this is a gen-
eral condition. I have thus far tried among the sea-urchins
Arbacia and Strongylocentrotus franciscanus and purpuratus.
Each of these forms is capable of osmotic parthenogenesis.
I am confident that the same is true for all species of sea-
urchins, although the optimal increase in the osmotic pressure
of the surrounding solution may vary for different forms.
But I consider it of more importance that with the same
methods I have been able to produce artificial partheno-
genesis in a starfish (Asterias Forbesii). By putting the
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unfertilized eggs of this starfish for about two hours into
a mixture of 88 c.c. of sea-water and 12 c.c. of a 24n NaCl
solution the eggs can be forced to develop and reach the
blastula stage, if put back afterward into normal sea-water.
T have not yet found the optimal condition for the partheno-
genetic development of Asterias, but the facts thus far ob-
tained suffice to state that a certain increase in the osmotic
pressure of the surrounding solution (and a loss of a certain
amount of water on the part of the egg) causes the egg of
this form to develop parthenogenetically.
I have mentioned in another place’ the precautions and
control experiments used to guard against the presence of
spermatozoa. I do not consider it necessary to repeat these
statements in this paper, but will mention one additional
precaution, for which I am indebted to the collector of the
Marine Biological Laboratory, Mr. Gray. Mr. Gray selects
the females of Arbacia for my experiments,so that in all
these later experiments I have not had one male in the labo-
ratory. Not one egg developed in the control material. All
the sea-water used in these experiments was heated to the
temperature of 70° C.
CONCLUSIONS
The results of my experiments are as follows:
1. Through a certain increase in the osmotic pressure of
the surrounding solution the unfertilized eggs of some
(probably all) Echinoderms (Arbacia, Strongylocentrotus,
Asterias) can be caused to develop into normal blastule
or even plutei.
2. This increase in osmotic pressure can be produced by
electrolytes as well as by non-conductors. It is therefore
probable that the parthenogenetic development is caused by
the egg losing a certain amount of water.
1 Part II, p. 576.
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XXXII
EXPERIMENTS ON ARTIFICIAL PARTHENOGENESIS IN
ANNELIDS (CHATOPTERUS) AND THE NATURE OF
THE PROCESS OF FERTILIZATION’
I. INTRODUCTION AND METHODS
My preceding papers on artificial parthenogenesis’ had
proved that by an increase in the osmotic pressure of the
sea-water the eggs of many, if not all, Echinoderms can be
caused to develop parthenogenetically. Two new problems
presented themselves for immediate consideration. The one
was to raise the parthenogenetic larvee until they were sex-
ually differentiated, in order to decide whether or not they
are of uniform sex. The second problem was to try whether
artificial parthenogenesis is confined to the group of Echino-
derms or whether it is a more general phenomenon. As the
means for the raising of sea-urchins were not available at
Woods Hole this year, the former problem had to be post-
poned. The solution of the second problem, however, was
possible, and yielded the result that the unfertilized eggs of
Cheetopterus, a marine Annelid, can be caused to develop into
swimming ciliated larve (trochophores). A short preliminary
report of this result has been published in Science.’
In experiments on parthenogenesis the greatest precau-
tions are necessary to exclude the possibility of a contamina-
tion of the eggs by spermatozoa. I purposely selected
Cheetopterus for my further experiments on account of the
possibility of discriminating between and separating the
females and males. If the experimenter handles females
and males in the same experiment or with the same instru-
1 American Journal of Physiology, Vol. IV (1901), p. 423,
2 Part II, pp. 539, 576, 624, and 638. 3 Science, Vol. XII (1900), p. 170.
646
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ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 647
ments, it is extremely hard to avoid an infection of the eggs
by sperm. I proceeded as follows in the experiments with
Chetopterus. As soon as the animals were brought into the
laboratory by the collector, the tubes in which they live were
opened and the worms removed. As soon as the first female
was found it was put into a special dish and thoroughly
washed off with sea-water, the water being renewed from six
to twelve times in succession. The sea-water in the labora-
tory was found to be absolutely free from spermatozoa of
Chetopterus. (The animals are found on the beach of an
island at some distance from the laboratory.) After the
female had undergone the process of washing, it was exposed
to a current of sea-water over night to remove as far as pos-
sible any spermatozoa that might have been left on the sur-
face. The next day the animal was ready to be used for an
experiment. On that day and before the experiment began,
the experimenter did not bring his hands in contact with
any other Chetopterus or with the aquarium that contained
such animals. His hands and instruments were sterilized
with fresh water. The posterior part of the animal which
contains the eggs was cut off and thoroughly washed for two
minutes in distilled or fresh water. Had any spermatozoon
been left on the surface of the animal, the distilled water
would have killed it. After this the part containing the eggs
was put into a vessel with sterilized sea-water, washed off
once more and then put into another dish containing steril-
ized sea-water. In this dish the single parapodia were
opened successively, the eggs sucked out from each with a
pipette, and then collected in another dish with sterilized
sea-water. After all the eggs had been collected they were
divided into two lots. The one lot remained in normal
(sterilized) sea-water, to serve as control material. The
other lot was distributed into the various solutions whose effect
T intended to test. In no case did I see a single egg of the
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648 STUDIES IN GENERAL PHYSIOLOGY
control material develop into a larva. I noticed only that
after from seven to ten hours some of these eggs may show
a beginning of a segmentation which, however, soon ceases.
This phenomenon seems to be quite common among many
marine animals. I mentioned in a former paper that O.
Hertwig had already noticed that it is a common occurrence
among Arthropods, Worms, and Echinoderms.’ If, however,
no such aseptic measures against spermatozoa were taken, a
number of eggs in the control material usually reached the
trochophore stage. The sea-water used in these experiments
was sterilized by heating it slowly to a temperature of from
60° to 80° C. In asmaller number of experiments I used
sea-water which had gone through a Pasteur (Chamberland)
filter which, of course, is absolutely impermeable to sperma-
tozoa.” If the eggs of more than one female were used for
an experiment, all the eggs were first gathered in one dish,
thoroughly mixed, and then divided into two lots, one to serve
as control material and one to be distributed into the various
solutions. Thus the control material and the material experi-
mented upon consisted always of the eggs of the same
females. It goes without saying that the same was the case
in all my previous experiments on Echinoderms.
Il. ARTIFICIAL PARTHENOGENESIS CAUSED BY AN INCREASE
IN THE OSMOTIC PRESSURE OF THE SEA-WATER
It was natural to try first whether or not the same means
that cause the parthenogenetic development in Echinoderms
are also sufficient to bring about the parthenogenetic devel-
opment of the eggs of Chetopterus.
First series. — When I received the first material, I at
10, HERTWIG, Die Zelle und die Gewebe, Vol. I (1893), p. 239.
2JTn almost all the experiments the sea-water used was sterilized. In a few
exceptions this precaution was purposely omitted in order to find out whether or not
the sea-water in the laboratory contained spermatozoa of Chetopterus. This, how-
ever, was not the case,
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once started an experiment, although I knew that it was
practically impossible to exclude contamination by sperma-
tozoa if I attempted to isolate the eggs immediately after
having handled a male. The female was washed off in steril-
ized sea-water, but of course I was aware that this would
not suffice to get rid of any spermatozoa that might be stick-
ing to the surface of the animal. The eggs, however, were
taken and distributed into the following five solutions:
(1) 6c. 24n KCl+94 c.c. sea-water
Q) 8 “ +92 “
(3) 10 “ +90 “
(4) 12 “ +88 “
(5) Normal sea-water (control)
One part of the eggs remained one hour and twenty-five
minutes, the rest one hour and forty minutes in the solu-
tions. The experiment was started in the afternoon. The
next morning’ I found numerous swimming larve (trocho-
phores) in the material that had been in the first four solu-
tions for one hour and twenty-five minutes. In the second
lot they were less numerous. But even in the control
material I found two swimming trochophores. It followed
that the Chetopterus were either naturally parthenogenetic or
the precautions against the entrance of spermatozoa had not
been sufficient.
Second series.—From now on I applied the rigid anti-
septic measures against spermatozoa described above in the
introduction. The following solutions were used:
(1) 8ec.24n KCl +92 c.c. sea-water
(2) 10cc.24n KCl +90 «“
(3) 12c.c.2kn KCl +88 «“
(4) 12e.e. 2in NaCl +88 “
(5) 20 cc. 24n MgCl, +80 ©
(6) Normal sea-water (control)
1] shall in the following descriptiou of the experiments consider only whether
or not swimming trochophores were formed. The morphological details will be
given insection v. It goes without saying that all the experiments deal with unfertil-
ized eggs, unless the contrary is distinctly stated.
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650 STUDIES IN GENERAL PHYSIOLOGY
All the sea-water had been sterilized the previous day by
heating it to a temperature of 80°; one part (a) of the eggs
remained one hour, a second part (6) one hour and twenty
minutes in these solutions.
The first four solutions yielded numerous swimming
trochophores; their number was greatest in the first two
solutions. Lot aof the MgCl, solution yielded no swimming
blastule, but lot b had a few. The control eggs were com-
pletely undeveloped, with the exception that after about ten
hours a few showed the beginning of a segmentation, which
in no case led to the formation of more than from 4 to 6
cells. During the next forty-eight hours no further develop-
ment occurred, and the eggs died and disintegrated. Accord-
ing to this experiment the unfertilized eggs of Cheetopterus
are not able to develop in normal sea-water. They can,
however, be caused to develop into trochophores if exposed
for about an hour to sea-water whose concentration has been
raised through the addition of the right quantity of KCl or
NaCl.
Third series.—The next task was to ascertain how much
the osmotic pressure of the sea-water must be raised in
order to bring about the parthenogenetic development, and
whether the increase in osmotic pressure necessary for this
purpose was the same in each case. The solutions used
were as follows:
(1) 10 ec. 24n KCl +90 c¢.c. sea-water
(2) 12h ec. 24n KCl +873 “
(3) 380 ee. 2 2 cane-sugar-+70
(4) 123 cc. 24 NaCl +874 $
(5) Normal sea-water (control)
The osmotic pressure in solutions 2, 8, and 4 was about
the same. The.eggs remained sixty-five minutes in these
solutions, and were then put back into normal sea-water.
While a great number of the eggs that had been in solutions
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ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 651
1 and 2 developed into trochophores, very few of the eggs of
solution 4 and none of solution 3 reached the trochophore
stage. The control eggs remained undeveloped.
Fourth series.—The results were obviously puzzling if
the increase of the osmotic pressure was the only factor that
brought about the development of the unfertilized eggs of
Cheetopterus. But they would be intelligible if there were,
in addition to the effect of an increase in the osmotic pres-
sure, a specific effect of the KCl or the K ions. In order
to decide this, the unfertilized eggs of a female were dis-
tributed into the following solutions:
(1) 5cc. 24x KCl +95 cc. sea-water
(2) 10 “ (79 +90 “
(8) 15 “ 6c + 85 (73
(4) 5 “ NaCl+ 95 «“
(5) 10 “ “ + 90 “
(6) 15 “ : “ce + 85 Ts
(7) Normal sea-water (control)
The eggs remained one hour in these solutions.
The next day the control eggs (7) were undeveloped.
The eggs that had been in the first three solutions were
teeming with trochophores. In lots 4 and 5 nota single
swimming trochophore was found, although many eggs had
begun to develop. The development stopped, however, in
an early stage. Of the eggs that had been in solution 6 a
large number had reached the trochophore stage and were
swimming. These results were as clear as could be desired.
In order to bring about artificial parthenogenesis through
the addition of NaCl, 15 ¢.c. of the 24m solution had to be
added, while 5 c.c. of a 24n KCl solution were sufficient.
Fifth series.—There was a possibility that the effect pro-
duced by NaCl was a specific Na effect, and not an effect of
the increase in osmotic pressure. An experiment with cane-
sugar could decide this question. My stock solution of
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cane-sugar was a 2n solution, while my NaCl solution was
24n. On account of the electrolytic dissociation, more than
30 c.c. of the cane-sugar solution were required to produce
the same increase of osmotic pressure as by 15 c.c. of the
24n NaCl solution. The following solutions were tried:
(1) 40 cc. 2n cane-sugar-+ 60 c.c. sea-water
(2) 20 (73 “ + 80 “
(8) 10 T3 (73 + 90 c
(4) 10 24nKCl +90 «
(5) Normal sea-water (control)
The eggs remained fifty-five minutes in these solutions.
Eight hours later swimming ciliated trochophores were found
in the eggs that had been in solutions 1 and 4. In 2 and 3
there were no swimming larve. In the control material all °
the eggs were still spherical and unsegmented. The next
morning about 25 per cent. of the eggs that had been in
solution 1 swam about in the most lively manner. |
ve
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FIG, 158
ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 671
on the right side the development of the unfertilized eggs.
At about the same time a drawing of the fertilized and
the unfertilized K eggs was made. At 12:45 some of the
fertilized eggs were found in the four- to eight-cell stage.
The unfertilized eggs were only amceboid at that time.
Some of them (see Fig. 158) at 12:55 showed an incision, as
if they were about to divide. At 1:20 some of the fertilized
eggs had reached the sixteen-cell stage, and at 1:30 only afew
eggs were found among the K eggs that seemed to be seg-
mented. At 2:10 the fertilized eggs were in an advanced
stage of cell-division, while the K eggs were not distinctly
segmented. At 3:40 the fertilized eggs had reached the
trochophore stage, with a clear edge and a dark center. At
that time the most differentiated eggs of the parthenogenetic
lot were in the condition that is represented at 3:15 in Fig.
158. At 4:30 we find these eggs still in the same condition,
and not until 7:40 did the parthenogenetic eggs reach the
beginning of the trochophore stage—clear edge and dark
center (Fig. 158). The fertilized eggs had formed their
cilia, and at about 5 o’clock were swimming around, while
the K eggs did not begin to swim until 8 or 9 o’clock. The
unfertilized control eggs which had remained in normal sea-
water during this time were at 8 o'clock still absolutely
spherical, and had given no signs of development or change.
Although the drawings in Fig. 158 give an idea of the
development of the parthenogenetic eggs, this idea has to be
supplemented by the statement that not all the eggs behaved
like those drawn. The majority of parthenogenetic eggs
never showed any higher degree of differentiation during
their development than those drawn in Fig. 157; many eggs
even remained spherical. The number of trochophores was
always considerably larger than the number of eggs that
became amceboid. The majority of parthenogenetic trocho-
phores are perfectly spherical. I have often wondered
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whether it was possible for the unfertilized K eggs to reach
the trochophore stage without any visible external signs of
cleavage.’ I shall have to postpone a definite answer to
this question until next year.
Another point worth mentioning is the fact that phenomena
‘of cleavage seem to be reversible in this form, inasmuch as
an egg divides
into two spheres
which very soon
fuse again. Such
8.041 8051 changes, which
occur very sud-
denly, may be
occasionally ob-
served inunfertil-
ized Cheetopterus
8.05} 8.06} eggs. Fig. 159
shows the succes-
sive stages which
were observed in
one egg within
er four minutes. I
had watched these
lively changes for several minutes before I decided to draw
them. The egg had been for an hour in a mixture of 95 ¢.c.
sea-water-+5 c.c. 24n NaCl,and had been back in sea-water for
eight hours. When I began to draw the egg, it had the appear-
ance of being in the two-cell stage (Fig. 159, 8:04). Ten
seconds later it changed suddenly into a three-cell stage, the
upper sphere breaking into two cells (8:044). A few seconds
after this the lower sphere began to flow into the right upper
sphere (8:05), and at 8:054 it had disappeared completely.
The egg was again in the two-cell stage (8:05). Then the
1 Professor F, Lillie in the following year confirmed this suggestion. [1903]
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ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 673
two spheres fused, and a small sphere or droplet appeared
above (8:054). This disappeared almost immediately, and a
new little droplet broke loose at the right lower side of the
egg (8:06). It disappeared in a few seconds, and the egg
once more divided, but with an altogether different position
of the cleavage plane (8:064, 8:074). In a few seconds
the two spheres fused into one cell, and
a number of small droplets appeared
below (8:08). Of course it is impos- 7 77
sible to tell whether or not these single
spheres or droplets contained nuclei.
These phenomena are of importance for
the mechanics of development, inasmuch as they show that the
bulk of the egg is liquid, and that in the case of Cheetopterus
its viscosity is very small, and less than in the case of the
sea-urchin’s egg. It is hard to understand what kind of
structure could be preformed in a liquid mass of such low
degree of viscosity beyond the differentiation into nuclear
and protoplasmic material and possibly centrosomes.
The appearance of the trochophores originating from un-
fertilized eggs is exactly like that of those arising from fer-
tilized eggs, if one compares equal stages of development.
Fig. 158 gives no good idea of the trochophore, inasmuca
as the latter is at first spherical. Fig. 160 shows two
parthenogenetic trochophores, drawn by the camera with
the exception of the cilia, which are more or less diagram-
matic. The eggs from which these trochophores originated
had been treated with KCl. It is hardly necessary to men-
tion that the appearance of the trochophores developing
from parthenogenetic eggs depends greatly upon the treat-
ment the egg had received. I mentioned this point in
connection with the artificial parthenogenesis of sea-urchins.
A point which must be discussed is the duration of
life of the parthenogenetic trochophores. All the Che-
FIG. 160
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674 STUDIES IN GENERAL PHYSIOLOGY
topterus larve, those that developed from fertilized eggs as
well as those that developed from unfertilized eggs, died
after two days. As the fertilized eggs developed faster
than the unfertilized eggs, the trochophores that had devel-
oped from the former eggs were in a more advanced stage
at the time of death than the parthenogenetic trochophores.
But to judge from the energy of their motion, the vitality
of the parthenogenetic trochophores equaled that of the
trochophores emanating from fertilized eggs.’ The cause of
death was apparently the development of micro-organisms
in the poorly aerated culture dishes. The parthenogenetic
larvee of Arbacia lived, under similarly unfavorable con-
ditions, as long as ten days.
VI. ON THE EFFECT OF VARIOUS IONS ON THE ARTIFICIAL
PRODUCTION OF PARTHENOGENETIC GIANT AND DWARF
EMBRYOS IN ARBACIA AND CHETOPTERUS
In a former paper on the artificial parthenogenesis of
sea-urchins I have mentioned the fact that as a rule more
than one embryo originates from one egg.” It was not un-
usual to see 3, 4, or even 6 blastule arise from one egg. Of
course each of these embryos was smaller than the normal
embryo of Arbacia in which the whole mass is utilized for
one embryo. In my first experiments I had caused the
parthenogenetic development of the eggs of Arbacia by
raising the osmotic pressure of the sea-water through the
addition of MgCl,. I have since found that it depends
upon the nature of the substance which is added to the sea-
water whether the parthenogenetic larvee are dwarfs or of
normal size. If the unfertilized eggs of Arbacia are put
1JIn the following year I found that the vitality of these parthenogenetic
larves is considerably lower than that of the larvees which came from fertilized
eggs. [1903]
2 Part II, p. 576.
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ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 675
into sea-water whose osmotic pressure has been raised by the
addition of KCl (e. g., 88 c.c. sea-water + 12 cc. 24n KCI),
and if after two hours they are put back into normal sea-
water, they will develop into swimming larvae. In this case,
as a rule, only one embryo develops from an egg, and dwarf
larvee are an exception. If, however, instead of KCl the
corresponding quantity of NaCl or MgCl, is added to the
sea-water, as a rule more than one embryo originates from
one egg, and larve of normal size are rare. I have not
made many experiments with CaCl,, but it seems to act
more like KCl than like NaCl. In the experiments in which
the osmotic pressure of the sea-water was raised by cane-
sugar, dwarf blastule were also observed.
I have already mentioned in an earlier paper that the
lack of a membrane favors the origin of more than one
embryo from the unfertilized egg. The fertilized egg has a
membrane which keeps the cleavage cells together. But if
the membrane be destroyed, the egg may give rise to more
than one embryo. In a small number of unfertilized eggs
the treatment with KCl gives rise to a very thin film, which
may act as a membrane and prevent the cleavage cells from
becoming separated. But such a fine film is lacking in the
majority of eggs treated with KCl (or CaCl,) in the right
proportions to produce parthenogenesis. And yet we do not
notice the falling apart of cleavage cells which in the case of
the NaCl eggs or MgCl, eggs leads to the formation of more
than one embryo from an unfertilized sea-urchin’s eggs.
The observation of the process of cleavage shows that the
treatment of the eggs with KCl increases their power of
adhesion. The various cleavage cells of a K egg stick to-
gether, while after a treatment with NaCl the cleavage cells
adhere much less to one another and fall apart. The same
tendency is produced by the addition of MgCl, to sea-water.
It is quite possible that the relative amount of the various
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676 STUDIES IN GENERAL PHYSIOLOGY
ions influences the degree of agglutination in the cleavage
cells. Herbst has observed that in sea-water without Ca the
cleavage cells of fertilized eggs show a tendency to fall
apart.’
It was to be expected that if KCl makes the cells of the
same egg stick together, it might also cause several eggs to
agglutinate. We know, from the experiments of Driesch?’
and Morgan’® on the eggs of sea-urchins and of Zur Strassen‘
on the eggs of Ascaris, that if two eggs stick together they
may give rise to a single embryo of larger dimensions. I
have never observed giant embryos in the parthenogenetic
eggs of sea-urchins. But I have seen them in almost every
experiment in which the Chetopterus eggs had been treated
with potassium. In such cases often two or more eggs would
stick together, and the result was either two or more trocho-
phores grown together or a single giant embryo of twice or
three times the mass of a normal trochophore. Of course
there were all kinds of transitions between the two extremes.
The formation of one giant embryo through the fusion of
two or more eggs is the more remarkable as the Chetopterus
eggs possess a membrane even in the unfertilized condition.
This membrane is evidently liquefied at the point of contact
of two eggs. This agglutination caused by K is not only
noticeable in unfertilized but also in fertilized eggs of Che-
topterus. Fig. 161 shows a number of trochophores which
originated from agglutinating fertilized eggs of Chetop-
terus. All these and many other specimens of this kind
were found in a few drops of the culture taken out with a
pipette. I have tried to make camera drawings of the
various types that occurred. The embryos were eight hours
old, and began to move. No. 1 (Fig. 161) is a trochophore
1 Hersst, Archiv fiir Entwickelungsmechanik, Vol. IX (1900), p. 424,
2 Driescu, ibid., Vol. X (1900), p. 411.
3 MorGAN, ibid., Vol. II (1895), p. 65.
4ZuR StRAssEn, ibid., Vol. VII (1898), p. 642.
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ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 677
developed from one egg; No. 2 shows two trochophores which
are grown together but are otherwise independent. In No.3
we notice the beginning of a common organization, inasmuch
as the clear peripheral areas (on the right side) are fused
together. In Nos. 4, 5, and 6 the clear areas are almost com-
FIG, 161
pletely fused together, and only the dark centers remain
separated. In No. 7 both eggs are fused completely and
form one giant embryo with one set of organs. Cases like
this are very frequent in the material treated with KCl.
Nos. 8 and 9 are examples of the fusion of more than two
eggs. I have seen four eggs form one giant embryo with one
common dark center and one common clear area. Such mon-
sters swam, but usually died sooner than the single embryos.
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The fact that the fusion of two eggs into one giant embryo
occurs so much more readily in Cheetopterus than in Arbacia
may be due to the difference in the viscosity of the two eggs,
The formation of one giant embryo from two eggs in
Cheetopterus is so very interesting for the reason that the
Cheetopterus egg possesses a characteristic cell-lineage. We
must conclude from this that the cell-lineage is either a sec-
ondary element in the formation of the embryo or that the
earlier processes of differentiation in the Chetopterus egg
are partly or wholly reversible (see section x).
I have made very few experiments with CaCl,, but in
these giant embryos were formed. Eggs that had been in a
solution of 90 c.c. sea-water + 10 c.c. 5n CaCl, for one hour
gave rise to a number of giant embryos. A sure way to
produce giant embryos in Cheetopterus is to put the unfertil-
ized eggs for about one hour into a mixture of 97 c.c. sea-
water +3 c.c. 24n KCL
I have occasionally, but very rarely, found that the fertil-
ized eggs of Cheetopterus show agglutination in normal sea-
water. The same phenomenon seems to occur in the eggs of
Ascaris, according to Zur Strassen.!
Dwarf embryos are rarely found in Chetopterus. I have
found them in the experiments with HCl. Perhaps the
existence of a membrane prevents the unfertilized eggs of
Cheetopterus from forming dwarf embryos as easily as the
unfertilized eggs of the sea-urchins.
VII. ON DIFFERENCES BETWEEN THE ARTIFICIAL PARTHENO-
GENESIS OF ECHINODERMS AND CHETOPTERUS AND THE
POSSIBILITY OF A HYBRIDIZATION BETWEEN THE TWO
It is impossible to hybridize Arbacia and Cheetopterus in
normal sea-water. I have tried a number of experiments
with negative results, as was to be expected. The negative
1ZuR STRASSEN, loc. cit.
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ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 679
result may be due to the impossibility of the spermatozoon
of the one species entering the egg of the second species, or
to the fact that the spermatozoon of Cheetopterus brings
about the development of the Chetopterus egg by substances
which are ineffective in the Arbacia egg, and vice versa, or
the spermatozoon of the one species is poisonous for the egg
of the other species, or vice versa.’ The second possibility
is of interest to us on account of the fact that we can bring
about the parthenogenetic development of the Cheetopterus
eggs by means which have no effect upon the Arbacia egg.
When we intend to produce artificial parthenogenesis in
the eggs of Echinoderms, it is only necessary to put them for
from one and one-half to two hours in sea-water, the osmotic
pressure of which has been raised about 374 to 75 per cent. ;
that is, into sea-water to which has been added 124 to 25 per
cent. of its volume of a 24” NaCl solution or of a solution
isosmotic with the latter. We have not yet determined the
osmotic pressure of the sea-water at Woods Hole, and on
indirect data assume that it is about isosmotic with a 3n
NaCl solution. The optimal increase of osmotic pressure
varies for different species and even for different females of
the same species. It may be that the temperature of the
water and the degree of maturity of the eggs play a rdle.
In making experiments of this kind, it is necessary to use
always a series of solutions of different osmotic pressure and
to take the eggs out at various intervals, from one-half to two
hours or more, until the optimum concentration and time
have been ascertained.
An increase in the osmotic pressure of the sea-water is
also able to cause artificial parthenogenesis in Chetopterus.
The chief difference between the Chetopterus and Arbacia
eggs is that at the same temperature the Chetopterus eggs
1Certain constituents of the blood (globulins, enzymes?) frequently destroy the
blood corpuscles of other species that are not closely related.
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680 STUDIES IN GENERAL PHYSIOLOGY
do not need to stay so long in the more concentrated solution
as the eggs of Arbacia.
Although in this regard the difference between Chetop-
terus and Arbacia is slight, a very striking difference exists
in regard to the specific effects of K ions upon the develop-
ment. While a pure KCl solution of lower osmotic pressure
than sea-water, or sea-water with a slight increase of K, e.g.,
a mixture of 98 c.c. sea-water + 2 c.c. 24” KCl, causes the
parthenogenetic development of the eggs of Cheetopterus that
have been exposed to such a solution only a few minutes,
such solutions are without any effect upon the unfertilized
eggs of sea-urchins (Arbacia). I left the unfertilized eggs
of Arbacia repeatedly in a mixture of 98 ¢.c. sea-water + 2
c.c. 24n KCl or 97 ¢.c. sea-water + 3 ¢.c. 24n KCl for from
three minutes to twenty-four hours without any develop-
ment following, with the exception of a few eggs that reached
the two-cell stage after about twenty hours. But this hap-
pens just as well in normal sea-water.
As far as the Arbacia eggs are concerned, I can only state
that if we increase the osmotic pressure of the sea-water by
adding KCI, a slightly smaller increase in the osmotic pres-
sure is required to bring about the parthenogenetic develop-
ment than if we add NaCl. I found regularly that while
90 c.c. sea-water + 10 c.c. 24n KCl sufficed to cause a great
many eggs to reach the blastula stage, a mixture of 90 c.c.
sea-water + 10 c.c. 24m NaCl was practically ineffective. I
had to take 874 c.c. sea-water +124 c.c. 2in NaCl. It is,
however, possible, that this difference is only apparent. As
the sea-water consists chiefly of NaCl, the addition of 10 cc.
of a 24 NaCl to 90 ¢.c. sea-water will increase the osmotic
pressure of the sea-water less than the addition of 10 e.c. of
a 24n KCl solution, as the degree of dissociation is less if
the concentration is higher. Further experiments with pure
NaCl and KCl solutions will have to decide whether the dif-
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ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 681
ference in the degree of dissociation is responsible for the
result. A second typical difference between the Arbacia
egg and the Chetopterus egg consists in the fact that the
latter can be caused to develop by a small addition of HCl
to sea-water. Any other inorganic acid would probably act
in the same way, as the addition of a small amount of Cl ions
has no such effect. This small addition of acid diminishes
or neutralizes the alkalinity of the sea-water, but I have
failed to test whether the latter is rendered acid.
The same treatment does not cause the Arbacia eggs to
develop beyond the two- or four-cell stage, even if they are
left in the solution for twenty-four hours. I have made a
number of new experiments this summer, but I have only
been able to confirm the experiments mentioned in a former
paper.’
I have pointed out that the experiments on artificial par-
thenogenesis force us to assume that the influence of the
spermatozoon upon the development and the transmission of
the qualities of the male depend upon different constituents
of the spermatozoon. On the basis of this assumption the
possibility of a successful hybridization between animals as
far apart as Worms and Echinoderms might be considered.
If we could cause the egg of Cheetopterus to develop by
treating it with KCl and at the same time force the sperma-
tozoon of an Arbacia (or a similarly distant animal) to enter
into the egg, we might carry Echinoderm qualities into an
Annelid egg.’ But in all my attempts at thus crossing the
female Cheetopterus with the male Arbacia perfect trocho-
phores without Echinoderm characteristics resulted. Al-
though the problem may not be capable of solution in these
two forms, I think that the experiments on artificial par-
1Part II, p.576.
2Provided the spermatozoon of the Echinoderm contains no poison for the
Annelid egg.
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682 STUDIES IN GENERAL PHYSIOLOGY
thenogenesis will ultimately make hybridizations possible
which otherwise would be impossible. J intend to continue
these experiments.
VIII. PRELIMINARY EXPERIMENTS ON PHASCOLOSOMA, FUNDU-
LUS, GONIONEMUS, AND PODARKE
I will report briefly on experiments which I began but
was not able to finish, partly from lack of material and partly
from lack of time. My experiments on Phascolosoma were
carried further than the rest. I began with putting the
unfertilized eggs of this form in mixtures of 90 c.c. sea-water
+10 c.c. 24n KCl and leaving them in this solution from
thirty to one hundred and fifty minutes. I never saw an
egg reach the two-cell stage. Then stronger solutions were
tried, and now some of the eggs began to segment. When
the eggs were put into a mixture of about 30 c.c. 24n KCl
+70 c.c. sea-water for about thirty minutes, they reached
a thirty- to sixty-cell stage. The appearance of the eggs
was so good that possibly in a continuation of these experi-
ments parthenogenetic larve will be produced. In these
experiments I received valuable advice from Dr. Gerould of
Dartmouth College, who is thoroughly familiar with the
biology and embryology of this form.
In Fundulus, a teleost fish, I succeeded in causing the
unfertilized eggs to reach the two-cell stage, but lack of
material prevented my carrying the experiments further.
Inmy experiments on Gonionemus, a Medusa, I was assisted
by Dr. Murbach, who was kind enough to select the females for
me. Dr. Murbach had observed that by putting these animals
into the dark they can at any time be caused to lay eggs.
My attempts (four experiments) to cause artificial par-
thenogenesis in these eggs have failed. All I was able to
accomplish was to force the eggs to become amceboid and
creep about, but no segmentation occurred.
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ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 683
In Podarke, an Annelid, I succeeded in producing the
first segmentation in unfertilized eggs. I interrupted these
experiments to go on with experiments on Cheetopterus which
were much more promising.
IX. NATURAL AND ARTIFICIAL PARTHENOGENESIS
In a definite although very small number of animals each
egg possesses the quality to develop parthenogenetically.
Instances of this are to be found in the bees, social wasps,
Bombyx, Psyche, Daphnia, plant lice and others. In all
these animals the egg can be fertilized also by a spermato-
zoon. How does it happen that in these forms, although
fertilization may occur, the egg is, under certain conditions
at least, able to develop parthenogenetically? Our experi-
ments show, that if the constitution of the sea-water were
only slightly different, that is, if it contained a little more
K, Chetopterus would have to be added to the list of nor-
mally parthenogenetic animals. What I stated in my pre-
liminary report is certainly true for Chetopterus, namely,
that it is the constitution of the sea-water which prevents
many or certain forms from being “naturally” parthenoge-
netic. By reversing this statement we may say that in the
naturally parthenogenetic animals it may be due to the con-
stitution of the blood (or the sea-water?) that the egg can
develop without fertilization.
The bridge between the phenomena of natural and artifi-
cial parthenogenesis is formed by those animals in which
physical factors decide whether or not their eggs develop
parthenogenetically. In plant lice parthenogenesis is the
rule only as long as the temperature is high or the plant has
plenty of water. If we lower the temperature or let the
plant dry out, sexual reproduction occurs. The drying out
of the plant causes the tissues of the lice to lose water. The
factor, loss of water, makes the artificial parthenogenesis of
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684 STUDIES IN GENERAL PHYSIOLOGY
Echinoderms and Chetopterus possible. In plant lice the
effect is of the same kind, only in the opposite direction.
I have read somewhere the statement that Artemia salina
is parthenogenetic, while Branchipus is not. Branchipus is
a fresh-water Crustacean which, if raised in concentrated
salt solutions (salt lakes), becomes smaller and undergoes
some other changes. In that case it is called Artemia. If
Artemia is parthenogenetic while Branchipus is not, it would
mean that the unfertilized eggs of the Branchipus cannot
develop in fresh water, while they are able to develop in
solutions of much higher osmotic pressure. This would be
identical with our observation on the artificial parthenogenesis
of Echinoderms and Cheetopterus.
As I have mentioned in a former paper, O. Hertwig makes
the statement that the unfertilized eggs of a number of marine
animals which deposit their eggs in sea-water begin to de-
velop after a number of hours, but do not develop beyond the
first cleavage stages. Arbacia eggs reach the two-cell stage
in about twenty hours; the egg of Cheetopterus may develop
as far as twelve or sixteen cells. According to Hertwig, not
only the eggs of Annelids and Echinoderms, but also those
of certain Crustaceans show this peculiarity. I have men-
tioned in a former paper the observation made by Janosik
that in the ovary of mammals occasionally eggs are found in
the process of cell-division. We shall make use of these
facts in the next section.
I finally wish to say a few words concerning experiments
published by Mr. Viguier of Africa, who maintains that the
eggs of Arbacia, Toxopneustes, and other sea-urchins are
naturally parthenogenetic.’ It would contradict neither my
experiments nor my views if his statement were correct, as
in all my papers I have assumed that these and many other
(if not all) eggs have a tendency to develop parthenogeneti-
1 VIGUIER, Comptes rendus de l’ Académie des Sciences, Paris, July 2, 1900.
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ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 685
cally, and that it is only due to the constitution of the sea-
water (or blood?) if they do not do so under natural con-
ditions.’ It might be that the constitution of the sea-water
at Algiers differs from that of the rest of the world, and
allows the eggs of the sea-urchin to develop parthenogenetic-
ally. The experiments of Mr. Viguier are, however, not
of such a character as to make this probable. They are few
in number, and he seems to have omitted no possibility
which could further the contamination of his eggs by sper-
matozoa. He always handled males and females together,
and opened males and females in the same experiment. No
mention is made of a sterilization of his hands or instruments.
Whenever males and females are in the same dish there is
danger that the water may be full of spermatozoa, especially if
the material is fresh. The sperm sticks to the surface of the
females and it is absolutely impossible to avoid fertilization
of the eggs. To be sure, Viguier mentions a precaution he
took, but this precaution shows that he is not familiar with
the methods of sterilization or disinfection. He washed the
females off in filtered sea-water. As everybody knows, the
spermatozoa go through filter paper, and, in addition, sea-
water does not remove the spermatozoa from the surface of
the female, for the latter stick to solid bodies, as Dewitz has
proved. In order to avoid this source of infection I washed
the surface of the female several minutes in distilled water, or
under a powerful stream of fresh water which kills the sper-
matozoa. Ihave in my former papers given a description of
the precautions necessary in experiments on parthenogene-
sis. These were by no means exaggerated if one wished to
guard absolutely against contamination. I did not even
succeed in excluding contamination by spermatozoa in my first
Cheetopterus experiment (see p. 649), although my precautions
were vastly superior to those taken by Viguier.
1 Part IT, p. 539,
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Another surprising fact in Viguier’s paper is that he does
not mention. whether or not his unfertilized eggs had a
membrane. In my researches on Arbacia I have considered
the lack or presence of a membrane the most important
criterion for deciding whether the development of the eggs
is due to the entrance of a spermatozoon or to the osmotic or
chemical treatment they have received. The fertilized eggs
form a thick membrane, while the unfertilized eggs generally
have no membrane (unless treated with certain salts in exces-
sive quantities and for a long time). The cleavage of the
parthenogenetic egg that has no membrane differs so radi-
cally from that of the fertilized egg within a membrane, that
it must arouse the interest or surprise of any morphologist.
These differences are most noticeable during the first hours
of the development. As soon as the egg approaches the
blastula stage the membrane very often begins to disintegrate.
Ido not think that any experienced observer would have
dared to publish the statement that the unfertilized eggs of
Arbacia reach the pluteus stage, without having convinced
himself that the “unfertilized” eggs had no membranes.!
Mr. Viguier makes the statement that he tried to repeat
my experiments but was not able to confirm them. This
does not surprise me, as he had not read my papers, and as
he did not even know how my solutions had been prepared.
My experiments have been repeated and confirmed by the
following authors: Dr. C. Herbst (Naples), Professor E. B.
Wilson (Columbia University), Dr. Hans Winkler (Tibin-
gen), and Dr. 8. Prowazek (Prague), and partly by Professor
A. Giard (Paris). In addition they were repeated with
success by all the members of the class in physiology and
embryolgy at Woods Hole last summer. As far as the state-
ment is concerned that the unfertilized eggs of Arbacia or
1Viguier’s paper has been criticised by A. GIARD, Comptes rendus de la Société
de Biologie, Vol. LII (1900), p. 761.
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ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 687
Strongylocentrotus are able to develop into plutei in normal
sea-water, I can say that this is most certainly not the case
at Woods Hole, in California (according to my own very
numerous observations), in Beaufort, N. C., and at Naples
and other places on the Mediterranean, that have been
visited by competent experimenters.
X. THE BEARING OF ARTIFICIAL PARTHENOGENESIS ON THE
THEORY OF FERTILIZATION AND OF LIFE PHENOMENA IN
GENERAL
The general opinion concerning the role of the sper-
matozoon in the process of fertilization is that it acts as a
stimulus, and that as such it starts the development of the
egg. This statement is certainly wrong for those eggs in
which we have been able to produce artificial partheno-
genesis. For these eggs, like many others, begin to segment
without any spermatozoon, if they are left long enoughin normal
sea-water. The only difference between these and the fertil-
ized eggs)is that the former begin to segment much later and
their development stops in the early segmentation stages (two
to sixteen cells at the most). The latter may be due to the
fact that the egg dies before it has time to develop further.
If we consider the fact that the eggs show at least a be-
ginning of a segmentation under ‘“‘normal” conditions, the
act of fertilization assumes a different aspect. The sper-
matozoon can no longer be considered the cause or the stimu-
lus for the process of development, but merely an agency
which accelerates a process that is able to start without i,
only much more slowly. Substances that accelerate chemical
or physical processes which would occur without them are
called catalyzers (Ostwald). According to this definition we
may assume that the spermatozoon carries a catalytic sub-
stance into the egg, which accelerates the process that would
start anyhow, but much more slowly.
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Through these facts and conceptions the phenomena of
artificial parthenogenesis assume a different aspect. It
would be wrong to say that the K ions are the stimulus that
causes the developmental process. They merely act as
catalyzers, accelerating a process that would otherwise
proceed too slowly. The loss of water on the part of the egg
cell must have a similar effect, but possibly a less direct one.
It may be that the loss of water alters the chemical processes
in the egg in such a way as to give rise to the formation of
a substance which acts catalytically.
Whether or not the catalytic substances introduced by
the spermatozoon are identical with those employed in my
experiments, I cannot say. I consider it probable that in
the case of Chetopterus the natural fertilization is not
brought about by K ions, inasmuch as the normal develop-
ment does not show the characteristics of a treatment of the
eggs with K.
I have made a series of experiments with various enzymes
to bring about the development of the unfertilized eggs of
Arbacia, thus far without any results. The only enzyme
that caused the egg to segment at all was papain. But
I cannot be certain whether this was not due to some acci-
dental constituent of the enzyme preparation used. The
other enzymes were absolutely without effect. If we wish to
find the active principle in the spermatozoon, we must make
experiments in the direction of those begun by Winkler.’
This author used extracts of the spermatozoon, and found
that such extracts caused the eggs of sea-urchins to reach
the two- or four-cell stage. As such a result can be brought
about by slight alterations in the osmotic pressure or con-
stitution of the sea-water, and as such alterations occurred
in Winkler’s experiment, I am not yet certain that these
1 WINKLER, Nachrichten der kiniglichen Gesellschaft der Wissenschaften, Got-
tingen, 1200.
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ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 689
results were actually due to the substances extracted from
the spermatozoon. But his experiments are certainly in the
right direction.
The idea that the spermatozoon and the substances which
cause parthenogenesis act only catalytically, has a great
bearing upon the theory of life phenomena. It means that
if we accelerate the processes of cell-division in the mature
egg (by specific catalyzers) the egg can live; but if these
processes occur too slowly at the ordinary temperature (as is
the case in the unfertilized egg in normal sea-water), the
egg dies. The introduction of the catalytic substances which
accelerate the processes of development saves the life of the
egg. This may be made intelligible on the following
assumption. Two kinds of processes are going on in the
mature egg after it has left the ovary. The one leads to the
formation of substances which kill the egg; the other leads
to the formation of substances which allow growth and cell-
division, and are not poisonous. We may use as an illustra-
tion Pasteur’s well-known experiments on the behavior of
yeast cells in the presence and absence of atmospheric
oxygen. In the presence of oxygen the yeast cells multiply
on a sugar solution, while the zymase effect is comparatively
small. In the absence of oxygen the multiplication of cells
is limited or may stop, while the zymase effect becomes more
prominent. The products of alcoholic fermentation are
comparatively harmless for the yeast cell, and for this reason
an increase in the fermentative activity of the cell does not
cause the death of the yeast. I imagine that matters are
similar in the mature egg cell after it has left the ovary,
with this difference, perhaps, that the substances formed (by
fermentation?) in the egg cell are more poisonous for the
egg than the alcohol and the other products of fermentation
are for the yeast. The process that causes the death of the
egg cell and the one that causes cell-division are at least
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690 STUDIES IN GENERAL PHYSIOLOGY
partly antagonistic. They are both inhibited by a low tem-
perature, so that in this case death does not occur, although
no cell-division is possible. If we succeed in finding a sub-
stance which accelerates the process of cell-division at the
normal temperature, this will at the same time lead toa
suppression or a reduction of the antagonistic process that
shortens life. In the case of the egg of Cheetopterus a trace
of K ions acts as such a catalytic substance; possibly a trace
of H ions; and perhaps certain substances that are formed
when the egg loses a certain amount of water. For the
Echinoderm egg we know at present only the last factor. In
addition there are the catalytic substances carried or pro-
duced by the spermatozoon (ions? enzymes?). But there are
certainly other catalytic substances, as is proved by tumors
and galls, in which the variety of structures corresponds to
an almost equal variety of parasites.’
It is very important to realize that the introduction of
catalytic substances into the egg does not. prolong its life
unless the egg has reached a critical point determined by
two sets of conditions. The one is the maturity of the egg,
the other the change of conditions connected with the egg
leaving the ovary. As long as the egg is immature it lives
without the introduction of these substances or the sperma-
tozoon, and this may be true for the mature egg as long as
it remains in the ovary. The fact that there is an age limit
for thedevelopmentof carcinoma may bea similar phenomenon.
The catalytic substances which are givenoff by thecancer para-
sitemay not be able to bring about cell-division in the epithelial
cells unless the latter have reached a critical point, which is
at least partly determined by the age of the individual.
1We do not need to assume a specific parasite for each kind of tumor. Tera-
tomas may be explained on the basis of the parthenogenetic tendency of the
mammalian egg in connection with some chemical change that furnishes the
catalytic substance. But it is not impossible that even in benign tumors, such as a
teratoma, the catalytic substance may be due to parasitic organisms.
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ARTIFICIAL PARTHENOGENESIS IN ANNELIDS 691
We generally consider development as a process which
can only occur in one direction, or, in other words, is irre-
versible. But this is certainly not generally the case. I
showed in a recent paper that the morphogenetic processes
in Hydroids are reversible. If the polyp of a Campanularia
is brought in contact with a solid body, it is transformed into
undifferentiated material and later into a stolon. If the
same organ is brought in contact with sea-water, it gives rise
toa polyp again.’ The same may be done with Margelis
and other Hydroids. In Antennularia a change in the
orientation of a branch with polyps will bring about the
transformation of this material into a stolon. Between the
two phases the material must pass through an undifferentiated
stage where it is neither polyp nor stolon. It will be the
task to determine how far in the animal kingdom the develop-
mental processes are found to be reversible. It is obvious
that in a form with a reversible development death will not
necessarily follow a certain stage of development (corre-
sponding to senility in man).
It is not impossible that ‘“‘natural” death is comparable
to the situation which is present in the mature egg after it
leaves the ovary. Nature has shown us the way by which
at this critical point death can be avoided in the case of the
egg.
1Part II, p. 627.
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XXXIV
ON AN APPARENTLY NEW FORM OF ABNORMAL IRRI-
TABILITY (CONTACT-IRRITABILITY ?) PRODUCED
BY SOLUTIONS OF SALTS (PREFERABLY SODIUM
SALTS) WHOSE ANIONS ARE LIABLE TO FORM
INSOLUBLE CALCIUM COMPOUNDS!
I. INTRODUCTION
A sERIES of papers published from my laboratory has
furnished the proof that the rhythmical contractions of
striped muscles, the swimming bell of jelly-fish, the heart
and the lymph hearts depend upon the presence of Na ions
in the surrounding solution. Calcium ions have a tendency
to diminish or inhibit the contractions altogether, although
a small number of them must exist in the tissues in order to
preserve contractility.” This point having been settled, I
next tried whether the sodium ions bring about these effects
directly or indirectly. I have not finished these researches
so far as the rhythmical contractions of the muscle are con-
cerned, but in pursuing this problem I have found a number
of facts which show that certain salts can bring about effects
indirectly by giving the muscle or nerve properties which
they do not possess normally and which to my knowledge
have not yet been described. If we put a fresh muscle
(gastrocnemius) of a frog for a short time (e. g., one to three
minutes) into a solution of a sodium salt whose anion is liable
to form insoluble calcium compounds (e. g., NaF], Na,CO,,
Na, HPO,, sodium oxalate, sodium citrate, etc.), the muscle
will as a rule not show any reaction except perhaps a slight
1American Journal of Physiology, Vol. V (1901), p. 362.
2Tt is possible that certain other ions may act as a substitute for the Ca ions for
this purpose.
692
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ABNORMAL IRRITABILITY PRODUCED By Sauts 693
shortening. But as soon as it is taken out of the solution
and comes in contact with air, it goes into tetanus or per-
forms a series of powerful contractions.
The tetanus or the
contractions cease at once and relaxation of the muscle
occurs when the muscle is put back into the solution.
It was found that not only the change of contact from
the above-mentioned solutions to air but
also toa number of other media produce "
these contractions. What the nature of
the stimulus in this case is I cannot say
definitely. Provisionally I will assume
that we are dealing with contact-irrita-
bility and I will call the above-mentioned
reaction of the muscle the contact-reac-
tion. It would seem as though the en-
trance of the anions of the above-men-
tioned solutions caused a change in the
superficial layer of the muscle or its
individual fibers, either by precipitating
calcium or by otherwise altering the con-
stitution of the protoplasm. This change
is intensified by the increase in Na ions
al
|
7
FIG, 162
in the same layer. In this condition the muscle is sensitive
to the nature of the substance with which it comes in contact.
In these experiments one end of the gastrocnemius of a
frog is tied to a glass rod, G (Fig. 162), and the other end
is tied to the lever, L. A dish, D, containing the solution
is raised from below when we wish to submerge the muscle,
and is lowered when we wish to bring the muscle into con-
tact with air.
In order to demonstrate the contact-irritability I used a
solution of 1 gram-molecule of sodium fluoride or sodium
citrate, etc., in about 8 or 10 liters. If the fresh gastrocne-
mius of a frog be put into such a solution for about one
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minute, the muscle will show a slight contraction when
taken out of the solution. If the process be repeated, a
stronger contraction will follow when the muscle is removed,
and after a series of submersions have occurred the muscle
will give one or a series of powerful contractions every time
it is taken out of the solution and brought into contact with
air. After a certain time, which may be an hour or more,
and which varies according to the solution, the reaction
becomes weaker and finally ceases.
If we use a stronger concentration than 1 gram-molecule
in 8 liters, we get more powerful contractions, but the irri-
tability of the muscle disappears sooner.
II. THE NATURE OF THE SOLUTIONS WHICH PRODUCE CONTACT-
IRRITABILITY IN MUSCLE
Solutions of cane-sugar or urea were unable to produce
the contact-reaction in muscle. I have tried these solutions
in all concentrations from 0 to normal or even 2n. A large
number of electrolytes were then tested. None of the salts
of Li, K, Ca, Mg, and NH, gave rise to the contact-reaction.
This statement is based upon experiments with LiCl, Li,SO,,
Li,CO,, KCl, K citrate, K oxalate, MgCl,, MgSO,, NH,Cl,
(NH,),CO,, and ammonium citrate. The degree of dilu-
tion used was as a rule 1 gram-molecule in about 8 or 10
liters. In some instances stronger solutions were tried, but
with the same negative result.
In my experiments on rhythmical contractions I have
shown that the sodium ions have a specific réle in the pro-
duction of these contractions. It seemed also possible that
they play such a réle in the production of the contact-irrita-
bility. But I found that or even stronger solutions of
8
NaCl, NaBr, NaI, NaNO, did not bring about the contact-
1Zoethout showed later in my laboratory that the addition of a trace of potas-
sium citrate to the sodium-citrate solution facilitates the production of contact-
irritability. [1903]
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irritability ; neither did sodium acetate nor other salts whose
anions form soluble calcium compounds.
But the sodium salts whose anions precipitate calcium
promptly produce these reactions. NaF, Na,CO,, Na,HPO,
sodium oxalate, sodium citrate,’ sodium tartrate give the
contact reaction in a dilution of 1 gram-molecule in 8 or 10
liters of water or even less. NaHCO, gives the reaction but
requires a higher concentration, e. g., 1 gram-molecule in 4
to 5 liters of water. If we put the muscle into a solution of
Na,PO,, it goes at once into a powerful tetanus. This
tetanus may be partly or wholly due to the high concentra- .
tion of HO ions in this solution. When a muscle goes into
tetanus in a solution, we cannot, as a rule, demonstrate the
contact-reaction. Thus I have never succeeded in producing
contact-reaction by a Na,PO, solution. NaH,PO, does
not cause contact-irritability, but this is in harmony with
our general result.
The HO and H ions deserve special attention. In my
experiments on rhythmical contractions I found that while
they are not able to produce rhythmical contractions directly,
they accelerate the beginning of these contractions in the
presence of Naions. In addition to such a catalytic action
common to both the HO and H ions, the former have another
effect which they do not share with the H ions. The muscle
produces constantly H,CO, and possibly other acids. These
acids will increase the solubility of Ca salts and increase
the number of Ca ions in the tissues. An addition of HO ions
will counteract this effect.
It is due to the presence of free HO ions that solutions
of Na valerianate and Na formate give rise to a slight degree
of contact irritability in muscle, although calcium formate
and calcium valerianate are soluble. If we diminish the
1The citrates require an alkaline reaction for the precipitation of calcium
This condition is fulfilled in the fresh normal muscle.
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alkalinity of a sodium formate and sodium valerianate solu-
tion by adding a small amount of free formic or valerianic
acid (without, however, rendering the solution entirely
neutral) they no longer produce the contact-irritability in
muscle. A small amount of alkali added to a NaCl solu-
tion may or may not produce a slight degree of contact-
irritability.
The solubility of CaSO, is comparatively high, and we
therefore cannot expect Na,SO, to be very effective for the
production of contact-irritability. In solutions of 1 gram-
molecule Na,SO, in 10 liters or less, I sometimes got and
sometimes failed to get the contact-reaction. May it not be
possible that the amount of free Ca ions in the muscle of
a frog varies at different periods of the year, and may not
this fact account for the seasonal variation in the irritability
of these animals? But if a Na,SO, solution fail to produce
contact-irritability in a muscle an addition of some HO ions
will produced the desired effect. As arule 4 cc. .% LiHO
or any other hydrate to 100 c.c. of the Na,SO, solution is
the optimum. We can produce the contact-reaction also
through the addition of a small amount of acid to the
Na,SO, solution, e. g., 4 c.c. of #", HNO, (or any other in-
organic acid) to 100 c.c. of the Na,SO, solution. The effects
are not so strong as if we add alkali.
The sulphates showed an exceptional behavior in still
another direction. With one exception only sodium salts
give rise to contact-irritability and this exception is a
sulphate, namely (NH,),SO,. It would almost seem that
the sulphates have physiological effects aside from their
effect upon calcium. This is in harmony with Miss Moore’s
experiments, in which she found that sulphates are as cap-
able of antagonizing the poisonous effects of a pure NaCl
solution as calcium salts.'
1 Moorz, American Journal of Physiology, Vol. V (1901), p. 87.
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It should finally be mentioned that sodium butyrate,
sodium succinate and sodium asparaginate did not produce
the contact-reaction.
Having thus proved that sodium salts, whose anions
precipitate calcium give rise to contact-irritability, it was to
be expected that solutions of calcium salts would prevent or
antagonize the contact-reaction. I found that by adding a
small amount of CaCl, toa Na-citrate solution the latter solu-
tion no longer produced the contact-reaction. The addition
of lec. of a 5n CaCl, solution to 100 cc. of an effective
sodium-citrate solution was sufficient to cause a muscle to
lose its contact-irritability at once. Only after a prolonged
stay in pure sodium-citrate solution does the contact-irrita-
bility return.
While all the facts thus seem to harmonize with the view
that a decrease in the amount of Ca ions in the tissues (and
possibly an increase in the amount of Na ions) is the essen-
tial condition for the production of the contact-reaction, it is
yet possible that the sodium salts whose anions form insoluble
calcium compounds may have a specific effect upon other
constituents of the protoplasm, e. g., proteids.
III. ON THE NATURE OF THE APPARENT CONTACT-REACTION
The reaction which we have provisionally called the con-
tact-reaction appears when a muscle, after having been sub-
merged in a sodium-citrate or any of the other above-
mentioned effective solutions, is brought into contact with air.
In this change from solution to air a number of conditions
change and it is now our task to determine which is the
essential one.
As soon as the muscle is taken out of the solution and
brought into air, more O, may diffuse into and more CO, may
diffuse from the muscle. These two conditions have, how-
ever, nothing to do with the reaction. The experiments
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were repeated in an almost pure atmosphere of CO,
instead of air and the contact-reaction was as powerful
as in air.
A second change is the sudden evaporation of water from
the surface of the muscle upon its leaving the solution.
The following experiment might suggest that this evapora-
tion is the cause of the contact-reaction. If we pack a
muscle, that gives powerful contact contractions, tightly in
moist filter paper the reaction will not occur when the muscle
is taken out of the solution, but will occur when the filter
paper is removed. Nevertheless, evaporation has nothing to
do with the reaction. We get the contact-reaction quite as
well in a moist chamber as in dry air. Furthermore we get
the reaction if we bring the muscle directly from the sodium-
citrate or fluoride solution into oil, without exposing it to
air. We can make this experiment in the following way.
The lower half of the dish, D (Fig. 162), is filled with the
effective sodium-citrate solution, the upper half with oil
(I used sperm and olive oil). The muscle is first brought
into the sodium-citrate solution and then, by lowering the
support 8, into the oil. Powerful contractions occur. Evap-
oration of water from the surface of the muscle is, there-
fore, not the cause of the contractions.
After this had been established it was to be expected that
changes in temperature were not responsible for the contact
reaction. Experiments in which the muscle was rapidly
cooled and heated yielded only negative results.
The next possible cause to be considered was electricity.
The fact that a change from the salt solution to a non-con-
ductor (air, oil) caused contractions suggested the possibility
that these contractions were in reality electrical break con-
tractions, the muscle itself acting as a battery. The only
fact which did not seem to accord with this explanation was
the lack of a make contraction when the muscle was put into
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the solution. A number of experiments excluded the assump-
tion that the contraction or tetanus of the muscle which
occurs when it leaves the sodium-citrate solution is due to a
break shock. I connected the two opposite ends of the
muscle by means of a thick copper wire. In this case the
muscle contracted just as powerfully as before when taken
out of the sodium-citrate solution, although no break shock
of any strength was possible. Another still more decisive
fact was found. After the muscle had been treated for some
time with a sodium-citrate solution the break contraction
could be produced by dipping the muscle for a short time,
e. g., thirty seconds, into a % or % solution of cane-sugar.
As soon as the muscle was brought into contact with air,
contractions occurred. The same was true for glycerin solu-
tions. Both the sugar and the glycerin solution are non-
conductors. The possibility of a mechanical stimulation as
the cause of the contact-reaction was next to be considered.
As long as the muscle is in the solution each of its elements
is under the hydrostatic pressure of the column of liquid
above it. If we expose the muscle to the air this pressure
ceases. This might suggest the idea that a decrease of the
hydrostatic pressure upon the muscle causes its contraction.
The dipping of the muscle into the solution causes a relaxa-
tion of the concentrated muscle, and the inference should be
drawn that an increase of the hydrostatic pressure causes
relaxation. The following experiments prove the erroneous-
ness of this view. The bottom of the dish was filled with
a liquid of much higher specific gravity than the sodium-
citrate solution, e. g., with chloroform, 2” cane-sugar solution,
or metallic mercury, and the sodium-citrate solution was put
carefully above the sugar solution or chloroform. The
muscle was then brought from the sodium-citrate solution
into the sugar solution by raising the dish D (Fig. 162). In
this case I noticed regularly one or more powerful contrac-
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tions, although the hydrostatic pressure on the surface of the
muscle was increased.
It thus seems to me that none of the known forms of
muscular irritability suffices to explain the phenomena
with which we are dealing. We have before us an appar-
ently new form of muscular irritability, probably contact-
irritability.
Contact-irritability is a very general form of irritability
among plants and lower animals. J need only to remind the
reader of the phenomena of stereotropism and of the fact
that by mere contact-effects a polyp of a campanularia can
be transformed into a stolon. But contact-irritability cer-
tainly exists among certain cells of vertebrates, for example,
the leucocytes. The nature of the body with which leuco-
cytes come into contact determines whether or not they give
off fibrin ferment and cause coagulation of the blood or other
liquids which contain fibrinogen. How the nature of the
contact can influence the leucocytes is still a mystery. One
might think of surface tension phenomena or the forma.ion
of double electric layers at the surfaces in contact.
If the phenomena described in this paper were really con-
tact-phenomena, a further search should reveal that only a
change of contact from certain bodies to other bodies can
cause contractions of the muscle.
I have begun experiments in this direction, and have thus
far found the following facts:
Contractions occur when the muscle passes:
; ( Air
{ Sodium-citrate solutions | CO,
| Sodium-fluoride solutions Oil :
From ; Sodium-oxalate solutions To ~ 2n sugar solution
| Sodium-carbonate solutions Glycerin
li etc. (see above) Chloroform
Toluol
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ABNORMAL IRRITABILITY PRODUCED By Satts TOL
Relaxation of the contracted muscle will occur when the
muscle passes from any medium in the right column above
to any medium in the left column.
After the muscle has been treated for some time with any
of the efficient solutions (Na citrate, etc.) the contractions are
also produced when the muscle passes:
From % or % sugar solution to air
From 3 or % glycerin to air
From any salt solution to air
A very interesting and theoretically important fact is that
the muscle loses this particular form of irritability very soon
when it remains in contact with the air, oil, sugar solution,
glycerin, or salt solutions different from those that produce
this specific irritability. In LiCl or NaCl solutions the
contact-irritability is lost as fast as, if not faster than, in a
sugar or glycerin solution. We can re-establish the irrita-
bility, however, if we put the muscle back into the sodium-
citrate solution for some time. This fact, together with those
mentioned before, suggests the following as the most prob-
able explanation of the peculiar phenomena of contraction
with which we have been dealing: the solutions which pro-
duce the contact-irritability possess anions that are liable to
form insoluble calcium compounds. They are all with one
exception —(NH,),SO,—Na salts. Whatever the effects
of these anions may be, the fact that in less than a minute
the contact effects are noticeable indicates that only the sur-
face layer of the muscle or, what is less probable, the surface
layer of each individual fiber, is altered. It is impossible for
the anions to migrate deeper into the muscle in so short a
time. In the surface layer of the muscle or the individual
fibers we have temporarily a diminution of Caions. We
have, then, a muscle, whose surface layer differs from that of
an ordinary excised muscle. If this layer is once established
the muscle contracts at any change from the media of the
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left column of the above list to those of the right column.
But it is obvious too that as soon as this change occurs the
surface layer gradually undergoes an alteration, for example,
in air, sugar solution, NaCl solution, etc. This change, in
which the contact-irritability is lost, occurs most rapidly in
a CaCl, solution. This suggests the following possibility.
The loss of contact-irritability of the muscle in air or oil,
etc., is due to the migration of Ca ions from the interior of
the fiber or the muscle to the surface, thus re-establishing
approximately the original normal surface condition. If we
then put the muscle back for a short time into a sodium-
citrate or sodium-fluoride, etc., solution, a diminution of
Ca ions will again occur in the surface layers and the con-
tact-irritability will be re-established, As is to be expected
the time the muscle remains in the solution is as important
as the concentration of the solution. If we dip a muscle for
a few seconds only into a sodium-citrate solution (1 gram-
molecule in 10 liters) the contact-irritability cannot be pro-
duced, as there is not time for a large enough number of
anions to diffuse into the muscle.
Still another fact harmonizes with our assumption. If we
lift only a piece of the muscle out of the sodium-citrate
solution, not the whole muscle contracts, but only the in-
dividual fibers that come in contact with the air. Similarly
amore powerful contraction occurs when we lift the thick
femur end of the gastrocnemius out of the solution than if
we expose the thin tendon-Achilles end to the air.
Finally it should be mentioned that the latent period is
somewhat long in these experiments. I have not measured
it yet exactly; but it may be a considerable fraction of a
second, especially when the contact-irritability is about to
disappear. This somewhat long latent period would harmo-
nize well with the assumption of contact-phenomena.
Although I have spoken chiefly of the diminution of
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Ca ions as the effect of the sodium-fluoride and similar
solutions, I wish to state that I consider it possible that
solution may have other effects which play a réle in these
phenomena.
Iv. THE EFFECTS OF SODIUM FLUORIDE AND CORRESPONDING
SOLUTIONS UPON THE NERVE
If we try the experiments described above on curarized
muscles we get little or no result. This would indicate that
the contact-reaction is not due to an effect of these solutions
upon the muscle but upon the nerve elements in the muscle.
There is a second possibility, namely that curare, although
it does not abolish the electrical irritability of muscle, may
yet alter its substance enough to prevent the effects of con-
tact stimuli, or prevent the formation of the hypothetical
surface layer.
It may be said with certainty that sodium-fluoride, sodium-
citrate, and the corresponding solutions act upon the nerve
in a way altogether different from that in which they act
npon muscle. If we put the nerve alone(without the muscle)
into one of these solutions which contains 1 gram-molecule
in about ten liters, as a rule nothing will happen during
the first five minutes. The removal of the nerve from the
solution will not call forth a contraction of the muscle. After
about five minutes the muscle will begin to twitch rhythmic-
ally, and very soon the muscle will shorten steadily until it
reaches a high degree of tetanic contraction. This twitching
continues as long as the nerve is in the solution. As soon
as the nerve is taken out of the solution and exposed to the
air the muscle relaxes more or less completely, and the
twitchings become less numerous. As soon as the nerve is
put back into the sodium-citrate solution the contraction in-
creases again and the twitchings become more powerful.
This may be repeated very often. It is obvious that the
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nerve behaves in exactly the opposite way from the muscle.
The latter contracts when taken out of the solution and ex-
posed to the air, and relaxes when put back into the solution.
If the nerve alone (without the muscle) be put into the
solution, contractions of the muscles occur while the nerve is
in the solution, and partial or complete relaxation is observed
when the nerve is taken out.
These experiments on the nerve give one the impression
that the sodium-citrate solution and the solutions of the
other sodium salts whose anions precipitate calcium stimu-
late the nerve chemically. Albert Mathews has recently
found that weak solutions of sodium salts can cause con-
tractions of the muscle when the nerve alone is put into the
solution, while the salts of the other metals can only produce
contractions when their osmotic pressure is considerably
higher than that of the tissues. I have confined my experi-
ments chiefly to those sodium salts whose anions precipitate
calcium. But I think I can show definitely that these salts
are not the direct stimulus that calls forth the contractions
of the muscle, but play only an indirect rdle, inasmuch as
they make the nerve more sensitive for another kind of
stimulus, either a mechanical- or a contact-stimulus. When
the nerve alone has been put into a sodium-citrate solution
(of 1 gram-molecule in about 10 liters) and the muscle has
begun to contract powerfully, a gradual relaxation of the
muscle is observed when the nerve is taken out of the solu-
tion and allowed to hang in the air. But at any time the
contractions and the final tetanus of the muscle will begin
again when the nerve is brought into contact with any solid
or liquid body, no matter whether it is a conductor or a non-
conductor. As soon as the contact ceases and the nerve is
surrounded by air again on all sides the muscle gradually
relaxes. This can be repeated quite often with the same
result. Among the substances whose contact causes con-
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traction I may mention hard rubber, glass, filter paper, var-
nished and unvarnished wood, bone, muscle, all kinds of
metals. Among the liquids tried were oil, glycerin, sugar
solutions and several salt solutions. It is thus obvious that
in a sodium-citrate solution two influences are united, first
the effects of the citrate ion which causes a modification or
an increase in the irritability of the nerve, and second, the
liquid character of the solution. The latter is the direct
cause for the contraction.
Another point is of interest in this connection. The
sodium-citrate or sodium-fluoride solution increases the elec-
trical irritability of the nerve so that it can easily be stimu-
lated by its own current of demarkation. This increase
occurs regularly before the twitchings of the muscle begin.
In my experiments on artificial parthenogenesis in Chee-
topterus I found that there are two ways by which the
unfertilized egg can be caused to develop —first, by certain
ions (K, H), and second, by causing the egg to lose water.
It follows from the facts of dissociation that a loss of water
on the part of the egg must alter the proportion of ions in
the egg. It thus becomes possible that the artificial par-
thenogenesis produced by the loss of water is in reality an
ion effect. In regard to the twitchings caused by putting
the nerve into solution Mathews has shown that two cases
must be distinguished —first, the effect of specific ions, and
second, the effect of loss of water. Any solution whose
osmotic pressure is high enough can cause contractions if
the nerve be put into it. Is it not possible that the loss of
water in the nerve acts in the same way as the citrate or
fluoride ions? The limited solubility of CaSO, would make
this possible. I tried whether a nerve after having been
put into a 2n sugar solution long enough to cause muscular
contractions would show the above-mentioned mechanical or
contact-irritability. This was indeed the case. If such a
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nerve is taken out of the sugar solution and brought into
contact with solid bodies, it gives rise to stronger contrac-
tions. But, as was to be expected, the nerve loses this irri-
tability again when put into 2 NaCl or Na citrate solution.
In such a solution water will enter the muscle and restore
the original condition, and only later will the entrance of
citrate ions show its effect.
It now remains to be seen how far these facts can throw
light upon the heart-beat. The fact that a heart which has
ceased to beat in a solution often begins to beat again when
taken out of the solution reminds us of the contact-reaction
of muscle described above.
Vv. SUMMARY
1. Certain salt solutions (1 gram-molecule in 8 or 10
liters) bring about an apparently new form of irritability in
muscles, which may be called provisionally contact-irrita-
bility. A muscle that has been treated in this way will
contract powerfully when it passes from the salt solution to
air, CO,, oil, sugar solution, etc., or from glycerin solutions,
sugar solutions to air.
2. The salts whose solutions produce this form of irrita-
bility are (with one exception) sodium salts, whose anions
are liable to precipitate calcium, namely:
Sodium fluoride Na.HPO, Sodium citrate
Sodium carbonate Sodium oxalate Sodium tartrate
3. If the nerve alone (without the muscle) be put into
one of these salt solutions (1 gram-molecule in 8 or 10 liters),
the muscle begins to twitch in about five minutes and finally
goes into tetanus. If the nerve be taken out of the solu-
tions, the contractions cease. Although this seems to indi-
cate that the salts or their ions stimulate the nerve directly,
it can be shown that they only modify or increase the irrita-
bility of the nerve. For when the same nerve is brought
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ABNORMAL IRRITABILITY PRODUCED By SaLts 707
into contact with any solid or liquid body (conductor or non-
conductor) the contractions of the muscle will be resumed,
while they will gradually cease or diminish when the nerve
is again surrounded by air on all sides.
4. The fact that certain ions are capable of bringing
about forms of irritability in nerves and muscles which do
not exist normally may perhaps furnish the explanation of a
number of certain morbid phenomena (neuroses, hysteria) in
which the motor and sensory reactions of the patient are
modified.
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XXXV
THE TOXIC AND THE ANTITOXIC EFFECTS OF IONS
AS A FUNCTION OF THEIR VALENCY AND POS-
SIBLY THEIR ELECTRICAL CHARGE’
I. INTRODUCTION
Five years ago I published a series of papers on the
physiological effects of the electric current which impressed
upon me the long-known fact that the galvanic current is the
most universal and effective stimulus for life-phenomena.
This fact suggested the idea that it should be possible to
influence life-phenomena just as universally and effectively
by the electrically charged molecules—the ions—as we can
influence them by the electric current.
My first aim was to find out whether or not it is possible
to alter the physiological properties of tissues by artificially
changing the proportion of ions contained in these tissues.
In this way originated the investigations on the effect of ions
upon the absorption of water by muscles,’ the effects of ions
upon the rhythmical contractions of muscles, and Meduse,’
the heart of the turtle,* and the lymph hearts® of the frog,
the réle of ions in chemotropic phenomena’ and the influence
of ions upon embryonic development,’ and the development
1 American Journal of Physiology, Vol. VI (1902), p. 411. A preliminary report
of these experiments appeared in Pyliigers Archiv fiir die gesammte Pysiologie,
Vol. LXXXVIII (1901), p. 68.
'2Part II, pp. 450, 501, and 510.
3Part II, pp. 518, 559, and 692; Archiv fiir die gesammte Physiologie, Vol. LXXX
(1900), p. 229.
4D. J. Lineuez, American Journal of Physiology, Vol. IV (1900), p. 265.
5 A. Moors, ibid., p. 386.
6 W. E. GARREY, ibid., Vol. III (1900), p. 291.
7 Part II, pp. 559 and 576.
708
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of unfertilized eggs (artificial parthenogenesis).' Those
who have followed my work on artificial parthenogenesis
may have noticed that from the start I aimed at bringing
about artificial parthenogenesis through ions. It seemed to
me that I could not find any better test for my idea that the
electrically charged ions influence life-phenomena most
effectively than by causing unfertilized eggs to develop
by slightly altering the proportion of ions contained in
them. I believe that all these experiments proved what I
expected they would prove, namely, that by slightly chan-
ging the proportion of ions in a tissue we can alter its physi-
ological properties.
The next step taken consisted in proving that it was in-
deed the electrical character of the ion that determined its
specific efficiency. I succeeded in doing this three years
ago. It was known that a frog’s muscle gives rise to twitch-
ings or rhythmical contractions when immersed in certain
solutions. I showed that such contractions occurred only in
solutions of electrolytes, and not in solutions of non-con-
ductors (distilled water, various sugars, glycerin, urea).”
Soon after I showed the same to be true also for the rhyth-
mical contractions of the Medusz.* From observations made
in my laboratory, the same fact was shown to hold for the
turtle’s heart by Mr. Lingle,’ and for the lymph hearts of
the frog by Miss Moore.’ I am confident that this fact
will be proved universally.
In the physiology of the heart one frequently encounters
the statement that calcium is the stimulus for the contraction
of the heart. I had found that a muscle is able to twitch
1 Part II, p. 646. Archiv fiir die gesammte Physiologie, Vol. LXXXVII (1901),
p. 594,
2 Part II, p. 518.
3 Part IT, pp. 553 and 559.
4 LINGLE, American Journal of Physiology, Vol. IV (1900), p. 265.
5 Moors, ibid., Vol. V (1900). p. 87.
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rhythmically when immersed in the solution of salts with a
monovalent kation—I obtained contractions in Na, Li, Rb,
and Cs salts—but that the addition of a small quantity
of a bivalent kation—Ca, Mg, Sr, Be, Mn, Co— inhibits
these rhythmical contractions.’ This seemed to be a direct
contradiction to the statement that calcium salts are the
‘“‘cause”’ of the heart-beat. The significance of the calcium
had to be looked for, then, in another direction. It was
soon found that the muscle, the apex of the heart, and a
Medusa contract rhythmically in a pure sodium-chloride so-
lution, but that they soon come to a standstill. If, however,
a trace of a soluble calcium salt is added to the sodium-
chloride solution, the contractions continue much longer. I
concluded from this that the pure sodium-chloride solu-
tion acts, in the long run, as a poison—that is to say, brings
about definite, but at present unknown, physical changes in
the protoplasm—but that a trace of a calcium salt anni-
hilates this toxic action. The amount of calcium neces-
sary for this antitoxic effect is, of course, much smaller than
the amount necessary to inhibit the rhythmical contractions.
Soon after I succeeded in demonstrating conclusively the
poisonous effect of a pure sodium-chloride solution, and the
annihilation of this effect by calcium.” The eggs of a
marine fish (Fundulus) develop normally in sea-water, but
they can develop just as well, as I had previously found, in
distilled water. The addition of ions from the outside is
consequently not necessary to the development of this animal.
I found, now, that if the freshly fertilized eggs of this fish
are put into a pure sodium-chloride solution having a con-
centration equal to the concentration of the sodium chloride
in the sea-water (about $m), not a single egg can develop
into an embryo. If, however, a trace of a calcium salt is
1 Part I, p. 518,
2 Part II, p. 559; Archiv fir die gesammte Physiologie, Vol. LXXX (1900), p. 229
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Toxto AND ANTITOXIC EFFrcts or Ions’ T11
added to a sodium-chloride solution, as many eggs develop,
and in just as normal a manner, as in ordinary sea-water.
The calcium ions in this case undoubtedly serve the purpose
of annihilating the poisonous effect of a pure sodium-
chloride solution.
In the meantime I had become familiar with the bril-
liant experiments of Hardy upon the influence of ions
and the galvanic current upon colloidal solutions.' They
indicated to me that the next step I had to take was to
see whether or not the valency and the sign of the elec-
trical charge of an ion determine its physiological effects.
I suspected that the antitoxic effect of the calcium ion in
the above-mentioned experiment was due to its electrical
charge and decided to investigate in a more systematic
way whether or not the sign and quantity of the electrical
charge influence life-phenomena. My experiments carried
on at Woods Hole this summer showed conclusively that
this is the case for the antitoxic effects of ions, and prob-
ably for the production of rhythmical contractions through
ions. It seems at least possible that it is true also for
artificial parthenogenesis.”
II. THE ANTITOXIC EFFECT OF IONS AS A FUNCTION OF THEIR
ELECTRICAL CHARGES AND VALENCY
1. The development of an embryo from the freshly fer-
tilized egg of the before-mentioned fish, Fundulus, served as
a test for the toxic and antitoxic effects of ions. I chose
this particular animal for two reasons. First, the process of
development in this form is to an astonishing degree inde-
pendent of the osmotic pressure of the surrounding solution.
The egg will develop not only in sea-water, the osmotic
1 Harpy, Proceedings of the Royal Society, Vol. LXVI (1900), p. 110.
2T have not altered this introduction, although I now think it probable that the
ions act chemically in all these cases. [1903]
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712 STUDIES IN GENERAL PHYSIOLOGY
pressure of which is about equal to that of a $m sodium-
chloride solution,’ but also in distilled water, or in sea-water
the concentration of which has been doubled (by the addi-
tion of NaCl). In the following experiments, therefore, we
need not at all consider the osmotic pressure of the sur-
rounding solution. Secondly, since enormous numbers
of the eggs can be obtained, it is an easy matter to per-
form the experiments upon hundreds or thousands of eggs
at once.
The eggs were artificially fertilized in the laboratory by
the addition of sperm, and then immediately distributed into
the various solutions. The embryo forms in from about
twenty-six to forty-eight hours—varying with the tempera-
ture —and twenty-four hours later the heart begins to beat,
and the circulation is established. Usually about two hun-
dred eggs were put into a solution, and after two or three
days the developed embryos were counted and the per-
centage of the eggs which had developed was determined.
The eggs were kept under observation as long as the
embryos remained alive. Usually when an embryo was
once formed, development went farther, and the circulation
was established.
2. First of all the toxic effects of a pure sodium-chloride
solution at various concentrations were tested. Ina NaCl
solution every egg produced an embryo which died, how-
ever, before, or immediately after, emerging from the egg.
(The embryo hatches from between twelve to twenty days
after fertilization.) On the other hand, in a 3m NaCl solu-
tion only a few of the eggs gave rise to embryos—about 1 to
5 per cent. Ina¢m NaCl solution an embryo forms but
rarely, and in a 3m NaClsolution the formation of embryos
is rendered impossible. The egg goes through the first
1m represents that degree of dilution of a solution which contains one gram-
molecule of the substance in one liter of the solution.
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Toxic AND ANTITOXIC Errects or Ions’ 713
stages of segmentation, but dies when it reaches the thirty-
two- or sixty-four cell stage. The concentration of a 3m
sodium-chloride solution is indeed so high above the point
of the fatal concentration of sodium chloride that a slight
decrease in the degree of dissociation of the NaCl solution
brought about through the addition of a small amount of
another salt having a common ion, could be entirely dis-
regarded. In the following experiments, however, salts with
different ions were combined, wherever this was possible.
If to a pure sodium-chloride solution a trace of a
calcium salt is added, as many eggs develop as in ordi-
nary sea-water, as shown by Table I.
TABLE I
7 Percentage of
Solution Eggs Aiciding
Mmbryos
100 c.c. §m NaCl 0
100 oe “ +4 e.c. 7 CaSO, 3
100 6c “ + 1 66 6 3
100 &e “ + 9 6 “c 20
100 ee it + 4 rT “ 75
100 6 6“ + 8 be 73 70
This series of experiments does not show whether it is
the Ca or the SO, ion that has the antitoxic effect. To
determine this point the same series of experiments was
twice repeated with certain modifications. In the first of
these Ca(NO,), was added to the 3 NaCl solution instead of
CaSO,. The result was practically that given in Table I.
In the second Na,SO, was added to the sodium-chloride
solution. The addition of Na,SO, did not inhibit the toxic
action of the sodium chloride, and the eggs developed no better
than in the pure sodium-chloride solution. We shall return
to this point later. However, in order to eliminate entirely
the effect of the anions in the antitoxic effects produced, a
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714 STUDIES IN GENERAL PHYSIOLOGY
series of experiments were instituted in which the toxic and
antitoxic salt both had the same anion.
TABLE II
Percentage
Solution of Eggs Yielding
Embryos
bere eee 100 c.c. § m NaNO, 8
Dis te Pete tanks LOO a “« ~+2ec. 7% Ca(NO,). 1
Biiackwenauls 100 ge £6 + 1 6s 6c 10
a osectessieieaun 100 “ 6 + 2 ‘“ ‘“ 15
Dist ag wise 100 ee 6“ +4 6“ “ 15
Cau aed ete 100 fe ee + 8 ‘“ “ 70
It is undoubtedly true, therefore, that the addition of
even a small amount of Ca ions diminishes the toxic
action of a pure sodium-chloride solution. It can further
be shown that the concentration of the Ca ions necessary
to abolish the poisonous effects of a sodium-chloride solu-
tion increases as the concentration of the latter increases
(see Table ITI).
Tables IT and IIT show clearly that the amount of calcium
necessary to annihilate the poisonous effect of a solution of
a sodium salt increases with the concentration of the sodium
salt in the solution.
The embryos formed in these solutions, rendered harmless
through the addition of calcium, developed a normal circula-
tion and lived several weeks. As a rule, however, they did
not hatch. It was further found that the addition of 5 c.c.
of a g” CaSO, solution could annihilate absolutely the toxic
effect of a 3m, 4m, or $m NaClsolution. These experiments
leave no room for doubt that the presence of a trace of Ca
ions is capable of rendering inert the poisonous effects of a
pure sodium-chloride solution.
3. It was next shown that Sr, Ba, and Mg ions are
also capable of annihilating the poisonous effects of a
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Toxio AND ANTITOXxIC EFFEOTS oF Ions’ 715
pure NaCl solution in a way similar to that of Ca ions
(see Table IV).
TABLE III
Percentage
Solution of Eggs Yielding
Embryos
100 c.c. $m NaNO, 5
100 ae +he.c. 7 Ca(NO,), 48
100 “ “ + 1 “ 6c 40
100 “ “ + 9 it “ 63
100 “ “ce + 4 “ o 66
100 “ iT3 + 8 “ “c 70
100 sf 1
100 “ ity + $ “ “ 8
100 6 “ + 1 6c oe 9
100 “ “ + 2 “c iy 45
100 “ 66 + 4 [79 it 42,
100 “c “ce + 8 “ “ 70
100 “ 0
100 it “ “ 6“
100 6c “ce a “ « :
100 6c 73 +2 6“ 7
100 7] “ + 4 “ “ 10
100 iT3 “ + 8 [77 ty 50
TABLE IV
Percentage
Solution of Eggs Yielding
Embryos
AL erative 100 c.c. £m NaCl 0
De barred ans 100 es “« +4¢.c.m BaCl, 75
® gon weds 100 & 90
Wate tes Benes 100 ff « +492 “ MgCl 75
ae 100 « « 149 ee. 4m SrCl, 90
nee 1002 4 aE «OF & Ga(NO,), 80
That the threshold for the antitoxic effects of Ba, Mg,
Sr has the same magnitude as that of Ca may be indicated
by a single experiment with Ba (Table V).
It can be seen that the threshold of the antitoxic effect
of barium is almost identical with that for Ca under similar
conditions.
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716 STUDIES IN GENERAL PHYSIOLOGY
TABLE V
Percentage
Solution of Eggs Yielding
Embryos
TD veatessisie- veg a 4 100 cc. §m NaCl 0
Dass 100 “ * +tee. 2% BaCl, 8
Di casaonods 10 « “© 4] « « 4
ERE LE 10 « «© 49 « 4 27
Gaee 100 “« «© +4 « & 76
EG 100“ “ +8 « & 5
Since all these ions are related chemically, the objection
was possible that we were dealing here, not with the effects
of the valence or the electrical charge of the ions, but with
a specific chemical effect. It was, therefore, necessary to
show that the same effect can be produced by bivalent kations
which lie outside of the calcium group. My first experi-
ments failed me, since I at first employed too large amounts
of the antitoxic salts. I discovered only gradually that the
poisonous effects of a sodium-chloride solution may be
annihilated by a bivalent kation in quantities much smaller
than are given in Table IV, which presents the results of one
of my first experiments. My experiments now succeeded.
4, A large number of experiments were performed with
ZnSO, as the antitoxic substance for NaCl. The NaCl
solution used was somewhat more concentrated than that
usually employed, namely 4} m instead of Sm.
TABLE VI
Percentage of
Solution Eggs Yielding
Embryos
EL Ae serge 100 c.c. }4m NaCl 0
Dre caeseey 100 ee “ + hee. pty ZnSO, h
ee oe 100 = oo +) se i 2
he Sa renecdrartusaate 100 e “ +2 « ss 22
Dike k-manes 100 e « 44 ee ee 50
Ox.dt eae 100 os “« +8 75
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Toxic AND ANTITOXIC EFrrects oF Ions’ 717
To supplement these results the following table dealing
with the effects of a more concentrated ZnSO, solution and
a more dilute NaCl solution than that of the previous table
may be given:
TABLE VII
Pp 4 re
Solution Boos atte
Embryos
a Nee eeere eee 100 c.c. $m NaCl 5
Ds aa Gatley af “ thee. J ZnSO, 90
Div done sess 100 ad Se dk 4 oe 80
A ealoitee hind 100 a © 412 “7 ee 86
Ogaxd saad 100 s ot 4 ss 88
The remaining experiments showed a similar agreement
in the results obtained. It is worthy of note that these
embryos remained alive over a week, possessed an entirely
normal circulation, and moved in the egg.
The experiments with freshly prepared FeSO, yielded as
striking results as the above. Only in these experiments the
transformation of the bivalent into the trivalent Fe ion
introduces a disturbing element. We shall see later that the
ferric ion is apparently extremely poisonous. The addition
of } cc. or 1 cc. of a freshly prepared % FeSO, solution
to 100 c.c. of 8m NaCl solution annihilates the poisonous
effect of the pure sodium-chloride solution just as com-
pletely as the addition of the Zn ions in the previous ex-
periment.
Then I tried whether cobalt ions are capable of anni-
hilating the antitoxic effects of a pure sodium-chloride solu-
tion. The results were very clear indeed.
Since the amount of the bivalent kation capable of exhib-
iting its antitoxic properties was so extraordinarily small, I
risked the attempt to annihilate the poisonous effects of a
pure sodium-chloride solution through the addition of Pb,
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718 STUDIES IN GENERAL PHYSIOLOGY
TABLE VIII
epleata o of
Solution Eggs Yielding
Embryos
Laces aos 100 c.c. §m NaCl 0
De dha wa ets 100 ss ee + lee x CoCl, 6
Boneneesicns 10 « « 49 °° “6 9
A sd recrag edo: 100 ee “ + 4 6c “ 9
Deca acne 100 se “ + 8 a “ 50
Grniindonnere 100 a “« +12 3 “ 88
heckstasitape wes 100 ce + 5 “ “ 62
Cu, and Hg ions. Had I not before demonstrated the anti-
toxic effects of so poisonous an icn as the zinc ion, such an
attempt would have appeared to me only ridiculous. With
copper acetate and mercuric chloride I obtained negative
results throughout, for these two ions are so poisonous indeed
that the small amount necessary to render inert the poison-
ous effects of a sodium-chloride solution are sufficient to
kill the egg or cause its coagulation. With lead ions, how-
ever, I had a distinct success. For the antitoxic salt lead
acetate was used, and for the toxic salt, sodium acetate. It,
was proved that the latter was slightly more toxic than NaCl.
TABLE IX
Percentage of
Solution Eggs Vielding
Embryos
1....] 100 ce. 4m CH, Co, Na 1
2....| 100 + hee. 7% Pb acetate 8
3....1100 « “ ae a “ 12
4....}100 « “ +9 « “ 23
5...., 100 « “ +4 “ 34
In another case 40 per cent. of the eggs formed embryos.
The objection was here again at hand that the decrease in
the degree of the dissociation of the sodium acetate had
played a rdle. Although lead chloride is only very slightly
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Toxic AND ANTITOXIC EFrFrrcts oF Ions’ 719
soluble, I tried to see if the few lead ions that go into solu-
tion when lead acetate is added to sodium chloride would
still suffice to weaken the poisonous effects of a pure NaCl
solution. Such was indeed the case.
TABLE X
| Percentage of
Solution Eggs Yielding
Embryos
Tete eens 100 c.c. $m NaCl 3
Deavees wees 100 se “ + tec. 7% Pb acetate 7
Dieescak sees 100 Be ee tel se oe 17
In the remaining solutions the number of embryos could
not be determined, since the eggs had been rendered opaque
by the precipitation of the lead salts.
5. Experiments were now made to see if it were possible
to annihilate the toxic effects of a sodium-chloride solution
through the addition of salts having a trivalent ion. AICl,,
Cr,(SO,), and FeCl, were used. The experiments with
FeCl, all yielded negative results. No concentration could
be found at which this salt exhibited antitoxic properties.
Perhaps the strongly acid character of this solution had
something to do with this result. The experiments with the
two other salts, however, yielded positive results.
TABLE XI
Percentage of
Solution Eggs Yielding
Embryos
US eldaasie Guo Get 100 c.c. §m NaCl 0
oceteeas 100. “* +he.c. 7%, AlCl, 0
Dieses wraiciats 100 ef ah se 4
A cuiaces ead 100 e « t+) es 25
Deicccescopor en 8 100 os « +12 is 39
G co ccciguasmreccin 100 ie “« +4 ee 25
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720 STUDIES IN GENERAL PHYSIOLOGY
Two other series of experiments yielded the same results.
It is worthy of note that the amount of a trivalent kation
capable of exerting a certain antitoxic effect is considerably
less than the amount of a bivalent kation necessary for the
same purpose. At the same time one notices, however, that
the number of eggs forming embryos is, even at the best,
lower than when bivalent kations are employed. The reason
for this lies, as I believe, in the fact that the trivalent ion
causes readily a coagulation of the egg contents, as direct
observation shows. But this coagulation is not exclusively
a function of the valency of the ions, for Cu, Hg, and to a
slight extent Pb have the same influence upon the egg.
The influence of the Cr ion in bringing about coagulation
is much more marked than is the case with Al, and its anti-
toxic effects are correspondingly slight, but yet definite.
TABLE XII
Percentage of
Solution Begs Yielding
mbryos
fh eeerroraree eae 100 c.c. §m NaCl 0
ee 100 “© 4 tee. % Cr,(SO4), 3
Bianediuwaass 100 is wo 44 is - 8
A dos cin teaetei 100 ae “ +1 - Ke 8
Mecgea a co seedy to 100 te ee + 2 66 &“ 10
Gh isin’ Danan 100 “ wot 4 “ “ 6
6. Since traces of trivalent kations and small amounts of
bivalent kations suffice thus to annihilate the poisonous
effects of a sodium-chloride solution, experiments were made
to ascertain if the same could also be brought about by
monovalent kations. The experiments have thus far led to
no positive results. I tried to see if the poisonous effects of
a pure sodium-chloride solution could be done away with by
the addition of potassium salts (KCl and K,SO,). Small
amounts of potassium salts were entirely without effect.
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Tox1o AND ANTITOXIC Errects or Ions’ 721
The addition of } to 2 ¢.c. of m KCl or K,SO, occasionally
yielded results, in that 1-5 per cent. of the eggs formed
embryos. Lithium salts showed themselves to be even less
active. I occasionally obtained a slight antitoxic action by
the addition of large amounts of NH, salts. Whether
hydrogen ions can yield better results must be determined
through further experiments.
7. Not only can the poisonous effects of a pure sodium-
chloride solution be annihilated through the addition of
small amounts of bivalent or trivalent kations, but it seems
as though the same holds for all salts which, like NaCl, havea
univalent kation and anion. No embryos develop ina 3m LiCl
solution. By the addition of small amounts of Ca(NO,),,
BaCl,, SrCl,, or MgCl,, 50-60 per cent. of the eggs were
caused to form embryos, which developed normally. Other
kations of a higher valency were not tested. I obtained
entirely similar results in regard to KCl. In a {m or even
a im KCl solution an egg may occasionally develop. When
a small quantity of MgCl,, Ca(NO,;),, SrCl,, BaCl, or
FeSO, was added, the poisonous effects of the pure KCl
solution were annihilated. Of salts having other bivalent
kations, only ZnSO, (a single experiment) was used. An
effect was obtained in this case also, but it was less striking
than in the case of the other bivalent kations.
NH,Cl seems to be the least toxic of all the salts men-
tioned thus far. Even in a ?m NH,Cl solution an embryo
could form occasionally. This immunity of the Fundulus
egg against NH,Cl is perhaps related to its great immunity
against urea. I cannot get rid of the suspicion that a per-
centage of the NH, ions is perhaps done away with in the
metabolism of the egg. I obtained striking antitoxic effects
with small amounts of SrCl, and, although less definite,
of FeSO,. Ca(NO,), increased the number of embryos
formed, though not as greatly as the other salts with a
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722 STUDIES IN GENERAL PHYSIOLOGY
bivalent kation, but the life of the embryos was very con-
siderably prolonged.
The shortness of the spawning season limited the number
of my experiments, so that I decided to bring my experi-
ments upon the annihilation of the poisonous effects of a
pure sodium-chloride solution to a close, and to carry the
remaining experiments only far enough to decide if we are
dealing here, in the main, with the same condition of affairs.
That, I believe, is undoubtedly the case, so that I feel my-
self justified in making the following statement: The salts
of monovalent kations (Na, Li, K, NH,) with monovalent
anions (Cl, NO,, CH,COO) exert a toxic effect at certain
concentrations. This toxic effect can be annihilated through
the addition of a small amount of a salt having a bivalent
kation. For NaCl, proof has been brought forward that
trivalent kations exhibit even a much more energetic anti-
toxic effect than bivalent kations. Further experiments
are yet to be made, to decide if the poisonous effects of the
other salts (LiCl, KCl, NH,Cl) can also be done away with
through the addition of such small amounts of trivalent ka-
tions as suffice for NaCl.
8. While the preceding experiments show an undoubted
influence of the valency of the ions upon their antitoxic
effects, it was now necessary to prove that the sign of the
electrical charge was the second determining variable. I
instituted a large number of experiments in which I
attempted to annihilate the poisonous effects of a £m NaCl
and a $m KCl solution by the addition of salts having a
univalent or bi- or trivalent anion. The antitoxic effects of
the following salts are investigated; KOH, NaBr, Nal,
NaHCO,, Na,CO,, NaSO,, Na,HPO,, sodium citrate,
K,SO,. Extensive quantitative experiments were made
with Na,SO,, K,SO,, NaHCO, and Na,HPO,. The
results were negative throughout. In the best cases 1 per
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Toxic AND ANTITOXIO Errrcts oF Ions 723
cent. of the eggs formed embryos. Jt followed from these
experiments that the toxic effects of salts with a monov-
alent kation and a monovalent anion can be annihilated
only by bi- or trivalent kations, but not by mono-, bi-, or triv-
alent anions. If we correlate this fact with that previously
found, that spontaneous, rhythmical contractions of muscles,
Medusee, and hearts are possible only in solutions of electro-
lytes, then the idea can certainly not be repudiated that the
antitoxic effect of salts in the above-mentioned experiments
may be a function of the magnitude and the sign of the
electrical charges of the ions.
9. If the toxicity of a pure CaCl,, MgCl,, BaCl, or
SrCl, solution is compared with the toxicity of a solution of
a chloride of a monovalent kation, then it is found that the
former are the more poisonous. In a ® Ca(NO,), solu-
tion no embryo develops. This same toxic concentration is
reached in a MgCl, solution at the dilution of ¥%. Can
the toxic effects of these solutions also be overcome? One
can indeed easily overcome the poisonous effects of a #
Ca(NO,), solution by adding large amounts of a KCl or
NH,Cl solution. NaCl and LiCl solutions are almost with-
out effect.
TABLE XIII
Percentage of
Solution Eggs Yielding
Embryos
sce ciety 100c.c. #? Ca(NO,). 0
Braces 100 © 4 ee. 23. m KCl 15
ee a 10 “ « 44 cnr 34
BOSCH 400 * « 42 wo« 40
Biss etd 100 “« «© 44 “4 55
Gy casamaae 100 a +8 ae Eats 67
As one can see, the number of embryos formed shows a
definite increase with an increase in the concentration of the
KCl. I tried still stronger solutions of KCl in further
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724 STUDIES IN GENERAL PHYSIOLOGY
experiments, and found that in a mixture of 100 cc. ™ Ca
(NO,), +20 c.c. 24 m KCl a still larger number of eggs
formed embryos than in the preceding experiments. On the
other hand, it could be shown that the addition of small
amounts of KCl was without effect.
TABLE XIV
Foreataee at
Solution Eges Yielding
Embryos
as natentoyea 100 c.c. # Ca(NO,). 0
Dies teaeettp 100 i ae +heec. ¥% KC 0
ee cae 100“ © 44 oe 0
Geese 100 *§ © 49 «4 « 0
eee 100“ & 44 « 4 0
Gscaomenan! 10 “ «© 4g « « 2
TP rescs sdeuetdeet 100 t ot +2 c.c. 24 m KCI 12
The size of the antitoxic dose of KCl in Ca(NO,), poi-
soning is, in fact, extraordinarily larger than the antitoxic
dose of Ca(NO,), in the case of KCl poisoning. Similar
relations exist for the antitoxic effect of NH,Cl upon CaCl,
poisoning.
TABLE XV
Percentage of
Solution Begs Yielding
mbryos
Li vsancis eet 100 c.c. # Ca(NO,). 0
Di abril 100 ne a +1 cc. 24m NH,Cl 9
Dsspstonasaet 10 # Oe QR :
Bowed eee 100 se as + 4 “ “ 16
De Siteiweees 100 He “6 + 8 “ “ 21
Gis cscituits ones 100 ee ee +16 “ “ 16
The antitoxic effects of NH,Cl are not as great as those
of KCl. Similar experiments with NaCl and LiCl as anti-
toxic substances were without positive result.
Similar experiments were then performed with MgCl.
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a
Toxic aND ANTITOXIC Errerots oF Ions’ 725
By the addition of MgSO, the toxic effects of MgCl, could
not be done away with. But through the addition of large
amounts of KCl, NH,Cl, or small amounts of SrCl, this was
possible, as also—to a slight extent—through the addition
of Ca(NO,),. The dilution at which a MgCl, solution
hinders the development of an embryo is },m MgCl,.
Table XVI shows a series of antitoxic experiments. NaCl
and LiCl were just as unable to annihilate the toxic effects
of the MgCl, solution as they were unable to annihilate the
poisonous effects of « Ca(NO,), solution. When less than
tcc. of a SrCl, solution was added, not a single egg
could develop.
10. If, in these experiments, only the kations have an
antitoxic effect, and this the greater, the greater their elec-
trical charge; and if in these antitoxic effects we are dealing
only with electrical effects, then it is to be logically expected
that the toxic effects which are inhibited in these cases are
also electrical effects, and indeed the effects of the negative
electrons. If the antitoxic ions are the strongly charged
positive ions, then the toxic ions in the sodium-chloride solu-
tion must be the Clions. But in a pure sodium-chloride
solution we have just as many kations as anions, and in con-
sequence just as many positive as negative electrical units.
It is therefore not at once intelligible why the negative
charges of the chlorine ions should be able to call forth
poisonous effects in a sodium-chloride solution. If it is
necessary for us to accept the fact that we are here dealing
with electrical effects, then we are forced further to conclude
that, for some reason or other, the negative charges of the
chlorine ions attain a greater activity than the positive
charges of the sodium ions. Nernst has pointed out the fact
that the metallic ions tend to bind their electrical charges
more strongly than the anions, and he brings this into con-
nection with the fact that we are acquainted with kathode
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726 STUDIES IN GENERAL PHYSIOLOGY
TABLE XVI
Percentage of
Solution Eges Yielding
Embryos
| eer 100 c.c. 5 m MgCl, 0
ye ee 100 a *“ "+ lec. 24m NH,Cl 16
eee ree 100 e “ + 2 66 “ 99
ee 100 - 6 + 4 “ “ 34
SD ecatetaia tine 100 i Ke + 8 “ “ 9
oe ee 100 i ee +16 “ “ 3
ieee 100 - “« + lee ¥ m SrCl, 25
Bus gsemmas 100 i “ +9 “ “ 22
Deve cisereGicens 100 Be ee + 4 “ “ 9
TOS sdetscaveys 100 . “ 48 “ “ 0
Ds cca etal 100 is « 116 “ “ 0
rays, but not with anode rays. Another possibility may be
thought of. The egg—and all protoplaam—is a system
with various phases; we have solid parts (membranes), and
liquid parts which are either rich or poor in colloids. It is
conceivable that the coefficient of distribution for the posi-
tive and negative ions is unequal in the various phases, and
that this fact leads to the toxic effects of the negative ions
which can be annihilated by the addition of a small number
of positive ions holding a double or triple charge.
I was long inclined to look upon the sodium ions as the
toxic ions in a pure sodium-chloride solution, and I have
upheld this view in my preliminary communication concern-
ing these experiments. What led me to this conclusion was
the following experiment: I tested the relative toxicity of H
and OH ions for the eggs of Fundulus. As was to be
expected, it came to light that the hydrogen and hydroxyl
ions differ in their toxicity. In a,”, KOH solution the
eggs developed and formed embryos, while a ;”,; HCl solu-
tion killed the eggs almost immediately. The hydrogen
ions are therefore at least as much as five times as poisonous
as the hydroxyl ions. But I do not believe that we are
forced to conclude from this that the poisonous effects of a
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Toxtco AND ANTITOXIO EFrFrects oF Ions 1727
sodium-chloride solution necessarily originate from the posi-
tively charged ions. Besides the electrical charge other fac-
tors may have to be considered in the toxicity of ions for the
determination of which physical chemistry and physics must
first furnish us the data. In this category belongs, for
example, the fact that the toxic effects of the sodium salts
of the halogens upon fish eggs (perhaps upon protoplasm in
general) increase in the following order: NaCl, NaBr, Nal,
NaF. In addition we find that the bivalent anions are in
general more poisonous than the monovalent, and the triv-
alent more poisonous than either. The same also holds true
to a certain extent for the poisonous effects of kations.
In order to make the bulk of this paper no greater than
it is already, I shall discuss my experiments on the toxic
effects of ions no further at this point.
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XXXVI
MATURATION, NATURAL DEATH, AND THE PROLON-
GATION OF THE LIFE OF UNFERTILIZED STAR-
FISH EGGS (ASTERIAS FORBESII) AND THEIR
SIGNIFICANCE FOR THE THEORY OF FERTILIZA-
TION!
I, INTRODUCTION
I HAVE pointed out in my earlier publications that fertili-
zation of the egg serves to prolong the life of the egg.’
The mature unfertilized egg dies in a comparatively short
time. Because of this fact the egg becomes of importance
as an object of experiment, to study the question of natural
death and the prolongation of life. For by no means has it
been decided that there is a “natural” death. We only
know that with an increase in age a critical period is reached
in which every living organism dies under the influence of
conditions which do not affect a younger organism. It may,
therefore, be of interest that we are able to show, as I believe,
that a critical period exists in the life of many eggs in which
they die a ‘‘natural” death, and that the life of the eggs can,
during this period, be saved or lengthened only through
various external conditions.
The egg of the starfish (Asterias Forbesii) serves as a
very favorable object of experiment in the study of this
question. When removed from the ovary this egg is gen-
erally “immature,” but as soon as it comes in contact with
sea-water it begins to “‘maturate.”
Morphologically, the immature state is characterized by
1 Biological Bulletin, Vol. IIT (1902), p. 295.
2Part II, p. 689; Lozs anp Lewis, American Journal of Physiology, Vol. VI
(1902), p. 305.
728
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NatTuRAL DEATH AND FERTILIZATION 729
avery large plainly visible nucleus.’ The process of matura-
tion consists morphologically in this, that the nucleus
becomes invisible and the polar bodies are thrown out.
This process is completed within one or two hours after
the eggs are removed from the ovaries and placed in sea-
water. Only when maturation is complete is it possible to
cause the egg to develop through the addition of sperm or
through the physical and chemical agencies that have been
described by me, Delage, Mathews, and Greeley.
Il. THE NATURAL DEATH OF THE MATURE UNFERTILIZED
STARFISH EGGS
The living eggs of Asterias are light yellow in color and
homogeneous. They retain this appearance during the pro-
cess of maturation as long as they are alive. They retain
this appearance also when they are made to develop through
the entrance of a spermatozoon or through the proper
chemical or physical means.
If, however, the mature eggs are not fertilized or do not
develop, they die in the course of four to twelve hours, and
this process of dying is accompanied by a characteristic
change in the color of the egg. The egg becomes at first
opaque, then almost black, and the homogeneous structure of
the protoplasm becomes granular. If such a culture of
unfertilized eggs is examined under the microscope after
twenty-four hours, two kinds of eggs are found, first, the
just-described dark, dead eggs which are mature, and
secondly, living, normally colored, but immature, eggs. For
usually not all the eggs that are removed from the ovaries of
a starfish mature at once; many mature very late, others not
at all. It is readily seen that the immature eggs remain
1The recent beautiful experiments of Delage have shown that, besides these
visible changes in the nucleus, chemical, but morphologically invisible, changes also
occur in the protoplasm. DELaGE, ‘“ Etudes expérimentales sur la maturation
cytoplasmique et sur la parthénogendse artificielle chez les Hchinodermes,” Arch. de
zoologie expériment., Vol. IX (1901).
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730 STUDIES IN GENERAL PHYSIOLOGY
alive for several days until they finally become the prey of
bacteria; while the mature eggs become opaque and die in
from four to twelve hours after maturation has been com-
pleted.
Is the death of the mature but undeveloped egg brought
about through internal conditions, or through the bacteria
contained in the sea-water ?
A trustworthy way of determining this consists in making
sterile culture of the eggs in sea-water. This is a relatively
simple procedure in the case of starfish. Hight flasks were
sterilized, filled with sterilized sea-water, and again heated
for twenty minutes on three successive days to 100°C. A
female starfish was thoroughly washed externally, an arm
was opened, and one of the ovaries removed with sterilized
forceps and placed in sterilized sea-water. From the thick
stream of eggs which at once flowed out of the ovary, a few
drops were quickly introduced with a sterilized pipette into
each of the sterilized flasks. A second series of eight flasks
contained normal, unsterilized sea-water, and a fewdrops of the
same eggs were introduced into these flasksalso. A third series
of flasks were filled with sea-water, to each of which were
added 2 c.c. of a putrid, foul-smelling culture of old starfish
eggs in order to bring about a rapid development of bacteria
from the beginning. Each of these flasks also contained
eggs from the same culture as those in the sterilized flasks.
That perfect sterilization had been attained in the first
eight flasks was proved by the fact that all the flasks remained
absolutely clear and cloudless during the course of the experi-
ment, and that three of the flasks which had not been opened
are even today (after six weeks) absolutely clear, and every
egg can be individually recognized. The flasks containing
the unsterilized sea-water became cloudy within as short a
time as twenty-four hours, and after two days the eggs had
become the prey of bacteria and no individual egg could be
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NaturaL DEATH AND FERTILIZATION 731
recognized. The sterilized flasks which were opened were
at all times free from foul odor, while the unsterilized flasks
gave off a penetrating stench, often after one, invariably
after two, days. The microscopic examination of the sea-
water for bacteria was always negative in the sterilized flasks,
always positive in the other flasks. In those flasks to which
2c.c. of the putrid culture of starfish eggs had been added,
bacteria and infusoria were exceedingly numerous from the
beginning.
Six hours after the beginning of the experiment one flask
of each of the three series was opened, and the eggs
examined microscopically. The picture was the same in all
three flasks. Nearly all the eggs were mature, and a small
number of them were opaque or black. But what is of the
greatest importance to us is the fact that the percentage of
opaque dead eggs was just as great in the sterile culture (if
not greater) than in the unsterilized or the infected sea-
water.
Twelve hours later, that is to say eighteen hours after the
beginning of the experiment, one of the flasks of each of the
three cultures was again opened. At this time nearly all the
eggs of the sterile culture were opaque or black, and a few
were already granular. In the two other cultures an equal
percentage of the eggs were opaque. The eggs, therefore,
die just as rapidly in the sterilized flasks which are abso-
lutely free from bacteria as in the flasks containing bacteria.
Death follows from internal causes, and so rapidly that the
few bacteria in the sea-water are scarcely able to accelerate
the death of the eggs. The eggs have already died from
internal causes before the bacteria can attack them in suffi-
cient numbers to threaten their existence.
The flasks which were opened later served only to corrob-
orate what has been said. The experiment was repeated
with the same result. Each of the flasks that were opened
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during the first few days also contained a small number of
living transparent eggs. The latter were, without exception,
immature. The experiment, therefore, shows that the mature
eggs of starfish die in the course of a few hours, and that
the cause of this death cannot be sought in the bacteria of
the sea-water; and further, that under exactly the same con-
ditions the immature eggs remain alive.
Ill. THE CHEMICAL CONDITIONS NECESSARY FOR MATURA-
TION IN STARFISH EGGS
Since the eggs of Asterias are usually immature in the
ovary, but, in part, at least, maturate in the course of one or
two hours when introduced into sea-water, the suspicion was
aroused that some of the substances contained in the sea-
water brought about the maturation. In order to determine
which substance this might be, a series of solutions were
prepared having approximately the osmotic pressure of the
sea-water. The result was so simple that it is not necessary
to describe all the experiments here. For it was found that
when the eggs are introduced into solutions which contain
free hydroxyl ions, maturation soon follows, but that this
does not occur in solutions containing no hydroxyl ions.
So, for example, the eggs retain their nucleus in a $m NaCl
solution, or in NaCl solutions to which some potassium or
calcium has been added. If, however, 0.5 to 2 c.c. 7)
NaOH is added to each 100 cc. of such solutions, matura-
tion soon follows: that is to say, the nucleus becomes in-
visible. Since sea-water contains free hydroxyl ions the
conclusion is justified that these are one of the causes for
the maturation of the starfish egg. It was possible to prove
this assumption through further experiments. If a small
amount of acid is added to sea-water, the free OH ions dis-
appear, and the water becomes acid in reaction (through the
addition of 1.5 ¢.c. or more ;", HCl to 100 c.c. sea-water).
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NaTuRAL DEATH AND FERTILIZATION 733
Immature eggs were introduced diréctly into sea-water to
which 1, 2, 3 and 4c.c. of a .”, HNO, solution had been
added to each 100 c.c. of sea-water. While, as is usual, a
large percentage of eggs soon maturated in the normal sea-
water, maturation did not occur at all in the vast majority or
in all the eggs contained in the sea-water to which 2 or more
e.c. acid had been added. The addition of even 1 c.c. of
acid diminishes the number of eggs that maturate. Butitis
not even necessary to keep the eggs permanently in neutral
or acid sea-water in order to inhibit maturation. If 4 or 5 c.c.
of a *, HNO, solution are added to 100 c.c. sea-water, and
immature eggs are introduced into such a solution for only
about fifteen minutes, relatively few eggs maturate when
they are returned to normal sea-water. Such acidified sea-
water does not kill the starfish eggs.
We shall see later that the procedure described here
which, when used upon immature eggs, prevents maturation,
brings about artificial parthenogenesis when used on mature
eggs."
I have, moreover, been able to convince myself of the
fact that the eggs which are introduced into acidified sea-
water in an immature state, can be fertilized by sperm if
they finally maturate. It is possibly in harmony with what
has just been said that the addition of NaHCOs, or larger
amounts of sodium citrate to the sea-water accelerates the
process of maturation. Free hydroxylions are present in the
solutions of both substances, and it is possible that their
addition to the sea-water increases the concentration of the
free hydroxy] ions in the sea-water.
But the hydroxyl ions are certainly not the only sub-
stances in the sea-water which favor or cause the maturation
of the starfish egg. I soon found that when different speci-
mens of eggs are taken from the same culture, and the per-
1 LOEB, FIscHER, AND NEILSON, Pfliigers Archiv, Vol. LXXXVIT (1901), p. 594,
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734 STUDIES IN GENERAL PHYSIOLOGY
centage of mature eggs is determined, this percentage is
subject to the greatest variations. The cause of these
variations was soon discovered. For it was found, where
the eggs lie together in a heap maturation occurs slowly,
but where they lie in a thin layer, maturation occurs quickly.
This fact suggested the importance of oxygen for matura-
tion. Where the eggs lie in a heap the appropriation of the
oxygen by the superticial layers of eggs prevents the diffu-
sion of the oxygen to those lying deeper.
Experiments were now made in which the oxygen of asmall
flask containing a small amount of sea-water was replaced
by hydrogen. When, in such experiments, all the oxygen
was entirely removed maturation did not occur in any, or at
least the majority of the eggs, in spite of the presence of
the hydroxyl ions in the sea-water. There are, therefore, at
least two substances in sea-water which cause or accelerate
maturation, oxygen and hydroxyl ions. Possibly other con-
stituents of the sea-water are also concerned in the process,
but NaCl, Ca, and K have apparently no beneficial effect upon
maturation.’
It seems, therefore, that the absence of oxygen and
hydroxyl ions in the ovaries belongs to the conditions which
inhibit maturation of the eggs in the ovary.
IV. THE PROLONGATION OF THE LIFE OF THE UNFERTILIZED
STARFISH EGG BY THE PREVENTION OF MATURATION
We have shown above that the mature eggs of a culture
of unfertilized starfish eggs die within a short time (which
decreases with an increase in temperature), while the imma-
ture eggs remain alive a relatively long time. It was
necessary now to show that when the maturation of a culture
of unfertilized egg of Asterias is prevented artificially, the
1 Professor Whitman informs me that the maturation of the eggs of Clepsine
does not begin until after they are laid. Possibly the oxygen contained in the water
is in this case also a necessary condition for maturation.
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Natura DEATH AND FERTILIZATION 735
eggs live longer. We begin with the experiment which is
technically most simple. The eggs streaming from the
ovary are divided into two portions. One portion of eggs
is carefully distributed without mechanical agitation, by
carefully tipping the vessel, in a thin layer over the bottom
of the vessel. The vessel must be low and the layer of sea-
water covering the eggs not too deep, so that the diffusion
of oxygen to the eggs can occur withease. A second portion
is introduced with just as great care into a small-calibered
glass tube sealed at one end. This glass tube is half filled
with eggs so that one is certain that the lower layers of the
eggs in the pipette receive little or no oxygen. It is self-
evident that the eggs must be introduced into the tube
immediately after being laid. When, after twenty-four
hours, the eggs which are distributed over the bottom of the
glass dish and which receive a large amount of oxygen are
compared with those at the bottom of the glass tube, a
striking difference is found between them. The eggs richly
supplied with oxygen contain a much larger percentage of
mature dead and black eggs than those kept in the lack of
oxygen. In the latter the living immature eggs are in the
majority, and a part of these maturate when spread out in a
thin layer over the bottom of a vessel. These experiments
are also well adapted to show that the rapid death of the
mature unfertilized sea-urchin eggs is determined through
internal conditions and not by the bacteria contained in the
sea-water. I will cite an example.
One portion of a lot of eggs was spread out in a thin
layer over the bottom of a dish; another was heaped ina
mass in the same dish. The sea-water was the same in both
cases. The first portion of eggs matured in a few hours and
were, in less than twelve hours, opaque and dead, while the
water was still absolutely clear and without odor of putrefac-
tion. After twenty-four hours the water became putrid and
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736 STUDIES IN GENERAL PHYSIOLOGY
contained many bacteria. Even after three days, when the
water was exceedingly foul and cloudy, a portion of the eggs
which had lain in a heap, that is to say, without oxygen,
were immature and living. They were introduced into fresh
water and spread out intoathin layer. They maturated and
developed into swimming larve upon the addition of sperm.
It is self-evident of course that even immature eggs finally
become the prey of bacteria, and so go to pieces in the sea-
water.
The same experiment can be made in a somewhat more
complicated way with pure oxygen and hydrogen. The
freshly laid eggs of a starfish were distributed into two
series of eight flasks. The one series of flasks was connected
with a hydrogen generator; the other with a tank contain-
ing pure oxygen. Before the beginning of the experiment
all the air in one of the series of flasks was driven out by
the current of hydrogen. During the course of the experi-
ment a vigorous current of hydrogen was maintained. Both
series of flasks contained freshly laid immature eggs of
Asterias. The experiment lasted three days, and from time
to time a flask was removed and its contents examined. The
eggs which had been exposed to the current of oxygen
maturated just as rapidly and as numerously as those in ordi-
nary sea-water, and the mature eggs soon died. In the cur-
rent of hydrogen maturation did not occur in the majority of
the eggs, and these remained alive. In the hydrogen
cultures a rapid development of bacteria occurred, while in
the oxygen cultures this occurred to a small degree.'
Treatment with acids which, as we have shown above,
prevents the maturation of the eggs (without killing them)
also prevents their death and disintegration.
Eggs which, without having been in contact with pure
1Care must be taken in these experiments tbat the air is thoroughly removed
from the sea-water in the hydrogen flasks before the eggs are introduced into them.
Of course the hydrogen apparatus must also be free from air.
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NATURAL DEATH AND FERTILIZATION 737
sea-water are introduced for ten or fifteen minutes into 100
c.c. sea-water plus 4 c.c. 7%; HCl maturate very slowly or
not at all when they are returned to normal sea-water. They
also retain, as long as they are immature, the transparent,
normal appearance of living eggs until they become the
prey of bacteria.
It seems to follow from these experiments that the same
processes which underlie the maturation of starfish eggs
also lead to their death (if they are not inhibited through
circumstances which we designate by the term fertilization).
I tried to see, now, whether it was also possible to maintain
the life of the mature egg through lack of oxygen. I
indeed obtained in a few cases positive results in this direc-
tion. The eggs of a starfish were spread in a thin layer
over the bottom of a dish. After three hours 75 per
cent. of the eggs were maturated. A portion of the mature
eggs was carefully introduced into the glass tube described
above, in which the deeper layers suffered from lack of
oxygen. A second portion was introduced into a small flask
through which a steady stream of pure oxygen was passed.
On the following morning, that is to say, fifteen hours after
the eggs were brought into the atmosphere of pure oxygen,
the various portions of the eggs were examined: The eggs
introduced into the current of oxygen showed in one vessel
98 per cent. mature and dark, dead eggs and 2 per cent.
immature living eggs. The eggs which had remained in
normal sea-water contained, as before, about 75 per cent.
mature eggs, all of which, however, were black and dead,
with the exception of a few eggs which had begun to divide,’
and were living.
The immature eggs were also still living. Upon the other
hand, the eggs which had been left in the glass tube in
1This cleavage was possibly brought about through mechanical agitation; I
had repeatedly shaken the dish to facilitate the introduction of oxygen into the
sea-water.
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738 STUDIES IN GENERAL PHYSIOLOGY
absolute or relatively high lack of oxygen, were nearly all
living! This observation seems to show that the same
processes which lead to the maturation of the egg bring
about its death if they are not inhibited at the right time.
In this way the process of fertilization becomes a life-saving
or life-prolonging act.
Vv. DO THESE FACTS HOLD FOR OTHER FORMS?
The question of the relation between maturation and
natural death can be studied most beautifully in the starfish
egg because it is possible to obtain it in an immature condi-
tion, and because maturation follows very rapidly. With
sea-urchin eggs conditions are much less favorable since the
egg maturates within the ovary, and since it is difficult to
obtain immature eggs during the spawning season. I have,
therefore, been unable to discover which chemical factors
determine the maturation of the sea-urchin egg, and to decide
whether the same circumstances cause the death of the sea-
urchin egg that bring about the death of the starfish egg;
and whether the life of the sea-urchin egg can be prolonged
through a prevention of these circumstances. In an indi-
rect way Lewis and I attempted to answer this question last
year when we assumed that the destructive processes which
bring about the death of the unfertilized egg are enzymatic
(autolytic?) processes which can be inhibited through poisons
such as KCN.’
We did in fact succeed in showing that the addition of a
small amount of KCN to the unfertilized sea-urchin eggs mark-
edly lengthens their life. Even after seven days such eggs
can be fertilized as soon as they are returned to normal sea-
water. We also pointed out that, because of the well-known
bactericidal properties of potassium cyanide, the experiments
on sea-urchin eggs were not in themselves decisive, and so
1 Logs AND LEwIs, American Journal of Physiology, Vol. VI (1902), p. 305.
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NatTuRAL DEATH AND FERTILIZATION 739
began experiments on starfish eggs’ which, however, we were
not able to complete at that time. In dealing with eggs
which are as long lived as sea-urchin eggs a great develop-
ment of bacteria in normal sea-water cannot be prevented,
since a few of the eggs always die and so serve as an excel-
lent culture medium for the further development of bacteria.
It need, therefore, surprise no one that the unfertilized eggs
of sea-urchins, as I was able to show this year, live in sterile
sea-water for five days, or possibly longer, while they die
much earlier in ordinary sea-water (about two days). The
very fact that the eggs of sea-urchins are found mature in the
ovary indicates that they are able to live a considerable time
after maturation and that they differ in this respect from
the starfish egg.
It is, however, a fact that in the same sea-water the fer-
tilized and developing sea-urchin eggs live longer than the
unfertilized eggs.
It almost seems as if in certain of the higher animals
there are eggs which develop only when they are fertilized
immediately after leaving the ovary. Under the direction of
Professor C. O. Whitman, Harper has shown that the eggs
of pigeons are fertilized the moment they leave the ovary.
The sperm lives in a gelatinous mass upon the surface of the
ovaries,’ so that provision is made for the necessary contact
between sperm and egg. This also does away with the diffi-
culty which many have found in explaining how the sper-
matozoon finds its way to the egg in animals in which fer-
tilization occurs within the body. Definite directive forces
are clearly not necessary, since a portion of the spermatozoa
must reach the ovary, through their ciliary motion, by way
of the uterus and Fallopian tubes. Experiments similar to
1 Tbid.
2Spermatozoa are in general much longer lived than mature eggs, even though
great differences exist in this regard in different animals. In the spermatic vesicles
of the queen bee spermatozoa are believed to remain alive more than a year after
copulation.
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740 STUDIES IN GENERAL PHYSIOLOGY
those made by Harper upon pigeons must yet be made upon
mammals. Yet there seems to be no doubt that the mam-
malian egg of many species is also fertilized before it reaches
the uterus. Cases of extra-uterine pregnancy also point to
the possibility that fertilization may occur at the surface of
the ovary.
VI. THE PROLONGATION OF LIFE AND THE THEORY OF
FERTILIZATION
Our experiments seem to have proved that the mature
unfertilized starfish egg dies within a few hours through
internal changes, but that the process of fertilization saves.
the life of the egg. This is true, not only of the fertiliza-
tion of the starfish egg by spermatozoa, but also for the
chemical fertilization through hydrogen ions. Mr. Neilson
succeeded this year in keeping the parthenogenetic larve of
starfish alive much longer than has thus far been the case
(over thirty days), and Dr. Fischer was able to accomplish
the same for the larvee produced osmotically from unfertil-
ized sea-urchin eggs. It is therefore possible that the chemi-
cal or osmotic fertilization of these eggs can give rise to as
long-lived larve as the fertilization of the egg through
sperm.
But how does the spermatozoon, or the physical and
chemical means substituted for it, save the life of the egg,
and why does the mature egg die when it is not fertilized
by sperm or artificial means? I believe that the answer
lies in this, that the fertilizing agencies accelerate metabolic
processes in the egg which, before fertilization, went on only
slowly. After fertilization by sperm or by the chemical or
physical means substituted for it, the egg divides and grows
which it did not do before fertilization occurred. Growth
is inconceivable without a preponderance of synthetical over
hydrolytical processes. I believe it possible that the deter-
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NatTurRAL DEATH AND FERTILIZATION 741
mining factor in the chemical forces set in motion within the
egg through fertilization consists in this that the synthetical
processes in the egg are accelerated. If these processes are
not inaugurated or accelerated the egg dies. The wasting
of the body in old age also indicates a decrease in syntheti-
cal processes. Whether the second critical period occurring
in old age is similar to the critical period of the egg cannot
yet be determined. Yet it is not impossible that the ques-
tion of the prolongation of life at this period should pass
over into the question of the possibility of accelerating
synthetical processes.
We, therefore, come to the conclusion that fertilization
accelerates a series of chemical changes (syntheses?) in the
eggs which do not occur sufficiently rapidly without spermatic,
chemical, or osmotic fertilization in the eggs of the majority
of animals. But why does the mature egg die when these
processes are not accelerated, and why does it remain alive
before it maturates? The egg must often exist for years in
the immature condition in the ovary. In answer I can
only suggest that the processes underlying maturation are
at least in some form of a destructive nature (one might
think of autolytic processes) which the egg cannot withstand
for an indefinite length of time without dying. In many
eggs the velocity of these destructive (autolytic?) processes
may be greater than in others and this may determine the
differences in the velocity with which the mature, unfer-
tilized egg dies. It is in harmony with this view that
when maturation is prevented, or the mature egg is put un-
der conditions which inhibit the process of maturation or the
chemical processes underlying it, the life of the egg is
lengthened. Lack of oxygen or the addition of an acid
works in this way in the case of starfish eggs; a slight addi-
tion of potassium cyanide, in the case of starfish and sea-
urchin eggs. But since all of these substances injure the
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742 STUDIES IN GENERAL PHYSIOLOGY
eggs indirectly and do not entirely do away with the
destructive (autolytic?) processes occurring within the egg,
life is not prolonged to the same extent by these means as
by fertilization in which case life is prolonged not only
through an inhibition of the destructive but also through
an acceleration of the synthetical processes.
That the chemical processes which underlie maturation are
not identical with those which bring about fertilization
seems to be supported by the observation made above, that
the same means—the treatment with acid—which causes
the mature egg to develop and live beyond the bipinnarian
stage, inhibits the maturation of the immature egg. When
the mature unfertilized eggs of a starfish are introduced
for fifteen to sixty minutes into a mixture of 100 cc. sea-
water plus 3 cc. %, HCl 90 per cent. of the eggs can,
under favorable conditions, develop into larve. If, how-
ever, the eggs are introduced into such a solution for the
same length of time before maturation, the maturation of
the eggs is prevented either permanently or for a long time.’
The difference is still more striking when the eggs are kept
for a shorter time in a mixture of 100 c.c. sea-water and 5 c.c.
#5; HCl. This shows that acid affects the process of devel-
opment and the process of maturation in opposite or at least
different ways.
We must now raise the question, How does the behavior
of naturally parthenogenetic eggs, such as the eggs of bees,
harmonize with these ideas?
In naturally parthenogenetic eggs it seems as if the
processes which underlie maturation pass over into those
underlying development. But it is possible that this is only
apparently the case, and that in reality it so happens that in
the processes underlying maturation a metabolic product is
1 The eggs which finally maturate in spite of the previous treatment with acid
often begin to cleave when maturation iscomplete and develop into larve, while the
control eggs kept in normal sea-water do not develop.
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NaturaL DEATH AND FERTILIZATION 743
formed in the parthenogenetic animals which favors the
processes of development. We know that an exceedingly
small amount of hydrogen ions suffices to bring about devel-
opment in unfertilized starfish eggs; that an exceedingly
small amount of calcium causes the unfertilized eges of
Amphitrite to develop; and that a trace of potassium ions
brings about the development of unfertilized Chetopterus
eggs.’ It is entirely possible that the specific ions or other
substances necessary to start the development of the eggs of
the bee are formed within the egg itself through the chemi-
cal changes taking place during or after maturation, and
that without the formation of these substances develop-
ment is impossible. In the case of sea-urchins and starfish
eggs one might also believe that the processes of maturation
and the processes of development pass over into each other.
For it has often been observed that the unfertilized eggs of
these forms after having resided in ‘‘normal” sea-water for
about twenty-four hours begin to cleave shortly before death.
This cleavage, however, never goes beyond the two- or four-
celled stage. This might be explained by the fact that the
eggs begin to die at this time. After I had found this year
that the eggs of sea-urchins can still be fertilized after a
residence of five days in sterilized sea-water (at summer tem-
perature), I decided to study this question of spontaneous
cleavage somewhat more closely. If it were true that indi-
vidual sea-urchin eggs begin to cleave in ordinary sea-water
after about twenty hours, and cease to develop any further
only because they soon die, it would be expected that many or
all should cleave when kept alive four or five days, and that
a number of them should reach a fairly advanced stage of
development. A lot of sea-urchin eggs were distributed
into a series of flasks containing sterile sea-water. One of
the flasks was opened every morning and a careful search
was made for developed eggs.
1 LoeB, FISCHER, AND NEILSON, loc. cit,
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In the course of five days I never found a single divided
egg, either in the two-celled stage or in later stages of devel-
opment. It is possible that during the last days of the
experiment a few eggs divided, and that the cleavage cells
fell apart. Lewis and I found last year that when eggs
are fertilized forty-eight or more hours after their removal
from the ovaries they form no membrane and _ the cleavage
cells fall apart. I have corroborated this fact this year.
Usually more than one embryo develops from such an egg,
because the cells drop apart. I kept this fact in mind and
will not deny that a few small eggs were present, which per-
haps represented only half the mass of an ordinary egg.
But nearly all the eggs were of normal size, and since small
eggs are occasionally found even under normal conditions,
the experiment shows that in sea-urchin eggs also the
processes of maturation are not continuous with those of
cleavage, and that entirely different conditions which we can
bring about through the abstraction of water or the entrance
of a spermatozoon are necessary that division may occur.
It cannot be urged that the sterilized water perhaps pre-
vented the cleavage. When at the conclusion of the experi-
ment these same eggs were fertilized in sterilized water by
adding a drop of sperm, they developed to the pluteus stage
in sterile sea-water. I, therefore, consider it possible that
where authors describe a cleavage of the unfertilized sea-
urchin eggs in ‘‘normal” sea-water, the sea-water or the
egg had in reality suffered some change which had escaped
the notice of the observers. One might think of evaporation
and increase in the osmotic pressure of the sea-water. A
very slight increase in the osmotic pressure of the sea-water
is sufficient to cause the sea-urchin egg to divide into two
cells in the course of twenty hours. One might also think
of a change in the sea-water brought about by the putre-
faction of the dead eggs. Finally it is possible that a sub-
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NaturaL Datu AND FERTILIZATION 745
stance is perhaps formed (for example, an acid) in the dying
eggs which brings about a single cleavage.
The relations which exist between maturation and natural
death upon the one hand, and fertilization and prolongation
of life upon the other, lead us to the conclusion that a “fer-
tilization” must perhaps come to pass in every egg, even in
those naturally parthenogenetic. Only, according to our
idea, the act of fertilization is not identical with the mor-
phological process which is designated fertilization. It is
rather a chemical or a physico-chemical act which accelerates
certain (synthetical?) metabolic changes in the egg, which
occur in the egg under ordinary conditions also, only much
too slowly, The difference between naturally partheno-
genetic eggs and the eggs which must be fertilized before
they can develop consists perhaps in this, that to the latter
the catalytically working substance or complexus of condi-
tions must be added from the outside in order to accelerate
the synthetical (?) processes, while in the naturally partheno-
genetic eggs these substances are formed within the eggs
(possibly in conjunction with the processes of maturation).
The connection between the prolongation of life and fer-
tilization clearly points out that every purely morphological
theory of fertilization is incomplete and that a correct theory
of this process must have a physico-chemical basis. The
means of reaching this basis I see in further attempts at
causing development of unfertilized eggs through unequiv-
ocal physical and chemical means.
VII. CONCLUSIONS
1. Our observations and experiments seem to show that
in the same sea-water and under otherwise identical con-
ditions, mature but unfertilized starfish eggs soon die, while
immature as well as mature but fertilized eggs live longer.
2. It seems certain that the rapid death of the mature
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unfertilized starfish eggs is determined by internal condi-
tions connected with maturation and not by the bacteria
contained in the sea-water. The proofs for this are: First,
mature eggs die just as rapidly in sterilized sea-water free
from bacteria as in unsterilized water, and secondly, when
maturation is prevented artificially the eggs may continue to
live in water containing many bacteria.
3. We have shown that oxygen and free hydroxyl ions
accelerate the maturation of starfish eggs; that lack of
oxygen and a neutral or faintly acid reaction of the sea-
water inhibit or prevent maturation. The fact that the eggs
which remain immature in the ovaries of the starfish
maturate when brought into sea-water seems to find its
explanation in part at least through this.
4. When the maturation of starfish eggs is prevented
artificially through lack of oxygen, or the addition of an
acid to the sea-water, they remain alive much longer than
when they maturate. The eggs in which maturation has
already begun, or has just been completed, seem also to be
saved from rapid death by these means.
5. It seems to follow from these facts that the same
chemical processes do not necessarily underlie the process
of maturation and the process of fertilization. Fertiliza-
tion by spermatozoa, chemical or physical agencies, lengthens
the life of the egg, while the changes following the matura-
tion of the egg lead, sooner or later, to death (through
autolysis?). Itis in harmony with what has been said that
the same treatment with acid which brings about artificial
parthenogenesis in mature starfish eggs inhibits the process
of maturation when used upon immature starfish eggs.
6. These facts corroborate a suggestion which I have
made before, that the fertilizing action of the spermatozoon
consists in this, that it carries into the eggs substances which
accelerate the course of certain (synthetical?) processes in
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the egg. Such an acceleration might, for example, be
brought about through certain ions (for example, the
hydrogen ions of nucleic acid), yet the possibility that
such catalytic effects might also be brought about through
enzymes or other substances is of course not shut out. Yet
this fact must be considered: that we have been able to
produce artificially normal embryos capable of development
through ions, while the careful experiments of Gies con-
ducted in my laboratory, in which he attempted to find the
same to hold for enzymes, have thus far failed.
In conclusion I wish to thank my assistant, Mr. Neilson,
for the assistance which he rendered me in these experi-
ments.
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XXXVII
ON THE PRODUCTION AND SUPPRESSION OF MUSCU-
LAR TWITCHINGS AND HYPERSENSITIVENESS OF
THE SKIN BY ELECTROLYTES’
Iv has been shown in former publications that a slight
variation in the proportion and character of the electrolytes
in a tissue is capable of imparting to that tissue properties
which it does not possess ordinarily, and it has been sug-
gested that this fact might help us in recognizing the nature
of a number of nervous and muscular diseases, and also
possibly furnish a means of curing or mitigating them.
This paper contains some further contributions to the same
subject. It deals with the determination of electrolytes which
are liable to produce and inhibit hyperactivity of muscles and
hypersensitiveness of the nerves of the skin; and tries to
answer the question whether or not the stimulating and
inhibiting effects of ions are a function of their valency and
electrical charge.
I. THE PRODUCTION AND SUPPRESSION OF MUSCULAR TWITCH-
INGS BY ELECTROLYTES
1. Our muscles do not normally contract or twitch rhyth-
mically, but they do so in certain diseases. The main
electrolyte in our blood is sodium chloride. When we put a
muscle into a pure sodium-chloride solution of the right
osmotic pressure (7. e., isotonic with the muscle), the muscle
soon begins to twitch rhythmically, and these twitchings may
last for several days, or about as long as the muscle lives.
But when we add a very small, though definite, amount of a
1 University of Chicago Decennial Publications (1902), Vol. X, p. 3.
2Part II, pp. 544, 559, and 692; Pyliigers Archiv, Vol. LXXXVIII (1901), p. 68.
748
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Muscunar Twitcuinas 749
soluble calcium salt, the twitchings will not occur, though
the muscle lives longer in such a solution than in a pure
sodium-chloride solution. I concluded from this that we
owe it to the calcium ions in the blood that our muscles do
not twitch or beat rhythmically like our heart.!
To test this idea further, Mr. W. E. Garrey and I under-
took, in 1889, a series of experiments, not yet published, on
the behavior of muscles in solutions of sodium salts whose
anions precipitate calcium. The muscle itself contains cal-
cium salts, and we considered it likely that these calcium
salts might help in preventing contractions. We therefore
thought that by putting the muscle into solutions of sodium
salts, which, by entering the muscle, precipitate the calcium
contained in it, we might produce still more powerful rhyth-
mical contractions than in a pure sodium-chloride solution.
This was found to be true. In solutions of sodium-fluoride,
-oxalate, -carbonate, -phosphate, etc., of the proper concen-
tration (1 gram-molecule in 8 liters of the solution), we
obtained similar, but more powerful, rhythmical contractions
than in sodium-chloride solutions of the same osmotic pres-
sure. Another series of observations confirms the idea that
it is due to the calcium salts in our body that our muscles do
not show any rhythmical contractions or twitchings. When
we inject into the body of an animal any salts that are liable
to precipitate calcium, we notice ‘almost immediately twitch-
ings of all the muscles.” It seems, therefore, rational that in
the pathology of muscular twitchings the concentration of
the calcium ions in the blood should be taken into considera-
tion. It is quite possible that abnormal conditions may arise
in the body which lead to an increase of such acids in the
circulation as diminish the amount of calcium ions in the
1Part II, p.518. See alsoS. RINGER, Journal of Physiology, Vol. VII (1886), p. 291
In this paper Ringer also mentions briefly the fact that Ba differs in its action from
Ca and Sr.
2FRIEDENTHAL, Engelmann’s Archiv, 1901, p. 145.
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body, e. g., oxalic acid, or others. The necessary outcome
would be muscular twitchings. In that case the administra-
tion of calcium salts might cure the disease.
2. In a recent paper I have shown that the antitoxic
effects calcium produces when added to a pure NaCl solution
are a function of its valency and the sign of its charge, inas-
much as similar effects can be produced by other bivalent or
trivalent kations (e. g., Mg, Sr, Ba, Zn, Fe, Co, Pb, Al, Cr),
but not by bivalent or trivalent anions.’ The question arises
whether or not the inhibiting effects of Ca ions in the case
of rhythmical contractions of muscles are also a function of
the valency and electrical charge of the Ca ion. My earlier
experiments were not opposed to such a conclusion. I had
found that in ™ solutions’ of LiCl, NaCl, RbCl, and CsCl
rhythmical contractions occur, while small amounts of the
chlorides of Ca, Mg, Sr inhibit these contractions. I have
since continued these experiments, with the following results:
When we put a muscle (the gastrocnemius of the frog was
used in these experiments) into a ™ sodium-acetate solu-
tion, the twitchings of the muscle begin at once. The addi-
tion of from 3 to 4 c.c. of am CaCl, solution to 100 c.c. of a
® sodium-acetate solution absolutely suppresses all twitch-
ings. But even half the amount suffices for practical pur-
poses, inasmuch as in this case only a few beats occur at the
beginning. MgCl, and SrCl, act like CaCl,. But BaCl,
acts altogether differently. An addition of 5 cc. of a m
solution of BaCl, to 10 c.c. % sodium-acetate solution not
only does not stop the rhythmical contractions, but makes
them more powerful. Instead of the rapid and rather weak
fibrillary twitchings which occur in a ™ sodium-acetate
solution, more tetanic and energetic contractions occur when
BaCl, is added. I then tried whether the muscle is able to
1Part II, p. 708.
2By a } solution is meant a solution which contains 1 gram-molecule in 8 liters.
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Muscuuar TwItcHINes 751
beat in a pure BaCl, solution. It goes without saying that
in pure solutions of MgCl,, CaCl,, and SrCl, a muscle does
not show any rhythmical contraction. Ina ™” BaCl, solu-
tion, however, the muscle beat for forty minutes; in a
solution, for one and a half hours; in a 7 BaCl, solution,
for over an hour; and in a 7 BaCl, solution, for about
half an hour. The beats showed the same tetanic form char-
acteristic of the presence of barium. The fact that the beats
stop sooner in a % BaCl, solution than in a ™ solution
is due to the poisonous effects of barium. The fact that the
beats stop also very soon in a 7% BaCl, solution is due to
the enormous absorption of water which occurs in such weak
solutions.’
Similar facts were found for Ba(NO,), and Ba (HO),.
The most striking fact is that the stimulating power of Ba
salts is greater than that of the corresponding Na salts.
In a 7% BaCl, solution the muscle may begin to beat in a
few minutes and may continue to do so for half an hour. I
have even occasionally noticed rhythmical contractions in a
zz, BaCl, solution. But I have never noticed any rhyth-
mical contractions of muscle in a #4 NaCl solution, and as
a rule even in a % NaCl solution the twitchings begin only
after a long latent period and last but a short time.
Barium is, however, not the only bivalent kation whose
chloride possesses a higher stimulating power than that of
the chloride of univalent kations. It was found that the
chlorides of the heavy metals are also capable of producing
rhythmical twitchings or beats of muscles in much lower
concentrations than those found effective in the case of NaCl
or LiCl. In ,%, solutions of ZnCl, strong beats occurred,
which, however, did not last long on account of the rapid
imbibition of the muscle with water, as well as on account
1The beats in BaCl, solution often do not begin at once, but after a latent period
of from two to fifteen minutes.
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752 STUDIES IN GENERAL PHYSIOLOGY
of the poisonous effects of Zn. In stronger solutions than
#z no beats occurred. The same was true for ZnSO,.
Solutions of CdCl, and Pb(No,), also gave rise to a few
contractions in the concentration of about 5%, to 7%.
The fact that the more concentrated solutions of the salts
of heavy metals did not act is probably due to their poison-
ous effect. Itis, therefore, evident that there are a number
of chlorides with bivalent kations which are able to produce
rhythmical contractions at a lower concentration than NaCl.
It would, therefore, be wrong to ascribe the inhibiting effect
of Ca salts upon rhythmical contractions to the double
valency and the positive charge of the Ca ions.
3. Does the effectiveness of salts for the production of
rhythmical muscular contractions increase with the valency
of the anion? This is decidedly not the case, as the fol-
lowing table shows. In this table are given the minimal
concentrations of the solutions of various sodium salts in
which rhythmical contractions occur:
Salt Minimal Effective
Concentration
( NaCl m
| NaBr mm
Univalont anions 3 0 ge
Na acetate mB
a io
Na formiate wm = ity
m
( Naz succinate
i
|
* ¥ ! Na, sO, 5
Bivalent anions NaHCO, m -#
Na, oxalate 2eo — 500
; : Na,HPO, Oe at
Trivalent anions Ne. cities = eee
It is obvious that the power of favoring rhythmical con-
tractions in muscles is not an unequivocal function of the
valency of the anion. It is likewise obvious that the sodium
salts whose anions precipitate calcium powerfully, like sodium
fluorides, sodium oxalate, and sodium phosphate, are among
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MuscuLar TWITCHINGS 753
the most favorable salts to produce rhythmical twitchings.
Sodium citrate does not precipitate calcium in the tissues, but
prevents its precipitating other compounds, and therefore
practically makes calcium inactive." But that the precipitation
and inactivation of calcium is perhaps not the only factor
involved is shown by the efficiency of sodium formiate. It
may be, however, that sodium formiate undergoes further
changes in the tissues, and that one of the products formed
acts upon calcium.
All these facts suggest that it might be worth while to
test the idea whether or not the pathological cases of mus-
cular hyperactivity and twitching are due to a lack of cal-
cium in the muscles (or blood), and whether the evil can be
mitigated by giving calcium salts to such patients. Experi-
ments must be made in animals to find out whether or not
such a treatment would do any harm before any therapeutical
experiments on patients should be attempted. It is our in-
tention to take up these experiments in the laboratory.
Il THE DIFFERENT EFFECTS OF CALCIUM IN THE CASE OF
MYOGENIC AND NEUROGENIC RHYTHMICAL CONTRACTIONS
1. While it seems easy to suppress, by the addition of
Ca, Sr, and Mg, rhythmical twitchings which originate in
the muscle itself, the question arises whether the same
means allow us to suppress, with equal ease, muscular twitch-
ings which originate through the central nervous system.
The simplest organism in which this can be tested is probably
the jelly-fish. These animals contract rhythmically. Their
central nervous system is contained in the margin of the
swimming-bell, while the center of the animal is said to con-
tain no nerve-elements except scattered neurons. By a cut
1 SABBATANTI has shown that although sodium citrate does not precipitate cal-
cium, it renders it inactive. In the presence of a sufficient quantity of sodium
citrate, calcium loses, e. g., its coagulating effect: Archives Italiennes de Biologie,
Vol. XXXVI (1901), p. 397.
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parallel to the edge we can divide the animal into a marginal
part, which contains the central nervous system, and a cen-
tral part, without a central nervous system.
When this operation is performed, the margin will go on
beating in sea-water, while the center will not beat. Ro-
manes, who was (as I believe) the first to make this experi-
ment, drew the conclusion that the central nervous system
was the originator of the automatic contractions of this
animal. From previous experiments of Aubert,’ Howell,’
and Greene*® on the heart, and my own experiments on the
muscles, I concluded that the center of a jelly-fish (Gonio-
nemus) did not beat in sea-water on account of the presence
of certain ions in sea-water, especially calcium, and I showed
that the center of a Medusa will beat rhythmically in pure
NaCl or NaBr solution. The center of a Medusa whose
margin is cut off seems then to behave, to a certain extent,
like the striped muscle. It was of some importance to
find out how far this analogy goes. The following six solu-
tions were prepared:
100 c.c. % NaCl
100 c.c. % NaCl-+ 4 c.c. +55 Ca(Nos3)2
100 c.c. # NaCl-+-1 c.c. +55 Ca(Nos)2
100 c.c. % NaCl-+ 2c.c. +3 Ca(Nos3)2
100 c.c. NaCl+4c.c. 1 Ca(No3)2
100 c.c. NaCl-+ 8 c.c. 75s Ca(Nos3)2
In solution 1 the center of a Medusa begins at once to con-
tract very rapidly. The velocity of contractions steadily in-
creases and very soon it becomes impossible to count the
contractions. Occasionally the same happens in solution 2.
But in the solutions 3 to 6 the center at first remains per-
fectly quiet. After a latent period of about ten minutes,
often, but not always, contractions begin in solutions 8 to 5,
OOP ON
1 AUBERT, Pfltigers Archiv, Vol. XXIV (1881), p. 361.
2 HOWELL, American Journal of Physiology, Vol. II (1898), p. 47.
3 GREENE, ibid., p. 82. 4 Part II, p. 559.
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MovscuLarR TWITCHINGS 755
or even in 6, which last sometimes as long as fifteen minutes.
These contractions are not as rapid as those observed in a
pure NaCl solution, and resemble more the normal contrac-
tions of a Medusa in sea-water. A series of experiments
was undertaken to find out the minimal amount of Ca re-
quired to prevent completely all contractions in a pure NaCl
solution. In a mixture of 100 c.c. of a % NaCl solution
+ 8c.c. of aSmCa(NO, ), solution no contractions occurred.
A series of experiments with a slightly greater amount of
CaCl, were made with the same result.
The same inhibitory effect can be produced if, instead
of Ca, Sr or Mg is used. But Ba behaves altogether differ-
ently. The following solutions were tested:
100 c.c. % NaNo;
100c.c.% NaNo;+ lec. m BaCl,
100c.c.% NaNos;+ 2c.c.m BaCl,
100 c.c.% NaNo;+ 4c.c, m BaCl,
100 c.c.% NaNo; + 8c.c. m BaCl,
100 c.c. % NaNo; + 16c.c.m BaCl,
When the center of a Medusa was thrown into any of
these solutions, the rhythmical contractions began at once.
The center behaved as if the Ba ion had not been present,
with this difference, however, that the solutions with a
larger amount of barium were more poisonous than a pure
NaCl solution. Ba has, therefore, little or no inhibitory
effect upon the center of a Medusa.’
The analogy between the effect of ions upon muscle and
the center of a Medusa goes still farther. I pointed out
that possibly the Ca ions in the sea-water and the tissues of
the Medusa prevent the isolated center from beating in sea-
1Since this was written I have received, through the kindness of Professor
Sabbatani in Cagliari, a paper published by his assistant, Dr. Regoli, in which the
latter shows that, while.Ca and Sr diminish the irritability of the cerebral
cortex, Ba has the onposite effect ; REGOLT, “Azione dei metalli alcalino-terrosi sulla
eccitabilita elettrica della corteccia cerebrale,” Bollentino d. Societate trait Cultoré
delle Scienze etc. in Cagliari, Torino, 1901.
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756 STUDIES IN GENERAL PHYSIOLOGY
water in the same way as the presence of Ca in the blood
seems to prevent our muscles from beating. In order to
- test this idea, I added to the sea-water various salts which
precipitate Ca, e. g., NaF and Na,HPO,. I found that
when a little more of these salts had been added than re-
quired to precipitate all the Ca in the sea-water, the center
behaved indeed in the same way as if it had been put into a
pure NaCl solution. When a little less Na,HPO, was
added, the beats began after a latent period, which varied
according to the amount of Na, HPO, added. Rapid con-
tractions began at once when 32 c.c. of a ® Na,HPO,
solution was added to 68 ¢.c of sea-water. The same result
was obtained when 16 c.c. of a normal NaF solution was
added to 100 c.c. of sea-water.
The addition of about 13 c.c. of m sodium-citrate solution
to 100 c.c. of sea-water also brought about immediate con-
traction of the isolated center. This salt does not bring
about a precipitation of Ca in the sea-water or the tissues,
but excludes the action of Ca ions in another way.
I did not succeed in bringing about such results with
the addition of Na,SO, to sea-water. Even the addition of
32 c.c of m Na,SO, to 100 cc. of sea-water did not give
rise to contractions, although the irritability of the center
was increased. Experiments with the addition of NaHCO,
remained also negative. But as only a few experiments
were made with Na,SO, and NaCHO,, it is possible that a
continuation of the work might lead to positive results.
It is, therefore, obvious that the centers can be caused to
beat through a diminution of the amount of Ca they contain,
and it may be further argued that the presence of Ca in the
sea-water is the cause, or at least one of the causes, that pre-
vent the centers from beating in sea-water.
It should, however, be added that, while a certain diminu-
tion of Ca in the center is necessary for the development of
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MuscunarR TWITCHINGS T57
rhythmical contractions, the diminution has its limit. It
appears that, if too much Ca is removed from the tissues,
the beats will also cease. This is demonstrated by the fol-
lowing facts: When we put the center of a Medusa into
sea-water to which enough sodium citrate has been added,
beats begin at once, last for a certain time, and then cease.
If at this time the centers are put back into sea-water with
less or no sodium citrate, beats will begin again. The ex-
planation of this phenomenon seems to be as follows: The
normal center of a Medusa contains too much Ca for sponta-
neous rhythmical contractions. If we put a center into sea-
water to which a large amount of NaF, Na, HPO,, or sodium
citrate has been added, so much of the salt will diffuse at
once into the organism that at least in the superficial cells
enough Ca will be eliminated from the field of action to
allow the spontaneous contractions to begin. Subsequently
the same will happen in the deeper cells. The process of
elimination of calcium in the cell proceeds, and very soon
a period comes when the loss of Ca in all the cells will be too
great for the contractions to go on. If, as soon as this
occurs, the center is thrown into normal sea-water, or sea-
water with only a little sodium citrate or phosphate, citrate
and phosphate anions will diffuse back from the tissues into
the sea-water, or Ca ions will diffuse into the cells, or both
phenomena will occur, and beats will again begin. The same
reasoning applies probably to the rhythmical contractions of
muscles and the apex of the heart.
2. When we put the margin containing the central
nervous system into a pure NaCl solution, it behaves very
much like the center, e. g., it begins to beat very rapidly,
and the rapidity of the beats increases, at first steadily, until
the poisonous effects of the pure NaCl solutions make them-
selves felt. But even the addition of large quantities of Ca
does not inhibit these contractions. For instance, when we
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add from 2 to 5c.c. of a $m solution of Ca(NO,), to 100 c.c.
of % NaCl solution, the margin at once begins its rapid
beats. The only effect the addition of calcium has is to make
the rate of the beats a little slower than without calcium. I
thought at first that the stimulus of the wound caused by
the cutting off of the margin might be responsible for these
contractions in the presence of calcium. But this is not the
case, for if we put a whole Gonionemus intact into any of
these solutions, it behaves like the isolated margin. The only
possible inference is that the margin is much more immune
toward the inhibiting effects of calcium than the center, a
fact which I have pointed out already in a former paper.’
In a pure CaCl, solution the margin will not beat.
Inasmuch as the essential difference between center and
margin which accounts for this difference in the effect of cal-
cium is the presence of the central nervous system in the mar-
gin, it may follow from these observations that for the supres-
sion of twitchings of a nervous origin larger doses of calcium
might be required than for the suppression of twitchings of
muscular origin. Preliminary experiments on the motor
nerves of frogs seem to harmonize with this idea. This sug-
gests the possibility that, while a calcium treatment might
be advisable for the cure of myogenic twitchings, for the
suppression of neurogenic twitchings so much calcium might
be required as to exclude the use of this remedy. This, too,
is a point which further experiments on animals must decide
before the matter may be tried in patients.
III. THE PRODUCTION OF HYPERSENSITIVENESS OF THE SKIN
BY ELECTROLYTES
1. In a former paper I have shown that, aside from the
rhythmical twitchings, the salts whose anions precipitate or
inactivate calcium also make muscles and motor nerves sensi-
1Part ITI, p. 692.
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Muscuuar TWITCHINGS 759
tive to stimuli which normally would not affect these organs.
For example, when we put a fresh muscle for one or more
minutes into a % solution of sodium citrate, a peculiar
form of irritability arises (contact-irritability)." Whenever
the muscle is taken out of the solution it goes into powerful
tetanic contractions, which cease at once and give way to
relaxation of the muscle as soon as the latter is put back into
the solution. When this hypersensitive condition is once
established, the contractions can be produced whenever the
muscle is changed from any aqueous solution to any other
non-aqueous medium, while the contractions cease when the
muscle is put back into an aqueous medium,no matter whether
the latter be a solution of an electrolyte or a non-conductor.
It is rather striking that these phenomena do not occur when
the above-mentioned solutions call forth at once the rhyth-
mical contractions mentioned in the previous part of this
paper. It almost looks as if there existed two allotropic
states of the muscle substance, the one giving rise to rhyth-
mical twitchings, the other to the peculiar tetanic contrac-
tions (contact-reactions) just referred to.’ Ultimately, how-
ever, in all cases, rhythmical twitchings are produced.
As far as motor nerves are concerned, I have shown in
the same paper that the same salts which produce this con-
tact-reaction produce hypersensitiveness of the nerve and
ultimately rhythmical contractions of the muscle when acting
upon the nerve alone.
It might be mentioned here in parenthesis that these facts
may throw some light upon the action of cathartics. All the
salts which give rise to the above-mentioned contact-reaction
or hypersensitiveness act as cathartics when introduced into
the intestine. The common explanation of their action is
1Part II, p. 692.
2This difference is emphasized by the fact, found by my pupil, Dr. Zoethout :
that an addition of potassium favors the contact-reaction. As far as rhythmical con-
tractions are concerned, K has an inhibiting effect.
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the one which, I believe, was first suggested by Schmiede-
berg, namely, that these salts prevent the absorption of
liquids from the intestine, and that this retention of liquids
causes the cathartic effect. I will not deny the effect of these
salts upon the phenomena of absorption of water from the
intestine,’ but it is obvious from our experiments that the
same salts must increase the irritability of the nerves and
muscles of the intestine, and that this must facilitate the
production of peristatic motions, possibly through the mechan-
ical or contact-stimuli of the faeces upon the nerve-endings
or the muscular wall of the intestine.
2. These experiments suggested the idea whether or not
electrolytes are capable of producing also a hypersensitive-
ness of the skin and conditions that may be comparable to
the conditions of hyperesthesia or hyperalgesia. It is well
known that when we suspend a pithed frog vertically so that
its legs hang down, the latter will be lifted at once when
they are dipped into an acid or alkali of a certain concentra-
tion, while no such reaction occurs when they are dipped
into water. The reaction of the animal to acid may be so
violent as to suggest to a layman the idea that it is suffering
intense pain. I wondered whether by an alteration of the
nature and proportion of ions in the skin the sensitiveness
could be increased or varied in such a way as to make the
skin as sensitive to the contact with pure water as it natur-
ally is to strong acid. The experiments resulted in my find-
ing certain solutions of electrolytes which did not seem to
affect the animal directly, but yet made it extremely sensi-
tive toward contact with water. The best solutions for this
purpose are, as far as my present experiments go, AICl,, and
sodium-citrate solutions. The way of proceeding is as fol-
lows: A number of solutions, say AICl,, are prepared,
namely, 7%, 7%, %, ", and possibly % Then the weakest
1CUSHNY AND WALLACE, American Journal of Physivlogy, Vol. I (1899).
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Muscuuar TwitcHInes 761
of the solutions is first brought in contact with the feet
of the frog. If the feet are not withdrawn, the next
stronger solution is used, and, if no reaction occurs, the next
stronger. If one thus succeeds in keeping the feet of the
animal for one minute or more in the AlCl, solution, subse-
quent contact of the feet with common tap-water or distilled
water makes the animal act as if the water caused the most
excruciating pain. The feet are violently withdrawn, rubbed
against each other in a way that one notices otherwise only
when the feet are dipped into strong acids. Ifthe AlCl,
solution chosen is too strong, the animal will not leave his
feet in the solution, but will try to withdraw them. But in
that case its attempts at withdrawing its feet from the solu-
tion are never as violent as the subsequent attempts at with-
drawing the feet when brought into contact with common
water. The stronger the solution of AlCl, is in which the
feet had been kept, and the longer they had been kept in
the solution, the stronger their sensitiveness toward water
will become.
Sodium citrate acts very similarly to aluminium chloride.
As the latter is slightly acid and sodium citrate slightly
alkaline, the possibility was suggested that the H and HO
ions are responsible for the hypersensitiveness. While it is
possible to produce occasionally a slight hypersensitiveness
toward common water by a pure solution of NaOH or HCl,
the results are very unreliable. It is practically the same if
one tries to use NaCl solutions to which slight and varying
quantities of HCl or NaOH have been added. Better results
can be obtained with solutions of oxalates, sulphates, car-
bonates, and phosphates. The sodium salts are preferable
to the potassium salts, for the animal withdraws its feet
much more rapidly from the solution of a potassium salt
than from the solution of the corresponding sodium salt.
This makes it difficult in the case of potassium salts to
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762 STUDIES IN GENERAL PHYSIOLOGY
saturate the foot with the sufficient number of ions to induce
the hypersensitiveness.
It goes without saying that the hypersensitiveness which
can be produced by AlCl, and sodium citrate does not make
itself felt toward water alone, but to salt solutions also.
One can find a minimal concentration for each solution of
an electrolyte at which a pithed frog almost instantly with-
draws its feet when they come in contact with the solution.
This minimal concentration is considerably lowered after a
treatment of the foot with an AIC1, or sodium-citrate solution.
The production of hypersensitiveness is only one side of
the problem. The mitigation of the hypersensitiveness is
the other side. The violent reactions of a frog when its
feet are dipped in tap-water after a treatment with AICI,
can be stopped instantly when the feet are put into a normal
solution of cane-sugar. When weaker solutions of cane-
sugar are used the feet are withdrawn, and the attempts at
withdrawing become more noticeable and violent the weaker
the sugar solution is. Very concentrated solutions of urea,
e. g., 2n solutions, act similarly, but not so powerfully as
cane-sugar. Glycerin solutions gave no such results;
neither have I been able to find as yet any solution of an
electrolyte which acted this way. The fact that only very
concentrated solutions of cane-sugar or urea inhibited the
hypersensitiveness gave rise to the idea that the diffusion of
water out of the foot might be the inhibiting factor, and
that a stream of diffusion in the opposite direction, namely,
from the outside into the skin, might give rise to a with-
drawal of the foot. The latter idea could be tested. When
the feet of a pithed frog are dipped into a normal solution
of cane-sugar, they are not withdrawn, no matter how long
they remain in the solation. But if subsequently (after
several minutes) the feet are put into pure water, after a
few (five to ten) seconds the feet are energetically withdrawn.
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MuscuLarR TWITcHINGS 763
In this case, obviously, water diffuses into the skin, which
previously had lost water.
There may be electrolytes which act similarly to cane-
sugar, but I have not yet found them. Every solution of
an electrolyte causes, above a certain concentration, an
immediate withdrawal of the feet, and this withdrawal is
the more energetic the more concentrated the solution.
This differs from the behavior of sugar and urea, which
above a certain concentration have the opposite effect.
The lowest concentration at which the solutions of various
electrolytes will cause a pithed frog to withdraw its feet
instantly or in from five to ten seconds, is about as follows:
HCl, am, or less MgCl,
NaOH, #3 or less CaCl, ( ;
AEN, B.vor'less SrCl, ™ or a little less
FeCl;, 2% or less BaCl,
CdCl, t ors KCl, ™ to
HgCl, § #2 O18 NH.Cl, ™ to 3m
AlCl;, %to® NaCl * a
Tier § Sees
It.almost looks as if the coagulating effect of the kations
upon proteids was of some importance. The powerful effects
of Ag, Cd, and Hg interfere somewhat with the conclusion
that we are dealing with a pure valency effect, which otherwise
seemstomakeitself felt. If,instead of thechlorides, thenitrates
or sulphates of the same metals are chosen, the order of effi-
ciency seems to remain practically the same, as far as can be
judged from an as yet incomplete series of experiments.
As far as the anions are concerned, the order of efficiency
is for the sodium salts about as follows:
Na, oxalate ” NaHCO;
Na; citrate, ” Na formiate tn toe
Na.SOu., ” Na, succinate ( °
NaHPO,, 2 NaCl
NaF, ? to ?
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764 STUDIES IN GENERAL PHYSIOLOGY
In this case, as in the case of rhythmical contractions,
the oxalates and citrates are the most powerful anions of
this series. It is clear that, in the determination of the
lowest concentration of a salt which is still able to cause the
immediate withdrawal of the foot, one must remember that
a number of solutions (e. g., AgNO,, AICI,, FeCl, , HCl,
NaOH, Na, citrate, etc.) have an after-effect which makes
itself felt in an increase of irritability. Other solutions
(e. g., those of calcium salts) may possibly have the opposite
effect, namely, to raise the threshold of stimulation for sub-
sequent tests.
It was of some interest to ascertain whether the results in
these experiments were produced through an action of the
electrolytes upon the nerve-endings, or upon the nerves
themselves. In experiments upon frogs whose skin had been
removed from the feet, the results described in this paper
could not be produced. The experiment of putting the nerves
themselves into the above-mentioned solutions remained prac-
tically without effect. It is possible that with solutions of
much greater concentration results may be obtained. It is,
therefore, certain that the results observed in our experiments
are due to an action of the electrolytes upon the nerve-endings
in the skin, and not to an action upon the sensory nerves.!
IV. CONCLUSIONS
The experiments mentioned in this paper were undertaken
with two aims in view, a practical and a theoretical one. As
far as the former is concerned, it follows from our investiga-
tions that abnormal muscular twitchings and contractions
may be brought about in an organism by a reduction in the
proportion of calcium (or magnesium) in the muscles or the
blood, or an increase in the proportion of Na and other
1The chemical irritability of muscles is, as far as electrolytes are concerned,
also greater than that of motor nerves. The reverse is true for electrical stimula-
tion.
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MuscuuarR TWITCHINGS 765
kations. In view of the fact that thus far no explanation
has been found for pathological phenomena of this kind, it
becomes of some importance to see whether or not in certain
of these diseases the relative amount of calcium ions in the
blood is diminished. If this should be the case, the adminis-
tration of calcium would be the cure for these diseases, which
thus far have been beyond medical control. It is also
apparent from our experiments that for the suppression of
neurogenic twitchings or contractions more calcium may
possibly be required than for the suppression of myogenic
twitchings. There has thus far been no clue as to the origin
of hypersensitiveness or hyperalgesia of the skin. Our ex-
periments show that slight variations in the proportion of
certain ions in the skin can cause an enormous hypersensi-
tiveness.
As far as the theoretical side of the paper is concerned,
it was our aim to test the idea whether or not the “stimulat-
ing” and inhibiting effects of ions are an unequivocal func-
tion of their electrical charge or valency. Over a year ago
I tested the same idea without being able to obtain positive
results, and nothing was said about the subject in the paper
in which the results were published." The test was con-
tinued in the above-mentioned experiments, with results _
which, in my opinion, are equally questionable, if not alto-
gether negative.
1Part II, p. 692.
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XXXVITI
ON THE METHODS AND SOURCES OF ERROR IN THE
EXPERIMENTS ON ARTIFICIAL PARTHENOGENESIS'
1. Because of various papers by European authors who
have encountered difficulties in repeating or continuing my
experiments on artificial parthenogenesis I wish to make a
few remarks on the methods and the sources of error in
these experiments. I do not need to dwell upon the impor-
tance of sterilizing the sea-water, the instruments, the hands,
and the animals themselves; it is self-evident. I wish
in this connection to mention only the greatest sources of
error, namely, the tendency of males, especially ripe sea-
urchins, to fill the sea-water in the pail in which they are
brought into the laboratory with sperm. It is therefore
advisable to keep the females isolated for twenty-four hours
or if possible even longer in sea-water free from sperm
before using them in the experiments on artificial partheno-
genesis. If one has taken the necessary precautions against
infection with sperm, the next step is to bring the unfertil-
ized eggs to development. In the eggs of sea-urchins the
only effective method which is known thus far by which
they can be made to develop parthenogenetically consists in
keeping the eggs for about one and one-half hours in sea-
water the osmotic pressure of which has been increased a
definite amount. In general it is immaterial how this
increase in osmotic pressure is brought about, whether
through evaporation of the sea-water or through the addition
of salt or sugar or urea to the sea-water. If we wish to
obtain many and, as nearly as possible, normal larve, the
choice of methods is somewhat more limited. I find after
1 Archiv fiir Entwickelungsmechanik der Organismen, Vol. XIII (1902), p. 481.
766
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EXPERIMENTS ON ARTIFICIAL PARTHENOGENESIS 767
all my experience that the addition of potassium or sodium
salts, especially potassium chloride and sodium chloride, is
perhaps the best. The degree of increase in concentration
is of great importance. If the correct concentration is not
struck, failure will result, and it is remarkable how greatly
the necessary concentration varies in different series of
experiments. Whether the variations are exclusively of an
individual character and correspond to the different states of
maturation of the eggs I will not endeavor tosay. Possibly
temperature also has some effect. In order to meet all these
possibilities I always work with a series of solutions. In
this way I am certain to obtain good results in at least one
of the solutions. I use as a stock solution a 24 normal
NaCl or KCl solution; that is, a solution which contains
about 186 g. of KCl in the liter of solution. In my experi-
ments the solutions were accurately titrated, but this is
superfluous for most purposes. I take six dishes, each con-
taining 100 c.c. sea-water, and add to these six dishes the
series of 8, 10, 12, 14, 16, 18 c.c. of the above 2$ normal
NaCl solution. The unfertilized eggs of the sea-urchin are
then distributed into these six dishes (and in addition into a
control dish containing pure sea-water).
Differences also exist regarding the time during which
the eggs must remain in these solutions. It is therefore
necessary to remove, not all the eggs at once, but at various
intervals after about one-half, one, one and one-half, and two
hours. In this way one will certainly strike the optimal
concentration and time of experiment. Potassium chloride
has the advantage that it leads usually to the formation of a
single embryo from each egg, while when sodium chloride
is used more than one embryo is usually formed from an egg.
The formation of the skeleton, however, probably occurs
somewhat better when sodium salts are employed than when
potassium salts are used. Mr. Hunter obtained very satis-
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factory parthenogenetic plutei by using sea-water the con-
centration of which had been increased 30 per cent. to 40
per cent. by evaporation. ‘When the eggs were introduced
for one to two hours into this concentrated sea-water they
developed beautifully when returned to normal sea-water.
A second important circumstance which perhaps plays a
role in these experiments is the temperature. The experi-
ments at Woods Hole (as well as Wilson’s experiments in
Beaufort) were all made at summer temperature when the
temperature of the water was 20° C. or higher. In Califor-
nia the temperature varied considerably in my experiments.
It was often pretty low and I was occasionally unsuccessful
in bringing about artificial parthenogenesis. I attributed this
at that time to the immaturity of the eggs. Possibly this was
right, and possibly this explains the negative results of most
of the European investigators who worked in winter. Since
then, however, I have thought that perhaps the temperature
affects the results of the experiments in such a way that
below a certain temperature artificial parthenogenesis does
not occur, or at least only with difficulty. This idea is
strengthened by a letter from Mr. Doncaster who has
worked in Naples, and who informs me that he at first
obtained only negative results, that he then suspected, how-
ever, that the temperature of the water in Naples was too
low, and so made experiments in water of the temperature
of about 20° C. In the latter case he obtained positive
results.
2. What has been said thus far refers only to experiments
on sea-urchin eggs, especially Arbacia. Especial care is
necessary when working with starfish eggs. A. Mathews
has observed that the unfertilized eggs of starfish (Asterias)
after maturation in sea-water can be made to develop by
shaking, and that a time exists at which the agitation con-
nected with transferring the eggs from one dish to another
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EXPERIMENTS ON ARTIFIOIAL PARTHENOGENESIS 769
is sufficient to obtain larve.' Eggs as sensitive as this must
be carefully handled in two directions if one does not wish
to obtain deceptive results. First it is necessary to transfer
the eggs from one dish to another in such a way that every
mechanical agitation is done away with. This is best done
by using pipettes with a wide opening for sucking up and
transferring the eggs. The latter manipulations must then
be made with the greatest care. The second precaution
consists in this, that whenever the experimental eggs are
transferred from one solution to another or into sea-water the
same mechanical manipulation must be repeated in exactly
the same way with the control eggs. In this way it can
be determined whether the parthenogenetic development in
individual cases is attributable to mechanical agitation, or to
other agents which one employs. With these precautions we
have made a series of experiments this summer on Asterias
eggs and have found up to the present time that, independ-
ently of mechanical agitation, only two methods lead to
artificial parthenogenesis in starfish eggs. First, the intro-
duction of the eggs for from three to twenty minutes into
sea-water to which 3 to 5 c.c. of a 34 normal HCl or some
other inorganic acid has been added to each 100 c.c. of sea-
water. The second method which was discovered by my
pupil, Mr. A. W. Greeley, consists in keeping the eggs, after
lying for a certain time in sea-water, on ice for a number of
hours. Other methods all gave negative results, especially
heating the eggs which Mr. Greeley also tried. Neither did
we succeed in obtaining clear results through the abstraction
of water from the egg, so that I suspect that in my earlier
experiments perhaps, in which I found starfish eggs to
develop through an increase in the concentration of the sea-
water, mechanical agitation really caused the development.
1I have since found that the eggs of the starfish can develop without any notice-
able external cause. [1903]
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770 STUDIES. IN GENERAL PHYSIOLOGY
I believe also that Delage has in part been led into error by
this circumstance when he asserts that about every physical
and chemical factor brings about artificial parthenogenesis.
I do not believe that such an assertion could be made on the
basis of experiments on sea-urchin eggs. In sea-urchin
eggs agitation does not act this way and this source of error
which is so inconvenient in working with starfish eggs does
not exist here. Nevertheless, I made it a rule from the
first to expose the control eggs to the same mechanical agita-
tion in the experiments with sea-urchin eggs as the experi-
mental eggs themselves.
3. The precautions necessary for the experiments on star-
fish eggs must also be used in the experiments on the eggs
of Annelids, Cheetopterus, and Amphitrite. In both these
forms it has been possible this summer to bring about arti-
ficial parthenogenesis through shaking and mechanical agi-
tation of the eggs. In Chetopterus, however, this result is
less certain than in Amphitrite. If the unfertilized eggs of
Annelids are allowed to remain in ordinary sea-water without
jarring the vessel, the eggs do not develop into larve any
more than do starfish eggs. We cannot speak of a “natural”
parthenogenesis of these forms. If, however, they are
allowed to remain for thirty minutes in the sea-water the
unfertilized eggs of Amphitrite can be made to develop into
larvee by squirting them from one vessel into another by
means of a pipette. This does not succeed with every culture,
but still very frequently.’ It is possible, however, to cause
the unfertilized eggs of Amphitrite to develop every time
without agitation, when they are introduced into sea-water to
which a small but definite amount of a soluble calcium salt
has been added. It is not necessary to return the Amphi-
trite eggs from such a solution to sea-water. They develop
1] suspect that the skaking affects the development of the egg only in an indrect
way. [1903]
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EXPERIMENTS ON ARTIFICIAL PARTHENOGENESIS 771
in such a solution to swimming larve. Just as hydrogen
ions bring about the development of larvee from starfish eggs
calcium ions bring about the development of Amphitrite
eggs. The addition of 2 to 5 c.c. of a normal calcium-nitrate
or calcium-chloride solution to 100 c.c. of sea-water is suffi-
cient for this purpose.
I repeated and confirmed this year my earlier experiments
on the specific effects of potassium ions on the development of
unfertilized Cheetopterus eggs. These eggs develop when
a small but definite amount of any soluble potassium salt
(KCl, KNO,, K,SO,) is added (about 1 to 2 c.c. of a 24
normal solution of one of these salts to 100 c.c.) to sea-water.
It is not necessary to remove the eggs from this solution and
to return them to normal sea-water. It may perhaps be well
to emphasize especially that calcium and potassium ions have
no specific effect upon starfish eggs, that potassium ions are
in the same way unable to cause the development of Amphi-
trite eggs, and that calcium ions are ineffective in the case of
Cheetopterus eggs.
4, The following facts are also indirectly of importance
for the methods of the experiments on artificial partheno-
genesis. All the ions which bring about parthenogenesis in
starfish, Amphitrite, and Cheetopterus, also bring about at the
same time agglutination of these eggs and the formation of
giant embryos. The problem at which Driesch once worked,
and which in sea-urchins is beset with great difficulties,
namely, to bring about the coalescence of the contents of
several eggs, succeeds beautifully in these experiments, and
to a large extent especially in the eggs of starfish. I need
scarcely emphasize the fact that it is of great theoretical
importance that the ions which bring about artificial parthen-
ogenesis also in a definite sense and at the same time alter
the physical state of the egg. I have not yet succeeded in
finding a specific ion which brings about the development of
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T72 STUDIES IN GENERAL PHYSIOLOGY
the unfertilized eggs of sea-urchins. It would be of interest
to determine whether such an ion when it is found also brings
about an agglutination of the sea-urchin eggs.
In other forms, Nereis, Podarke, and Phascolosoma, the
experiments have been carried far enough so that we can say
that artificial parthenogenesis (swimming larve) is possible in
these. The experiments, however, have not yet been worked
out sufficiently in order to allow them to be published.
We can say with certainty of the methods given here that
they lead to successful results in the American forms on the
Atlantic ocean. In the attempt to discover new methods it
may perhaps be well to keep the following (theoretical) con-
siderations in view, which I have discussed in greater detail
in various earlier papers. The artificial methods for obtain-
ing parthenogenesis must be able, first of all, to favor the
liquefaction or other destruction of the nuclear membrane.
Secondly, they must also alter in a definite way the physical
properties of the protoplasm (viscosity, etc.). It seems that
in the eggs in which artificial parthenogenesis has succeeded
thus far (and possibly in many, if not all, other eggs) chemical
changes take place under natural circumstances in the unfer-
tilized egg, which endeavor to alter the egg in the two direc-
tions mentioned above; that these, however, under ordinary
conditions occur so slowly that the egg dies before it under-
goes actual cell-division. Those circumstances which are
able to accelerate these natural processes will also bring about
the development of the unfertilized egg.
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INDICES
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AUTHORS’ INDEX
Allman, 116, 117, 118, 128, 129.
Andrews, E, A., 289.
Araki, 372.
Arrhenius, 450.
d’Arsonval, 496.
Aubert, 187, 371, 404, 754.
Baer, 345.
Bardeen, 363.
Béclard, 426.
Bemmelen, van, 466.
Bert, Paul, 9, 378.
Berthold, 506.
Bickford, E., 336.
Bickford, Miss, 629.
Biedermann, 420, 518.
Birukoff, 449.
Bonnet, Charles, 118, 119, 228, 250, 627.
Boveri, 325, 331.
Breuer, 186, 189.
Budgett, 507, 636,
Bumpus, 282, 597.
Bunge, 310, 370,
Bunsen, 236.
Castell, 404.
Chauveau, 496.
Chun, 290.
Claus, 336.
Conklin, Dr., 576.
Contarini, N., 167, 171.
Cooke, Miss, 450, 469.
Cremer, 499.
Cunningham, J. T., 190.
Cushny, 511, 760.
Maisons 116, 117, 127, 128, 129, 136, 175, 250,
7.
Danilewsky, 482, 491.
Darwin, 77, 81, 286, 358.
Delage, 187, 729, 770.
Demoor, 384.
De Vries, 231.
Dewitz, J., 23, 111, 580.
Diquemare, 167.
Dohrn, A., 338.
Doncaster, 768.
Driesch, 589, 629, 676, 771.
Driesch, Hans, 105, 148, 306, 307, 328, 332,
Dubrochet, 378.
Digitized by
Duclaux, 635.
Duhamel, 119.
Dutrochet, 426.
Edwards, 426.
Engelmann, 14, 15, 73, 77.
Faraday, 489, 496.
Farkas, 535,
Fernet, 236.
Fischer, 597, 733, 740.
Forchhammer, 225, 242.
Fraenckel, 535.
Friedenthal, 535, 749.
Friedlander, 438,
Friedlander, B., 221, 357 ff.
Galileo, 81.
Garrey, W. E., 708, 749.
Gemmill, 597.
Geppert, 479.
Giard, A., 686.
Gleichen-Russwurm, 88.
Goltz, 189, 368, 437, 438.
Graber, 12, 13, 16, 358.
Greely, 729.
Greeley, A. W., 769.
Greene, 530, 535, 754.
Groom and Loeb, 89, 180, 272, 290.
Gruber, 508,
Hardy, 711.
Hammarsten, 623.
Harper, 739.
Hegel, 38.
Heider, A. von, 201, 250.
Hempel, 310.
Herbst, 205, 307, 575, 589, 590, 595, 627, 686,
Hermann, 370.
Hertwig, 255, 325, 580, 595.
Hertwig, O., 684.
Hertwig, R., 581.
Hilger, 229.
Hoeber, 535.
Hoek, 340,
Hoff, Van ’t, 450, 496, 499.
Hoffmeister, 77, 78, 358, 513.
Hogies, 88.
Hoppe-Seyler, 225, 244, 245, 371, 644,
Howell, 513, 519, 533, 754.
Huxley, 286.
775
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STUDIES IN GENERAL PHYSIOLOGY
Jacobsen, 241.
Janésik, 543.
Kahlenberg, 451, 452, 474.
Koblrausch, 456.
Kuhne, 372, 440, 448.
Kulagin, 580.
Lewis, 728, 738.
Lingle, D. J., 708.
Locke, 526, 533, 549.
Lubbock, 10, 11, 55.
Ludwig, 238.
Lyon, 87.
Mach, Ernst, 69, 81, 87, 88, 187, 189, 202, 499.
Marshall, W., 117, 283.
Massart, 283.
Mathews, 579, 729, 768.
Maxwell, 447.
Mead, 540, 591, 656.
Metschnikoff, 637.
Miescher, 637.
Mingazzini, P., 217, 222,
Moore, A., 708.
Moore, Miss, 696.
Morgan, 577, 591, 607, 618, 644, 676.
Miller, Johannes, 9.
Miller, Wilhelm, 43.
Neilson, 733, 740.
Noll, 202, 203.
Norman, 540, 577, 578, 607, 618, 644.
Norman, W. W., 399.
Nussbaum, 117, 205, 321, 322, 323, 336, 341,
508, 580
Ostwald, 450, 456, 459, 476, 490.
Ostwald, Wolfgang, 292.
Pasteur, 636, 689,
Pauli, Dr. W., 548, 622.
Pemsel, 547.
Pfliger, 202, 323, 371, 404.
Plateau, 110.
Pleasanton, General, 426.
Preyer, 184, 185.
Prowazek, Dr. 5S., 686.
Rad], 69, 87.
Ranke, 450.
Réaumur, 7, 37, 38, 41, 56.
Regoli, 755.
Ringer, 513, 519, 533, 549, 749.
Romanes, 37, 80, 81, 541, 560.
Roth, 226.
Roux, 448.
Sabbatani, 753, 755,
Sachs, 2, 4, 5, 6, 7, 14, 42, 73, 80, 89, 96, 97,
A , 176, 179, 207, bare 212, 215, 249, 342, ‘343,
Saussure, 378.
Schrader, 88, 186, 347. :
Schmankewitsch, 237, 238, 239, 240.
Schwann, 378.
Semper, 56, 229, 230, 248,
Sewall, 186.
Spallanzani, 370, 378, 627.
Spencer, Herbert, 229,
Spiro, 546, 547.
Spitzer, 506.
Stahl, 6, 14, 73, 75.
Steiner, 187, 345, 346.
Stieglitz, 546.
Strasburger, 5, 73, 283.
Strassen, zur, 676, 678.
Tichomirof, 580,
Tiedemann, 404.
Tornier, Gustav, 436,
Torrey, 167.
Traube, 506.
Trembley, 7, 8, 73, 116, 118.
True, 451, 452, 534.
Verworn, 506, 507, 508.
Vignier, 684,
Voit, 499.
Wallace, 511, 760.
Weismann, 319.
Wilson, E. B., 686, 768.
Winkler, Hans, 686, 688.
Wheeler, 287, 288.
Whitman, C, O., 734, 739.
Wortmann, 6, 212, 231.
Willner, 238.
Young, Emil, 427.
Zoethout, 694, 759,
Zuntz, 236, 237, 309.
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SUBJECT INDEX
ABSORPTION OF WATER: relation of, to
regeneration, 223 tf.; a basis for judg-
ing effects of ions, 453; effect of H.
and OH. ions on, 464 ff.; by muscles
and soaps, 510 ff.
AcIps: physiological effects of, 453 {f.,
501 tf.
ACTINIA CARA: heteromorphosis in, 166,
ACTINIA DIAPHENA: heteromorphosis in,
171.
ACTINIA EQUINA: heteromorphosis in,
66 ff.; stereotropism in, 170.
ACTINIA MESEMBRYANTHEMUM: geotro-
pism in, 183; basal end preferred in new
growth, 201.
ACTINOSPHERIUM: exception to Pfli-
ger’s law, 440; liquefaction of proto-
plasm of, 448.
ADAMSIA RONDELETTI: heteromorphosis
in, 166.
ADVENTITIOUS ROOTS: stereotropism of,
136.
AGLAOPHENIA PLUMA: heteromorphosis
in, 115, 130; morphology of, 130; deter-
minants of heteromorphosis in, 132;
stereotropism in, 135; longitudina.
growth of, 136; larvee of, 137; geotro-
pism in, 176.
ALG#: influence of direction of rays of
light on, 5, 89; heliotropism of swarm-
spores of, 283
AMBLYSTOMA: influence of central nery-
ous system on development of, 436 ff.;
galvanotropism in, 440 ff.
AMMONIUM CHLORIDE: effects on regen-
eration and growth, 246.
AMMOTHEA: segmentation in, 340.
AMOEBA: spontaneity in, 321.
AMPHIpPyRA : heliotropism in, 21; stereot-
ropism in, 21; geotropism in, +:
AMPHITRITE: artificial parthenogenesis
in, 770.
ANEMONIA SULCATA:
in, 166.
ANISOTROPY: influence of intense light
on, 62.
ANTENNULARIA: heieromorphosis in,
628 ff.
heteromorphosis
ANTENNULARIA ANTENNINA: geotropism
in, 177; growth in, dependent on geot-
ropism, 191-204; variation in, 204; irri-
tability and growth in, 213; hetero-
morphosis in, 249.
Ants: influence of less refrangible rays
of light on wingless form, 11; heliot-
ropism of winged form, 19, 113; rela-
tion between heliotropism and sexu-
ality of, 52, 113; factors determining
nuptial flight of, 53.
ARBACIA (SEA-URCHIN) : effect of change
of concentration of sea-water on cleav-
age of eggs of, 253 ff.; twin production
in, 303 ff.; limit of divisibi ity of em-
bryo of, 323 ff.; lack of oxygen and seg-
mentation of eggs of, 400 f. effect of
ions on unfertilized ogRs of, 576 ff.; on
fertilized eggs of, 581 ff.; artificial par-
thenogenesis in, 624 ff,
ARTEMIA MULHAUSENIT: artificial con-
version into Branchipus, 237 ff.
ARTEMIA SALINA: conversion into Mal-
hausenii, 237 ff.
ARTISTIC IMPULSE:
conception of, 165.
ASCARIS: agglutination of eggs of, 678.
ASSOCIATIVE MEMORY: definition of, 365.
ASTERIAS (STAR-FISH): geotropism in
183 ff., 291; artificial parthenogenesis
by shaking eggs of, 768,
ASTERIAS ForBgEsII: artificial partheno-
genesis in, 644; prolongation of life of
unfertilized eggs of, 728 ff.
ASTERINA GIBBOSA: geotropism in, 183.
ASTERINA TENUISPINA: heliotropism in
183.
anthropomorphic
AUDITORY NERVES: relation of, to orien-
tation, 188,
AURELIA AURITA: relation of concentra-
tion of water to contractions of, 561.
Bacteria: heliotropism of, 15; orienta-
tion influenced by assimilation, 15,
BALANUS PERFORATUS: negative stereot-
ipl ioe in, 111; change of sense of
heliotropism in, 113, 272, 417: depth-
migrations of, 290.
BARANA CASTELLI: regeneration of, 338.
Baszs: physiological effects of, 461.
BATHOMETRIC DISTRIBUTION: of pelagic
animals, 178: physiological conditions
determining, 289 ff.
BEETLES: heliotropism of, 56, 70, 85.
BLOOD-VESSELS: development of, 297 ff.
BomByx: parthenogenesis in, 580.
BomMByYx LANESTRIS: heliotropism in, 38.
BoMBYX NEUSTRA: geotropism in, 85.
BRAIN: regeneration of, 251; physiology
of, in worms, 345 ff.
BRANCHIOMMA: explanation of eye on
gill of, 97.
Brancuipus: Schmankewitsch’s experi-
ment on, 237.
Bryopsis: organization controlled by
external forces, 202.
BursaRiA: heliotropism in, 15.
. BUTTERFLIES: sleep of, 37; heliotropism
in, 20, 37.
7717
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778
STUDIES IN GENERAL PHYSIOLOGY
CAMPANULARIA: heteromorphosis in, 629.
CATALYTIC SUBSTANCES: importance in
oxidation, 505.
CATERPILLARS: heliotropism in, 20, 42,
CELL-DIVISION: mechanics of, 389.
CEREACTIS AURANTIACA: heteromorpho-
sis in, 166.
CEREBRATULUS MARGINATUS:
physiology of, 356 ff.
CERIANTHUS MEMBRANACEUS: secretion
of, due to friction, 99; lack of hetero-
morphosis in, 116; formation of ten-
tactes of, 145; heteromorphosis in, 150;
relation between form and irritability
of, 152; life phenomena of, 159; impor-
tance of turgor in, 162 ff; external con-
ditions in formation of tubes of, 165.
CH#TOPTERUS: artificial parthenogene-
sis in, 540, 579, 646, 674, 770; specific
effects of K ions on, 656.
CHEMICAL STIMULI:
Musca toward, 66.
CHLOROPHYLL-BEARING PLANTS: influ-
ence of light on movements of proto-
plasm of, 6.
CHROMATOPHORES: distribution in Fun-
dulus, 300.
CIONA INTESTINALIS: organization of,
215; regeneration of nervous system of,
217; threshold of stimulation of, 219.
CLADocoRA: geotropism in, 201; hetero-
morphosis in, 250.
CLEAVAGE: general remarks, 253 ff.;
lack of oxygen and, 373 ff; carbon di-
oxide and, 393 ff.; pure oxygen and, 394.
CLEPSINE: oxygen necessary for matu-
ration of eggs of, 734.
CoccINELLA: geotropism in, 85, 179.
CocKROACH: geotropism in, 86, 182.
COLOR PREFERENCE: anthropomorphic
idea of, 16.
COMPENSATORY MOVEMENTS:
of inner ear on, 186.
ConTACT-STIMULI: effect on orientation,
193; effect on organization of, 214.
CopErops: heliotropism of, 282; change
of sense of heliotropism, 283 ff., 417.
CryProps: stereotropism in, 110.
CTENOLABRUS: influence of lack of oxy-
gen on, 878 ff.; influence of carbon di-
oxide on, 393 ff,
CEERI CUCUMIS: geotropism in,
brain
orientation of
influence
CUMA RATHEII: heliotropism in, 73 ff.
Cycuas: effect of hydrogen on heart-beat
of, 417,
DAPANIA PULEX: Bert’s anthropomor-
phic idea of, 9; Lubbock’s idea of
“preference” in, 10.
DEPTH DISTRIBUTION AND MIGRATION:
conditions determining, 289 ff.
DEVELOPMENT OF ORGANS: influence of
light on, 425 ff.
DIVISIBILITY OF MATTER: orientation of
particles, 117; limits of, 321 ff.
DrosERA: reactions of, 286.
Eartuworms: heliotropism in, 73, 77;
brain physiology of, 357 ff.
EcHINUS: segmentation not influenced
by light, 427.
ELECTRICAL WAVES: physiological ef-
fects of, 482 ff.
EMBRYONIC TISSUE: elfects of ions on,
EPHEMERID#: geotropism in, 44.
EUDENDRIUM RACEMOSUM: heliotropism
in, 106; heteromorphosis in, 140, 172;
influence of light on growth of, 428.
EvUGLENA: influence of direction of rays
on, 14; influence of refrangibility of
rays on, 14; sensitive spot of, 77.
Tages relation between irritability and,
FERTILIZATION: nature of, 539, 620, 638,
646; theory of, 683, 740.
ae (LARVZ): heliotropism of, 20, 68,
FORFICULA AURICULARIA: heliotropism
of, 22; stereotropism of, 110, 158.
FREEZING: effects of, 225.
FRICTION: a cause of movement, 107, 110.
Funpuuus: development of embryo of,
295 ff. ; sensitiveness to lack of oxygen,
309, 397; effects of lack of oxygen on
cardiac activity of embryos, 404; in-
fluence of light on embryos of, 434;
effects of ions on, 550 ff.; partheno-
genesis in, 682,
Fusion: of cleavage cells through lack
of oxygen, 383.
GALVANOTROPISM: of Amblystoma, 440
in theory of, 440 ff.; of Crustaceans,
GAMMARUS LOcUSTA: heliotropism in, 73.
GASTROSTYLA: divisibility of, 321.
GEOMETRA PINIARIA: heliotropism in, 40.
GEOTROPISM: in Porthesia chrysorrhcea,
33; in Lepidoptera, 43; in Amphipyra,
44; in Bombyx, 85; theory of, 102; in
Hydroids, 174 ff.; in free-swimming
animals, 178; in Asterina, 183; depend-
ence on inner ear, 185; influence of, on
axis of eyes, 186; in Antennularia an-
tennina, 251; relation of, to heliotro-
eae 285; in Loligo, 292; in star-fish,
GONIONEMUS: effects of ions on 553, 559;
parthenogenesis in, 682, 754.
GONOTHYREA LOVENII: abnormality of
erent in, 144; heteromorphosis in,
GRAVITATION: relation to light, 95, 105;
effects of, on Cerianthus, 107, 109, 154
ff.; effect of, on orientation of body,
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Supyect INDEX
779
186; effect on position of eyes, 186; rela-
tion to heteromorphvsis in Antennula-
ria, 191 ff.
GREEN SLIPPER ANIMALCUL: heliot-
ropism of, 15.
Growta: phenomena of importance in,
175; dependence of, on geotropism, 191;
relation between concentration of sea-
water and, 228 ff.; relation of, to quan-
tity of water, 247; and regeneration,
pas a
aa heliotropism in, 114,
Hesars action of potassium salts on,
HELIoTROPISM: identity of, in animals
and plants, 1 ff., 89; influence of direc-
tion of rays on, 2, 4, 5, 16, 53, 91, 108, 265 ;
in plants, 4; in Hydra, 8; in Euglena,
14; in Infusoria, 14; in bacteria, 15;
in Bursaria, 15; methods of experi-
mentation, 17; negative form of, 17, 56;
positive form of, 17, 24; effect of col-
ored rays on, 18, 29 ff.; in ants, 19, 523
in caterpillars, 20, 42, 74; in fly larve,
20; inversion of sense of, 20, 68, 265 ff.,
417 ff.; in Amphipyra, 21; in Forficula
auricularia, 22; effect of temperature
on 37; in butterflies, 37; in Bombyx
lanestris, 38; in Moths, 38; in Sphinx
euphorbie, 38; in Geometra piniaria,
40; periodic variations in, 40; in Papi-
jio machaon, 42; in plant lice, 45; in
beetles, 56, 70, 85; in Mesocarpus, 56,
113; character of protoplasm in, 57;
in Musca, 57; in Tenebrio molitor, 70;
in June bugs, 71; in Melolontha vul-
garis, 71; in Cuma rathkii, 73 ff.; in
earthworms, 73, 77; in Gammarus lo-
custa, 73; in leeches, 73; in, planaria,
73; in snails, 73; distribution of, in
animal kingdom, 73; variations of, ac-
cording to pole of animal, 79, 83; not
dependent on central nervous system,
84, 221; in sessile animals, 89; in Spiro-
graphis Spallanzanii, 90-110; Sach’s
theory of, 102; in hydroids, 103; in Ser-
tularia, 103, 266; relation of, to instinct,
109; sexuality in relation to, 113; in
Balanus perforatus, 113, 272, 417; lack
of, in Tubularia mesembryanthemum,
426; in Eudendria, 141; effect of, on
heteromorphosis, 174; in Asterina ten-
uispina, 183; in Ciona, 221; in Limulus
polyphemus, 267; theory of, 270; in
Polygordius, 273; intensty of, 273; effect
of concentration of sea-water on, 279,
982; in Temora longicornis, 282; in
Copepods, 283; difference in locomo-
tion under influence of, 284; liberation
of energy in, 286; in Loligo, 291; lack
of oxygen in, 417.
HEREDITY: theory of, 319.
HerrromorpHosis: in Aglaophenia
pluma, 115, 130; definition, 120; in Tu-
bularia mesembryanthemum. 120 f.;
in Plumularia pinnata, 137; in Euden-
drium, 138, 140, 172; effects of aration
on, 140, 144; in Sertularia, 142; in Go-
nothyrea lovenii, 144, 173; in Cerian-
thus membranaceus, 150; in Actinia
cara, 166; in Actinia equina, 166; in
Adamsia rondeletti. 166; in Anemonia
sulcata, 166; in Cereactis aurantiaca,
166; in Actinia diaphena, 171; law gov-
erning, 173; influence of geotropism on,
191 ff,; in uninjured organs, 196; casu-
istic ideas of, 249; in Antennularia an-
tennina, 249; in Cladocora, 250: in
horse-shoe crab, 267; in Hirudine. 341;
in Hirudo, 343; in Crustaceans, 627; in
Hydroids, 627 ff.; in Antennularia, 628
ff.; in Campanularia, 629.
HYBRIDIZATION: possibility of, between
Cheetopterus and Echinoderms, 678.
Hypra: heliotropism of, 8, 73; theory of
polarity in, 117, 118, 216; regeneration
in, 149, 150, 178, 205; amount of sub-
stance necessary for regeneration of,
Hyprorps: heliotropism in, 103-5; stere-
otropism in, 111; heteromorphosis in,
115, 116, 627 ff.; geotropism in, 177.
HypromeEpus#: influence of ions on con-
traction of, 541; Romanes’s ideas of, 541.
HYDROSTATIC PRESSURE: importance of,
for growth of tentacles of Cerianthus,
175; relation to geotropism, 181 ff.
Hyprorropism: in Plasmodia, 179, 182.
HYPERSENSITIVENESS OF SKIN: produc-
tion and suppression of, by electro-
lytes, 748 ff.
InFusorIA: heliotropism in, 14, 73; re-
generation in, 205: importance of nu-
cleus in, 321; influence of lack of oxy-
gen, 636,
Instinct: inherited, 61; _ movements
called instinctive due to physical laws,
107; physical laws in, 107-110; anthro-
pomorphic idea of, 165.
IoN-PROTEIDS: importance in absorp-
tion of water by muscles, 510 ff,; role
in life-phenomena, 544 tf.; relation to
ciliary movements, 535 ff.; in parthe-
nogenesis, 646 ff.
Ions: physiological effects of, 450 ff., 501
ff.; in relation to absorption of water
by muscles, 464 ff.; velocity of migra-
tion of. in relation to toxicity of, 474
ff.; and rhythmical contractions of
muscles, 518, 559; effects of, on devel-
opment of muscle, 565 ff.; effect of, on
undifferentiated embryonic tissue, 565
ff.; in artificial parthenogenesis 576
ff.; effect of, on fertilized eggs of Ar-
pacia, 581 ff.; effects of, in artificial
parthenogenesis of giant and dwarf
embryos of Arbacia and Cheetopterus,
674 ff.; effect of, on nerves, 703 ff. ; toxic
and antitoxic effects of. 708 ff.
TRRITABILITY: a. function of tempera-
ture, 36; sex-differences in, 56; relation
between structure of body and, 76;
distribution of, 80; and form, 152; and
organization, 213; dependence of, on
concentration of medium, 262.
Isopops: change of sense of heliotro-
pism of, 419.
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STUDIES IN GENERAL PHYSIOLOGY
JUNE-BUG LARV2: heliotropism of, 20, 71.
Lack OF OXYGEN: influence of, on fish
embryos, 309 ff.; and Perca fluviatilis,
320; hysiological effects of, 370 ff.;
and &o pepods, 373; and Ctenolabrus
eggs, 374; influence of, on Fundulus
eggs, 375, 397; and segmentation of egg,
400 ff. ; and cardiac activity of fish em-
bryox, 404; and Cyclas larvew, 417; and
heliotropism, 417; and pigment cells,
Leecues: heliotropism in, 73: oral end
more sensitive to light, 78; stereotro-
pism in, 79; lack of regeneration i in, 341
ff.; brain physiology of, 361.
LexDOPTRRA: heliotropism, in, 7, 37, 40,
54, 56, 61, 74, 86; chemotropism in, 112.
LeEucocytEs: migration of, due to
stereotropism, 111.
Licut: effect of direction of ray_on
movements, 2, 4, a 16, 53. 91, 108. 265;
effective rays of, 3,5 ; effect of change
in intensity of, 3 27 276; effect of, on
swarm-spores ot ‘alee, 33 effect of, on
Lepidoptera, 7; effect of, on Protozoa,
1s mechanical effects of, 7; effect of,
on water fleas, 8; effect, of, on Daphnia,
9; influencing ‘instinctive move-
ments, 107, 109; effect of, on develop-
ment of organs, 423 ff.; "effect of, on
Fundulus embryos, 434 ‘ft.
LIMULUS POLYPHEMUS: heliotropism in,
267. 288%; locomotion dependent on
sense of heliotropism of, 284.
Louieo: heliotropism in, 291; geotro-
pism in, 292.
Loss oF WATER: effect of, on cleavage,
258: influence of, on embryos, 309, 314..
ge a F@TIDUS: brain physiology
of, 39
LYMNUS STAGNALIS: growth in, 229.
Macu#riTeEs: blindness of, as related
to sex, 56.
MARGELIS: heteromorphosis in, 628 ff.
MATURATION: of unfertilized eggs of
star-fish, 728 ff.
Mepusa: periodic migration of, 366; ion-
proteids in, 544; locomotion of, 553;
effects of Ca ions on, 753.
MELOLONTHA VULGARIS:
in, 71
MesocarPvs: influence of light on, 6.
MeEramorPHOSIS: influence of central
nervous system on, 436.
MICELLA: conceptions of, 334.
MIGRATIONS OF ANIMALS: influence of
gr otrop(sm on, 180; physiological con-
itions determining, 289 ff
Morus: heliotropism in, 7, 38.
MULTIPLE EMBRYOS: development of, 303.
Musca voMIToriA: helitropism in, 56,
113; stereotropism in, 64, 68; effect of
heat on orientation of, 63; relation of
orientation of, to chemical stimuli, 66:
movements due to sum of stimuli, 1.
heliotropism
Muscue: absorption of water by, 510 ff.;
rhythmical contractions of, ‘518 ff.;
contact-irritability of, 692 ff.; produc-
tion and suppression of twitchings of,
by electrolytes, 748 ff.
Mysip#: stereotropism in, 110.
NEMERTINES: brain physiology of, 356 ff.
NEREIS: brain physiology of, 358; artifi-
cial parthenogenesis in, 773,
NERVE: regeneration of, 217, 252; effects
of fluorides on, 703 ff.
Nucueus: effect of, on growth. 321 ff.;
the organ of oxidation, 508.
OcELLA: formation of, in Ciona, 216.
CEpEMA: influence of osmotic pressure
on, 471; increase in osmotic pressure of
muscle causing, 515.
ORAL PLATES: in Cerianthus, 159, 160.
ORBITOLITES: changes of enucleated
pieces of, 506, 507.
ORGANIZATION: dependent on orienta-
tion, 191, 192; relationship of, to
growth, tou, 251; internal causes of,
205; Sach’s theory of, 207, 211, 212, 215;
Loeb’s theory of, 208; relationship of.
to irritability, 213; in Ciona, 215; em-
bryonal, 319.
ORIENTATION: influence of direction of
rays on, 2, 4, 5, 16, 53, 91, 108, 265; influ-
ence of temperature, 3; influence of in-
tensity of light on, 3, 4, 32; influence of
physical laws on, 51: . influence of form
of body on, 75; of plants, 109; com-
position of forces in, 112; forms of irri-
tability influencing, 174, 251; influence
of gravitation on, 176. 177, 178; governs
regeneration and heteromorphosis,
ORTHOTROPISM: of organs, 2; in dorsi-
ventral animals, 97.
OsMOTIC PRESSURE: relation of, to
growth, 228, 240; in bursting of egg-
membrane, 308 ; relation of, to absorp-
tion by muscle, 466; in parthenogenesis,
640, 648 ff.
OTOLITHS:
187, 189
OxyGEN (see Lack of Oxygen): rdle of,
in heliotropism, 15; relation of, to re-
generation, 240, 252: Hoppe- -Seyler’s
theory of action’ of, Bil: relation of, to
cleavage, 394; relation’ of, to growth,
relation of, to geotropism,
PANTOPODS: regeneration of, 338.
PAPILIO MACHAON: heliotropism in, 42.
PARTHENOGENESIS: artificial, 539 ff.,
638 ff.; in Arbacia, 576 ff., ce ff.; in
Annelids (Chastopterus), 46 ff.; in
Fundulus, 682; in Gocimeniie. 682;
in Podarke, 682; in Phascolosoma,
682,772; in Crustaceans, 684; sources of
error in, 765 ff.
PERCA FLUVIATILIS:
oxygen on, 320.
effect of lack of
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SuBsJEctT INDEx
781
PHASCOLOSOMA: artificial parthenogen-
esis in, 682, 772.
PHOTOKINETICS: definition, 265; reac-
tions influencing, 286 ff.
PHOXICHILIDIUM MAXILLARA: regenera-
tion of, 338 ff.
PHYSIOLOGICAL PROBLEMS: discussion
of, 497 ff.
PHYSIOLOGIC UNIT: idea of, 322.
PIGMENT: relation of, to gravitation, 190;
in Tubularia, 210; influence of lack of
oxygen on, 420,
PLAGIOTROPIC ORGANS: definition, 2.
PuaANarta: heliotropism in, 73,77; pho-
tokinesis in, 287; heteromorphosis in,
344; brain physiology of, 352 i.
PLANoRBIS: influence of light on seg-
mentation of, 427.
PLANT LICE: heliotropism in, 45, 49, 50,
112; geotropism in, 50; temperature
effects on, 51, 108: flight movements of,
54; movements of, not voluntary, 109.
PLANULA: influence of light on, 433.
PLASMODIA: geotropism in, 179.
PLUMULARIA PINNATA: heteromorpho-
sis in, 187, 172.
PopARKE: Parthenogenesis in, 682, 772.
POLARITY: heliotropism as a factor in,
76; Allmann’s theory of, 117, 129; as
regards Tubularia mesembryanthe-
mum, 128; in Cerianthus, 206, 215.
PoLyGorDIvs; change of sense of heliot-
zopem in, 273, 293, 417; locomotion in,
Po.yps: formation of, 124.
PORTHESIA CHRYSORRH@A : heliotropism
in, 19, 24-37, 112; stereotropism in, 21.
Porassium: effects of, on regeneration,
242, 252; effects of, on Fundulus, 296, 297.
Pressure: effects of, on so-called in-
stinct, 110.
ProtTopLasM: influence of light on, 6;
movements of, 212; isotropy in, 334.
Protozoa: heliotropism in, 7.
PYcNOGONIDES: regeneration of, 338 ff.
QUANTITY OF WATER: influence on re-
generation and growth, 247, 252.
RANA: influence of light on develop-
ment of, 427.
REFLEXEs: persistence of, in Ciona, 218;
in Planaria, 355 ff.
REGENERATION: Bonnet’s theory of, 119;
definition of, 120; in Tubularia, 122; in
Ciona intestinalis, 217; influence of
concentration of sea-water on, 222;
necessity of oxygen for, 240; relation
of, to inorganic substances, 242, 245;
influence of quantity of water on, 247,
252; influence of magnesium on, 252;
quantitative limits of, 336; general
ideas on, 338; in Pantopods, 338; in
Chetopods, 341; and nucleus, 505; of
organs in Hydroids, 627 ff.
RESEGMENTATION: of Ctenolabrus eggs
without oxygen, 37%.
RESPIRATORY ORGAN: effect of light on,
RHEOTROPISM: in Eudendria, 141; in
Plasmodia, 179, 182.
RHIZOPODS: regeneration in, 507.
SACCHAROMYCES CEREVISI®: a ferment
for glucoses, 498.
ScYLLIUM CANICULA: geotropism in, de-
pendent on inner ear, 187.
SEA-WATER: regeneration in, 222; con-
centration of, and influence on longi-
tudinal growth, 228; analysis of, 242;
influence of concentration of, on seg-
mentation, 257; effect of concentration
of, on heliotropism, 279-82.
SEGMENTATION: of nucleus without seg-
mentation of protoplasm, 258.
SERPULA: heliotropism in, 101, 106: pho-
tokinesis in, 289,
SERTULARIA HALECINA: heliotropism in,
SERTULARIA (POLYZONIAS): _heliotro-
pism in, 103; heteromorphosis in, 142,
173, 174, 249; change of sense of heliot-
ropism in, 266
SESSILE ANIMALS:
90-106. ; ;
SEXUALITY: relation of, to heliotropism,
52, 68, 118.
SHARKS: geotropism in, 186.
SIAMESE TWINS: formation of, 304 ff.
SnAILs: heliotropism in, 73.
SoDIUM CHLORIDE: poisonous character
of solution of, 544 ff.
SPERMATOZOA: stereotropism of, 23, 111.
oe EUPHORBIZ: heliotropism in,
38 ff.
heliotropism in,
SPIROGRAPHIS SPALLANZANII: heliotro-
pism in, 90-100; movements of, not in-
stinctive, 107; change of sense of heli-
otropism, 266.
STENTOR VIRIDIS: influence of oxygen
on, 15.
STEREOTROPISM: definition of, 23; in
Porthesia chrysorrhoea, 34; in Lepi-
doptera, 54; in plant lice, 54; in ants,
55: in June-bugs, 71: relation of, to in-
stinct, 109, 111; in Tubularia mesem-
bryanthemum, 121, 124; in Aglaophe-
nia pluma, 139; in Plumularia, 148; in
Sertularia, 143: in Hydroids 174; in
Antennularia antennina, 251; in Lum-
bricus foetidus, 360.
STIMULATION: in heteromorphosis, 140;
threshold of, 219: latent period of, 221.
STRONGYLOCENTRATUS FRANCISCANUS:
artificial parthenogenesis in, 626, 63%,
STRONGYLOCENTRATUS PURPURATUS: ar-
tificial parthenogenesis in, 626, 638, 644.
SunLiIcuT: effect on Lepidoptera, 48;
effect on Polygordius, 277.
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STUDIES IN GENERAL PHYSIOLOGY
TEMORA LONGICORNIS: change of sense
of heliotropism in, 282.
TEMPERATURE: effects of, on Porthesia,
36; on Musca larve, 65; on Polygor-
dius, 274; on depth-migrations, 293.
TENEBRIO MOLITOR: heliotropism in, 70;
stereotropism in, 71.
TENTACLES: formation of, in Cerianthus,
145-152, 160.
THYSANOZOON BROCCHII: regeneration
in, 221; brain physiology of, 347 ff.
TOXOPNEUSTES: natural parthenogene-
sis in, 684.
TRADESCANTIA: influence of oxygen on
formation of cell wall, 384.
TRANSFORMATION OF ORGANS: in Hy-
droids, 627 ff.; basis of, 634 ff.
TuBEs: formation of, in Cerianthus, 164,
TUBULARIA: heteromorphosis in, 118,
215; stereotropism in, 174; irritability
and organization of, 211; regulation
and _ concentration of sea-water in,
222 ff. ; effect of salts on growth of, 230,
242 ff.; regeneration of, 336.
TUBULARIA CROCEA: organization of, 214,
ea a INDIVISA: growth of polyps
in, 127.
TUBULARIA MESEMBRYANTHEMUM: heter-
omorphosis in, 120-27; morphology of,
121; laws governing reactions of, 125;
conditions of growth of polyps of, 127;
internal causes of organization in, 205;
stereotropism in, 213.
TuUNICATES: heteromorphosis in, 627.
TurRGoR: importance of, in growth, 162
f., 175.
”
VARIATION: in irritability, 40.
VASCULAR SYSTEM: development of, 297.
VIOLET RAYS: influence of, on growth,
426 ff.
ViEAG FORCE: anthropomorphic idea,
114.
VOLUNTARY MOVEMENTS: influence of
physical laws on, 107, 109, 110.
Wasps: Lubbock’s experiments on, 11.
WATER FLEAs: effect of light on, 8.
WATER RIGOR: influence of dilute sea-
water on, 327.
WILL: subject to physical influences,
107, 109. 110.
Worms: brain physiology of, 345 ff.
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