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The

Organism as a Whole

From a Physicochemical Viewpoint

Jacques Loeb, M.D., Ph.D., Sc.D.

Member of the Rockefeller Institute for Medical Research

With 51 Illustrations

G. P. Putnam’s Sons

New York and London

The ‘Knickerbocker Press
1916

JACQUES LOEB

The Knickerbocker Press, Rew Work

To
THE MEMORY OF

DENIS DIDEROT

Of the Encyclopédie and the Systéme de la nature

“He was one of those simple, disinterested,
and intellectually sterling workers to
whom their own personality is as nothing
in the presence of the vast subjects that
engage the thoughts of their lives.”

Joun Mortey.
(Article Diderot, Encyclopedia Britannica.)

_PREFACE

It is generally admitted that the individual physio-
logical processes, such as digestion, metabolism, the
production of heat or of electricity, are of a purely
physicochemical character; and it is also conceded that
the functions of individual organs, such as the eye or
the ear, are to be analysed from the viewpoint of the
physicist. When, however, the biologist is confronted
with the fact that in the organism the parts are so
adapted to each other as to give rise to a harmonious
whole; and that the organisms are endowed with
structures and instincts calculated to prolong their life
and perpetuate their race, doubts as to the adequacy of
a purely physicochemical viewpoint in biology may
arise. The difficulties besetting the biologist in this
problem have been rather increased than diminished
by the discovery of Mendelian heredity, according to
which each character is transmitted independently of
any other character. Since the number of Mendelian
characters in each organism is large, the possibility
must be faced that the organism is merely a mosaic of

independent hereditary characters. If this be the case
Vv

vi Preface

the question arises: What moulds these independent
characters into a harmonious whole?

The vitalist settles this question by assuming the
existence of a pre-established design for each organism
and of a guiding ‘‘force” or ‘“‘principle’”’ which directs
the working out of this design. Such assumptions
remove the problem of accounting for the harmonious
character of the organism from the field of physics or
chemistry. The theory of natural selection invokes
neither design nor purpose, but it is incomplete since
it disregards the physicochemical constitution of living
matter about which little was known until recently.

In this book an attempt is made to show that the
unity of the organism is due to the fact that the egg
(or rather its cytoplasm) is the future embryo upon
which the Mendelian factors in the chromosomes can
impress only individual characteristics, probably by
giving rise to special hormones and enzymes. We can
cause an egg to develop into an organism without a
spermatozo6n, but apparently we cannot make a sperm-
atozo6n develop into an organism without the cyto-
plasm of an egg, although sperm and egg nucleus
transmit equally the Mendelian characters. The con-
ception that the cytoplasm of the egg is already the
embryo in the rough may be of importance also for the
problem of evolution since it suggests the possibility
that the genus- and species-heredity are determined by
the cytoplasm of the egg, while the Mendelian heredi-

Preface vii

tary characters cannot contribute at all or only to a
limited extent to the formation of new species. Such
an idea is supported by the work on immunity, which
shows that genus- and probably species-specificity are
due to specific proteins, while the Mendelian characters
may be determined by hormones which need neither be
proteins nor specific or by enzymes which also need
not be specific for the species or genus. Such a con-
ception would remove the difficulties which the work on
Mendelian heredity has seemingly created not only for
the problem of evolution but also for the problem of
the harmonious character of the organism as a whole.

Since the book is intended as a companion volume
to the writer’s former treatise on The Comparative
Physiology of the Brain a discussion of the functions of
the central nervous system is omitted.

Completeness in regard to quotation of literature
was out of the question, but the writer notices with
regret, that he has failed to refer in the text to so
important a contribution to the subject as Sir E. A.
Schafer’s masterly presidential address on ‘‘Life ”’ or
the addresses of Correns and Goldschmidt on the de-
termination of sex. Credit should also have been given
to Professor Raymond Pearl for the discrimination be-
tween species and individual inheritance.

The writer wishes to acknowledge his indebtedness
to his friends Professor E. G. Conklin of Princeton,
Professor Richard Goldschmidt of the Kaiser Wilhelm

Vili Preface

Institut of Berlin, Dr. P. A. Levene of the Rockefeller
Institute, Professor T. H. Morgan of Columbia Univer-
sity, and Professor Hardolph Wasteneys of the Univer-
sity of California who kindly read one or more chapters
of the book and offered valuable suggestions; and he
wishes especially to thank his wife for suggesting many
corrections in the manuscript and the proof.

The book is dedicated to that group of freethinkers,
including d’Alembert, Diderot, Holbach, and Voltaire,
who first dared to follow the consequences of a mechan-
istic science—incomplete as it then was—to the rules of
human conduct and who thereby laid the foundation of
that spirit of tolerance, justice, and gentleness which
was the hope of our civilization until it was buried
under the wave of homicidal emotion which has swept
through the world. Diderot was singled out, since to
him the words of Lord Morley are devoted, which,
however, are more or less characteristic of the whole

group.
jJ. L.
THE ROCKEFELLER INSTITUTE

FoR MEDICAL RESEARCH,
August, 1916

CONTENTS

CHAPTER I

INTRODUCTORY REMARKS : : i ‘

CHAPTER II

Tue SPECIFIC DIFFERENCE BETWEEN LIVING AND
DEAD MATTER AND THE QUESTION OF THE
4 ORIGIN OF LIFE. : : ‘ P

CHAPTER III

Tue CHEMICAL Basis OF GENUS AND SPECIES:
I.—Twe INCOMPATIBILITY OF SPECIES NOT
CLOsELY RELATED . ; , :
II.—Tue CHeEmIcAL Basis oF GENUS AND ecuaes
AND OF SPECIES SPECIFICITY . . ‘

CHAPTER IV

SPECIFICITY IN FERTILIZATION. é 255
CHAPTER V,

ARTIFICIAL PARTHENOGENESIS. aS ne “Ua
CHAPTER VI

DETERMINISM IN THE FORMATION OF AN ORGANISM
FROM AN EGG ; . - ‘ 5 :

PAGE

128

x Contents

CHAPTER VII

REGENERATION . . F . ; ‘

CHAPTER VIII

DETERMINATION OF SEX, SECONDARY SEXUAL

CHARACTERS, AND SEXUAL INSTINCTS:

I.—Tue CytotocicaL Basis or SEX DETER-

MINATION

IIl.—Twue PuHysioLtocicAL Basis oF SEx

TERMINATION .

CHAPTER IX

MENDELIAN HEREDITY AND ITS MECHANISM .

CHAPTER X

ANIMAL INSTINCTS AND TROPISMS

CHAPTER XI

Tue INFLUENCE OF ENVIRONMENT < ‘

CHAPTER XII

ADAPTATION TO ENVIRONMENT

CHAPTER XIII

EVOLUTION . : ‘ - ‘ A

CHAPTER XIV

DEATH AND DISSOLUTION OF THE ORGANISM

INDEX 3 i , 3 ‘ ‘3 5

DeE-

PAGE

153

198

214

229

253

286

346

349

The Organism as a Whole

CHAPTER I
INTRODUCTORY REMARKS

I. The physical researches of the last ten years have
put the atomistic theory of matter and electricity on
a definite and in all probability permanent basis. We
know the exact number of molecules in a given mass
of any substance whose molecular weight is known to
us, and we know the exact charge of a single electron.
This permits us to state as the ultimate aim of the
physical sciences the visualization of all phenomena
in terms of groupings and displacements of ultimate
particles, and since there is no discontinuity between
the matter constituting the living and non-living world
the goal of biology can be expressed in the same way.

This idea has more or less consciously prevailed for
some time in the explanation of the single processes
occurring in the animal body or in the explanation of

the functions of the individual organs. Nobody, not
I

2 Introductory Remarks

even a scientific vitalist, would think of treating the
process of digestion, metabolism, production of heat,
and electricity or even secretion or muscular contrac-
tion in any other than a purely chemical or physico-
chemical way; nor would anybody think of explaining
the functions of the eye or the ear from any other
standpoint than that of physics.

When the actions of the organism as a whole are con-
cerned, we find a totally different situation. The same
physiologists who in the explanation of the individ-
ual processes would follow the strictly physicochemi-
cal viewpoint and method would consider the reactions
of the organism as a whole as the expression of non-
physical agencies. Thus Claude Bernard,t who in
the investigation of the individual life processes was a
strict mechanist, declares that the making of a har-
monious organism from the egg cannot be explained
on a mechanistic basis but only on the assumption of
a ‘‘directive force.’’ Bernard assumes, as Bichat and
others had done before him, that there are two opposite
processes going on in the living organism: (1) the pheno-
mena of vital creation or organizing synthesis; (2) the
phenomena of death or organic destruction. It is only
the destructive processes which give rise to the physical
manifestations by which we judge life, such as respira-
tion and circulation or the activity of glands, and so on.

* Bernard C., Legons sur les Phénoménes de la Vie. Paris, 1885, i.,
22-64.

Introductory Remarks 3

The work of creation takes place unseen by us in the
egg when the embryo or organism is formed. This
vital creation occurs always according to a definite
plan, and in the opinion of Bernard it is impossible
to account for this plan on a purely physicochemical
basis.

There is so to speak a pre-established design of each being
and of each organ of such a kind that each phenomenon by
itself depends upon the general forces of nature, but when
taken in connection with the others it seems directed by
some invisible guide on the road it follows and led to the
place it occupies. .. .

We admit that the life phenomena are attached to physico-
chemical manifestations, but it is true that the essential
is not explained thereby; for no fortuitous coming together
of physicochemical phenomena constructs each organism
after a plan and a fixed design (which are foreseen in ad-
vance) and arouses the admirable subordination and har-
monious agreement of the acts of life... .

We can only know the material conditions and not the
intimate nature of life phenomena. We have therefore
only to deal with matter and not with the first causes or
the vital force derived therefrom. These causes are inacces-
sible to us, and if we believe anything else we commit an
error and become the dupes of metaphors and take figura-
tive language as real. . . . Determinism can never be but
physicochemical determinism. The vital force and life,
belong to the metaphysical world.

In other words, Bernard thinks it his task to account
for individual life phenomena on a purely physico-
chemical basis—but the harmonious character of the

4 Introductory Remarks

organism as a whole is in his opinion not produced by
the same forces and he considers it impossible and
hopeless to investigate the ‘‘design.”” This attitude
of Bernard would be incomprehensible were it not for
the fact that, when he made these statements, the
phenomena of specificity, the physiology of develop-
ment and regeneration, the Mendelian laws of heredity,
the animal tropisms and their bearing on the theory
of adaptation were unknown.

This explanation of Bernard’s attitude is apparently
contradicted by the fact that Driesch? and v. Uexkull,?
both brilliant biologists, occupy today a standpoint
net very different from that of Claude Bernard. Driesch
assumes that there is an Aristotelian ‘‘entelechy”’
acting as directing guide in each organism; and
v. Uexkill suggests a kind of Platonic ‘‘idea” as a
peculiar characteristic of life which accounts for the
purposeful character of the organism.

v. Uexkull supposes as did Claude Bernard and as
does Driesch that in an organism or an egg the ulti-
mate processes are purely physicochemical. In an
egg these processes are guided into definite parts
of the future embryo by the Mendelian factors of
heredity—the so-called genes. These genes he compares
to the foremen for the different types of work to be

* Driesch, H., The Science and Philosophy of the Organism. 2 vols.
The Gifford Lectures, 1907 and 1908.

av. Uexkull, J., Bausteine 2u einer biologischen Weltanschauung.
Manchen, 1913.

Introductory Remarks 5

done in a building. But there must be something
that makes of the work of the single genes a harmonious
whole, and for this purpose he assumes the existence
of ‘‘supergenes.”* v. Uexkill’s ideas concerning the
nature of a Mendelian factor and of the ‘‘super-
genes’’ are expressed in metaphorical terms and the
assumption of the “‘supergenes”’ begs the question.
The writer is under the impression that this author
was led to his views by the belief that the egg is entirely
undifferentiated. But the unfertilized egg is not homo-
geneous, on the contrary, it has a simple but definite
physicochemical structure which suffices to determine
the first steps in the differentiation of the organism.
Of course, if we suppose as do v. Uexkull and Driesch
that the egg has no structure, the development of
structure becomes a difficult problem—but this is not
the real situation.

2. Claude Bernard does not mention the possibility
of explaining the harmony or apparent design in the
organism on the basis of the theory of evolution, he
simply considers the problem as outside of biology.
It was probably clear to him as it must be to everyone
with an adequate training in physics that natural
selection does not explain the origin of variation.
Driesch and v. Uexkull consider the Darwinian theory
a failure. We may admit that the theory of a forma-

a
ty. Uexkill, J., Bausteine zu einer biologischen Weltanschauung.
Mianchen, 1913, p. 216.

6 Introductory Remarks

tion of new species by the cumulative effect of aimless
fluctuating variations is not tenable because fluctuating
variation is not hereditary; but this would only demand
a slight change in the theory; namely a replacement of
the influence of fluctuating variation by that of equally
aimless mutations. With this slight modification which
is proposed by de Vries,* Darwin’s theory still serves
the purpose of explaining how without any pre-estab-
lished plan only purposeful and harmonious organisms
should have survived. It must be said, however,
that any theory of life phenomena must be based on
our knowledge of the physicochemical constitution of
living matter, and neither Darwin nor Lamarck was
concerned with this. Moreover, we cannot consider
any theory of evolution as proved unless it permits us
to transform at desire one species into another, and
this has not yet been accomplished.

It may be of some interest to point out that we do
not need to make any definite assumption concerning
the mechanism of evolution and that we may yet be
able to account for the fact that the surviving organ-
isms are to all appearances harmonious. The writer
pointed out that of all the 100,000,000 conceivable
crosses of teleost fish (many of which are possible)
not many more than 10,000, 7. e., about one-hundredth
of one per cent., are able to live and propagate. Those
that live and develop are free from the grosser type

1 de Vries, H., Die Mutationstheorie. Leipzig, 1901.

Introductory Remarks rj

of disharmonies, the rest are doomed on account of a
gross lack of harmony of the parts. These latter we
never see and this gives us the erroneous conception
that harmony or ‘‘design”’ is a general character of
living matter. If anybody wishes to call the non-
viability of 99:%% per cent. of possible teleosts a pro-
cess of weeding out by ‘‘natural selection’’ we shall
raise no objection, but only wish to point out that our
way of explaining the lack of design in living nature
would be valid even if there were no theory of evolu-
tion or if there had never been any evolution.

3. v. Uexkill is perfectly right in connecting
the problem of design in an organism with Mendelian
heredity. The work on Mendelian heredity has shown
that an extremely large number of independently
transmissible Mendelian factors help to shape the
individual. It is not yet proven that the organism
is nothing but a mosaic of Mendelian factors, but no
writer can be blamed for considering such a possibility.
If we assume that the organism is nothing but a mosaic
of Mendelian characters it is difficult indeed to under-
stand how they can force each other into a harmonious
whole’; even if we make ample allowance for the law

« This difficulty is also felt by mechanistic writers like Child, who
on page 12 of his recent book on Senescence and Rejuvenescence
(Chicago, 1915) makes the following remarks: ‘These theories of Weis-
mann do not account satisfactorily for the peculiarly constant course
and character of development and morphogenesis. If we follow them
to their logical conclusion, which their authors have not done, we find
ourselves forced to assume the existence of some sort of controlling and’

8 Introductory Remarks

of chance and the corresponding wastefulness in the
world of the living. But it is doubtful whether this
idea of the réle of Mendelian factors is correct. The
facts of experimental embryology strongly indicate
the possibility that the cytoplasm of the egg is the
future embryo (in the rough) and that the Mendelian
factors only impress the individual (and variety) char-
acters upon this rough block. This idea is supported
by the fact that the first development—in the sea
urchin to the gastrula stage inclusive—is independent
of the nucleus, which is the bearer of the Mendelian
factors. Not before the skeleton or mesenchyme is
formed in the sea urchin egg is the influence of the
nucleus noticeable. This has been shown in the ex-
periments of Boveri in which an enucleated fragment of
an egg was fertilized with a spermatozoon of a foreign
species. If this is generally true, it is conceivable that
the generic and possibly also the species characters
of organisms are determined by the cytoplasm of the
egg and not by the Mendelian factors.

co-ordinating principle outside the units themselves and superior to
them. If the units constitute the physicochemical basis of life, as
their authors maintain, then this controlling principle, since it is an
essential feature of life, must of necessity be something which is not
physicochemical in nature. In short these theories lead us in the final
analysis to the same conclusion as that reached by the neovitalists.
If we are not content to accept this conclusion we must reject the
theories.” These last sentences do not exhaust all the possibilities,
since the writer is trying to show in this book that the widest accept-

ance of the chromosome theory of heredity is compatible with a con-
sistent physicochemical conception of the organism as a whole.

Introductory Remarks 9

In any case, we can state today that the cytoplasm
contains the rough preformation of the future embryo.
This would show then that the idea of the organism
being a mosaic of Mendelian characters which have
to be put into place by ‘‘supergenes” is unnecessary.
If the egg is already the embryo in the rough we can
imagine the Mendelian factors as giving rise to specific
substances which go into the circulation and start or
accelerate different chemical reactions in different
parts of the embryo, and thereby call forth the finer
details characteristic of the variety and the individual.
The idea that the egg is the future embryo is supported
by the fact that we can call forth a normal organism
from an unfertilized egg by artificial means; while it is
apparently impossible to cause the spermatozoon to
develop into an organism outside the egg.

4. The influence of the whole on the parts is no-
where shown more strikingly than in the field of re-
generation. It is known that pieces cut from the plant
or animal may give rise to new growth which in many
cases will restore somewhat the original organism.
Instead of asking what is the cause of this so-called
regeneration we may ask, why the same pieces do not
regenerate as long as they are parts of the whole. In
this form the mysterious influence of the whole over
its parts is put into the foreground. We shall see that
growth takes place in certain cells when certain sub-
stances in the circulation can collect there. The

10 Introductory Remarks

mysterious influence of the whole on these parts con-
sists often merely of the fact that the circulating speci-
fic or non-specific substances—we cannot yet decide
which—will in the whole be attracted by certain spots
and that this will prevent them from acting on other
parts of the organism. If such farts are isolated the
substances can no longer flow away from these parts
and the parts will begin to grow. It thus becomes
utterly unnecessary to endow such organisms with a
‘directing force’’ which has to elaborate the isolated
parts into a whole.

5. The same difficulty which we have discussed in
regard to morphogenesis exists also in connection with
those instincts which preserve the life of the organism
and of the race. The reader need only be reminded of
all the complicated instincts of mating by which sperm
and eggs are brought together; or those by which the
young are prevented from starvation to realize the
apparently desperate problems in store for a mechanist,
to whom the assumption of design is meaningless.
And yet we are better off in regard to our knowledge
of the instincts than we are in regard to morphogenesis,
as in the former we can show that the apparent instincts
in some cases obey simple physicochemical laws with
almost mathematical accuracy. Since the validity of
the law of gravitation has been proved for the solar
system the idea of design in the motion of the planets
has lost its usefulness, and this fact must serve us as

Introductory Remarks II

a guide wherever we attempt to put science beyond the
possibility of mysticism. As soon as we can show that
a life phenomenon obeys a simple physical law there
is no longer any need for assuming the action of non-
physical agencies. We shall see that this has been
accomplished for one group of animal instincts; namely
those which determine the relation of animals to light,
since these are being gradually reduced to the law of
Bunsen and Roscoe. This law states that the chemical
effect of light equals the product of intensity into dura-
tion of illumination. Some authors object to the
tendency toward reducing everything in biology to
mathematical laws or figures; but where would the
theory of heredity be without figures? Figures have
been responsible for showing that the laws of chance
and not of design rule in heredity. Biology will be
scientific only to the extent that it succeeds in reducing
life phenomena to quantitative laws.

Those familiar with the theories of evolution .know
the extensive réle ascribed to the adaptations of organ-
isms. The writer in 1889 called attention to the fact
that reactions to light—e. g., positive heliotropism—
are found in organisms that never by any chance make
use of them; and later that a great many organisms
show definite instinctive reactions towards a galvanic
current—galvanotropism—although no organism has
ever had or ever will have a chance to be exposed to
such a current except in laboratory experiments. This

12 Introductory Remarks

throws a different light upon the seemingly purposeful
character of animal reactions. Heliotropism depends
primarily upon the presence of photosensitive sub-
stances in the eye or the epidermis of the organism,
and these substances are inherited regardless of whether
they are useful or not. It is only a metaphor to call
reactions resulting from the presence of photosensitive
substances ‘‘adaptation.”” In this book other examples
are given which show that authors have too often
spoken of adaptation to environment where the en-
vironment was not responsible for the phenomena.
The blindness of cave animals and the resistance of
certain marine animals to higher concentrations of sea
water are such cases. Cuénot speaks of ‘‘preadapta-
tion” to express this relation. The fact is that the
“adaptations” often existed before the animal was
exposed to surroundings where they were of use. This
relieves us also of the necessity of postulating the
existence of the inheritance of acquired characters,
although it is quite possible that the future may furnish
proof that such a mode of inheritance exists.

6. We have mentioned that according to Claude
Bernard two groups of phenomena occur in the living
organism: (1) the phenomena of vital creation or or-
ganizing synthesis (especially in the egg and during
development); (2) the phenomena of death or organic
destruction. These two processes are briefly discussed
in the first and last chapters.

Introductory Remarks 13

These introductory remarks may perhaps make it
easier for the reader to retain the thread of the main

ideas in the details of experiments and tables given in
this book.

CHAPTER II

THE SPECIFIC DIFFERENCE BETWEEN LIVING AND DEAD
MATTER AND THE QUESTION OF THE ORIGIN
OF LIFE

1. Each organism is characterized by a definite
form and we shall see in the next chapter that this form
is determined by definite chemical substances. The
same is true for crystals, where substance and form are
definitely connected and there are further analogies
between organisms and crystals. Crystals can grow
in a proper solution, and can regenerate their form in
such a solution when broken or injured; it is even
possible to prevent or retard the formation of crystals
in a supersaturated solution by preventing ‘‘germs”’
in the air from getting into the solution, an observation
which was later utilized by Schroeder and Pasteur in
their experiments on spontaneous generation. How-
ever, the analogies between a living organism and a
crystal are merely superficial and it is by pointing out
the fundamental differences. between the behaviour of
crystals and that of living organisms that we can best

The Origin of Life 15

understand the specific difference between non-living
and living matter. It is true that a crystal can grow,
but it will do so only in a supersaturated solution of
its own substance. Just the reverse is true for living
organisms. In order to make bacteria or the cells of
our body grow, solutions of the split products of the
substances composing them and not the substances
themselves must be available to the cells; second, these
solutions must not be supersaturated, on the contrary,
they must be dilute; and third, growth leads in living
organisms to cell division as soon as the mass of the
cell reaches a certain limit. This process of cell divi-
sion cannot be claimed even metaphorically to exist in _
a crystal. A correct appreciation of these facts will
give us an insight into the specific difference between
non-living and living matter. The formation of living
matter consists in the synthesis of the proteins, nucleins,
fats, and carbohydrates of the cells, from the split pro- .
ducts. To give an historical example, Pasteur showed
that yeast cells and other fungi could be raised on the
following sterilized solution: water, 100 gm., crystallized
sugar, IO gm., ammonium tartrate, 0.2 gm. to 0.5 gm.,
and fused ash from yeast, 0.1 gm.’ He undertook this
experiment to disprove the idea that protein or organic
matter in a state of decomposition was needed for the
origin of new organisms as the defenders of the idea
of spontaneous generation had maintained.
t Pasteur, L., Annal. d. Chim. et d. Physique, 1862, 3 sét., Ixiv., I.

16 Living and Dead Matter and

2. That such a solution can serve for the synthesis
of all the compounds of living yeast cells is due to the
fact that it contains the sugars. From the sugars
organic acids can be formed and these with ammonia
(which was offered in the form of ammonium tartrate)
may give rise to the formation of amino acids, the
“‘building stones” of the proteins. It is thus obvious
that the synthesis of living matter centres around the
sugar molecule. The phosphates are required for the
formation of the nucleins, and the work of Harden and
Young suggests that they play also a réle in the alco-
holic fermentation of sugar.

Chlorophyll, under the influence of the red rays of
light, manufactures the sugars from the CO, of the air.
This makes it appear as though life on our planet should
have been preceded by the existence of chlorophyll,
a fact difficult to understand since it seems more natural
to conceive of chlorophyll as a part or a product of
living organisms rather than the reverse. Where then
should the sugar come from, which is a constituent of
the majority of culture media and which seems a pre-
requisite for the synthesis of proteins in living organ-
isms?

The investigations of Winogradsky on nitrifying,*
sulphur and perhaps also on iron bacteria have to all
appearances pointed a way out of this difficulty. It

* Winogradsky, S., “Die Nitrification,” Handb. d. tech. Mykol., 1904-
06, iii., 132.

The Origin of Life 17

seemed probable that there were specific micro-organ-
isms which oxidized the ammonia formed in sewage
or in the putrefaction of living matter, but the attempts
to prove this assumption by raising such a nitrifying
micro-organism on one of the usual culture media, all
of which contained organic compounds, failed. Led
by the results of his observations on sulphur bacteria
it occurred to Winogradsky that the presence of organic
compounds stood in the way of raising these bacteria,
and this idea proved correct. The bacteria oxidizing am-
monia to nitrites were grown on the following medium;
I gm. ammonium sulphate, I gm. potassium phosphate,
I gm. magnesium carbonate, to I litre of water. From
this medium, which is free from sugar and contains
only constituents which could exist on the planet before
the appearance of life, the nitrifying bacteria were able
to form sugars, fatty acids, proteins, and the other
specific constituents of living matter. Winogradsky
proved, by quantitative determination, that with the
nitrification an increase in the amount of carbon com-
pounds takes place. ‘‘Since this bound carbon in the
cultures can have no other source than the CO, and
since the process itself can have no other cause than
the activity of the nitrifying organism, no other alter-
native was left but to ascribe to it the power of assimi-
lating CO,.’* ‘‘Since the oxidation of NH, is the
only source of chemical energy which the nitrifying

t Winogradsky, Joc. cit., p. 163 and ff.

18 Living and Dead Matter and

organism can use it was clear a priori that the yield
in assimilation must correspond to the quantity of oxi-
dized nitrogen. It turned out that an approximately
constant ratio exists between the values of assimi-
lated carbon and those of oxidized nitrogen.”” This is
illustrated by the results of various experiments as
shown in Table I.

TABLE I
No. § No. 6 No.7 No. 8
mg. meg. mg. mg.
Oxidized N........... 722.0 506.1 928.3 815.4
Assimilated C......... 19.7 15.2 26.4 22.4
Ratio N: C........... 36.6 33.3 35.2 36.4

It is obvious that 1 part of assimilated carbon
corresponds to about 35.4 parts oxidized nitrogen or
96 parts of nitrous acid.

These results of Winogradsky were confirmed in very
careful experiments by E. Godlewski, Sr.*

The nitrites are further oxidized by another kind of
micro-organisms into nitrates and they also can be
raised without organic material.

Winogradsky had already previously discovered that

* Godlewski, E., Anz. d. Akad. d. Wissersch. in Krakau, 1892, 408;
1895, 178.

The Origin of Life 19

the hydrogen sulphide which is formed as a reduction
product from CaSO, or in putrefaction by the activity
of certain bacteria can be oxidized by certain groups
of bacteria, the sulphur bacteria. Such bacteria, e. g.,
Beggiatoa, are also commonly found at the outlet of
sulphur springs. They utilize the hydrogen sulphide
which they oxidize to sulphur and afterwards to
sulphates, according to the scheme:

(1) 2H.S+0, =2H,0+S,
(2) $.+30.+2H,0 =2H,S0,

The sulphuric acid is at once neutralized by car-
bonates.

Winogradsky assumes that the oxidation of H.S by
the sulphur bacteria is the source of energy which plays
the same réle as the oxidation of NH, plays in the
nitrifying bacteria, or the oxidation of carbon compounds
—sugar and others—in the case of the other lower and
higher organisms. Winogradsky has made it very
probable that sulphur bacteria do not need any organic
compounds and that their nutrition may be accom-
plished with a purely mineral culture medium, like
that of the nitrite bacteria. On the basis of this assump-
tion they should also be able to form sugars from the
CO, of the air.

Nathanson? discovered in the sea water the existence

t Nathanson, Mitteil. d. zool. Station, Neapel, 1902.

20 Living and Dead Matter and

of bacteria which oxidize thiosulphate to sulphuric
acid. They will develop if some Na.S.O, is added to
sea water. These bacteria can only develop if CO,
from the air is admitted or when carbonates are pre-
sent. For these organisms the CO, cannot be replaced
by glucose, urea, or other organic substances. Such
bacteria must therefore possess the power of producing
sugar and starch from CO, without the aid of chloro-
phyll. Similar observations were made by Beijerinck
on a species of fresh-water bacteria. *

Finally the case of iron bacteria may briefly be
mentioned though Winogradsky’s views are not accepted
by Molisch.

We may, therefore, consider it an established fact
that there are a number of organisms which could
have lived on this planet at a time when only mineral
constituents, such as phosphates, K, Mg, SO,, CO.,
and O, besides NH;, or SH., existed. This would
lead us to consider it possible that the first or-
ganisms on this planet may have belonged to that
world of micro-organisms which was discovered by
Winogradsky.

If we can conceive of this group of organisms as
producing sugar, which in fact they do, they could
have served as a basis for the development of other
forms which require organic material for their develop-
ment.

1 Beijerinck, M., Folia Microbiologica, 1914, iii., 91.

The Origin of Life 21

In 1883 the small island of Krakatau was destroyed
by the most violent volcanic eruption on record. A
visit to the islands two months after the eruption
showed that ‘‘the three islands were covered with
pumice and layers of ash reaching on an average a
thickness of thirty metres and frequently sixty metres.’’!
Of course all life on the islands was extinct. When
Treub in 1886 first visited the island, he found that
blue-green algze were the first colonists on the pumice
and on the exposed blocks of rock in the ravines on
the mountain slopes. Investigations made during
subsequent expeditions demonstrated the association
of diatoms and bacteria. All of these were probably
carried by the wind. The alge referred to were accord-
ing to Euler of the nostoc type. Nostoc does not re-
quire sugar, since it can produce that compound from
the CO, of the air by the activity of its chlorophyll.
This organism possesses also the power of assimilating
the free nitrogen of the air. From these observations
and because the Nostocacee generally appear as the
first settlers on sand the conclusion has been drawn
that they or the group of Schizophycee to which they
belong formed the first settlers of our planet.? This
conclusion is not quite safe since in the settlement of
Krakatau as well as in the first colonizing of sand

tErnst, A., The New Flora of the Volcanic Island of Krakatau,
Cambridge, 1908.
2 Euler, H., Pflanzenchemie, 1909, ii. and iii., 140.

22 Living and Dead Matter and

areas the nature of the first settler is determined
chiefly by the carrying. power of wind (or waves and
birds).

We may now return from this digression to the real
object of our discussion, namely that the nutritive
solutions of organisms must be very dilute and consist
of the split products of the complicated compounds
of which the organisms consist. The examples given
sufficiently illustrate this statement.

The nutritive medium of our body cells is the blood,
and while we take up as food the complicated com-
pounds of plants or animals, these substances undergo
a digestion, 7. ¢., a splitting up into small constituents
before they can.diffuse from the intestine into the blood.
Thus the proteins are digested down to the amino
acids and these diffuse into the blood as demonstrated
by Folin and by Van Slyke. From here the cells take
them up. The different proteins differ in regard to
the different types of amino acids which they contain.
While the bacteria and fungi and apparently the higher
plants can build up all their different amino acids from
ammonia, this power is no longer found in the mammals
which can form only certain amino acids in their body
and must receive the others through their food. As
a consequence it is usually necessary to feed young
animals on more than one protein in order to make
them grow, since one protein, as a rule, does not contain
all the amino acids needed for the manufacture of all

The Origin of Life a3

the proteins required for the formation of the material
of a growing animal.*

3. The essential difference between living and non-
living matter consists then in this: the living cell syn-
thetizes its own complicated specific material from
indifferent or non-specific simple compounds of the
surrounding medium, while the crystal simply adds
the molecules found in its supersaturated solution.
This synthetic power of transforming small “‘building
stones” into the complicated compounds specific for
each organism is the ‘‘secret of life” or rather one of
the secrets of life.

What clew have we in regard to the nature of this
synthetic power? We know that the comparatively
great velocity of chemical reactions in a living organism
is due to the presence of enzymes (ferments) or to
catalytic agencies in general. Some of these catalytic
agencies are specific in the sense that a given catalyzer
can accelerate the reaction of only one step in a com-
plicated chemical reaction. While these enzymes are
formed by the action of the body they can be separated
from the body without losing their catalytic efficiency.
It was a long time before scientists succeeded in isolat-
ing the enzyme of the yeast cell which causes the alco-
holic fermentation of sugar; and this gave rise to the

t This fact was thoroughly established by Mendel and Osborne. A
summary of their work is given in Underhill, F. P., Phystology of the
Amino Acids, 1916.

24 Living and Dead Matter and

premature statement that it was not possible to isolate
this enzyme since it was bound up with the life of the
yeast cell. Such a statement was even made by a
man like Pasteur, who was usually a model of restraint
in his utterances, and yet the work of Buchner proved
him to be wrong.

The general mechanism of the action of the hydro-
lyzing enzymes is known. The old idea of de la Rive,
that a molecule of enzyme combines transitorily with
a molecule of substrate; the further idea, which may
possibly go back to Engler, that the molecule of sub-
strate is disrupted in the ‘“‘strain’”’ of the new combina-
tion and that the broken fragments fall off or are easily
knocked off by collision from the ferment molecule
which is now ready to repeat the process, seems to be
correct. On the assumption that the velocity of en-
zyme reaction is proportional to the mass of the enzyme
and that de la Rive’s idea was correct, Van Slyke and
Cullen were able to calculate the coefficients of the
velocity of enzyme reactions for the fermentation of
urea and other substances, and the agreement between
calculated and observed values was remarkable. *

While the hydrolytic action of enzymes is thus clear
the synthesis in the cell is still a riddle. An interesting
suggestion was made by van’t Hoff, who in 1898 ex-
pressed the idea that the hydrolytic enzymes should

*Van Slyke, D. D., and Cullen, G.E., Jour. Biol. Chem., 1914, xix.,
141.

The Origin of Life 25

also act in the opposite direction, namely synthetically.
Thus it should not only be possible to digest proteins
with pepsin but also to synthetize them from the pro-
ducts of digestion with the aid of the same enzyme.
This expectation was based on the idea that the enzyme
did not alter the equilibrium between the hydrolyzed
and non-hydrolyzed part of the substrate but only
accelerated the rate with which the equilibrium was
reached. Van’t Hoff’s idea omitted, however, the pos-
sibility that in the transitory combination between
enzyme molecule and substrate a change in the molec-
ular configuration of the substrate or in the distribution
of intramolecular strain may take place. The first
apparently complete confirmation of van’t Hoff’s sug-
gestion appeared in the form of the synthesis of maltose
from grape sugar by the enzyme maltase, which decom-
poses maltose into grape sugar. By adding the enzyme
maltase from yeast to a forty per cent. solution of
glucose Croft Hill' obtained a good yield of mal-
tose. It turned out, however, that what he took for
maltose was not this compound but an isomer, name-
ly isomaltose, which has a different molecular con-
figuration and cannot be hydrolyzed by the enzyme
maltase.

Lactose is hydrolyzed from kephyr by an enzyme
lactase into galactose and glucose; by adding this
enzyme to galactose and glucose a synthesis was

« Hill, C., Jour. Chem. Soc., 1898, Ixxiii., 634.

26 Living and Dead Matter and

obtained not of lactose but of isolactose; the latter,
however, is not decomposed by the enzyme lactase.

E. F. Armstrong has worked out a theory which
tries to account for this striking phenomenon by assum-
ing ‘‘that the enzyme has a specific influence in pro-
moting the formation of the biose which it cannot
hydrolyze. The theory is very ingenious and seems
supported by fact. This then would lead to the result
that certain hydrolytic enzymes may have a synthetic
action but not in the manner suggested by van’t Hoff.

The principle enunciated by Armstrong, that in the
synthetic action of hydrolytic enzymes not the origi-
nal compound but an isomer is formed which can
not be hydrolyzed by the enzyme, may possibly be
of great importance in the understanding of life phe-
nomena. It shows us how the cell can grow in the
presence of hydrolytic enzymes and why in hunger the
disintegration of the cell material is so slow. It was
at first thought that the formation of isomers con-
tradicted the idea of the reversible action of enzymes,
but this is not the case; on the contrary, it supports it
but makes an addition which may solve the riddle of
what Claude Bernard called the creative action of
living matter. We shall come back to this problem
in the last chapter.

Kastle and Loevenhart demonstrated the synthesis
of a trace of ethylbutyrate by lipase if the latter enzyme

+ Armstrong, E. F., Proc. Royal Soc., 1905, B. lxxvi., 592.

The Origin of Life 27

was added to the products of the hydrolysis of ethyl-
butyrate, ethyl alcohol, and butyric acid by the same
enzyme.* Taylor? obtained the synthesis of a slight
amount of triolein

by the addition of the dried fat-free residue of the castor
bean to a mixture of oleinic acid and glycerine. . . . No
synthesis occurred with acetic, butyric, palmitic, and
stearic acids with glycerine, mannite, and dulcite, and the
experiments with the last two alcohols and oleinic acid
likewise yielded no synthesis.

This suggests possibly a specific action of the enzyme.
If this slight reversible action had any biological signi-
ficance (which might be possible, since in the organism
secondary favourable conditions might be at work
which are lacking in vitro) there should be a parallelism
between masses of lipase in different kinds of tissues and
fat synthesis. Loevenhart indicated that this might
be a fact, but a more extensive investigation by H. C.
Bradley has made this very dubious.

Very little is known concerning the reversible action
of the hydrolytic protein enzymes. A. E. Taylor
digested protamine sulphate with trypsin and found
that after adding trypsin to the products of digestion
a precipitate was formed after long standing; and we

Kastle, J. H., and Loevenhart, A. S., Am. Chem. Jour., 1900, xxiv.,

4ol.

2Taylor, A. E., Univ. Cal. Pub., 1904, Pathology, i., 33; Jour. Biol.
Chem., 1906, ii., 87.

3 Bradley, H. C., Jour. Biol. Chem., 1913, xiii., 407.

28 Living and Dead Matter and

may also refer to experiments of Robertson with pepsin
on the products of caseinogen to which we shall return
in the next chapter. It therefore looks at present as
if van’t Hoff’s idea of reversible enzyme action might
hold in the modification offered by Armstrong. It
remains doubtful, however, whether this reversibility
can explain all the synthetic processes in the cell. No
objection can be offered at present if any one makes
the assumption that each cell has specific synthetic
enzymes or some other synthetic mechanisms which
are still unknown.

The mechanisms for the synthesis of proteins must
have one other peculiarity: they must be specific in
their action. We shall see in the next chapter that
each species seems to possess one or more proteins not
found in any other but closely related species. Each
organism develops from a tiny microscopic germ and
grows by synthetizing the non-specific building stones
(amino acids) into the specific proteins of the species.
This must be the work of the yet unknown synthetic
enzymes or mechanisms. The elucidation of their
character would seem one of the main problems of
biology. Needless to say crystallography is not con-
fronted with problems of such a nature.

The fact that the living cell grows after taking up
food has given rise to curious misunderstandings.
Traube has shown that drops of a liquid surrounded
with a semipermeable membrane may increase in

The Origin of Life 29

volume when put into a solution of lower osmotic
pressure. This has led and is possibly still leading to
the statement that the process of growth by a living
cell has been imitated artificially. Only one feature has
been imitated, the increase in volume; but the essential
feature of the process in the living cell, 7. é., the forma-
tion of the specific constituents of the living cell from
non-specific products, has of course not been imitated.

4. The constant synthesis then of specific material
from simple compounds of a non-specific character is
the chief feature by which living matter differs from
non-living matter. With this character is correlated
another one; namely, when the mass of a cell reaches
a certain limit the cell divides. This is perhaps most
obvious in bacteria which on the proper nutritive me-
dium take up food, grow, and divide into two bacteria,
each of which takes up food, divides, and grows ad
infinitum, as long as the food lasts, provided the harm-
ful products of metabolism are removed. If it be
true that specific synthetic ferments exist in each cell
it follows that the cell must synthetize these also,'

t This would lead to the idea that the enzymes in the cell also syn-
thetize molecules of their own kind, or that, in other words, the syn-
thetic processes in the cell are of the nature of autocatalysis. Loeb,
Der chemische Character des Befruchtungsvorgangs, Leipzig, 1908. Ro-
bertson, T. B., Arch. f. Entwicklngsmech., 1908, xxv., 581; Xxvi., 108;
1913, XXXVii., 497; Am. Jour. Physiol., 1915, xxxvii., 1; Robertson and
Wasteneys, H., Arch. f. EntwickIngsmech., 1913, Xxxvii., 485. Ostwald,
Wo., Uber die zeitlichen Eigenschaften der Entwicklungsvorgénge, Leipzig,
1908.

30 Living and Dead Matter and

as otherwise the synthesis of specific proteins would
have to come to a standstill.

This problem of synthesis leads to the assumption
of immortality of the living cell, since there is no @ priort
reason why this synthesis should ever come to a stand-
still of its own accord as long as enough food is avail-
able and the proper outside physical conditions are
guaranteed. It is well known that Weismann has
claimed immortality for all unicellular organisms and
for the sex cells of metazoa, while he claimed the neces-
sity of death for the body cells of the latter. Leo Loeb
was led by his investigations on the transplanta-
tion of cancer to assume immortality not only for
the cancer cell but also for the body cell of the
organism. He had found in transplanting a malignant
tumor from one individual to another that the tumor
grew; that it was not the cells of the host but the
transplanted tumor cells of the graft which grew and
multiplied, and that this process could be repeated appar-
ently indefinitely so that it was obvious that the trans-
planted tumor cells outlived the original animal. Such
experiments have since been carried on so long that
we may now say that an individual cancer cell taken
from an animal and transplanted from time to time
on a new host lives apparently indefinitely. Leo Loeb
had found that these tumor cells are simply modified
somatic cells. He therefore suggested that the somatic
cells might be considered immortal with the same right

The Origin of Life 31

as we speak of the immortality of the germ cells of such
animals."

This view receives its support first from the fact that
certain trees like the Sequoia live several thousand years
and may therefore be considered immortal; and second,
from the method of tissue culture. The method of
cultivating tissue cells in a test tube, in the same way
as is done for bacteria, was first proposed and carried
out by Leo Loeb, in 1897,? but his test-tube method
did not permit the observation of the transplanted cell
under the microscope. This was made possible by a
modification of the method by Harrison, who established
the fact that the axis cylinder grows out from the gan-
glionic cell. Harrison and Burrows then perfected
the method for the cultivation of the cells of warm-
blooded animals, and with the aid of these methods
Carrel succeeded in keeping connective-tissue cells
of the heart of an early chick embryo alive more
than four years, and these cells are still growing
and dividing. Only very tiny masses of cells can
be kept alive in this way since all the cells in the
centre of a piece die on account of lack of oxygen;

t Loeb, Leo, Jour. Med. Res., 1901, vi., 28; Arch. f. Entwicklngsmech.,
1907, xxiv., 655.

2Loeb, Leo, Uber die Entstehung von Bindegewebe, Leucocyten und
rothen Blutkérperchen aus Epithel und tiber eine Methode tsolierte Gewebs-
teile zu ztichten. Chicago, 1897.

3 While this has been demonstrated thus far only for connective-
tissue cells it may be true also for other cells.

32 Living and Dead Matter and

and every two days a few cells from the margin of
the piece have to be transferred to a new culture
medium.

This effect of lack of oxygen explains also why the
immortality of the somatic cells is not obvious. Death
in a human being consists in the stopping of heart
beat and respiration, which also terminates the action
of the brain or at least of consciousness. Immediately
after the cessation of heart beat and respiration the cells
of muscle and of the skin and probably many or most
other organs are still alive and might continue to live
if transferred to another body with circulation and
respiration. As a consequence of the lack of oxygen
supply in the dead body they will, however, die com-
paratively rapidly. It may be stated that hearts taken
out of the body after a number of hours can still beat
again when put into the proper solutions and upon
receiving an adequate oxygen supply.

The idea that the body cells are naturally immortal
and die only if exposed to extreme injuries such as
prolonged lack of oxygen or too high a temperature
helps to make one problem more intelligible. The
medical student, who for the first time realizes that
life depends upon that one organ, the heart, doing its
duty incessantly for the seventy years or so allotted to
man, is amazed at the precariousness of our existence.
It seems indeed uncanny that so delicate a mechanism
should function so regularly for so many years. The

The Origin of Life 33

mysticism connected with this and other phenomena
of adaptation would disappear if we could be certain
that all cells are really immortal and that the fact which
demands an explanation .is not the continued activity
but the cessation of activity in death. Thus we see
that the idea of the immortality of the body cell if it
can be generalized may be destined to become one of
the main supports for a complete physico-chemical
analysis of life phenomena since it makes the durability
of organisms intelligible.

5. This generalized idea of the immortality of some
or possibly most or all somatic cells has a bearing upon
the problem of the origin of life on our planet. The
experiments of Spallanzani, Schwann, Schroeder, Pas-
teur, Tyndall, and all those who have worked with pure
cultures of micro-organisms, have proved that no spon-
taneous generation of living from non-living matter
can be demonstrated; and the statements to the con-
trary were due to experimental errors inasmuch as the
new organisms formed were the offspring of others
which had entered into the culture medium by mistake.

In the last chapter of that most fascinating book
Worlds in the Making,* Arrhenius discusses the possi-
bility of life being eternal and of living germs of very
small dimensions—e. g., the spores of micro-organisms—
being carried through space from one planet to another

t Arrhenius, S, Worlds in the Making, London and New York, 1908,
p. 212.
3

34 Living and Dead Matter and

or even from one solar system to another. If it be
true that there is no spontaneous generation; if it be
true that all cells are potentially immortal, we may
indeed seriously raise the question: May not life after
all be eternal? Such ideas were advocated by Richter
in a rather phantastic way and more definitely by
Helmholtz as well as Kelvin. The latter authors
assumed that in the collision of planets or worlds on
which there is life, fragments containing living organ-
isms will be torn off and these fragments will move as
seed-bearing stones through space. ‘‘If at the present
instant no life existed upon this earth, one such stone
falling upon it might . . . lead to its becoming covered
with vegetation.”” Arrhenius points out the difficulties
which oppose such a view, as, e. g., the fact ‘‘that the
meteorite in its fall towards the earth becomes incan-
descent all over its surface and any seeds on it would
therefore be deprived of their germinating power.”

Arrhenius suggests another and much more ingenious
idea based on the fact that for particles below a certain
size the mechanical pressure produced by light waves—
the radiation pressure—can overcome the attractive
force of gravitation.

Bodies which according to Schwarzschild would undergo
the strongest influence of solar radiation must have a dia-
meter of 0.00016 mm. supposing them to be spherical. The
first question is therefore: Are there any living seeds of
such extraordinary minuteness? The reply of the botanist

The Origin of Life 35

is that spores of many bacteria have a size of 0.0003 or
0.0002 mm., and there are no doubt much smaller germs
which our microscopes fail to disclose.

This assumption is undoubtedly correct.

We will, in the first instance, make a rough calculation
of what would happen if such an organism were detached
from the earth and pushed out into space by the radiation
pressure of our sun. The organism would first of all have
to cross the orbit of Mars; then the orbits of the smaller
and of the outer planets. . . . The organisms would cross
the orbit of Mars after twenty days, the Jupiter orbit after
eighty days, and the orbit of Neptune after fourteen months.
Our nearest solar system would be reached in nine thousand
years.

For the assumption of eternity of life only the transfer-
ence of germs from one solar system to another would
have to be considered and the question arises whether
or not germs can keep their vitality so many thousands
of years. Arrhenius thinks that this is possible on
account of the low temperature (which must be below
—220° C.) at which no chemical reaction and hence
no decomposition and deterioration are possible in the
spores; and on account of the absence of water vapour.

The question then arises: Have we any facts to war-
rant the assumption that spores may remain alive for
thousands of years under such conditions and retain
their power of germination? We know that seeds
have a very limited vitality, and the statement that

36 Living and Dead Matter and

grain found in the Egyptian tombs was still able to
germinate has long been recognized asa myth. Miss
White’ found that in wheat grains, there appeared a
well-marked drop in their germinating power after
about the fourth year, reaching zero in eleven to seven-
teen years. In a drier climate they last longer than
in a moist climate. It is of importance that the hydro-
lyzing enzymes in the seeds, such as diastase, erepsin,
remained unimpaired even after the germinating power
of the seeds had disappeared. The seeds were able to
resist for two days the temperature of liquid air, though
the subsequent germination was delayed by this treat-
ment. Macfadyen? exposed non-sporing bacteria, viz.,
B. typhosus, B. coli communis, Staphylococcus pyogenes
aureus, and a Saccharomyces to liquid air.

The experiments showed that a prolonged exposure of
six months to a temperature of about — 190° has no ap-
preciable effect on the vitality of micro-organisms. Tojudge
by the results there appeared no reason to doubt that the
experiment might have been successfully prolonged for a
still longer period.

Paul Becquerel found that seeds which possess a very
thick integument may live longer than the grain in
Miss White’s experiments. The thickness of the in-
tegument prevents the exchange of gases between air

t White, J., Proc. Roy. Soc., 1909, B, 1xxxi., 417.

2 Macfadyen, A., Proc. Roy. Soc., 1903, Ixxi., 76.
3 Becquerel, P., Revue générale des Sciences, 1914, XXV., 559.

The Origin of Life 37

and seed. Thus seeds of leguminoses (Cassia bicapsu-
laris, Cytisus biflorus, Leucena leucocephala, and Tri-
folium arvense) had retained their power of germination
for eighty-seven years. Becquerel has shown that the
dryness of the membrane is very essential for such a
duration of life, since when dry it is impermeable for
gases and the slow chemical reactions inside the grain
become impossible.

In the cosmic space there is no water vapour, no
atmosphere, and a low temperature, and there is hence
no reason why spores should lose appreciably more of
their germinating power in ten thousand years than in
six months. We must therefore admit the possibility
that spores may move for an almost infinite length
of time through cosmic space and yet be ready for ger-
mination when they fall upon a planet in which all
the conditions for germination and development exist,
e. g., water, proper temperature, and the right nutritive
substances dissolved in the water (inclusive of free
oxygen).

While thus everything is favourable to Arrhenius’s
hypothesis, Becquerel raises the objection that the
spores going through space would yet be destroyed by
ultraviolet light. This danger would probably exist
only as long as the germ is not too farfromasun. The
difficulty is a real one since the ultraviolet rays have
a destructive effect even in the absence of oxygen. It
is possible, however, that there are spores which can

38 Living and Dead Matter and

resist this effect of ultraviolet light. Arrhenius’s
theory can not of course be disproved and we must
agree with him that it is consistent not only with the
theories of cosmogony but also with the seeming poten-
- tial immortality of certain or of all cells.

The alternative to Arrhenius’s theory is that living
matter did originate and still originates from non-living
matter. If this idea is correct it should one day be
possible to discover synthetic enzymes which are cap-
able of forming molecules of their own kind from a
simple nutritive solution. With such synthetic en-
zymes as a starting point the task might be undertaken
of creating cells capable of growth and cell division,
at least in the apparently simple form in which these
phenomena occur in bacteria; viz., that after the mass
has reached a certain (still microscopic) size it divides
into two cells and so on. If Arrhenius is right that
living matter has had no more beginning than matter
in general, this hope of making living matter artificially
appears at present as futile as the hope of making
molecules out of electrons.

The problem of making living matter artificially
has been compared to that of constructing a perpetuum
mobile; this comparison is, however, not correct. The
idea of a perpetuum mobile contradicts the first law of
thermodynamics, while the making of living matter
may be impossible though contradicting no natural law.

Pasteur’s proof that spontaneous generation does

The Origin of Life 39

not occur in the solutions used by him does not prove
that a synthesis of living from dead matter is impossible
under any conditions. It is at least not inconceivable
that in an earlier period of the earth’s history radio-'
activity, electrical discharges, and possibly also the
action of volcanoes might have furnished the combina-.
tion of circumstances under which living matter might |
have been formed. The staggering difficulties in
imagining such a possibility are not merely on the
chemical side—e. g., the production of proteins from
CO, and N—but also on the physical side if the neces-
sity of a definite cell structure is considered. We shall
see in the sixth chapter that without a structure in the
egg to begin with, no formation of a complicated organ-
ism is imaginable; and while a bacterium may have a
simple structure, such a structure as it possesses is as |
necessary for its existence as are its enzymes. ;
Attempts have repeatedly been made to imitate the
structures in the cell and of living organisms by colloidal
precipitates. It is needless to point out that such
precipitates are of importance only for the study of the
origin of structures in the living, but that they are not
otherwise an imitation of the living since they are
lacking the characteristic synthetic chemical processes.

CHAPTER III
THE CHEMICAL BASIS OF GENUS AND SPECIES

1. It is a truism that from an egg of a species an
organism of this species only and of no other will arise.
It is also a truism that the so-called protoplasm of an
egg does not differ much from that of eggs of
other species when looked at through a microscope.
The question arises: What determines the species of
the future organism? Is it a structure or a specific
chemical or groups of chemicals? In a later chapter
we shall show that the egg has a simple though
definite structure, but in this chapter we shall see
that the egg must contain specific substances and
that these substances which determine the ‘‘species’”’
and specificity in general are in all probability proteins.
Since solutions of different proteins look alike under a
microscope we need not wonder that it is impossible
to discriminate microscopically between the protoplasm
of different eggs.

The idea of definiteness and constancy of species, a

matter of daily observation in the case of man and
40

Chemical Basis of Genus and Species 41

higher animals in general, was not so readily accepted
in the case of the micro-organisms, which on account
of their minuteness and simplicity of structure are not
so easy to differentiate. There existed for a long time
serious doubt whether or not the simplest organisms,
the bacteria, possessed a definite ‘‘specificity” like
the higher organisms, or whether they were not en-
dowed, as Warming put it, with an “unlimited plasti-
city,’ which forbade classifying them according to
their form into definite species as Cohn had done. An
interesting episode in this discussion, which was settled
about twenty-five years ago arose concerning the sulphur
bacteria, which often develop in large masses on parts
of decaying plants or animals along the shore. Sir E.
Ray Lankester found collections of red bacteria cover-
ing putrefying animal matter in a vessel and forming
a continuous membrane along its wall. These red
bacteria were of very different shape, size, and group-
ing, but they seemed to be connected by transition forms.
They had a common character, however, namely, their
peach-coloured appearance. This common character,
together with their association in the same habitat,
led Lankester to the then justifiable belief that they
all belonged to one species which was protean in char-
acter and that the different forms were “only to be
considered as phases of growth of this one species.
The presence of the same red pigment ‘‘ Bacterio-pur-
purin” seemed justly to indicate the existence of

42 Chemical Basis of Genus and Species

common chemical processes. Cohn, on the contrary,
considered the different forms among these red bacteria
(they are today called sulphur bacteria since they
oxidize the hydrogen sulphide produced by bacteria
of putrefaction to sulphur and sulphates) as definite
and distinct species, inspite of their common colour
and their association. Later observations showed that
Cohn was right. Winogradsky* succeeded in proving
by pure culture experiments that each of these different
forms of sulphur bacteria was specific and did not give
rise to any of the other forms of the same colour found
in the same conditions.

The method of pure line breeding inaugurated by
Johannsen? has shown that the degree of definiteness
goes so far that apparently identical forms with only
slight differences in size may breed true to this size;
but for reasons which will become clear later on we
may doubt whether they are to be considered as definite
species.

The fact of specificity is supported by the fact of
constancy of forms. de Vries has pointed out that
regardless of the possible origin of new species by muta-
tion the old species may persevere. Walcott has found
fossils of annelids, snails, crustaceans, and alge in a
precambrian formation in British Columbia whose age

*Winogradsky, S., Beitrdge zur Morphologie und Physiologie der
Bacterien. Leipzig, 1888.
* Johannsen, W., Elemente der exacten Erblichkeitslehre. 2d ed., 1913.

Chemical Basis of Genus and Species 43

(estimated on the rate of formation of radium from
uranium) may be about two hundred million years and
estimated on the basis of sedimentation sixty million
years. And yet these invertebrates are so closely
related to the forms existing today that the systematists
have no difficulty in finding the genus among the modern
forms into which each of these organisms belongs. W.
M. Wheeler, in his investigations of the ants enclosed
in amber, was able to identify some of them with forms
living today, though the ants observed in the amber
must have been two million years old. The constancy.
of species, 7. e., the permanence of specificity may there-
fore be considered as established as far back as two or
possibly two hundred millions of years. The definite-
ness and constancy of each species must be deter-
mined by something equally definite and constant in
the egg, since in the latter the species is already fixed
irrevocably.

We shall show first that species if sufficiently sepa-
rated are generally incompatible with each other and
that any attempt at fusing or mixing them by grafting
or cross-fertilizing is futile. In the second part of the
chapter we shall take up the facts which seem destined
to give a direct answer to the question as to the cause
of specificity. It is needless to say that this latter
question is of paramount importance for the problem
of evolution, as well as for that of the constitution of

living matter.

44 Chemical Basis of Genus and Species

I. The Incompatibility of Species not closely Related

2. It is practically impossible to transplant organs
or tissues from one species of higher animals to an-
other, unless the two species are very closely related;
and even then the transplantation is uncertain and
the graft may either fall off again or be destroyed.
This specificity of tissues goes so far that surgeons
prefer, when a transplantation of skin in the human is
intended, to use skin of the patient or of close blood
relations. The reason why the tissues of a foreign
species in warm-blooded animals cannot grow well on
a given host has been explained by the remarkable
experiments of James B. Murphy of the Rockefeller
Institute. Murphy discovered that it is possible to
transplant successfully any kind of foreign tissue upon
the early embryo of the chick. Even human tissue
transplanted upon the chick embryo will grow rapidly.
This shows that at this early stage the chick embryo
does not yet react against foreign tissue. This lack
of reaction lasts until about the twenty-first day in
the life of the embryo; then the growth of the graft not
only ceases but the graft itself falls off or is destroyed.
Murphy noticed that this critical period coincides with
the development of the spleen and of lymphatic tissue
in the chick and that a certain type of migrating cells,

* Murphy, J. B., Jour. Exper. Med., 1913, xvii., 482; 1914, xix., 1813
xix., 513; Murphy and Morton, J. J., Jour. Exper. Med., 1915, xxii., 204.

Chemical Basis of Genus and Species 45

the so-called lymphocytes, which develop in the lym-
phatic tissue, gather at the edge of the graft in great
numbers, and he suggested that these lymphocytes (by
a secretion of some substance?) rid the host of the
graft. He applied two tests both of which confirmed
thisidea. First he showed that when small fragments of
the spleen of an adult chicken are transplanted into the
embryo the latter loses its tolerance for foreign grafts.
The second proof is still more interesting. It was known
that by treatment with Roentgen rays the lymphocytes
in an animal could be destroyed. It was to be expected
that an animal so treated would have lost its specific
resistance to foreign tissues. Murphy found that this
was actually the case. On fully grown rats in which
the lymphocytes had been destroyed by X-rays (as
ascertained by blood counts) tissues of foreign species
grew perfectly well. These experiments have assumed
a great practical importance since they can also be
applied to the immunization of an animal against
transplanted cancer of its own species. Murphy found
that by increasing the number of lymphocytes in an
animal (which can be accomplished by a mild treatment
with X-rays) the immunity against foreign grafts as
well as against cancer from the same species can be
increased. It is quite possible that the apparent im-
munity to a transplantation of cancer produced by
Jensen, Leo Loeb, and Ehrlich and Apolant through
the previous transplantation of tissue in such an animal

46 Chemical Basis of Genus and Species

was due to the fact that this previous tissue transplan-
tation led to an increase in the number of lymphocytes
in the animal. The medical side, however, lies outside
of our discussion, and we must satisfy ourselves with
only a passing notice. The facts show that each warm-
blooded animal seems to possess a specificity whereby
its lymphocytes destroy transplanted tissue taken
from a foreign species.

A lesser though still marked degree of incompatibility
exists also in lower animals for grafts from a different
species.‘ The graft may apparently take hold, but
only for a few days, if the species are not closely
related. Joest apparently succeeded in making a per-
manent union between the anterior and posterior ends
of two species of earthworms, Lumbricus rubellus and
Allolobophora terrestris. Born and later MHarrison
healed pieces of tadpoles of different species together.
An individual made up of two species Rana virescens
and Rana palustris lived a considerable time and went
through metamorphosis. Each half regained the char-
acteristic features of the species to which it belonged.
It seems, however, that if species of tadpoles of two
more distant species are grafted upon each other no
lasting graft can be obtained, e. g., Rana esculenta and
Bombinator igneus. These experiments were made at
a time when the nature and bearing of the problem of

*The reader is referred to Morgan’s book on Regeneration (New
York, 1901), for the literature on this subject.

Chemical Basis of Genus and Species 47

specificity was not yet fully recognized. The rdéle of
lymphocytes in these cases has never been investigated.
The grafted piece always retained the characteristics
of the species from which it was taken.

Plants possess no leucocytes and we therefore see
that they tolerate a graft of foreign tissues better than
is the case in animals. Asa matter of fact heteroplastic
grafting is a common practice in horticulture, although
even here it is known that indiscriminate heteroplastic
grafting is not feasible and that therefore the specificity
is not without influence. The host is supposed to fur-
nish only nutritive sap to the graft and in this respect
does not behave very differently from an artificial nutri-
tive solution for the raising of a plant. The law of
specificity, however, remains true also for the grafted
tissues: neither in animals nor in plants does the graft
lose its specificity, and it never assumes the specific
characters of the host, or vice versa. The apparent
exceptions which Winkler believed he had found in the
case of grafts of nightshade on tomatoes turned out to
be a further proof of the law of specificity. Winkler,
after the graft had taken, cut through the place of
grafting, after which operation a callus formation oc-
curred on the wound. In most cases either a pure
nightshade or a pure tomato grew out from this callus.
In some cases he obtained shoots from the place
where graft and host had united, which on one side
were tomato, on the other side nightshade. What

48 Chemical Basis of Genus and Species

really happened was that the shoots had a growing
point whose cells on the one side consisted of cells of
nightshade, on the other side of tomato. We know of
no case in which the cell of a graft has lost its specificity
and undergone a transformation into the cell of the host.

3. Another manifestation of the incompatibility
of distant species is found in the domain of fertiliza-
tion. The eggs of the majority of animals cannot
develop unless a spermatozoén enters. The entrance
of a spermatozo6n into an egg seems also to fall under
the law of specificity, inasmuch as in general only the
sperm of the same or a closely related species is able
to enter the egg. The writer? has found, however, that
it is possible to overcome the limitation of specificity
in certain cases by physicochemical means, and by the
knowledge of these means we may perhaps one day be
able to more closely define the mechanism of specificity
in this case. He found that the eggs of a certain Cali-
fornian sea urchin, which cannot be fertilized by the
sperm of starfish in normal sea water, will lose their
specificity towards this type of foreign sperm if the
sea water is rendered a little more alkaline, or if a little
more Ca is added to the sea water, or if both these
variations are effected. Godlewski has confirmed the
efficiency of this method for the fertilization of sea-
urchin eggs with the sperm of crinoids.

* Baur, E., Einfithrung in die experimentelle Vererbungslehre. Berlin,
IQII, p. 232.
2 Literature on this subject in Chapter IV.

Chemical Basis of Genus and Species 49

If such heterogeneous hybridizations are carried out,
two striking results are obtained. The one is that

Fic. 1. Five-days-old larve from a sea urchin (Strongy-
locentrotus purpuratus) 2 and a starfish (Asterias) o’.
(Front view.)

the resulting larva has only maternal characteristics
(Figs. 1 and 2), as if the sperm had contributed no he-

Fic. 2. Five-days-old larve of Strongylocentrotus purpur-
atus produced by artificial parthenogenesis. (Side view.)
The larve in Figs. 1 and 2 are identical in appearance,
proving that heterogeneous hybridization leads to alarva
with purely maternal characters.

reditary material to the developing embryo. This result

could not have been predicted, for if we fertilize the

egg of the same Californian sea urchin, Strongylocen-

trotus purpuratus, with the sperm of a very closely
4

50 Chemical Basis of Genus and Species

related sea urchin, S. franciscanus, the hereditary
effect of the spermatozodn is seen very distinctly in the
primitive skeleton formed by the larva.’ (Fig. 3.)
In the case of the heterogeneous hybridization the
spermatozoén acts practically only as an activating
agency upon the egg and not asa transmitter of paternal
qualities.

The second striking fact is that while the sea-urchin

Fic. 3. Five-days-old larve of two closely related forms of sea
urchins (S. purpuratus @ and S. franciscanus o’). In this
case the larva has also paternal characters as shown by the
skeleton.

eggs fertilized with starfish sperm develop at first
perfectly normally they begin to die in large numbers
on the second and third day of their development, and
only a very small number live long enough to form a
skeleton; and these are usually sickly and form the
skeleton considerably later than the pure breed. It is
not quite certain whether the sickliness of these hetero-
geneous hybrids begins or assumes a severe character

t Loeb, J., King, W. O. R., and Moore, A. R., Arch. f. Entwicklngs-
mech., 1910, XXix., 354.

Chemical Basis of Genus and Species 51

with the development of a certain type of wandering
cells, the mesenchyme cells; it would perhaps be worth
while to investigate this possibility. The writer was
under the impression that this sickliness might have
been brought about by a poison gradually formed in
the heterogeneous larve.

He investigated the effects of heterogeneous hybridi-
zation also in fishes, which are a much more favourable
object. The egg of the marine fish Fundulus hetero-
clitus can be fertilized with the sperm of almost any
other teleost fish, as Moenkhaus' first observed.
This author did not succeed in keeping the hybrids
alive more than a day, but the writer has kept many
heterogeneous hybrids alive for a month or longer,?
and found the same two striking facts which he had
already observed in the heterogeneous cross between
sea urchin and starfish: first, practically no transmis-
sion of paternal characters, and second, a sickly con-
dition of the embryo which begins early and which
increases with further development. The heterogene-
ous fish hybrids between, e. g., Fundulus heteroclitus 9
and Menidia @ have usually no circulation of blood,
although the heart is formed and beats and blood-
vessels and blood cells are formed; the eyes are often
incomplete or abnormal though they may be normal
at first; the growth of the embryo is mostly retarded.

t Moenkhaus, W. J., Am. Jour. Anat., 1904, iti., 29.
2 Loeb, J., Jour. Morphol., 1912, xxiii., I.

52 Chemical Basis of Genus and Species

In exceptional cases circulation may be established
and in these a normal embryo may result, but such an
embryo is chiefly maternal.

This incompatibility of two gametes from different
species does not show itself in the case of heterogeneous
hybridization only, but also though less often in the
case of crossing between two more closely related
forms. The cross between the two related forms S.
purpuratus 2 and S. franciscanus & is very sturdy and
shows no abnormal mortality as far as the writer’s ob-
servations go. If, however, the reciprocal crossing is
carried out, namely that of S. franciscanus ? and S.
purpuratus &', the development is at first normal, but
beginning with the time of mesenchyme formation
the majority of larve become sickly and die; and again
the question may be raised whether or not the begin-
ning of sickliness coincides with the development of
mesenchyme cells. If we assume that the sickliness
and death are due to the formation of a poison, we
must assume that the poison is formed by the proto-
plasm of the egg, since otherwise we could not under-
stand why the reciprocal cross should be healthy.

All of these data agree in this one point, that the
fusion by grafting or fertilization of two distant species
is impossible, although the mechanism of the incompat-
ibility is not yet understood. It is quite possible that
this mechanism is not the same in all the cases men-
tioned here, and that it may be different when two

Chemical Basis of Genus and Species 53

different species are mixed and when incompatibility
exists between varieties, as is the case in the graft on
mammals.

II. The Chemical Basis of Genus and Species and of
Species Specificity

4. Fifty or sixty years ago surgeons did not hesitate
to transfuse the blood of animals into human beings.
The practice was a failure, and Landois* showed by
experiment that if blood of a foreign species was intro-
duced into an animal the blood corpuscles of the trans-
fused blood were rapidly dissolved and the animal into
which the transfusion was made was rendered ill and
often died. The result was different when the animals
whose blood was used for the purpose of transfusion
belonged to the same species or a species closely related
to the animal into which the blood was transfused.
Thus when blood was exchanged between horse and
donkey or between wolf and dog or between hare and
rabbit no hemoglobin appeared in the urine and the
animal into which the blood was transfused remained
well.2. This was the beginning of the investigations
in the field of serum specificity which were destined to
play such a prominent réle in the development of medi-
cine. Friedenthal was able to show later that if to

Landois, L., Zur Lehre von der Bluttransfusion. Leipzig, 1875.
2 This is probably true only within the limits of exactness used in

these experiments.

54 Chemical Basis of Genus and Species

10 c.c, of serum of a mammal three drops of defibrinated
blood of a foreign species are added and the whole is
exposed in a test tube to a temperature of 38°C. for
fifteen minutes the blood cells contained in the added
blood are all cytolyzed; that this, however, does not
occur so rapidly when the blood of a related species is
used. He could thus show that human blood serum
dissolves the erythrocytes of the eel, the frog, pigeon,
hen, horse, cat, and even that of the lower monkeys
but not that of the anthropoid apes. The blood of the
chimpanzee and of the human are no longer incom-
patible, and this discovery was justly considered by
Friedenthal as a confirmation of the idea of the evolu-
tionists that the anthropoid apes and the human are
blood relations. *

This line of investigation had in the meanwhile
entered upon a new stage when Kraus, Tchistowitch,
and Bordet discovered and developed the precipitin re-
action, which consists in the fact that if a foreign serum
(or a foreign protein) is introduced into an animal the
blood serum of the latter acquired after some time
the power of causing a precipitate when mixed with
the antigen, 7. e., with the foreign substance originally
introduced into the animal for the purpose of causing
the production of antibodies in the latter; while, of
course, no such precipitation occurs if the serum of a

* Friedenthal, H., ‘Experimenteller Nachweis der Blutverwandt-
schaft.” Arch. f. Physiol., 1900, 494-

Chemical Basis of Genus and Species 55

non-treated rabbit is mixed with the serum of the blood
of the foreign species.

In 1897 Kraus discovered that if the filtrates from
cultures of bacteria (e. g., typhoid bacillus) are mixed
with the serum of an animal immunized with the same
serum (¢. g., typhoid serum) it causes a precipitate; and
that this precipitin reaction is specific. This fact
was confirmed and has been extended by the work
of many authors.

Tchistowitch in 1899 observed that the serum of
tabbits which had received injections of horse or eel
serum caused a precipitate when mixed with the serum
of these latter animals.

Bordet found in 1899 that if milk is injected into a
rabbit the serum of such a rabbit acquires the power
of precipitating casein, and Fish found that this reac-
tion is specific inasmuch as the lactoserum from cow’s
milk can precipitate only the casein of cow’s milk but
not that of human or goat milk. Wassermann and
Schiitze reached the same result independently of each
other.

Myers and later Uhlenhuth showed that if white of
egg from a hen’s egg is injected into a rabbit, precipitins
for white of egg are found in the serum of the latter,
and Uhlenhuth' found, by trying the white of egg of
different species of birds, that the precipitin reaction

®Uhlenhuth, P., and Steffenhagen, K., Kolle-Wassermann, Handb.
d. pathol. Mikroorg., 2nd Ld., 1913, iii., 257.

56 Chemical Basis of Genus and Species

called forth by the blood of the immunized animal is
specific, inasmuch as the proteins from a hen’s egg
will call forth the formation of precipitins in the blood
of the rabbit which will precipitate only the white of
egg of the hen or of closely related birds.

To Nuttallt belongs the credit of having worked out
a quantitative method for measuring the amount of
precipitate formed, and in this way he made it possible
to draw more valid conclusions concerning the degree
of specificity of the precipitin reaction. He found
by this method that when the immune serum is
mixed with the serum or the protein solution used for
the immunization a maximum precipitate is formed,
but if it is mixed with the serum of related forms a
quantitatively smaller precipitate is produced. In
this way the degree of blood relationship could be
ascertained. He thus was able to show that when the
blood of one species, e. g., the human, was injected into
the blood of a rabbit, after some time the serum of the
rabbit was able to cause a precipitate not only with the
serum of man, or chimpanzee, but also of some lower
monkeys; with this difference, however, that the pre-
cipitate was much heavier when the immune serum was
added to the serum of man. The method thus shows
the existence of not an absolute but of a strong quanti-
tative specificity of blood serum. This statement may

* Nuttall, George H. F., Blood Immunity and Blood Relationship,
Cambridge Univ. Press, 1904.

Chemical Basis of Genus and Species 57

be illustrated by the following table from Nuttall.
The antiserum used for the precipitin reaction was
obtained by treating a rabbit with human blood serum.
The forty-five bloods tested had been preserved for
various lengths of time in the refrigerator with the
addition of a small amount of chloroform.

TABLE II

QUANTITATIVE TESTS wITH ANTI-PRIMATE SERA

Tests with Antthuman Serum

Precipitum

Bioop oF niet Percentage
Primates
Mian: i’. cue einen aakneies swe -031 100
Chimpanzee............... 04 130 (loose precipitum)
Gorilla..... Es ssitegeche ina tcareina 021 64
QUTANE. fo oisc dei nas susie ne O13 42
Cynocephalus mormon...... O13 42
Cynocephalus sphinx....... .009 29
Ateles geoffroyi............ .009 29
Insectivora
Centetes ecaudatus......... 0 fe)
Carnivora
Canis auretis.............. 003 10 (loose precipitum)
Canis familiaris............ -OOI 3
Dutra; vilgans) sja05.6 gies cane .003, 10 (concentrated serum)
Ursus tibetanus............ 0025 8
Genetta tigrina............ -OOL 3
Felis domesticus........... .OOI 3
Felis caracal............... 0008 3
Felis tigris......... 000-0 0005 2
Ungulata
Kk cars Ge ada wel aniogants .003 10
Sheepie ine sqeeatiow ve sda news 003 10
Cobus unctuosus........... .002 7
Cervus porcinus..........- 002 7
Rangifer tarandus.......... .002 7
Capra negaceros........... 0005 2
Equus caballus............ .0005 2
Sus scrofa.c.as.saess sero xe) to)

58 Chemical Basis of Genus and Species

Precipitum

BLoop oF ‘Amount Percentage
Rodentia
Dasyprocta cristata........ .002 7 (concentrated serum
clots)
Guinea-pig...........0.005- Ke) o
Rabbity ga aac ve cwew es xtems Re) o
Marsupialia

Petrogale xanthopus......
Petrogale penicillata......
Onychogale frenata.......
Onychogale unguifera..... 0 o
Onychogale unguifera.....
Macropus bennetti.......
Thylacinus cynocephalus. .

Among the Primate bloods that of the Chimpanzee
gave too high a figure, owing to the precipitum being floc-
culent and not settling well, for some reason which could
not be determined. The figure given by the Ourang is
somewhat too low, and the difference between Cynocepha-
lus sphinx and Ateles is not as marked as might have
been expected in view of the qualitative tests and the series
following. The possibilities of error must be taken into
account in judging of these figures; repeated tests should be
made to obtain something like a constant. Other bloods
than those of Primates give small reactions or no reactions
at all. The high figures (10%) obtained with two Car-
nivore bloods can be explained by the fact that one gave
a loose precipitum, and the other was a somewhat concen-
trated serum."

We have mentioned that even the proteins of the
egg are specific according to Uhlenhuth. Graham
Smith, one of Nuttall’s collaborators, applied the lat-

* Nuttall, Blood Immunity and Blood Relationship, pp. 319 and 320.

Chemical Basis of Genus and Species 59

ter’s quantitative method to this problem and confirmed

the results of Nuttall.
an illustration.

A few examples may serve as

TABLE III

Tests wita AntI-Duck’s-Ecc SERUM

Material tested peeietad Percentage
Duck's egg-albumin.............. 0384, 100
Pheasant’s cr ee ee .0328 85
Fowl’s mS “aiathya eevee 0234 61
Silver Pheasant’s Sh Ses eae atte a ancuues -0140 36
Blackbird’s Bid Achy anata eta conteatate .0065 15
Crane’s er re ee 0051 14
Moorhen’s a ee .0046 12
Thrush’s Sh Sede mae ye aemes £0046 12
Emu's Oo canna sara es 0018 5
Hedge-Sparrow’s rere trace ?
Chaffinch’s Nh (SGU Maem taal ey °
Tortoise serum hae eee cece trace i
Turtleserum ka eee eee ee ?
Alligator serum °

Frog, Amphiuma, and Dogfish sera, as well as Tortoise and
Dogfish egg-albumins, were also tested, with negative results.

TABLE IV
Tests wItH ANTI-FowL’s-Ecc SERUM
Material tested pelea Percentage
Fowl’s egg-albumin (old)......... O159 100
Fowl’s ee (fresh)........ 0140 88
Silver Pheasant’s - 0075 47
Pheasant’s o 0075 47
Crane’s - 0046 29
Blackbird’s ne 0046 29
Duck’s “ 0037 23
Moorhen’s i 0028 18

60 Chemical Basis of Genus and Species

Thrush, Emu, Greenfinch, and Hedge-sparrow egg-albu-
mins were tested and gave traces of precipita, as also did
Tortoise and Turtle sera. The egg-albumins of the Tor-
toise, Frog, Skate, and two species of Dogfish did not react.
Alligator, Frog, Amphiuma, and Dogfish sera also yielded
no results.*

By improving the quantitative method in various
ways, Welsh and Chapman? were able to explain why
the precipitin reaction with egg-white was not strictly
specific but gave also, though quantitatively weaker,
results with the egg-white of related birds. They
found that by a new method devised by them ‘“‘it is
possible to indicate in an avian egg-white antiserum
the presence of a general avian antisubstance (pre-
cipitin) together with the specific antisubstance.”’

The Bordet reaction was not only useful in indicating
the specificity and blood relationship for animals but
also among plants. Thus Magnus and Friedenthal;
were able to demonstrate with Bordet’s method the
relationship between yeast (Saccharomyces cerevisi@)
and truffle (Tuber brumale).

5. We must not forget, while under the spell of
the problem of immunity, that we are interested at
the moment in the question of the nature of the speci-
ficity of living organisms. It is only logical to conclude

t Nuttall, pp. 345 and 346.

? Welsh, D. A., and Chapman, H. G., Jour. Hygiene, 1910, x., 177.

3 Magnus, W., and Friedenthal, H., Ber. d. deutsch. bot. Gesellsch.,
1906, xxiv., 601.

Chemical Basis of Genus and Species 61

that the fossil forms of invertebrate animals and of algze
and bacteria, which Walcott found in the Cambrian and
which may be two hundred million years old, must
have had the same specificity at that time as they or
their close relatives have today; and this raises the
question: What is the nature of the substances which
are responsible for and transmit this specificity? It is
obvious that a definite answer to this question brings
us also to the very problem of evolution as well as that
of the constitution of living matter.

There can be no doubt that on the basis of our present
knowledge proteins are in most or practically all cases
the bearers of this specificity. This has been found
out not only with the aid of the precipitin reaction but
also with the anaphylaxis reaction, by which, as the
reader may know, is meant that when a small dose of
a foreign substance is introduced into an animal a
hypersensitiveness develops after a number of days
or weeks, so that a new injection of the same substance
produces serious and in some cases fatal effects. This
hypersensitiveness, which was first analysed by Richet,*
is specific for the substance which has been injected.
Now all these specific reactions, the precipitin reaction
as well as the anaphylactic reaction, can be called forth
by proteins. Thus Richet, in his earliest experiments,
showed that only the protein-containing part of the
extract of actinians, by which he called forth anaphy-

tRichet, C., L'anaphylaxie. Paris, 1912.

62 Chemical Basis of Genus and Species

laxis, was able to produce this phenomenon, and later
he showed that it was generally impossible to produce
anything resembling anaphylaxis by non-protein sub-
stances, ¢. g., cocain or apomorphin.* Wells isolated
from egg-white four different proteins (three coagulable
proteins and one non-coagulable) which can be distin-
guished from each other by the anaphylaxis reaction,
although all come from the same biological object.?
Michaelis as well as Wells found that the split products
of the protein molecule are no longer able to call forth
the anaphylaxis reaction. Since peptic digestion has
the effect of annihilating the power of proteins to call
forth anaphylaxis, we are forced to the conclusion that
the first cleavage products of proteins have already
lost the power of calling forth immunity reactions.

A pretty experiment by Gay and Robertson? should
be mentioned in this connection. Robertson had shown

that a substance closely resembling paranucleins both in
its properties and its C, H, and N content can be formed
from the filtered products of the complete peptic hydro-
lysis of an approximately four per cent. neutral solution
of potassium caseinate by the action of pure pepsin at
36°C.

He considered this a case of a real synthesis of proteins
from the products of its hydrolytic cleavage. This

* Quoted from Wells, H. G., Jour. Infect. Diseases, 1908, v., 449.
2 Ibid., 1911, ix., 147.
3 Gay, F. P., and Robertson, T. B., Jour. Biol. Chem., 1912, xii., 233.

Chemical Basis of Genus and Species 63

interpretation was not generally accepted and received
a different interpretation by Bayliss and other workers.
Gay and Robertson were able to show that paranuclein
when injected into an animal will sensitize guinea-pigs
for anaphylactic intoxication for either paranuclein
or casein and apparently indiscriminately. The pro-
ducts of complete peptic digestion of casein had no
such effect, but the synthetic product of this diges-
tion obtained by Robertson’s method has the same
specific antigenic properties as paranuclein, thus
making it appear that Robertson had indeed suc-
ceeded in causing a synthesis of paranuclein with the
aid of pepsin from the products of digestion of casein
by pepsin.

There are a few statements in the literature to the
effect that the specificity of organisms might be due
to other substances than proteins. Thus Bang and
Forssmann claimed that the substances (antigens)
responsible for the production of hemolysis were of a
lipoid nature, but their statements have not been con-
firmed, and Fitzgerald and Leathes' reached the con-
clusion that lipoids are non-antigenic. Ford claims
to have obtained proof that a glucoside contained in
the poisonous mushroom Amanita phalloides can act
as an antigen. But aside from this one fact we know
that proteins and only proteins can act as antigens and

t Fitzgerald, J. G., and Leathes, J. B., Univ. Cal. Pub., 1912, ‘ Patho-
logy,” ii, 39.

64 Chemical Basis of Genus and Species

are therefore the bearers of the specificity of living
organisms.

Bradley and Sansum' found that guinea-pigs sensi-
tized to beef or dog hemoglobin fail to react or react but
slightly to hemoglobin of other origin. The hemoglo-
bins tried were dog, beef, cat, rabbit, rat, turtle,
pig, horse, calf, goat, sheep, pigeon, chicken, and
man.

6. It would be of the greatest importance to show
directly that the homologous proteins of different
species are different. This has been done for hemo-
globins of the blood by Reichert and Brown,* who have
shown by crystallographic measurements that the
hemoglobins of any species are definite substances for
that species.

The crystals obtained from different species of a genus
are characteristic of that species, but differ from those of
other species of the genus in angles or axial ratio, in optical
characters, and especially in those characters comprised
under the general term of crystal habit, so that one species
can usually be distinguished from another by its hemoglobin
crystals. But these differences are not such as to preclude
the crystals from all species of a genus being placed in an
isomorphous series (p. 327).

t Bradley, H. C., and Sansum, W. D., Jour. Biol. Chem., 1914, xviii.,
497.

2 Reichert, E. T., and Brown, A. P., ‘‘ The Differentiation and Speci-
ficity of Corresponding Proteins and other Vital Substances in Relation
to Biological Classification and Organic Evolution.” Carnegie Insti-
tution Publication No. 116, Washington, 1909.

Chemical Basis of Genus and Species 65

As far as the genus is concerned it was found that
the hemoglobin crystals of any genus are isomorphous.

In some cases this isomorphism may be extended to
include several genera, but this is not usually the case,
unless as in the case of dogs and foxes, for example, the
genera are very closely related.

The most important question for us is the following:
Are the differences between the corresponding hemo-
globin crystals of different species of the same genus
such as to warrant the statement that they indicate
chemical differences? If this were the case we might
say that blood reactions as well as hemoglobin crystals
indicate that differences in the constitution of proteins
determine the species specificity and, perhaps, also
species heredity. The following sentences by Reichert
and Brown seem to indicate that this may be true for
the crystals of hemoglobin.

The hemoglobins of any species are definite substances
for that species. But upon comparing the corresponding
substances (hemoglobins) in different species of a genus it is
generally found that they differ the one from the other to a
greater or less degree; the differences being such that when
complete crystallographic data are available the different
species can be distinguished by these differences in their
hemoglobins. As the hemoglobins crystallize in isomor-
phous series the differences between the angles of the
crystals of the species of a genus are not, as a rule, great;
but they are as great as is usually found to be the case with

66 Chemical Basis of Genus and Species

minerals or chemical salts that belong to an isomorphous
group (p. 326).

As Professor Brown writes me, the difficulty in
answering the question definitely, whether or not the
hemoglobins of different species are chemically different,
lies in the fact that there is as yet no criterion which:
allows us to discriminate between a species and a Men-
delian mutation except the morphological differences.
It is not impossible that while species differ by the con-
stitution of some or most of their proteins, Mendelian
heredity has a different chemical basis.

It is regrettable that work like that of Reichert and
Brown cannot be extended to other proteins, but it
seems from anaphylaxis reactions that we might expect
results similar to those in the case of the hemoglobins.
The proteins of the lens are an exception inasmuch as,
according to Uhlenhuth, the proteins of the lens of
mammals, birds, and amphibians cannot be discrimi-
nated from each other by the precipitin reaction.*

7. The serum of certain humans may cause the
destruction or agglutination of blood corpuscles of
certain other humans. This fact of the existence of
““soagglutinins” seems to have been established for
man, but Hektoen states that he has not been able to
find any isoagglutinins in the serum of rabbits, guinea-
pigs, dogs, horses, and cattle. Landsteiner found the

tUhlenhuth, Das biologische Verfahren zur Erkennung und Unter-
Scheidung von Menschen und Tierblut, Jena, 1905, p. 102.

Chemical Basis of Genus and Species 67

remarkable fact that the sera of certain individuals of
humans could hemolyze the corpuscles of certain other
individuals, but not those of all individuals. A system-
atic investigation of this variability led him to the
discovery of three distinct ‘groups of individuals, the
sera of each group acting in a definite way towards
the corpuscles of the representatives of each other
group. Later observers, for example Jansky and Moss,
established four groups. These groups are, according
to Moss,* as follows:

Group 1. Sera agglutinate no corpuscles.
Corpuscles agglutinated by sera of Groups 2, 3, 4.

Group 2. Sera agglutinate corpuscles of Groups 1, 3.
Corpuscles agglutinated by sera of Groups 3, 4.

Group 3. Sera agglutinate corpuscles of Groups I, 2.
Corpuscles agglutinated by sera of Groups 2, 4.

Group 4. Sera agglutinate corpuscles of Groups 1, 2, 3.
Corpuscles agglutinated by no serum.

The relative frequency of the four groups follows
from the following figures. Of one hundred bloods
tested by Moss in series of twenty there were found:

10 belonging to Group I.
40 belonging to Group 2.

7 belonging to Group 3.
43 belonging to Group 4.

Groups 2 and 4 are in the majority and in over-
whelming numbers, which indicates that, as a rule, the

1 Moss, W. L., Johns Hopkins Hospital Bulletin, 1910, xxi., 62.

68 Chemical Basis of Genus and Species

sera agglutinate the blood corpuscles of individuals of
the other groups, but not those of individuals belong-
ing to the same group. The phenomenon that a serum
agglutinates no corpuscles (Group 1), or that the cor-
puscles are agglutinated by no serum (Group 4), are
the exceptions. It is obvious that, as far as our problem
is concerned, only Groups 2 and 3 are to be considered.
There is no Mendelian character which refers only to
one half of the individuals except sex. Since nothing
is said about a relation of Groups 2 and 3 to sex such
a relation probably does not exist.

8. The facts thus far reported imply the suggestion
that the heredity of the genus is determined by proteins
of a definite constitution differing from the proteins of
other genera. This constitution of the proteins would
therefore be responsible for the genus heredity. The
different species of a genus have all the same genus
proteins, but the proteins of each species of the same
genus are apparently different again in chemical con-
stitution and hence may give rise to the specific bio-
logical or immunity reactions.

We may consider it as established by the work of
McClung, Sutton, E. B. Wilson, Miss Stevens, Morgan,
and many others, that the chromosomes are the carriers
of the Mendelian characters. These chromosomes
occur in the nucleus of the egg and in the head of the
sperm. Now the latter consists, in certain fish, of
lipoids and a combination of nucleinic acid and pro-

Chemical Basis of Genus and Species 69

tamine or histone, the latter a non-coagulable protein,
more resembling a split product of one of the larger
coagulable proteins.

A. E. Taylort found that if the spermatozca of the
salmon are injected intoa rabbit, the blood of the animal
acquires the power of causing cytolysis of salmon sper-
matozoa. When, however, the isolated protamines
or nucleinic acid or the lipoids prepared from the
same sperm were injected into a rabbit no results of this
kind were observed. H. G. Wells more recently tested
the relative efficiency of the constituents of the testes
of the cod (which in addition to the constituents of
the sperm contained the proteins of the testicle).
From the testicle he prepared a histone (the protein
body of the sperm nucleus), a sodium nucleinate,
and from the sperm-free aqueous extract: of the testi-
cles a protein resembling albumin was separated by
precipitation.?

The albumin behaved like ordinary serum albumin or
egg albumin, producing typical and fatal anaphylactic re-
actions and being specific when tried against mammalian
sera. The nucleinate did not produce any reactions when
guinea-pigs were given small sensitizing and larger intoxicat-
ing doses (0.1 gm.) in a three weeks’ interval; a result to
be expected, since no protein is present in the preparation.
The histone was so toxic that its anaphylactic properties
could not be studied.

x Taylor, A. E., Jour. Biol. Chem., 1908, v., 311.
a Wells, H. G., Jour. Infect. Diseases, 1911, ix., 166.

70 Chemical Basis of Genus and Species

It is not impossible that protamines and histones
might be found to act as specific antigens if they
were not so toxic. The positive results which Taylor
observed after injection of the sperm might have
been due to the proteins contained in the tail of the
spermatozoa, which in certain animals at least does
not enter the egg and hence can have no influence on
heredity.

It is thus doubtful whether or not any of the con-
stituents of the nucleus contribute to the determination
of the species. This in its ultimate consequences
might lead to the idea that the Mendelian characters
which are equally transmitted by egg and spermatozo6n,
determine the individual or variety heredity, but not
the genus or species heredity. It is, in our present
state of knowledge, impossible to cause a spermatozo6én
to develop into an embryo,’ while we can induce the
egg to develop into an embryo without a spermatozoén.
This may mean that the protoplasm of the egg is the
future embryo, while the chromosomes of both egg and
sperm nuclei furnish only the individual characters.

t Loeb, J., and Bancroft, F. W., Jour. Exper. Zodél., 1912, xii., 381.

CHAPTER IV
SPECIFICITY IN FERTILIZATION

1. We have become acquainted with two character-
istics of living matter: the specificity due to the specific
proteins characteristic for each genus and possibly
species and the synthesis of living matter from the
split products of their main constituents instead of
from a supersaturated solution of their own substance,
as is the case in crystals. We are about to discuss in
this and the next chapter a third characteristic, name‘y,
the phenomenon of fertilization. While this is not
found in all organisms it is found in an overwhelming
majority and especially the higher organisms, and of
all the mysteries of animated nature that of fertiliza-
tion and sex seems to be the most captivating, to judge
from the space it occupies in folklore, theology, and
“literature.”’ Bacteria, when furnished the proper
nutritive medium, will synthetize the specific material
of their own body, will grow and divide, and this process
will be repeated indefinitely as long as the food lasts

and the temperature and other outside conditions are
71

72 Specificity in Fertilization

normal. It is purely due to limitation of food that
bacteria or certain species of them do not cover the
whole planet. But, as every layman knows, the major-
ity of organisms grow only to a certain size, then die,
and the propagation takes place through sex cells or
gametes: a female cell—the egg—containing a large
bulk of protoplasm (the future embryo) and reserve
material; and the male cell which in the case of the
spermatozo6n contains only nuclear material and no
cytoplasmic material except that contained in the tail
which in some and possibly many species does not enter
the egg. The male element—the spermatozoé6n—enters
the female gamete—the egg—and this starts the de-
velopment. In thecase of most animals the egg cannot
develop unless the spermatozo6n enters. The question
arises: How does the spermatozo6n activate the egg?
And also how does it happen that the spermatozoén
enters the egg? We will first consider the latter ques-
tion. These problems can be answered best from ex-
periments on forms in which the egg and the sperm are
fertilized in sea water. Many marine animals, from
fishes down to lower forms, shed their eggs and sperm
into the sea water where the fertilization of the egg
takes place, outside the body of the female.

The first phenomenon which strikes us in this con-
nection is again a phenomenon of specificity. The sper-
matozo6n can, as a rule, only enter an egg of the same
or a closely related species, but not that of one more

Specificity in Fertilization 73

distantly related. What is the character of this speci-
ficity? The writer was under the impression that a
clue might be obtained if artificial means could be
found by which the egg of one species might be fertil-
ized with a distant species for which this egg is natu-
rally immune. Such an experiment would mean that
the lack of specificity had been compensated by the
artificial means. It is well known that the egg of the
sea urchin cannot as a rule be fertilized with the sperm
of a starfish in normal sea water. The writer tried
whether this hybridization could not be accomplished
provided the constitution of the sea water were changed.
He succeeded in causing the fertilization of a large
percentage of the eggs of the Californian sea urchin,
Strongylocentrotus purpuratus, with the sperm of various
starfish (e. g., Asterias ochracea) and Holothurians by
slightly raising the alkalinity of the sea water, through
the addition of some base (NaOH or tetraethylammo-
niumhydroxide or various amines), the optimum being
reached when 0.6 c.c. N/10 NaOH is added to 50 c.c.
of sea water. It is a peculiar fact that this solution is
efficient only if both egg and sperm are together in the
hyperalkaline sea water. If the eggs and sperm are
treated separately with the hyperalkaline sea water and
are then brought together in normal sea water no fer-
tilization takes place as a rule, while with the same
sperm and eggs the fertilization is successful again if
both are mixed in the hyperaikaline solution. From

74 Specificity in Fertilization

this the writer concluded that the fertilizing power
depends on a rapidly reversible action of the alkali on
the surface of the two gametes. It was found that
an increase of the concentration of calcium in the sea
water also favoured the entrance of the Asterizas sperm
into the egg of purpuratus; and that if CCa was in-
creased it was not necessary to add as much NaOH.

The spermatozo6n enters the egg through the so-
called fertilization cone, 7. e., a protoplasmic process
comparable to the pseudopodium of an amceboid cell.
The analogy of the process of phagocytosis—i. e., the
taking up of particles by an amceboid cell—and that of
the engulfing of the spermatozoén by the egg presents
itself. We do not know definitely the nature of the
forces which act in the case of phagocytosis—although
surface tension forces and agglutination have been
suggested; both are surface phenomena and are rapidly
reversible.

We should then say that the specificity in the process
of fertilization consists in a peculiarity of the surface
of the egg and spermatozoén which in the case of S.
purpuratus @ and Asterias & can be supplied by a
slight increase in the COH or Ca.

By this method fifty per cent. or more of the eggs of
purpuratus could be fertilized with the sperm of the
starfish Asterias ochracea, capitata, Ophiurians, and
Holothurians, while with the sperm of another starfish,
Pycnopodia spuria, only five per cent., and with the

Specificity in Fertilization 75

sperm of A sterina only one per cent. could be fertilized.*
Godlewski succeeded by the same method in fertilizing
the eggs of a Naples starfish with the sperm of a crinoid.?
The writer did not succeed in bringing about the ferti-
lization of the egg of another sea urchin in California,
Strongylocentrotus franciscanus, with the sperm of a
starfish. Although these eggs formed a membrane
in contact with the sperm, the latter did not enter the
egg; nor has the writer as yet succeeded in causing the
sperm of Asterias to enter the egg of Arbacia.

Kupelwieser? observed that the spermatozoon of
molluscs may occasionally enter into the egg of S.
purpuratus in normal sea water and later, at Naples,
he observed the same for the sperm of annelids. In
these cases no development took place. In teleost
fishes the spermatozoén can enter the eggs of widely
different species but with rare exceptions all the embryos
will die in an early stage of development.‘

2. The fact that an increase in the alkalinity or in
the concentration of calcium allowed foreign sperm to
enter the egg of the sea urchin, suggested the idea that
a diminution of alkalinity or calcium in the sea water

* Loeb, J., Arch. f. d. ges. Physiol., 1903, xcix., 323; 1904, civ., 325;
Arch. f. Entwckingsmech., 1910, xxx., II., 44; 1914, xl., 310; Science,
1914, xl., 316.

2 Godlewski, E., Arch. f. Entwcklngsmech., 1906, xX., 579.

3 Kupelwieser, H., Arch. f. Entwekingsmech., 1909, xxvii., 434; Arch.
f. Zellforsch., 1912, Viii., 352.

4 See Chapter IT.

76 Specificity in Fertilization

might block the entrance of the sperm of sea urchin into
eggs of their own species. This was found to be cor-
rect; when we put eggs and sperm of the same species
of sea urchin into solutions whose concentration of Ca
or of OH is too small, the sperm, although it may be
intensely active, cannot enter the egg.

For the purpose of these experiments the ovaries
and testes of the sea urchins were not put into sea
water, but instead into pure m/2NaCl and after several
washings in this solution were kept in it (they remain
alive for several days in pure m/2 NaCl). Several
drops of such sperm and one drop of eggs were in one
series of experiments put into 2.5.c.c. of a neutral mix-
ture of m/2 NaCl and 3/8 m MgCl, in the proportion in
which these two salts exist in the sea water. In such
a neutral solution eggs of Arbacia or purpuratus are
not fertilized no matter how long they remain in it,
although the spermatozoa swim around the eggs very
actively. That no spermatozoén enters the eggs can
be shown by the fact that the eggs do not divide (al-
though they can segment in such a solution if previously
fertilized in sea water or some other efficient solution).
When, however, eggs and sperm are put into 2.5 c.c.
of the same solution of NaCl+MgCl., containing in
addition one drop of a N/100 solution of NaOH (or NH,
or benzylamine or butylamine) or eight drops of m/100
NaHCO,, most, and often practically all of the eggs
at once form fertilization membranes and segment at

Specificity in Fertilization res

the proper time, indicating that fertilization has been
accomplished. The same result can be obtained if the
eggs are transferred into a neutral mixture of NaCl+
MgCl,+CaCl, (in the proportion in which these salts
exist in the sea water) or into a neutral mixture of
NaCl+MgCl,+KCl+CaCl,. In such neutral mix-
tures the eggs form fertilization membranes and begin
to segment. The eggs are not fertilized in a neutral
solution of NaCl or of NaCl+KCL.?*

It is, therefore, obvious that if we diminish the al-
kalinity of the solution surrounding the egg and deprive
this solution of CaCl, we establish the same block to the
entrance of the spermatozoén of Arbacia into the egg
of the same species as exists in normal sea water for
the entrance of the sperm of the starfish into the egg of
purpuratus.

The “block” created in this way, to the entrance of
the sperm of Arbacia into the egg of the same species
is also rapidly reversible.

We reach the conclusion, therefore, that the speci-
ficity which allows the sperm to enter an egg is a sur-
face effect which can be increased or diminished by an
increase or diminution in the concentration of OH as
well as of Ca. The writer has shown that an increase
in the concentration of both substances may cause an
agglutination of the spermatozoa of starfish to the

t Loeb, J., Sctence, 1914, xl., 316; Am. Naturalist, 1915, xlix.,
257.

78 Specificity in Fertilization

jelly which surrounds the egg of purpuratus.* It is
thus not impossible that the specificity which favours
the entrance of a spermatozoén into an egg of its own
species may consist in an agglutination between sper-
matozo6on and egg protoplasm (or its fertilization cone);
and that this agglutination is favoured if the Con or
Cca or both are increased: within certain limits.

Godlewski discovered a very interesting form of block
to the entrance of the spermatozo6én into the egg which
takes place if two different types of sperm are mixed.
He had found that the sperm of the annelid Chetopterus
is able to enter the egg of the sea urchin and that in
so doing it causes membrane formation. The egg,
however, does not develop but dies rapidly, as is the
case when we induce artificial membrane formation, as
we shall see in the next chapter.

Godlewski found that if the sperm of Chetopterus and
the sperm of sea urchins are mixed the mixture is not
able to induce development or membrane formation,
since now neither spermatozo6én can enter; blood has the
same inhibiting effect as the foreign sperm. The mix-
ture does not interfere with the development of the
eggs if they are previously fertilized.?

The phenomenon was further investigated by Her-
lant’ who found that if the sperm of a sea urchin is

‘Loeb, Arch. f. Entwckingsmech., 1914, xl., 310.
2 Godlewski, E., Arch. f. Entwcklngsmech., 1911, xxxiii., 196.
3 Herlant, M., Anat. Anzeiger, 1912, xlii., 563.

Specificity in Fertilization 79

mixed with the sperm of certain annelids (Chetopterus)
or molluscs, and if after some time the eggs of the
sea urchin are added to the mixture of the two kinds
of sperm no egg is fertilized. If, however, the solution
is subsequently diluted with sea water or if the egg
that was in this mixture is washed in sea water, the
same sperm mixture in which the egg previously re-
mained unfertilized will now fertilize the egg. From
these and similar observations Herlant draws the con-
clusion that the block existed at the surface of the egg,
inasmuch as a reaction product of the two types of
sperm is formed after some time which alters the sur-
face of the egg and thereby prevents the sperm from
entering. This view is supported not only by all the
experiments but also by the observation of the writer
that foreign sperm or blood is able to cause a real agglu-
tination after some time if mixed with the sperm of a
sea urchin or a starfish. We can imagine that the
precipitate forms a film around the egg and acts as a
block for. the agglutination between egg and spermato-
zoon. The block can be removed mechanically by
washing.

3. The fact has been mentioned that the most
motile sperm will not be able to enter into the egg if
certain other conditions (specificity or CoH or Cca)
are not fulfilled. On the other hand, living but immo-
bile sperm cannot enter the egg under any conditions.

«Loeb, J., Jour. Exper. Zoél., 1914, xvii., 123.

80 Specificity in Fertilization

If we add a trace of KCN to the sperm of Arbacia so
that the spermatozoén becomes immobile no egg is
fertilized even if the eggs and the sperm are thoroughly
shaken together; while the same spermatozoa will
fertilize these eggs as soon as the HCN has evaporated
and they again become motile. It was formerly
thought that the spermatozo6n had to bore itself into
the egg, being propelled by the movements of the
flagellum. It is, however, more probable that only
a certain energy of vibration is needed on the part of
the spermatozo6n to make the latter stick to the surface
of the egg and agglutinate and that later forces of a
different character bring the spermatozodn into the
egg. The fact that under normal conditions a very
slight degree of motility on the part of the spermatozo6n
allows it to enter the egg of its own species seems to
favour such a view.

It is a common experience that spermatozoa become
very active when they reach the neighbourhood of an
egg. v. Dungern assumed that only foreign sperm
became thus active, but F. R. Lilliet has pointed out
that this may be a specific effect. The writer tested
this idea on the sperm and eggs of two species of star-
fish and of sea urchins. It should be mentioned that
the eggs of the starfish used in this experiment were
completely immature and could not be fertilized, while
the eggs of the sea urchins were mature. The testicles

* Lillie, F. R., Jour. Exper. Zodl., 1914, xvi., 523.

Specificity in Fertilization

and ovaries had been kept in NaCl and all the sperm

was immotile.

Eggs and sperm were mixed together

in a pure m/2 NaCl solution where the sperm was

The spermatozoa of starfish show a marked speci-

ficity inasmuch as they are strongly activated only by
the eggs of their own species, although in this experi-
ment these were immature, and to a slight degree only
by the eggs of the sea urchin purpuratus. But it is
also obvious that the specificity is far from exclusive
since the immature eggs of Asterina activate the sperm
of the sea urchin franciscanus as powerfully as is done

tLoeb, J., Am. Naturalist, 1915, xlix., 257.
6

82 Specificity in Fertilization

by the mature eggs of the sea urchin purpuratus and
franciscanus. In studying these results the reader must
keep in mind first that all these experiments were made
in a NaCl solution and second that it requires a stronger
influence to activate the spermatozoa of the starfish,
which are not motile at first even in sea water, than
the sea urchin spermatozoa which are from the first
very active in such sea water, and which may there-
fore be considered as being at the threshold of activity
in pure NaCl solution.

Wasteneys and the writer (in experiments not yet
published) did not succeed in demonstrating an activat-
ing effect of the eggs of various marine teleosts upon
sperm of the same species.

4. F.R. Lillie? has studied the very striking phe-
nomenon of transitory sperm agglutination which
takes place when the sperm of a sea urchin or of
certain annelids is put into the supernatant sea water
of eggs of the same species. If we put one or more
drops of a very thick sperm suspension of the Cali-
fornian sea urchin S. purpuratus carefully into the
centre of a dish containing 3 c.c. of ordinary sea water
and let the drop stand for one-half to one minute and
then by gentle agitation mix the sperm with the sea
water the mass of thick sperm which is at first rather
viscous is distributed equally in sea water in a few

t Lillie, F. R., Science, 1913, xxxviii., 524; Jour. Exper. Zoél., 1914,
xvi., 523; Biol. Bull., 1915, xxviii., 18.

Specificity in Fertilization 83

seconds and the result is a homogeneous sperm sus-
pension. When, however, the same experiment is
made with the sea water which has been standing for
a short time over a large mass of eggs of the same spe-
cies, the thick drop of sperm seems to be less miscible
and instead of a homogeneous suspension we get, as a
result, the formation of a large number of ‘distinct
clusters which are visible to the naked eye and which
may possess a diameter of 1or2mm. The rest of the
sea water is almost free from sperm. These clusters
of spermatozoa may last for from two to ten minutes
and then dissolve by the gradual detachment of the
spermatozoa from the periphery of the cluster.

This phenomenon seems to occur in sea urchins and
annelids. The writer has vainly looked for it in differ-
ent forms of the Californian starfish or molluscs and
in fish at Woods Hole. Lillie failed to find it in the
starfish at Woods Hole.

The writer found that the sperm of the Californian
sea urchin Strongylocentrotus purpuratus will form clus-
ters with the egg sea water of purpuratus but not with
that of franciscanus; while the sperm of franciscanus will
agglutinate with the egg sea water of both species,
but the clusters last a little longer with the eggs of
its own species.

He also found that the clusters are more durable in
a neutral than in a slightly alkaline solution and that
the agglutination disappears the more rapidly the

84 Specificity in Fertilization

more alkaline the solution. The presence of bivalent
cations, especially Ca, also favours the agglutination.

It was also found that this agglutination occurs
only when the spermatozoa are very motile; thus if a
trace of KCN is added to a mass of thick sea-urchin
sperm so that the spermatozoa become immotile a
drop of this sperm will not agglutinate when put in egg
sea water of the same species; while later, after the
HCN has evaporated, the same sperm will agglutinate
when put into such sea water.

The writer suggests the following explanation of
the phenomenon. The egg sea water contains a sub-
stance which forms a precipitate with a substance on
the surface of the spermatozoén whereby the latter
becomes slightly sticky. This precipitate is slowly
soluble in sea water and the more rapidly the more
alkaline (within certain limits). Only when the
spermatozoa run against each other with a certain im-
pact will they stick together, as Lillie suggested. Lillie
assumes that this agglutinating substance contained
in egg sea water is required to bring about fertilization
and he therefore calls it ‘‘fertilizin.
tion seems to go beyond the facts inasmuch as the
existence of such an agglutinating substance can only

But this assump-

be proved in a few species of animals (sea urchins and

annelids); and as, moreover, sea-urchin sperm can

fertilize eggs which will not cause the sperm to agglu-
t Lillie, F. R., Joc. cit.

Specificity in Fertilization 85

tinate, e. g., the egg of franciscanus can be fertilized
by sperm of purpuratus, although the egg sea water
of franciscanus causes no agglutination of the sperm of
purpuratus. When the jelly surrounding the egg of
the Californian sea urchin S. purpuratus is dissolved
with acid and the eggs are washed, the eggs will not
cause any more sperm agglutination; and yet one hun-
dred per cent. of such eggs can be fertilized by sperm.?

5. It is well known that if an egg is once fertilized
it becomes impermeable for other spermatozoa. This
cannot well be due to the fact that the egg develops;
for the writer found some time ago that eggs of Stron-
gylocentrotus purpuratus which are induced to develop
by means of artificial parthenogenesis can be fertilized
by sperm. The following observation leaves no doubts
in this respect. When the unfertilized eggs of pur-
puratus are put for two hours into hypertonic sea water
(50c.c. of sea water+8 c.c. 214m NaCl) and then trans-
ferred into sea water it occasionally happens that a
certain percentage of the eggs will begin to divide into
2, 4, 8 or more cells, without developing any further.
When to such eggs after they have remained in the
resting stage for a number of hours or a day, sperm is
added, some or all of the blastomeres form a fertiliza-
tion membrane and now begin to develop into larve;
and if the spermatozo6n gets into a blastomere of the

t Loeb, J., Jour. Exper. Zoél., 1914, xvii., 123; Am. Naturalist, 1915,
xlix., 257-

86 Specificity in Fertilization

2- or 4-cell stage normal plutei will result. When the
sperm is added while the eggs are in active partheno-
genetic cell division the individual blastomeres into
which a spermatozo6n enters will also form a fertiliza-
tion membrane, but such blastomeres perish very
rapidly. It is not yet possible to state why it should
make such a difference for the possibility of develop-
ment whether the spermatozoén enters into a blasto-
mere when at rest or when it is in active nuclear division,
although.the idea presents itself that in the latter case
an abnormal mix-up and separation of chromosomes
and other constituents may be responsible for the fatal
result. Whatever may be the explanation of this
phenomenon it proves to us that it is not the process
of development in itself which acts as a block to the
entrance of a spermatozo6n into an egg which is already
fertilized.*

When the spermatozoén enters the egg of the sea
urchin it calls forth the formation of a membrane—
the fertilization membrane. It might be considered
possible that this membrane formation or the alteration
underlying or accompanying it is responsible for the
fact that an egg once fertilized becomes immune against
a spermatozoén. We shall see in the next chapter
that it is possible to call forth the membrane in an
unfertilized sea-urchin egg by treating it with butyric

¥ Loeb, J., Arch. f. Entwckingsmech., 1907, xxii., 479; Artificial Par-
thenogenesis and Fertilization, Chicago, 1913, p. 240. ;

Specificity in Fertilization 87

acid. This membrane is so tough in the egg of Stron-
gylocentrotus that no spermatozodn can get through
it; in the egg of Arbacia the membrane is occasionally
replaced by a soft gelatinous film. If no second treat-
ment is given to such eggs they will disintegrate in a
comparatively short time, but when sperm is added
some or most of the eggs will develop in the way charac-
teristic of fertilized eggs.‘ When the membrane is too
tough to allow the spermatozoén to enter the egg it
can be shown that if the membrane is torn mechanically
the egg can still be fertilized by sperm.

Should it be possible that the spermatozoén can no
longer agglutinate with the fertilized egg or that those
phagocytotic reactions which we suppose to play a
réle in the entrance of the spermatozoén into the egg
are no longer possible after a spermatozo6n has entered?
The mere fact of development is apparently not the
cause which bars a spermatozoén from entering an
egg already fertilized by sperm.

Lillie assumes that the egg loses its ‘‘fertilizin” in
the process of membrane formation since the sea water
containing such eggs no longer gives the agglutinin
reaction with sperm, and he believes that the lack of
“‘fertilizin’’ in the fertilized egg or in the egg after
membrane formation is the cause of the block in the
fertilized egg. But we have seen that the artificial

rLoeb, J., Science, 1913, Xxxviii., 749; Arch. f. Entwcklngsmech.,
1914, Xxxviii., 277; Wasteneys, H., Jour. Biol. Chem., 1916, xxiv., 281.

88 Specificity in Fertilization

membrane formation does not create such a block al-
though it puts an end to the “‘fertilizin” reaction. In
the egg of purpuratus the ‘‘fertilizin” reaction ceases
when the jelly surrounding the egg is dissolved by an
acid and the eggs are repeatedly washed; yet such eggs
can easily be fertilized by sperm.

Lillie does not assume that the ‘‘fertilizin” causes
an agglutination between egg and spermatozo6én—we
should assent to such an assumption—but that the
“‘fertilizin”’ acts like an ‘“‘amboceptor’”’ between egg
and spermatozo6n, the latter being the complement,
the former the antigen. The pathologist would prob-
ably object to this interpretation since no “ambocep-
tor’’ is needed for agglutination. The writer has had
some doubts concerning the value of Ehrlich’s side-
chain theory which, besides, can only be applied in a
metaphorical sense to the mechanism of the entrance
of the spermatozoén into the egg.*

t Loeb, J., Am. Naturalist, 1915, xlix., 257.

The writer may be permitted to illustrate by a special case his reason
for declining to accept Ehrlich’s side-chain theory. Ehrlich and Sachs
found that if to a given mass of toxin small quantities of antitoxin are
added successively the first fraction added neutralized more than the
later fractions; and on the basis of this reasoning Ehrlich concluded
that ten different toxins were contained in the diphtheria toxin. Ar-
rhenius showed that the same phenomenon can be obtained when a
weak base like NH,OH is neutralized by a weak acid (e. g., boric
acid); hence we should assume that NH,OH consists of ten different
forms of ammonia. Both cases, the saturation of toxin with anti-
toxin and ammonia with boric acid are equilibrium phenomena.

(Arrhenius, S., Quantitative Laws in Biological Chemistry, London,
1915.)

Specificity in Fertilization 89

6. The reason that an egg once fertilized with
sperm cannot be fertilized again may be found in a
group of facts which we will now discuss, namely, the
self-sterility of many hermaphrodites. The fact that
hermaphrodites are often self-sterile, while their eggs
can be fertilized with sperm from a different individual
of the same species has played a great rdle in the
theories of evolution. We are here only concerned
with the mechanism which determines the block to
the entrance of a spermatozoén into an egg of the
same hermaphroditic individual.

Castle’ observed and studied the phenomenon of
self-sterility in an Ascidian, Ciona intestinalis, which is
hermaphroditic. Animals which were kept isolated
discharged both eggs and sperm into the surrounding
sea water. Often no egg was fertilized, but in some
cases five, ten, or as many, as fifty per cent. of the eggs
could be successfully fertilized with sperm from the
same individual; while if several individuals were put
into the same dish as a rule one hundred per cent. of
the eggs which were discharged segmented. Morgan?
found that the eggs of various females differ in their
power of being fertilized by sperm of the same in-
dividual while one hundred per cent. could usually be
fertilized with sperm of a different individual. He

t Castle, W. E., Bull. Mus. Comp. Zoél., Harvard, 1896, xxvii., 203.
2 Morgan, T. H., Jour. Exper. Zodl., 1904, i., 135; Arch. f. Entweklngs-
mech., 1910, XXX., 206.

90 Specificity in Fertilization

found in addition that if the eggs of Ciona are put for
about ten minutes into a two per cent. ether solution:
in sea water in a number of cases the percentage of
eggs fertilized by sperm of the same individual shows
a slight increase. Fuchs" has reported results similar
to those of Castle and Morgan.

A new point of attack has been introduced into the
work of self-sterility in plants by the consideration
of heredity. Darwin found that in Reseda which is
moncecious (or hermaphroditic) certain individuals are
either completely self-sterile or completely self-fertile;
and Compton showed that apparently self-fertility
is a Mendelian dominant to self-sterility.?

According to Jost this self-sterility in hermaphroditic
plants is due to the fact that if pollen of the same plant
is used the normal growth of the pollen tube is inhibited,
while this inhibition does not exist for pollen from a
different individual. Correns calls these substances
which prevent the adequate growth of pollen, ‘‘inhibit-
ory’’ substances, and finds that they can apparently
be transmitted to the offspring. He made experiments
on Cardamine pratensis which is self-sterile.3 He fertil-
ized two individuals of Cardamine crosswise and raised
sixty plants of the first generation. He compared the
fertility of these F, plants toward (a) their parents, and

«Fuchs, H. M., Jour. Genet., 1915, iv., 215.
2 Quoted from Fuchs.
3 Correns, C., Biol. Centralbl., 1913, XXxiii., 389.

Specificity in Fertilization gI

(0) foreign plants. All the fertilizations with the foreign
plants were successful, but the fertilizations with the
parents were only partly successful. According to
their reaction they could be divided into four groups:

(A) fertile with both parents. Type bg

(B) fertile with one (B), sterile with the other parent (G).
(a) fertile with B, sterile with G. Type bG
(b) fertile with G, sterile with B. Type Bg

It was found that approximately fifteen of the sixty
children belonged to each of the four groups. This
should be expected if the inhibitory substance to each
parent is transmitted to the children independently.
Half of the children will thus inherit the inhibitory
substance of one parent and the other half will inherit
the inhibitory substance of the other parent. This
agrees with the assumption that there are definite
determiners for the inhibitory substances in the child-
ren which will be transmitted to half of the children.
Rather complicated assumptions are needed to explain
all the facts observed by Correns on this basis and since
the subject is still under investigation we need not go
further into the details.

To us the assumption and experimental support of
the idea that self-sterility is caused by the presence of
a substance inhibitory to the entrance of a spermato-
zoon isimportant. Should it be possible that the block

92 Specificity in Fertilization

created by the entrance of a spermatozodn into the
egg is also due to an inhibitory substance carried by a
spermatozo6n into the egg; and furthermore that the
effect of the inhibitory substance should be the pre-
vention of further agglutination of the spermatozoén
with the egg or of the growth of the pollen tube in
plants? On such an assumption self-sterility would be
due to a lack of agglutination between the egg of a
nermaphrodite and a spermatozoén of the same indiv-
idual. The experiments on the agglutinins have shown
that while isoagglutinins (. e., agglutinins for other
individuals of the same species) are common auto-
agglutinins (2. e., agglutinins for cells of the same indi-
vidual) rarely if ever occur.

7. A positive chemotropism of the spermatozoa
toward an egg of the same species has been demon-
strated in a few cases, but it seems that this pheno-
menon is not determined by that type of substances
which give rise to species specificity. The famous
experiment of Pfeffer on the spermatozoa of ferns
inaugurates this line of investigation. He found that
such spermatozoa when moving in a straight line
through the water will be deviated in their course
if they come near an archegonium; they will then
turn toward it, enter it, and enter the egg. Pfeffer
showed that 0.01 per cent. malic acid if put into
a capillary tube will attract the spermatozoa of
ferns.

Specificity in Fertilization 93

When the liquid in the tube contains only 0.01 per cent.
malic acid the spermatozoa of ferns very soon move toward
the opening of the capillary tube and within from five to
ten minutes many hundreds of spermatozoa may accumu-
late in the tube. The malic acid acts as well in the form
of a free acid as in the form of salts.?

These experiments were continued and amplified by
Shibata. Bruchmann? found that the spermatozoa
of Lycopodium are positively chemotactic to citric acid
and salts of this acid, although no citric acid could be
shown in the contents of the archegonia. They are
also positively chemotactic to the watery extract from
archegonia.

Dewitz, Buller, and the writer have vainly tried to
prove the existence of a positive chemotropism of
spermatozoa to eggs of the same species. Lillie claims
to have proved a positive chemotropism of the sperm
of sea urchins to ‘‘fertilizin,’’ but such a conclusion is
only justified if a method similar to that of Pfeffer’s
with capillary tubes, gives positive results; such a
method was not used in Lillie’s experiments. It seems
that the fertilization of the egg by sperm is rendered
possible by two facts; first that where fertilization takes
place outside the body egg and sperm are shed simul-
taneously by the two sexes. This can be easily ob-

t Pfeffer, Untersuchungen aus dem botanischen Institut 2u Tubingen,
1881-1885, i., 363.
2 Bruchmann, H., Flora, 1909, ic., 193-

94 Specificity in Fertilization

served in the case of fish. But it is also the case in
invertebrates. Thus the writer has observed that the
sea urchins Strongylocentrotus purpuratus at the shore
of Pacific Grove all spawn simultaneously. The
examination extended over several miles of shore.
At such spawning seasons the sea water becomes a
suspension of sperm.

The second fact guaranteeing the fertilization of the
eggs is the overwhelming excess of spermatozoa over
eggs. The enormous waste in animated nature is in
agreement with the idea of a lack of purpose; since
in this case the laws of chance must play a great
role; and the origin of durable organisms by laws of
chance is only comprehensible on the basis of an
enormous wastefulness, for which evidence is not
lacking.

CHAPTER V
ARTIFICIAL PARTHENOGENESIS

1. The majority of eggs cannot develop unless they
are fertilized, that is to say, unless a spermatozoén
enters into the egg. The question arises: How does
the spermatozo6n cause the egg to develop into a new
organism? The spermatozoén is a living organism
with a complicated structure and it is impossible to
explain the causation of the development of the egg
from the structure of the spermatozoén. No progress
was possible in this field until ways were found to
replace the action of the living spermatozoén by well-
known physicochemical agencies.‘ Various observers
such as Tichomiroff, R. Hertwig, and T. H. Morgan
had found that unfertilized eggs may begin to segment
under certain conditions, but such eggs always disin-
tegrated in their experiments without giving rise to
larve. In 1899 the writer succeeded in causing the

t The substitution of well-known physicochemical agencies for the
mysterious action of the spermatozoén was the task the writer set
himself in this work and not the explanation of natural parthenogenesis,

as the author of a recent text-book seems to assume.
95

96 Artificial Parthenogenesis

unfertilized eggs of the sea urchin Arbacia to develop
into swimming larve, blastule, gastrule, and plutei,
by treating them with hypertonic sea water of a definite
osmotic pressure for about two hours. When such
eggs were then put back into normal sea water many
segmented and a certain percentage developed into
perfectly normal larve, blastule, gastrule, and plutei.*
Soon afterward this was accomplished by other methods
for the unfertilized eggs of a large number of marine
animals, such as starfish, molluscs, and annelids. None
of these eggs can develop under normal conditions
unless a spermatozoén enters. These experiments
furnished proof that the activating effect of the sper-
matozo6n upon the egg can be replaced by a purely
physicochemical agency.’

The first method used in the production of larve
from the unfertilized eggs did not lend itself to an
analysis of the activating effect of the spermatozo6n
upon the egg, since nothing was known about the action
of a hypertonic solution, except that it withdraws
water from the egg; and there was no indication that
the entrance of the spermatozo6én causes the egg to lose
water. No further progress was possible until another
method of artificial parthenogenesis was found. When
a spermatozoén enters the egg of a sea urchin or starfish

t Loeb. J., Am. Jour. Physiol., 1899, iii., 1353 1900, iii., 434.
2Loeb, J., Artificial Parthenogenesis and Fertilization, Chicago, 1913.
The reader is referred to this bock for the literature on the subject.

Artificial Parthenogenesis 97

or certain annelids, the surface of the egg undergoes
a change which is called membrane formation; and
which consists in the appearance of a fine membrane
around the egg, separated from the latter by a liquid
(Figs. 4 and 5). O. and R. Hertwig and Herbst had

Fic. 4 Fic. 5

Fic. 4. Unfertilized egg surrounded by spermatozoa (whose
‘flagellm is omitted in the drawing).

Fic. 5. The same egg after a spermatozo6n has entered. The
fertilization membrane is separated from the egg by a clear
space.

observed that such a membrane could be produced in
an unfertilized egg if the latter was put into chloroform
or xylol, but such eggs perished at once. It was gene-
rally assumed, moreover, that the process of membrane
formation was of no significance in the phenomenon of
fertilization, except perhaps that the fertilization mem-
brane guarded the fertilized egg against a further
invasion by sperm. However, since the fertilized egg
is protected against this possibility by other means

the membrane is hardly needed for such a purpose.
%

98 Artificial Parthenogenesis

‘In 1905 the writer found that membrane formation,
or rather the change of the surface of the egg under-
lying the membrane formation, is the essential feature
in the activation of the egg by a spermatozoén. He
observed that when unfertilized eggs of the Californian
sea urchin Strongylocentrotus purpuratus are put for
from one and a half to three minutes into a mixture of
50 c.c. of sea water+2.6 c.c. N/Io acetic or propionic
or butyric or valerianic acid and are then put into
normal sea water all or the majority of the eggs form
membranes; and that such eggs when the temperature
is very low will segment once or repeatedly and may
even—if the temperature is as low as 4°C. or less—
develop into swimming blastule?; but they will then
disintegrate. On the other hand, if they are kept at
room temperature they will develop only as far as the
aster formation and nuclear division and then begin
to disintegrate. It should be mentioned that the time
which elapses between artificial membrane formation
and nuclear division is greater than that between the
entrance of a spermatozo6n and nuclear division.

It was obvious, therefore, that artificial membrane
formation induced by butyric acid initiates the processes
underlying development of the egg but that for some
reason the egg is sickly and perishes rapidly.

When, however, such eggs were given a short treat-

« The reader will find a description of the development of this egg in
the next chapter.

Artificial Parthenogenesis 99

ment with hypertonic sea water or with lack of oxygen
or with KCN they developed into normal larve.
This new or improved method of artificial partheno-
genesis is as follows: The eggs are put for from two to
four minutes into 50 c.c. sea water containing a certain
amount of N/1o butyric acid (2.6 c.c. in the case of 5S.
purpuratus in California and 2.0 c.c. in the case of
Arbacia in Woods Hole). Ten or fifteen minutes later
the eggs are put into hypertonic sea water (50 c.c. sea
water+8 c.c. 2144 m NaCl or Ringer solution or cane
sugar) in which they remain, at 15° C. from thirty-five
to sixty minutes in the case of purpuratus, and from
1744 minutes to 2214 minutes at 23° in the case of
Arbacia at Woods Hole. If the eggs are then trans-
ferred to normal sea water they will develop. In
making these experiments, which have been repeated
and confirmed by numerous investigators, it should be
remembered that this effect of the hypertonic solution
has a high temperature coefficient (about two for
10° C.) and that a slight overexposure to the hyper-
tonic sea water injures the eggs so that development is
abnormal. By this method it is possible to imitate the
activating effect of the living spermatozoén upon the
egg in every detail and eggs treated in this way will
develop in large numbers into perfectly normal larve.
We shall see later that they can also be raised to the
adult state.

2. The next task was to find out the nature of the

100 Artificial Parthenogenesis

action of the two agencies upon the development of the
egg. It soon became obvious that the membrane
formation (or the alteration underlying membrane
formation) was the more important of the two, since in
the eggs of starfish and annelids this was sufficient for
the production of larve; and that the second treatment
had only the corrective effect, of overcoming the sickly
condition in which mere membrane formation had left
the eggs. It was, therefore, of great interest to ascer-
tain what substances or agencies caused membrane
formation in the egg, since it now became clear that
the spermatozo6n could only cause membrane formation
by carrying one such substance into the egg. These
investigations led the writer to the result that all those
substances and agencies which are known to cause
cytolysis or hemolysis (see Chapter III) will also induce
membrane formation, and that the essential feature in
the causation of development is a cytolysis of the
superficial or cortical layer of the egg. As soon as
this layer is destroyed the development of the egg can
begin.

The substances and agencies which cause cytolysis and
hence, if their action is restricted to the surface of the
egg, will induce development are, besides the fatty acids:
(1) saponin or solanin or bile salts; (2) the solvents of
lipoids, benzol, toluol, amylene, chloroform, aldehyde,
ether, alcohols, etc.; (3) bases; (4) hypertonic or hypo-
tonic solutions; (5) rise in temperature, and (6) certain

Artificial Parthenogenesis IOI

salts, e. g., BaCl, and SrCl, in the case of the egg of
purpuratus, and according to R. Lillie, Nal or NaCNS
in the egg of Arbacia. Whenever we submit an unfer-
tilized sea-urchin egg to any of these agencies and
restrict the cytolysis to the superficial or cortical layer
of the egg (i. e., if we transfer the egg to normal sea
water before the cytolytic agent has had time to diffuse
into the main egg) the egg will form a membrane and
behave as if the membrane formation had been called
forth by a fatty acid, with this difference only, that
the various agencies are not all equally harmless for the
egg.*

If the idea was correct that the change underlying
membrane formation was essentially a cytolysis of the
cortical layer of the egg, it was to be expected (from the
data contained in Chapter III) that the blood serum
or the cell extracts of foreign species would also cause
membrane formation and thus induce the development
of the unfertilized egg, while serum of animals of the
same species or genus would have no such effects. This
was found to be correct. In 1907 the writer showed
that the blood serum of a Gephyrean worm, Dendro-
stoma, was able to cause membrane formation in the
egg of the sea urchin. When added in a dilution of
I c.c. of serum to 500 or 1000 c.c. of sea water to eggs of
purpuratus a certain number formed fertilization mem-
branes. It was found later that the serum and tissue

1 The reader is referred for details to the writer’s book on the subject.

102 Artificial Parthenogenesis

extracts of a large number of animals, especially of
mammals (rabbit, pig, ox, etc.), had the same effect,
though it was necessary to use higher concentrations,
one-half sea water and one-half isotonic blood serum.
The eggs of every female sea urchin, however, did not
give the reaction and not all the eggs even of sensitive
females formed membranes. The writer found, how-
ever, that it was possible to increase the susceptibility
of the eggs against foreign blood serum by putting them
into a 3/8 m solution of SrCl, for from five to ten
minutes (or possibly a little longer) before exposing
them to the foreign blood serum. BaCl, acts similarly.
The fact that SrCl, alone can cause membrane forma-
tion in unfertilized eggs if they are left long enough in
the solution suggests that the sensitizing effect of the
substance consists in a modification of the cortical
layer similar to that underlying membrane formation;
and that the subliminal effect of a short treatment
with SrCl, and the subliminal effect of the foreign
serum when combined suffice to bring about the
membrane formation.

Not only the watery extract of foreign cells but also
that of foreign sperm, induces membrane formation
in the sea-urchin egg. The watery extract of sperm
of starfish is especially active, but the degree of activity
varies considerably with the species of starfish from
which the sperm is taken. The eggs of different species
of sea urchins also show a different degree of suscepti-

Artificial Parthenogenesis 103

bility for the sperm of foreign species. Thus the eggs
of Strongylocentrotus purpuratus require a higher con-
centration of sperm extract than the eggs of S. fran-
ciscanus. For the latter the amount of foreign cell
constituents which suffices to call forth membrane
formation is so small that contact with almost any
foreign living spermatozoén produces this effect; and
as a rule no previous sensitizing action of SrCl: is
required. When we bring the unfertilized eggs of S.
franciscanus into contact with the living sperm of star-
fish or shark or even of fowl, the eggs form a fertiliza-
tion membrane without previous sensitization. A
specific substance from the foreign spermatozoén
causes membrane formation before the spermatozoén
has time to enter the egg. The effect is the same as if
artificial membrane formation had been called forth
with butyric acid, 7. e., they begin to develop and
then disintegrate unless they receive a second short
treatment.

When, however, we treat the eggs with the watery
extracts from the cells of their own or closely related
species we find that these extracts are utterly inactive,
even if used in comparatively strong concentrations.
This agrees with the results given in Chapter III.

These phenomena lead to a very paradoxical result;
namely that while in the case of foreign sperm we can
cause membrane formation by both the living and the
dead spermatozoén, only the living spermatozoén of

104, Artificial Parthenogenesis

the same species can induce membrane formation.
This might find its explanation on the assumption
that the active substance contained inthe foreign
sperm or serum is water-soluble and a protein, while
the activating or membrane-forming substance in the
spermatozo6n is insoluble in water but soluble in the
egg (or in lipoids). If this assumption is correct
the two substances are essentially different.

Robertson? has succeeded in extracting a substance
from the sperm of the sea urchin which causes mem-
brane formation of the sea-urchin egg after the latter
has been sensitized by a treatment with SrCl,. It
seems to the writer that if the substance extracted by
Robertson were the real fertilizing agent contained in
the spermatozoén it should fertilize the egg without a
previous sensitization of the egg with SrCl, being
required.

3. The action of acids in the mechanism of artificial
parthenogenesis provides some interesting physiological
problems. When unfertilized sea-urchin eggs are left
in sea water containing any of the lower fatty acids up
to capronic, the eggs will form no membranes, while
in such sea water, and they will show no outer signs of
cytolysis (swelling). When, however, the eggs are
left in sea water containing any of the fatty acids from
heptylic upward the eggs will form membranes while
im the acid sea water and soon afterward will cytolyze

* Robertson, T. B., Arch. f. Entwckingsmech., 1912, Xxxv., 64.

Artificial Parthenogenesis 105

completely and swell enormously. In solutions of the
mineral acids no membranes are formed and none are
formed as a rule when the eggs are transferred back to
sea water. When both a mineral and a lower fatty acid,
é. g., butyric, are added to sea water the mineral acid
acts as if it were not present, 7. e., the eggs form mem-
branes when transferred back to sea water if the con-
centration of the butyric acid is high enough. All
these data are comprehensible if we assume that only
that part of the acid causes membrane formation which
is lipoid soluble, while the water soluble part is not
involved in the process of membrane formation; and
that the cytolysis or swelling of the whole egg can only
take place in the higher fatty acids (heptylic or above)
which are little soluble in water and very soluble in
lipoids, while the lower fatty acids, whose water solubil-
ity is comparatively high, can only bring about a cyto-
lysis and swelling in the cortical layer but not in the
rest of the egg. This makes it appear as though the
part undergoing an alteration in membrane formation
was a lipoid; and this would harmonize with the assump-
tion that the specific membrane-inducing substance
in the spermatozo6n is not soluble in water, but soluble
in fat.

4. These and other observations led the writer to
the view that the essential process which causes de-
velopment might be an alteration of the surface of the
egg, in all probability an alteration of the superficial

106 Artificial Parthenogenesis

layer probably of the nature of a superficial cytolysis.
The question remains: What could be the physico-
chemical nature of this cytolysis? The writer had
suggested in former papers that in the cytolysis under-
lying membrane formation lipoids were dissolved, and
he supposed that the substance to be dissolved might
be a calcium-lipoid compound which might form a
continuous layer under the surface of the egg.' v.
Knaffl, working on the cytolysis of eggs in the writer’s
laboratory, gave the following idea of the process:

Protoplasm is rich in lipoids; probably it is mainly an
emulsion of these and proteins. Any physical or chemical
stimulus which can liquefy the lipoids causes cytolysis of
the egg. The protein of the egg can really only swell or
be dissolved if the condition of aggregation of the lipoid
is altered by chemical or physical agencies. The mechan-
ism of cytolysis consists in the liquefaction of the lipoids
and thereupon the lipoid-free protein swells or is dissolved
by taking up water. . . . Hence this supports Loeb’s view
that membrane formation is induced by the liquefaction
of lipoids.?

The writer suggested that the destruction of an
emulsion in the cortical layer might possibly be the
essential feature of the alteration leading to membrane
formation and development. It had been long ob-
served that unfertilized starfish eggs may begin to

"Loeb, J., Uber den chemischen Charakter des Befruchtungsvorgangs,

etc., Leipzig, 1908.
av. Knaffl, E., Arch. f. d. ges. Physiol., 1908, cxxiii., 279.

Artificial Parthenogenesis 107

develop apparently without any outside ‘‘stimulus,”’
and A. P. Mathews found that slight mechanical agita-
tion of these eggs in sea water increased the number
which developed. It has been shown in numerous
experiments by Delage, R. S. Lillie, and the writer,
that the substances causing development in the
starfish egg are identical or closely related to those
which bring about this effect in the egg of the sea ur-
chin and in both cases the development is preceded
by a membrane formation.

But how can membrane formation be produced by mere
agitation? It seems to me that this can be understood if
we suppose that it depends upon the destruction of an
emulsion in the cortical layer of the egg. It is conceivable
that in the egg of certain forms the stability of this emulsion
is so small that mere shaking would be enough to destroy
it and thus induce membrane formation and development.

The durability of emulsions varies, and where an emul-
sion is very durable shaking has no effect, while where
it is at the critical point of separating into two con-
tinuous phases a slight shaking will bring about the
separation, and where the emulsion is still less durable
we observe the phenomenon of a ‘“‘spontaneous”’
parthenogenesis. Eggs like those of most sea urchins
belong to the former, eggs like those of some starfish
and annelids belong to the second or third type.

It is impossible to state at present whether the fertil-

t Loeb, J., Artificial Parthenogenesis and Fertilization, p. 255.

108 Artificial Parthenogenesis

ization membrane is preformed in the fertilized egg and
merely lifted off from the egg or whether its formation
is due to the hardening of a colloidal substance sepa-
rated from the emulsion (or excreted) and hardened in
touch with sea water. But we can be sure of one thing,
namely, that the liquid between egg and membrane
contains some colloidal substance which determines
the tension and spherical shape of the membrane. The
membrane is obviously permeable not only to water but
also to dissolved crystalloids, while it is impermeable
to colloids. When we add some colloidal solution
(e. g., white of egg, blood serum, or tannic acid) to the
sea water containing fertilized eggs of purpuratus, the
membrane collapses and lies close around the egg;
while if the eggs are put back into sea water or a sugar
solution the membrane soon assumes its spherical
shape. This is intelligible on the assumption that in
the process of membrane formation (or in the destruc-
tion of the emulsion in the cortical layer) a colloidal
substance goes into solution which cannot diffuse into
the sea water since the membrane is impermeable to
the colloidal particles. The membrane is, however,
permeable to the constituents of sea water or to sugar.
Consequently sea water will diffuse into the space
between membrane and egg until the tension of the
membrane equals the osmotic pressure of the colloid
dissolved in the space between egg and the membrane.
If we add enough colloid to the outside solution so that

Artificial Parthenogenesis 109

its osmotic pressure is higher than that of the colloidal
- solution inside the membrane the latter will collapse.

It should also be stated that the unfertilized eggs
of many marine animals are surrounded by a jelly
(chorion) which is dissolved when the egg is fertilized. ‘l
The writer has shown that the same chemical substances
which will induce membrane formation and artificial
parthenogenesis will as a rule also cause a swelling and’
liquefaction of the chorion.

We have devoted so much space to the mechanism
of membrane formation since it is likely to give a clearer
insight into the physicochemical nature of physiological
processes than the phenomena of muscular stimulation
and contraction or nerve stimulation, upon which the
majority of physiologists base their conclusions con-
cerning the mechanism of life phenomena.

Before we come to the discussion of the second factor
in the activation of the egg it should be stated more
definitely that for the eggs of some forms the first
factor, the process underlying membrane formation,
suffices for the development of the egg into a larva and
that no second factor is required in these cases. This
is true for the eggs of starfish and certain annelids.

t It has been stated by several writers that the eggs of the sea urchin
can no longer form the fertilization membrane when the jelly surround-
ing the egg is dissolved. The writer has found that if the jelly sur-
rounding the eggs of Strongylocentrotus purpuratus is dissolved by acid
the eggs still form a fertilization membrane upon the entrance of a
spermatozoon.

10 Artificial Parthenogenesis

Thus in 1901 Loeb’ and Neilson showed that a short
treatment with HCl and HNO, sufficed to cause some
eggs of Asterias in Woods Hole to develop into larve
without a second treatment being needed, and Delage?
showed the same for CO,; and in 1905 the writer found
that the eggs of the Californian starfish Asterina can
be induced to form a membrane by butyric acid treat-
ment and that ten per cent. of these eggs developed
into normal larve. Quite recently R. S. Lillie ob-
served that the eggs of Asterias at Woods Hole can be
caused to form membranes and develop into larve by
a treatment with butyric acid and that the time of ex-
posure required to get a maximal number of larve varies
approximately inversely with the concentration of the
acid, within a range of 0.0005 to 0.006 N butyric acid.
If the exposure is too short membrane formation will
occur without normal development. :

All this leads us to the conclusion that the main
effect of the spermatozo6n in inducing the development
of the egg consists in an alteration of the surface of the
latter which is apparently of the nature of a cytolysis
of the cortical layer. Anything that causes this altera-
tion without endangering the rest of the egg may induce
its development. The spermatozoén, therefore, causes

tLoeb, J., Artificial Parthenogenesis and Fertilization, 1913, p. 250
er Y., Arch. d. Zodl. expér. et gén., 1902, X., 213; 1904, ii., 273

1905, iii., 104.
3 Lillie, R. S., Jour. Biol. Chem., 1916, xxiv., 233.

Artificial Parthenogenesis Ill

the development of the egg by carrying a substance
into the latter which effects an alteration of its surface
layer.

5. We will now discuss the action of the second,
corrective factor, in the inducement of development.
When we cause membrane formation in a sea-urchin
egg by the proper treatment with butyric acid it will
commence to develop and segment but will disinte-
grate rapidly if kept at room temperature and the
more rapidly the higher the temperature. If, however,
the eggs are treated afterward for a certain length of
time (from thirty-five to sixty minutes at 15° C. for
purpuratus and 1714 to 2214 minutes for Arbacia at
23° C.) in a solution which is isosmotic with 50 c.c. sea
water +8 c.c. 214 m NaCl,‘ they will develop into larve,
many of which may be normal. Any hypertonic solu-
tion of this osmotic pressure, sea water, sugar, or a single
salt, will suffice provided the solution does not contain
substances that are too destructive for living matter.
The hypertonic solution produces its corrective effect
only if the egg contains free oxygen; and in a slightly
alkaline medium more rapidly than in a neutral medium.
The time of exposure in the hypertonic solution dimin-

t It is necessary to call attention to the fact that sugar solutions of a
high concentration (e. g., m solutions) have a much higher osmotic
pressure than that which they should have theoretically (Lord Berkeley
and Hartley). Delage by ignoring this fact has misinterpreted his
experiments with sugar solutions. See Lloyd, D. J., Arch. f. Entwcklng:-
mech., 1914, XXXviii., 402.

112 Artificial Parthenogenesis

ishes in certain limits with the concentration of OH
ions in the solution.

It is strange that in the eggs of purpuratus the cor-
rective effect can also be brought about by exposing
the eggs after the artificial membrane formation for
about three hours to normal sea water free from oxygen;
or to sea water in which the oxidations have been re-
tarded by the addition of KCN. This method is not
so reliable as the treatment with hypertonic solution.

What does the hypertonic solution do to prevent the
disintegration of the egg after the artificial membrane
formation? The writer suggested in 1905 that the arti-
ficial membrane formation alone starts the develop-
ment but leaves the eggs usually in a sickly condition
and that the hypertonic solution or the lack of oxygen
allows them to recuperate from such a condition. The
second factor is, according to this view, merely a cor-
rective or curative factor. The following observations
will explain the reasons for such an assumption.

The writer found that if we keep the unfertilized
eggs after artificial membrane formation in sea water
deprived of oxygen the disintegration of the egg fol-
lowing artificial membrane formation is prevented for
a day at least. The same result can be obtained by
adding ten drops of 4 per cent. KCN to 50 c.c. of
sea water, and certain narcotics, e. g., chloral hydrate,
act in the same way. Wasteneys and the writer found
that chloral hydrate (and other narcotics) in the con-

Artificial Parthenogenesis 113

centration required do not suppress or even lower the
oxidations in the egg to any considerable extent,* but
they prevent the processes of cell division. Hence it
seems that the egg disintegrates so rapidly after arti-
ficial membrane formation because it is killed by those
processes leading to nuclear division or cell division
which are induced by the artificial membrane forma-
tion. If we suppress these phenomena of development
(for not too long a time) we give the egg a chance to
recover and if now the impulse to develop is still active
we notice a perfectly normal development. If the egg
is kept too long without oxygen it suffers for other
reasons and cannot develop; the writer has shown that
if eggs fertilized by sperm are kept for too long a time
without oxygen they also will no longer be able to
develop normally. The short treatment with a hyper-
tonic solution supplies the corrective factor required, so
that the egg can then undergo cell division at room
temperature without disintegrating.

The correctness of this interpretation, which is in
reality mainly a statement of observations, is proved
by the two following groups of facts. The older ob-
servers had already noticed that the unfertilized eggs
of the sea urchin when lying in sea water will die after
a day or more, and that occasionally such eggs show
nuclear division or even the beginning of cell division

«Loeb, J., and Wasteneys, H., Jour. Biol. Chem., 1913, xiv., 517;
Biochem. Ztschr., 1913, lvi., 295.
8

114 Artificial Parthenogenesis

shortly before disintegration sets in. The writer has
studied this phenomenon in the unfertilized eggs of
purpuratus and found that only the eggs of certain
females show this cell division before disintegration
and that the cell division is preceded by an atypical
form of membrane formation; the eggs surrounding
themselves by a fine gelatinous film comparable to
that produced in the egg of Arbacia by a treatment with
butyric acid. It is difficult to state what induces the
alteration of the surface in the eggs that lie so long in
sea water. It may be due to the CO, formed by the
eggs—since we know that CO, may induce membrane
formation—or it may be due to the alkalinity of the
sea water or to a substance originating from the jelly
surrounding the eggs. It was found that if such eggs
are kept without oxygen their disintegration (and cell
division) will be delayed considerably. The presum-
able explanation for this is that the lack of oxygen pre-
vents the internal changes underlying cell division and
thus prevents the disintegration of the egg. The direct
proof that an egg in the process of cell division is more
endangered by abnormal solutions than an egg at rest
has been furnished by numerous observations of the
writer. He showed in 1906 that the fertilized egg of
purpuratus dies rather rapidly in a pure m/2 NaCl
or any other abnormal isotonic solution, while the unfer-
tilized egg can live for days in such solutions. In
* Loeb, J., Biochem. Zischr., 1906, ii., 81.

Artificial Parthenogenesis 115

a series of papers, beginning in 1905, he showed that
the fertilized egg will live longer in hypertonic, hypo-
tonic, and otherwise abnormally constituted solutions
when the cell divisions are suppressed by lack of oxygen
or by the addition of KCN or of chloral hydrate.* It
is thus obvious that coincident with the changes under-
lying nuclear division or cell division alterations occur
in the sensitiveness of the egg to salt solutions of ab-
normal concentration or constitution, e. g., NaCl+CaCl,
isotonic with sea water, hypertonic, or hypotonic
solutions. 4

We must, therefore, conclude that artificial mem-
brane formation induces development but that it
leaves the egg in a sickly condition in which the very
processes leading to cell division bring about its de-
struction; that if it is given time it can recover from
this condition and that the treatment with the hyper-
tonic solution also brings about this recovery rapidly
and reliably.

Herlant? suggested that the corrective effect of the
hypertonic solution consisted in the proper develop-
ment of the astrospheres required for cell division.
According to this author mere membrane formation
does not lead to the formation of sufficiently large
astrospheres and hence cell division may remain im-

«Loeb, J., Arch. f. d. ges. Physiol., 1906, cxiii., 487; Biochem. Ztschr.,
IgI0, xxvi., 279, 289; XXVii., 304; xxix., 80; Arch. f. Entwckingsmech.,
1914, xl., 322. 2 Herlant, M., Arch. de Biol., 1913, Xxviii., 505.

116 Artificial Parthenogenesis

possible.t The writer has no a priori objection to this
suggestion which agrees with earlier observations by
Morgan except that it is at present difficult to harmonize
it with all the facts. Why should it be possible to
replace the treatment with the hypertonic solution by
a suspension of the oxidations in the egg for three hours
while we know that lack of oxygen suppresses the for-
mation of astrospheres in the fertilized eggs? What
becomes of the astrospheres if the treatment with the
hypertonic solution precedes the membrane formation
by a number of hours or a day (which is possible as
we shall see), and why do they not induce cell division,
if Herlant’s idea is correct? Nevertheless the sugges-
tion of Herlant deserves to be taken into serious con-
sideration.

6. How can an alteration of the surface of the egg
—e. g.,a cytolytic or other destruction of the cortical
layer—lead to a beginning of development? The
answer is possibly given in the relation of oxidation to
development. The writer found in 1895 that if oxygen
is withdrawn from the fertilized sea-urchin egg it can
not segment and this seems to be the case for eggs in
general.? In 1906 he found that the rapid disintegra-
tion of the eggs of the sea urchin which follows artificial

It is also important to remember that the formation of astrospheres
after mere membrane formation occurs considerably more slowly than
if the egg has also received a treatment with a hypertonic solution.

? The writer found that the eggs of Fundulus will segment a number
of times even if all the oxygen has apparently been removed.

Artificial Parthenogenesis 117

membrane formation could be prevented when the
eggs were deprived of oxygen or when the oxidations
were suppressed in the eggs by KCN. This suggested
a connection between the disintegration of the egg
after artificial membrane formation and the increase
in the rate of oxidations; and he found further that the
formation of acid is greater in the fertilized than in the
unfertilized egg. He, therefore, expressed the view
in 1906 that the essential feature (or possibly one of the
essential features) of the process of fertilization was
the increase of the rate of oxidations in the egg and
that this increase was caused by the membrane forma-
tion alone.t These conclusions have been since amply
confirmed by the measurements of O. Warburg as
well as those of Loeb and Wasteneys, both showing that
the entrance of the spermatozoén into the egg raises
the rate of oxidations from 400 to 600 per cent., and that
membrane formation alone brings about an increase
of similar magnitude. Loeb and Wasteneys found
that the hypertonic solution does not increase the rate
of oxidations in a fertilized egg. It does do so, how-
ever, in an unfertilized egg without membrane forma-
tion, but merely for the reason that in such an egg the
hypertonic solution brings about the cytolytic change in
the cortex of the egg underlying membrane formation. ?

t Loeb, J., Biochem. Zischr., 1906, ii., 183.
2 Thus the treatment of an unfertilized egg without membrane with
a hypertonic solution combines two effects, first the general cytolytic

118 Artificial Parthenogenesis

According to Warburg it is probable that the oxi-
dations occur mainly if not exclusively at the surface
of the egg since NaOH, which does not diffuse into the
egg, raises the rate of oxidations more than NH,OH
which does diffuse into the egg. And finally, the same
author showed that the oxidations in the sea-urchin
egg are due to a catalytic process in which iron acts
as a catalyzer.* In view of all these facts and their
harmony with the methods of artificial parthenogenesis
the suggestion is justifiable that the alteration or
cytolysis of the cortical layer of the egg is in some
way connected with the increased rate of oxidations.

The question remains then: How can membrane for-
mation or the alteration of the cortical layer underlying
membrane formation cause an increase in the rate of oxi-
dations? One possibility is that the iron (or whatever
the nature of the catalyzer may be) exists in the cortex
of the egg in a masked condition—or in a condition in
which it is not able to act—while the alteration of the
cortical layer makes the iron active. It might be that
either the iron or the oxidizable substrate is contained
in the lipoid layer in the unfertilized condition of the egg
and that the destruction or cytolysis of the cortical
layer brings both the iron and the oxidizable substrate
into the watery phase in which they can interact.

alteration of the cortical layer of the membrane and the corrective
effect of the hypertonic solution. The former effect raises the rate of
oxidations in the egg, the latter does not.

* Warburg, O., Sitengsber. d. Heidelberger Akad. d. Wissnsch., B. 1914.

Artificial Parthenogenesis 119

Another possibility is that the act of fertilization in-
creases the permeability of the egg. This idea, which
seems attractive, was first suggested and discussed
by the writer in 1906.7 He had found that when
fertilized and unfertilized eggs were put into ab-
normal salt solutions, e. g., pure solutions of NaCl,
the fertilized eggs died more rapidly than the unfer-
tilized eggs and he pointed out that these experiments
suggested the possibility that fertilization increases
the permeability of the egg for salts. The reason for
his hesitation to accept this interpretation was, that
the fertilized egg is also more easily injured by lack
of oxygen than the unfertilized egg and in this case
the greater sensitiveness of the fertilized egg was ob-
viously due to its greater rate of metabolism. Later
experiments by the writer showed that the fertilized
egg can be made more resistant to abnormal salt solu-
tions if its development is suppressed by lack of oxygen
or by KCN or by certain narcotics. With our present
knowledge it does not seem very probable that lack
of oxygen diminishes the permeability of the egg, but
we know that it inhibits the developmental processes.
Warburg has made it appear very probable that the
fertilized egg is impermeable for NaOH and if this is
the case it should also be impermeable for NaCl.?

t Loeb, J., Biochem. Zischr., 1906, ii., 87.
2 Unless the egg is left so long in the pure NaCl solution that its
permeability is increased.

120 Artificial Parthenogenesis

The idea that fertilization and membrane formation
cause an increase in the permeability of the egg was
later accepted and elaborated by R. Lillie. This
author assumes that the unfertilized egg cannot de-
velop because it contains too much CO, but that the
CO, can escape from the egg as soon as its permeability
is increased through the destruction of the cortical
layer of the egg.* After the CO, has escaped, the
excessive permeability must be restored to its normal
value and this is the réle of the hypertonic treatment.
It is, however, difficult to harmonize the assumption
of an impermeability of the unfertilized egg for CO,
with the fact that if the unfertilized sea-urchin egg is
cut into two, as is done in merogony, no development
takes place, while such pieces will develop when a
spermatozoén enters. The cortical layer is removed
along the cut surface and there is no reason why the
CO, should not escape. Besides, the experiments of
Godlewski and the writer prove that the cortical layer
of the unfertilized sea-urchin egg is apparently very
permeable for CO, since the latter causes membrane
formation if contained in the sea water in sufficiently
high concentration.

Lillie assumes that the hypertonic treatment restores
the permeability raised to excess by the butyric acid
treatment, but this assumption is not in harmony with

t Lillie, R. S., Jour. Morphol., 1911, xxii., 695; Am. Jour. Physiol.,
IQII, XXVii., 289.

Artificial Parthenogenesis 121

the following facts. The writer has shown that it is
immaterial whether the eggs are treated first with the
hypertonic solution and then with butyric acid or the
reverse, if only the eggs remain longer in the hypertonic
solution when the hypertonic treatment precedes the
butyric acid treatment. It was stated in the beginning
of this chapter that the development of the egg can
be induced by hypertonic sea water, and we know the
reason since hypertonic sea water is a cytolytic agency.
The writer found that when we expose unfertilized
eggs of purpuratus for from two to two and a half-
hours to hypertonic sea water they will often not de-
velop and only a few eggs will undergo the first cell
divisions, then going into a condition of rest. When
these eggs, both the segmented and unsegmented, were
treated twenty-four or thirty-six hours later with
butyric acid, so that they formed a membrane, they
all developed into larvee without further treatment.
It is impossible to apply Lillie’s theory to these facts,
for the simple reason that the treatment with hyper-
tonic sea water was just long enough to induce de-
velopment in some eggs and hence according to Lillie’s
ideas must have increased the permeability of these
eggs. Yet these same eggs were induced to develop
normally when subsequently treated with butyric
acid, which according to Lillie also acts by increasing
the permeability. Nothing indicates that the treat-
ment of the eggs with a hypertonic solution diminishes

122 Artificial Parthenogenesis

their permeability; the reverse would be much more
probable.

Lillie’s theory also fails to explain that mere treat-
ment of the eggs with a hypertonic solution can bring
about their development into larve. This, however,
is intelligible on the assumption that the hypertonic
solution in this case has two different effects, first a
cytolysis of the cortical layer of the egg and second an
entirely different effect, possibly upon the interior of
the egg, which represents the second or corrective effect.

McClendon* has shown that the electrical conduc-
tivity of the egg is increased after fertilization, and J.
Gray? has found that this increase in conductivity is
only transitory and disappears in fifteen minutes.
This might indicate that the egg becomes transitorily
more permeable for salts after the entrance of the
spermatozoén or after membrane formation; although
an increase in conductivity might be caused by other
changes than a mere increase in permeability of the
egg. The writer is of the opinion that it is necessary
to meet all these and other difficulties before we can
state that the alteration of the cortical layer, which is
the essential feature of development, acts chiefly or ex-
clusively by an increase in the permeability of the egg.

McClendon, J. F., Publications of the Carnegie Institution, No. 183,
125; Am. Jour. Physiol., 1910, xxvii., 240.

2Gray, J., Proc. Cambridge Philosophical Society, 1913, xvii., 1.

3h. Lillie has recently shown that in a hypotonic solution water
dituses more rapidly into a fertilized than into an unfertilized egg.

Artificial Parthenogenesis 123

7. When the experiments on artificial partheno-
genesis were first published they aroused a good deal
of antagonism not only among reactionaries in general
but also among a certain group of biologists. O.
Hertwig had defined fertilization as consisting in the
fusion of two nuclei, the egg nucleus and the sperm
nucleus. No such fusion of two nuclei takes place in
artificial parthenogenesis since no spermatozo6én enters
the egg, and it became necessary, therefore, to abandon
Hertwig’s definition as wrong. The objection raised
that the phenomena are limited to a few species soon
became untenable since it has been possible to produce
artificial parthenogenesis in the egg of plants (Fucus,
according to Overton) as well as of animals, from echino-
derms up to the frog; and it may possibly one day be
accomplished also in warm-blooded animals. A second
objection was that the eggs caused to develop by the
methods of artificial parthenogenesis could never reach
the adult stage and that hence the phenomenon was
merely pathological. There was no basis for such a
statement, except that it is extremely difficult to raise
marine invertebrates. Delage' was courageous enough

This is exactly what one should expect since the unfertilized egg is not
only surrounded by the cortical layer but also by a thick layer of jelly
both of which are lacking in the fertilized egg. It is difficult to under-
stand how this observation can throw any light on the mechanism of
development, since water diffuses rapidly enough into the unfertilized
egg.

t Delage, Y., Compt. rend. Acad. Sc., 1909, cxlviii., 453.

124 Artificial Parthenogenesis

to make an attempt to raise parthenogenetic larve of
the sea urchin beyond the larval stage and he succeeded
in one case in carrying the animal to the mature stage.
It proved to be a male.

Better opportunities were offered when a method was
discovered which induced the development of the un-
fertilized eggs of the frog. In 1907, Guyer made the
surpriseng observation that if he injected lymph or
blood into the unfertilized eggs of frogs he succeeded
in starting development and he even obtained two
free-swimming tadpoles. ‘‘Apparently the white rather
than the red corpuscles are the stimulating agents
which bring about development, because injections of
lymph which contains only white corpuscles produce
the same effects as injections of blood.” Curiously
enough, Guyer thought that probably the cells which
he introduced and not the egg were developing. In
1910, Bataillon showed that a mere puncture of the
egg with a needle could induce development but he
believes that for the full development the introduction
of a fragment of a leucocyte is required. Bataillon
has called attention to the analogy with the writer’s
results on lower forms, the puncturing of the egg
corresponding to the cytolysis of the surface layer of the
egg and the introduction of a leucocyte as the analogue
of the second or corrective factor. The method of
producing artificial parthenogenesis by puncturing
the egg has thus far been successful only in the egg of

FIG. 6.

Artificial Parthenogenesis 125

the frog. The writer has tried it in vain on the eggs
of many other forms. He has at present seven par-
thenogenetic frogs over a year old, produced by merely
puncturing the eggs with a fine needle (Fig. 6).
These frogs have reached over half the size of the adult
frog. They can in no way be distinguished from the
frogs produced by fertilization with a spermatozoén.
This makes the proof conclusive that the methods of
artificial parthenogenesis can result in the production
of normal organisms which can reach the adult stage.

Bancroft and the writer tried to determine the sex
of a parthenogenetic tadpole and of a frog just carried
through metamorphosis. Since in early life the sex
glands of both sexes in the frog contain eggs it is not
quite easy to determine the sex, except that in the male
the eggs gradually disappear and from this and other
criteria we came to the conclusion that both partheno-
genetic specimens, which were four months old, were
males.

The writer has recently examined the gonads of a
ten months old parthenogenetic frog. Here no doubt
concerning the sex was possible since the gonads were
well-developed testicles containing a large number of
spermatozoaof normal appearance,and noeggs.' (Figs.
7and8.) This would indicate that the frog belongs to
those animals in which the male is heterozygous for sex.

t Since this was written, two more of the parthenogenetic frogs over
a year old died. Both were males.

126 Artificial Parthenogenesis

8. The fact that the egg of so high a form as the
frog can be made to develop into a perfect and normal
animal without a spermatozodn—although normally
the egg of this form does not develop unless a sperma-
tozoén enters—corroborates the idea expressed in
previous chapters that the egg is the future embryo
and animal; and that the spermatozoén, aside from its
activating effect, only transmits Mendelian characters
to the egg. The question arises: Is it possible to cause
a spermatozo6n to develop into an embryo? ‘The idea
has been expressed that the egg was only the nutritive
medium on which the spermatozoén developed into
an embryo, but this idea has been rendered untenable
by the experiments on artificial parthenogenesis. Never-
theless the question whether or not the spermatozoén
can develop into an embryo on a suitable culture
medium remains, and it can only be decided by direct
experiments. It was shown by Boveri, Morgan, Delage,
Godlewski, and others, that if a spermatozo6én enters
an enucleated egg or piece of egg it can develop into
an. embryo, but since the cytoplasm of the egg is the
future embryo this experiment proves only that the
egg nucleus may be replaced by the sperm nucleus;
and also that the sperm nucleus carries into the egg
the substances which induce development: Inciden-
tally these experiments on merogony also prove that
the mere mechanical tearing of the cortical layer,—
which must happen in the separation of the unfertilized

Artificial Parthenogenesis 127

egg into parts with and without a nucleus,—by dissection
or by shaking, is not sufficient to start development
in the sea-urchin egg.

J. de Meyer put the spermatozoa of sea urchins
into sea water containing an extract of the eggs of the
same species but found only that the spermatozoa
swell in such a solution. Loeb and Bancroft made
extensive experiments in cultivating spermatozoa of
fowl in vitro on suitable culture media. In yolk and
white of egg the head of the spermatozoén underwent
transformation into a nucleus, but no mitosis or aster
formation was observed.'!| These experiments should
be continued.

t Loeb, J., Artificial Parthenogenesis and Fertilization, Chicago, 1913.

CHAPTER VI

DETERMINISM IN THE FORMATION OF AN ORGANISM
FROM AN EGG

I. The writer in a former book (Dynamics of Living
Matier, 1906, p. 1), defined living organisms as chemical
machines consisting chiefly of colloidal material and
possessing the peculiarity of preserving and reproducing
themselves. Some authors like Driesch, and v. Uex-
ktill seem to find it impossible to account for the devel-
opment of such machines from an undifferentiated egg
ona purely physicochemical basis. A study of Driesch’s
very interesting and important book* shows that he
assumes the eggs of certain animals, e. g., the sea urchin,
to consist of homogeneous material; and he concludes
that nature has solved, in the formation of highly differ-
entiated organisms from such undifferentiated material,
a problem which does not seem capable of a solution
by physicochemical agencies alone. But the supposi-
tion of a structureless egg is wrong, since Boveri has

tDriesch, H., Science and Philosophy of the Organism. London,
1908 and 1909.
128

Organisms from Eggs 129

demonstrated the existence of a very simple but definite
structure in the unfertilized egg of the sea urchin; and
a similar simple structure has been demonstrated by
other authors, especially Conklin, in the eggs of other
forms.

In this chapter we shall attempt the task among
others of showing how, on the basis of the simple physi-
cochemical structure of the unfertilized egg, the main
organ of self-preservation of the organism, the intestine,
is formed through the mere ptocess of cell division and
growth. Cell division is the most general of the specific
functions of living matter and it is the basis underlying
the differentiation of the comparatively simple struc-
ture of the egg into a more complex organism. If cell
division and growth were equal in all parts of the egg
no differentiation would be possible, but the different
regions of the unfertilized egg contain different consti-
tuents and these, probably on account of their chemical
difference, do not all begin to grow or divide simulta-
neously and equally.

Boveri? found that in the unfertilized egg of the sea
urchin Strongylocentrotus lividus at Naples a definite
structure is indicated by the fact that the yellowish-red
pigment is not equally distributed over the whole
surface of the egg but is arranged in a wide ring from
the equator almost to one of the poles. Thus three

« Boveri, Th., Verhandl. d. physik.-med. Gesellsch., Wirzburg, 1901,
xxxiv., 145+
9

130 Organisms from Eggs

zones can be recognized in the egg (Fig. 9), a small clear
cap A at one pole, a pigmented ring B, and the
rest again un-
pigmented C.
Observation
has shown that
each one of
these regions
gives rise to a
definite con-
stituent of
the egg: A fur-
nishes the
mesenchyme
from which the
skeleton and

Fic. 9

the connective tissue originate; B is the material for
the formation of the intestine, and C gives rise to the
ectoderm.

The pigment is only at the surface of the egg, and its
collection at B indicates only that the material in B
differs physicochemically from A and C. The real
determiners of the three different groups of organs are
three different groups of substances whose distribution
is approximately but probably not wholly identical
with the regions indicated by distribution of pigment.
The intestine-forming material is probably not entirely
lacking in C but is contained here in a lower concen-

Organisms from Eggs 131

tration and probably the more so the greater the dis-
tance from B; and the same may probably be said for
the substances determin-
ing mesenchyme and
ectoderm formation.
Hence the unfertilized
egg contains already a
rough preformation of
the embryo inasmuch as
the main axis of the em-
bryo and the arrange-
ment of its first organs

Fic. 10
are determined.

After the egg is fertilized the cell divisions begin.
The first division is as a rule at right angles to the
stratification of the
egg, each of the two
cells contains one-half
of the pigment ring
(and of each of A and
C) (Fig. 10), and after
the next division each
contains one-fourth of
the pigmented part.
Each of the four cells
is a diminutive whole
egg since each contains
the three layers in the normal arrangement (Fig. 11).

132 Organisms from Eggs

The next divisions bring about an unequal division
of the material. Four cells will be formed of ectoderm
material C and only
little intestine ma-
terial B, the other
four cells containing
BandA. These lat-
ter form at the next
division four very
small colourless
cells, the so-called
micromeres, A (Fig.
Fic. 12 12), from which the

mesenchyme, skele-

ton, and connective tissue are formed, four larger cells,
B, from which the intestine is formed, and eight cells,
C, from which the ectoderm will arise. The separa-
tion of the three groups of substances is probably not
as complete as our purely diagrammatic drawing (Fig.

12) indicates.

Thecell division proceeds and the cells become smaller
and smaller and all gather at the surface of the egg,
thus forming a hollow sphere. It is not known what
brings about this gathering of the cells at the surface,
whether it is protoplasmic creeping or streaming or
whether the cells are held by a jelly-like layer which
covers the surface of the egg (hyaline membrane) (Fig.
13). Then the cilia are formed at the external surface

Organisms from Eggs

133

of these cells and the egg begins to swim; we say it has
reached the first larval, the so-called blastula stage.

This happens according
to Driesch after the
tenth series of cell divi-
sions, when the number
of cells is theoretically
1024, in reality not quite
so many (between 800
and goo). The next step
consists in the cells de-
rived from the material
A (mesenchyme and mi-
cromeres) gliding into

the hollow sphere, where they form a ring, the physico-

chemical process respon-
sible for this gliding being
yet unknown. At the

Bp Opening of this ring an ac-

tive growing of the cells
of the entoderm into the
© hollow sphere takes place
and the hollow cylinder
formed by this growth
is the intestine (Fig. 14).
Why the cells grow into

the hollow sphere and not into the opposite direction

is unknown.

The next step is the formation of a

134 Organisms from Eggs

skeleton by the formation of crystals consisting of the
CaCO, by the mesenchyme cells surrounding the intes-
tine. For the establishment of the principle in which
we are interested the description of morphogenesis
need not be carried farther.

This principle which is under discussion here is the
development of a purposeful arrangement of organs
out of the egg. If we assume that the egg consists
of homogeneous material we are indeed confronted with
a riddle. Since the facts contradict such an assump-
tion but show, as Boveri has pointed out, a prearrange-
ment which allows us to indicate in the unfertilized
egg already the exact spot where the intestine will grow
into the blastula cavity, we are on solid physicochemical
ground, although many questions of detail cannot yet
be answered. Such a preformation as Boveri has de-
monstrated is only conceivable if the material of the
egg has not too high a degree of fluidity; we may con-
sider it as consisting essentially of a semi-solid gel
which is not homogeneous throughout the egg but
divided into three strata.

2. Lyon' tried to ascertain whether by centrifuging
the sea-urchin egg it was possible to modify its struc-
ture and thereby affect the later embryo. He and
subsequent experimenters found that it only is pos-

t Lyon, E. P., Arch. f. Entwcklngsmech., 1907, xxiii., 151; Morgan,
T. H., and Spooner, G. B., ibid., 1909, xxviii., 104; Morgan, Jour.
Exper. Zoél., 1910, ix., 594; Conklin, E. G., tbid., 1910, ix., 417; Lillie,
F.R., Biol. Bull., 1909, xvi., 54.

Organisms from Eggs 135

sible to change the position of the nucleus and the
distribution of the pigment in the egg. It follows from
this that the nucleus and the pigment are suspended
in rather fluid material, the former in the centre, the
pigment at or near the surface. The position of the
nucleus determines the first plane of segmentation,
since the nuclear division precedes the division of the
cytoplasm of the egg and the plane of nuclear division
becomes also the plane of the division of the whole egg
—a point which need not be discussed here. It was
found, however, by Lyon and the subsequent investi-
gators that the place where the micromeres are formed
and where the intestine of the embryo later originates
is little influenced by the centrifuging of the egg. The
localization of this spot must therefore be determined
by a structure sufficiently solid not to be shifted by the
centrifugal force. The intestinal stratum in the egg
contains the forerunners of the tissues which secrete hy-
drolyzing enzymes, e. g., trypsin into the digestive tract.

When the surrounding solution is altered in consti-
tution or when the temperature is too high, the intestine
instead of growing into the hollow sphere grows outside,
we get an evagination instead of an invagination of the
intestine. Such larve may live for a few days but they
cannot grow into a living organism. The forces which
make the intestine grow into the hollow sphere are
unknown; it may possibly be only the difference between
the tension on the external and internal surfaces of the

136 Organisms from Eggs

hollow sphere; under normal conditions, the resistance
on the inner surface being smaller, the intestine grows
into the hollow sphere.

The intestine is one of the organs required for the
self-preservation of a more complicated organism, in
fact a higher organism without a digestive tract is not
capable of living for any length of time. In the gastrula
—i. e., the blastula with an intestine—we have an
organism which is durable, but the processes leading up
to the formation of the intestine are so simple that it
is difficult to understand why the assumption of a
“‘supergene”’ should be required in this case.

3. Driesch* was the first to show that if we isolate
one of the first two cells of a dividing egg each develops
into a whole embryo of half size. This is perfectly
intelligible, since each of the two cells contains all the
three layers in the normal arrangement (Fig. 10). The
cells divide and the cells having the tendency to creep
to the surface of the mass arrange themselves in a hollow
sphere, the blastula. Since micromeres and intestine
material are present and in their normal position an
intestine will grow into the blastula and a whole or-
ganism will result. All of this is as necessary as is the
formation of one embryo from the whole egg material.
Yet the two half-embryos betray their origin from two
cleavage cells of the same egg, in that the two gastrule
formed are often if not always symmetrical to each

* Driesch, H., Ztschr. f. wissnsch. Zoél., 1891, liii., 160.

Organisms from Eggs 137

other (Fig. 15), as the writer had a chance to observe
in the egg of Strongylocentrotus purpuratus* in the fol-
lowing experiment. The eggs of the sea urchin Strongy-
locentrotus purpuratus are put soon after fertilization
into solutions which differ from sea water in two
points; namely that they are neutral or very faintly acid
(through the CO,

absorbed from the

air) instead of being

faintly alkaline, and

second, that. one of

the following three

constituents of the

sea water is lacking;

namely: K, Na, or Ca. When the eggs are allowed to
segment in such a solution the first two cleavage cells
are as a rule in a large percentage of cases—often as
many as ninety per cent.—separated from each other,
and when the eggs are put into normal sea water (about
twenty minutes after the cell division) each cell develops
into a normal embryo. In a number of cases the em-
bryos remained inside the egg membrane and did not
move until after the invagination of the intestine was
far advanced; in such cases it was found quite often
that the invagination began at the plane of cleavage at
symmetrical points of the two embryos, and the growth
of the intestine was symmetrical in both embryos.

t Loeb, J., Arch. f. Entwckingsmech., 1909, xxvii., 119.

Fic. 15

138 Organisms from Eggs

This symmetry is probably due to the following fact:
the first cleavage plane goes through that spot where
the intestine grows into the blastula cavity. If the
micromere material does not change its position after
the two cleavage cells are separated and the new blas-
tule do not become completely spherical the symmetry
which we observed is bound to occur. The occurrence
is a confirmation of Boveri’s observation. It is natural
that Driesch also found that each cell in the four-cell
stage should give rise to a full embryo, since each of these
cells is in reality a diminutive egg containing the three
strata in the right arrangement. When, however, the
cells of the eight- or sixteen-cell stage were isolated
Driesch’s results were different. In this case the isolated
cells from the ectoderm material did no longer all form
a gastrula; when such a cell still formed a gastrula it
was probably due to the fact that it contained some
entoderm material; while the cells taken from the
entoderm region all formed embryos and therefore
contained ectoderm material. The isolated ectoderm
cells of a blastula could no longer form an intestine;
they were lacking the entoderm material. It looks as
if a gradual migration of all the entoderm material
from the ectoderm into the entoderm took place during
the blastula formation.

When the contents of the egg are displaced by pres-
sure the result will be determined by the location of

* Driesch, H., Arch. f. Entwckingsmech., 1900, x., 361.

Organisms from Eggs 139

the main mass of the intestine-forming material; where
the main mass of this body is located the invagination
of the intestine will take place. In his earlier work
Driesch assumed from pressure experiments that the
egg had a great power of ‘‘regulation.’’ In a later
paper? he expressed to a large extent his agreement
with Boveri who denied this power of ‘‘regulation”’ and
showed that the existence of the structure of the egg—
7. €., a division into three strata, one forming the ecto-
derm, the second the entoderm, and the third the
mesoderm—was sufficient to explain the various pheno-

mena of apparent ‘‘regulation.’’ Driesch’s idea of a
regulation in this case has often been used to insist
upon the non-explicability of the phenomena of de-
velopment from a purely physicochemical view-point.
It is, therefore, only fair to point out that Boveri?
has furnished the facts for a simpler explanation, which
seems to have escaped the notice of antimechanists. 3
The objection may be raised that in accepting Boveri’s
facts and. interpretation we pushed the miracle only
one step farther and that we now have to explain the
origin of the structure in the unfertilized egg. This

Driesch, H., Arch. f. EntswckIngsmech., 1902, xiv., 500.

2 Boveri, Th., Verhandl. d. physik. med. Gesellsch., Warzburg, N.F.,
T1901, xxxiv., 145.

3v. Uexkill makes in his last book (Bausteine zu einer biologischen
Weltanschauung, Miinchen, 1913, p. 24) the following statement:
“Driesch suceeded in showing that the germ cell has no trace of a’
machine-like structure but consists entirely of equivalent parts.’ This
is not correct.

140 Organisms from Eggs

Boveri has done by showing that the egg grows from
the wall of the ovary and that that part of the egg
which is connected with the wall of the ovary gives
rise to the ectoderm layer, while the opposite part gives
rise to the mesenchyme and the intestine. This shows
a connection between the orientation of the egg in the
wall of the ovary and its stratification. While this
does not solve the problem of stratification in the egg
it gives the clue to its solution.

The ultimate origin of stratification probably goes
back to the fact of the presence of watery and water-
immiscible substances, such as fats. The experiments
by Beutner and the writer have shown that the electro-
motive forces which are observed in living tissues
originate at the boundaries between a watery and a water-
immiscible phase, like oleic acid or lecithin.’ In his
earlier writings? the writer had thought that the colloids
had special significance and this idea seems to prevail
today; but the actual observations have shown that
the phase boundary fat-water is of greater importance.
Needless to say the fats if not present in the cell
from the beginning can be formed in the metabolism.

4. All the ‘‘regulation” in the egg is of a purely
physicochemical character; it consists essentially of a
flow of material. If this idea is correct, the apparent

* Loeb, J., and Beutner, R., Biochem. Ztschr., 1912, xli., 1; xliv., 3033
1913, li., 288; li., 300; 1914, lix., 195.

2 Loeb, J., The Dynamics of Living Matter. New York, 1906. Intro-
ductory Remarks.

Organisms from Eggs 141

power of ‘‘regulation”’ of the blastomeres should differ
according to the degree of fluidity and the possibility
of different layers separating, and this assumption
is apparently supported by facts. The first plane of
segmentation of the egg is usually the plane of sym-
metry of the later organism and where the degree of
fluidity is less than in the sea-urchin egg, a separation
of the two first blastomeres should easily result in the
formation of two half-embryos instead of two whole
embryos.

This is the case for the frog’s egg as Roux showed in
a classical experiment. Roux destroyed one of the
two first cleavage cells of a frog’s egg with a hot needle
and found that as a rule the surviving cell developed
into only a half-embryo.' The frog’s egg consists of
two substances, a lighter one which is on top and a
heavier one below. Although viscous, the two sub-
stances are not too viscous to prevent a flow if the egg
is turned upside down. O. Schultze found that if a
normal egg is turned upside down in the two-cell stage
and held in that position, two full embryos arise, one
from each of the two blastomeres. Through the flow
of the lighter liquid in the egg upwards the two halves
of the protoplasm on top become separated and de-
velop independently into two whole embryos instead
of into two half-embryos. In Roux’s experiment this
flow of protoplasm was avoided. Morgan showed that

t Roux, W., Virchow’s Archiv, 1888, cxiv., 113.

142 Organisms from Eggs

if Roux’s experiment is repeated with the modification
that the egg is put upside down after the destruction
of the one cell, the intact cell will give rise not to a half
but to a whole embryo.*? These experiments prove
that each of the first two cleavage cells of the frog’s
egg represents one-half of the embryo and that a whole
embryo can develop from each half only when a re-
distribution of material takes place, which in the egg
of the frog can be brought about by gravitation since
the egg consists of a lighter and a heavier mass.

When, therefore, in the egg of the sea urchin each
of the first two blastomeres naturally gives rise to a
whole embryo it is due to a greater degree of fluidity
of the protoplasm and not to a lack of preformation
of the embryoin the cytoplasm. This idea is confirmed
by the observations on the egg of Ctenophores whose
cytoplasm seems to be more solid than that of most
other eggs. Chun found that the isolated blastomere
of the first cell division produced a half-larva, possessing
only four instead of the eight locomotor plates of the
normal animal.

It seems that in the egg of molluscs, also, the simple
symmetry relations of the body are already preformed.
It is well known that there are shells of snails which
turn to the right while others turn in the opposite
direction. The shells of Lymneus turn to the right,
those of Planorbis to the left. It was observed by

«Morgan, T. H., Embryology of the Frog. New York.

Organisms from Eggs 143

Crampton’, Kofoid, and Conklin that the eggs of
right-wound snails do not segment in a symmetrical,
but in a spiral, order, and that in left-handed snails
the direction of the spiral segmentation is the reverse
of that of the segmentation in the right-handed snails.
Conklin was able to show that the asymmetrical spiral
structure is already preformed in the egg before cleav-
age. The asymmetry of the body in snails is therefore
already preformed in the egg.?

E. B. Wilson’ has found a marked differentiation in
the eggs of some annelids and molluscs, He isolated
the first two blastomeres of the egg of Lanice, an Anne-
lid. These two blastomeres are somewhat different
in size; from the larger one of the first two blastomeres,
the segmented trunk of the worm originates. Wilson
found that

when either cell of the two-cell stage is destroyed, the re-
maining cell segments as if it still formed a part of an entire
embryo.4 The later development of the two cells differs
in an essential respect, and in accordance with what we
should expect from a study of the norma! development.
The posterior cell develops into a segmented larva with a
prototroch, an asymmetrical pre-trochal or head region, and

«Crampton, H. E., New York Academy of Sciences, 1894; Kofoid,
C. A., Proc. Am. Acad. Arts and Sciences, 1894, xxix.

2Conklin, E. G., Anat. Anzeig., 1903, xxiii., 577; Heredity and
Environment in the Development of Man. Princeton, 1915, p. 171.

3 Wilson, E. B., Science, 1904, xx., 748; Jour. Exper. Zodl., 1904, iy
I, 197.

4 The reader will notice the absence of “‘regulation.”

144 _ Organisms from Eggs

a nearly typical metameric seta-bearing trunk region, the
active movements of which show that the muscles are
normally developed. The pre-trochal or head region bears
an apical organ, but is more or less asymmetrical, and, in
every case observed, but a single eye was present, whereas
the normal larva has two symmetrically placed eyes. The
development of the anterior cell contrasts sharply with that
of the posterior. This embryo likewise produces a proto-
troch and a pre-trochal region, with an apical organ, but
produces no post-trochal region, develops no trunk or setz,
and does not become metameric. Except for the presence
of an apical organ, these anterior embryos are similar in
their general features to the corresponding ones obtained
in Dentalium. None of the individuals observed developed
a definite eye, though one of them bore a somewhat vague
pigment spot.

This result shows that from the beginning of develop-
ment the material for the trunk region is mainly localized
in the posterior cell; and, furthermore, that this material
is essential for the development of the metameric struc-
ture. The development of this animal is, therefore, to
this extent, at least, a mosaic work from the first cleavage
onward—a result that is exactly parallel to that which I
earlier reached in Dentalium, where I was able to show that
the posterior cell contains the material for the mesoblast,
the foot, and the shell; while the anterior cell lacks this
material. I did not succeed in determining whether, as
in Dentalium, this early localization in Lanice pre-exists in
the unsegmented egg. The fact that the larva from the
posterior cell develops but a single eye, suggests the possi-
bility that each of the first two cells may be already specified
for the formation of one eye; but this interpretation remains
doubtful from the fact that the larva from the anterior
cell did not, in the five or six cases observed, produce any
eye.

Organisms from Eggs 145

Conklin has established the existence of a definite
structure in the unfertilized eggs of Ascidians, Amphi-
oxus, and many molluscs. In all cases the results of
the isolation of the first blastomeres seem to agree with
the demonstrable structure of the unfertilized egg.

5. These examples may suffice to show that the
egg has from the beginning a simple structure, and
we will now point out by which means further differen-
tiation may come about. Sachs suggested that all
differentiation and the formation of every organ pre-
supposes the previous existence of specific substances
responsible for the formation. These substances which
are now called internal secretions or hormones develop
gradually during embryonic development. What ex-
ists first is a jelly-like block of protoplasmic material
with a varying degree of viscosity and with just enough
differentiation to indicate head and tail end, a right
and left, and a dorsal and ventral side of the future
embryo.

Aside from such simple differences phenomena of
protoplasmic streaming contribute to the further differ-
entiation. Such streaming begins, according to Conk-
lin,’ in the egg just before fertilization when the surface
layer of the egg protoplasm

Conklin, E. G., Heredity and Environment in the Development of
Man. Princeton University Press, 1915. The reader is referred to
this book for the literature and main facts on the structure of the egg;

it should also be stated that Conklin's book is one of the best introduc-
tions to modern biology in the English literature.

146 Organisms from Eggs

streams to the point of entrance of the sperm, and these
movements may lead to the segregation of different kinds
of plasma in different parts of the egg and to the unequal
distribution of these substances in different regions of the
egg.

One of the most striking cases of this is found in the
Ascidian Styela in which there are four or five different
kinds of substances in the egg which differ in colour, so that
their distribution to different regions of the egg and to
different cleavage cells may be easily followed and even
photographed while in the living condition. The peripheral
layer of protoplasm is yellow and when it gathers at the
lower pole of the egg where the sperm enters it forms a
yellow cap. This yellow substance then moves following
the sperm nucleus, up to the equator of the egg on the poste-
rior side and there forms a yellow crescent extending around
the posterior side of the egg just below the equator. On
the anterior side of the egg a grey crescent is formed ina
somewhat similar manner and at the lower pole between
these two crescents is a slate-blue substance, while at the
upper pole is an area of colourless protoplasm. The yellow
crescent goes into cleavage cells which become muscle and
mesoderm, the grey crescent into cells which become ner-
vous system and notochord, the slate-blue substance into
endoderm cells, and the colourless substance into ectoderm
cells.

Thus within a few minutes after the fertilization of the
egg and before or immediately after the first cleavage, the
anterior and posterior, dorsal and ventral, right and left
poles are clearly distinguishable, and the substances which
will give rise to ectoderm, endoderm, mesoderm, muscles,
notochord, and nervous system are plainly visible in their
characteristic positions."

* Conklin, E. G., loc. cit., p. 117.

Organisms from Eggs 147

We may finally allude briefly to the fact that when
once a number of tissues are differentiated each one
may influence the other by calling forth tropistic re-
actions. Thus the writer showed that in the yolk sac
of the fish Fundulus the pigment cells lie at first without
any definite order but that they gradually are compelled
to creep entirely on the blood-vessels and form a sheath
around them with the result that the yolk sac assumes
a tiger-like marking.* Driesch? has pointed out that
the mesenchyme cells are directed in their migration;
and it seems that the direction of the growth of the
axis cylinder is determined by the tissues into which
it grows. The idea of tropistic reactions in the forma-
tion of organs has been discussed by Herbst.

6. As a consequence of further changes definite
anlagen or buds originate later in the embryo which
are destined to give rise to definite organs. Thus in
the tadpole early mesenchyme cells are formed which
are the anlagen for the four legs, which will grow out
under the proper conditions. These anlagen are
specific inasmuch as from the anlage of a foreleg only
a foreleg, and from the anlage for a hindleg only a hind-
leg, will develop. Braus* has proved this by trans-

«Loeb, J., Jour. Morphol., 1893, xiii., 161; The Mechanistic Con-
ception of Life. Chicago, 1912, p. 106.

2 Driesch, H., Science and Philosophy of the Organism, i., p. 104.

3Herbst, C., Formative Reize in der tierischen Ontogenese. Leipzig,
1901.

+Braus, H., Miinchener Med. Wochnschr., 1903, 1 (II.), No. 47, p.
2076,

148 Organisms from Eggs

planting the anlage of a foreleg to different parts of
the body. No matter into which part of the body they
are transplanted the mesenchyme cells for the foreleg
will give rise to a foreleg only; even if they are trans-
planted into the spot from which the hindlegs grow out
under natural conditions. There is therefore nothing
to indicate ‘‘regulation.”’

The same is true for the formation of the eye and
probably in general. We have to consider the forma-
tion of the various organs of the body as being due to
the development of specific cells in definite locations
in the organisms which will grow out into definite
organs no matter into which part of the organism they
are transplanted. It is at present unknown what
determines the formation of these specific anlagen.
They may lie dormant for a long time and then begin
to grow at definite periods of development. We shall
see later that we know more about the conditions which
cause them to grow.

7. The fact that the egg, and probably every cell,
has a definite structure should determine the limits of
the divisibility of living matter. In most cases the
complete destruction of a cell means the cessation of
life phenomena. A brain or kidney which has been
ground to a pulp is no longer able to perform its func-
tions; yet we know that such pulps can still perform
some of the characteristic chemical processes of the
organ; é. g., the alcoholic fermentation characteristic

Organisms from Eggs 149

of yeast can be caused by the press juice from yeast;
or characteristic oxidations can be induced by the
ground pulp of organs. The question arises as to how
far the divisibility of living matter can be carried with-
out interfering with the total of its functions. Are
the smallest particles of living matter which still exhibit
all its functions of the order of magnitude of molecules
and atoms, or are they of a different order? The first
step toward obtaining an answer to this question was
taken by Moritz Nussbaum,* who found that if an
infusorian be divided into two pieces, one with and
one without a nucleus, only the piece with a nucleus
will continue to live and perform all the functions of
self-preservation and development which are character-
istic of living organisms. This shows that at least two
different structural elements, nucleus and cytoplasm,
are needed for life. We can understand to a certain
extent from this why an organ after being reduced to
a pulp, in which the differentiation into nucleus and
protoplasm is definitely and permanently lost, is unable
to accomplish all its functions. ?

The observations of Nussbaum and those who re-
peated his experiments showed that although two differ-
ent structures are required, not the whole mass of an

tNussbaum, M., Arch. f. mikroscop. Anat., 1886, xxvi., 485.

2It must not be overlooked that in bacteria and the blue alge no

distinct differentiation into nucleus and protoplasm can be shown. To
these organisms, therefore, the experiments of Nussbaum cannot be

applied.

150 Organisms from Eggs

infusorian is needed to maintain its life. The question
then arose: How small a fraction of the original cell
is required to permit the full maintenance of life? The
writer tried to decide this question in the egg of the
sea urchin. He had found a simple method by which
the eggs of the sea urchin (Arbacia) can easily be divided
into smaller fragments immediately after fertilization.
When the egg is brought from five to ten minutes after
fertilization (long before the first segmentation occurs)
into sea water which has been diluted by the addition
of equal parts of distilled water, the egg takes up water,
swells, and causes the membrane to burst. Part of the
protoplasm then flows out, in one egg more, in another
less. If these eggs are afterward brought back into
normal sea water those fragments which contain a
nucleus begin to divide and develop. It was found
that the degree of development which such a fragment
reaches is a function of its mass; the smaller the piece,
the sooner as a rule its development ceases. The small-
est fragment which is capable of reaching the pluteus
stage possesses the mass of about one-eighth of the
whole egg. Boveri has since stated that it was about
one twenty-seventh of the whole mass. Inasmuch
as only the linear dimensions are directly measurable,
a slight difference in measurement will cause a great
discrepancy in the calculation of the mass. Driesch’s

1 Loeb, J., Arch. d. f. ges. Phystiol., 1893, lv., 525.

Organisms from Eggs I51

results disagree with the statement of Boveriand support
the observation of the writer.

If we raise the question why such a limit exists in
regard to the divisibility of living matter, it seems
probable that only those fragments of an egg are
capable of development into a pluteus which contain a
sufficient amount of material of each of the three layers.
If this be correct, it would certainly not suffice to mix
the chemical constituents of the egg in order to produce
a normal embryo; this would require besides the proper
chemical substances a definite arrangement or structure
of this material. The limits of divisibility of a cell
seem therefore to depend upon its physical structure
and must for this reason vary for different organisms .
and cells. The smallest piece of a sea-urchin egg that
can reach the pluteus stage is still visible with the naked
eye, and is therefore considerably larger than bacteria
or many alge, which also may be capable of further
division.

8. The most important fact which we gather from
these data is that the cytoplasm of the unfertilized egg
may be considered as the embryo in the rough and that
the nucleus has apparently nothing to do with this
predetermination. This must raise the question sug-
gested already in the third chapter whether it might
not be possible that the cytoplasm of the eggs is the
carrier of the genus or even species heredity, while the
Mendelian heredity which is determined by the nucleus

152 Organisms from Eggs

adds only the finer details to the rough block. Sucha
possibility exists, and if it should turn out to be true
we should come to the conclusion that the unity of the
organism is not due to a putting together of a number
of independent Mendelian characters according to a
‘‘pre-established plan,” but to the fact that the organ-
ism in the rough existed already in the cytoplasm of
the egg before the egg was fertilized. The influence
of the hereditary Mendelian factors or genes consisted
only in impressing the numerous details upon the rough
block and in thus determining its variety and individ-
uality; and this could be accomplished by substances
circulating in the liquids of the body as we shall see
* in later chapters.

CHAPTER VII
REGENERATION

I. The action of the organism as a whole seems
nowhere more pronounced than in the phenomena of
regeneration, for it is the organism as a whole which
represses the phenomena of regeneration in its parts,
and it is the isolation of the part from the influence
of the whole which sets in action the process of regenera-
tion. The leaf of the Bermuda ‘‘life plant’’—Bryo-
phyllum calycinum—behaves like any other leaf as long
as it is part of a healthy whole plant, while when isolated
it gives rise to new plants. The power of so doing was
possessed by the leaf while a part of the whole, and it
was the ‘‘whole” which suppressed the formative forces
in the leaf. When a piece is cut from the branch of a
willow it forms roots near the lower end and shoots at
the upper end, so that a tolerably presentable ‘‘whole”’
is restored. How does the ‘‘whole’’ prevent the basal
end of the shoot from forming roots as long as it is part
of the plant? A certain fresh-water flatworm has the
mouth and pharynx in the middle of the body. Whena

153

154 Regeneration

piece is excised between the head and the pharynx a
new head is formed at the oral end, a new tail at the
opposite end, and in the middle of the remaining old
tissue a new mouth and pharynx is formed. How does
the ‘“‘whole’’ suppress all this formative power in the
part before the latter is isolated? It almost seems as
if the isolation itself were the emancipation of the part
from the tyranny of the whole. The explanation of this
tyranny or of the correlation of the parts in the whole
is to be found, however, in a different influence. The
earlier botanists, Bonnet, Dutrochet, and especially
Sachs,* pointed out that the phenomena of correlation
are determined by the flow of sap in the body of a
plant. These authors formulated the idea that the
formation of new organs in the plant is determined by
the existence of specific substances which are carried
by the ascending or descending sap. Specific shoot-
producing substances are carried to the apex, while
specific root-producing substances are carried to the
base of a plant. When a piece is cut from a branch of
willow the root-forming substances must continue to
flow to the basal end of the piece, and since their further
progress is blocked there they induce the formation of
roots at the basal end. Goebel? and de Vries have

ty* Sachs, J., “Stoff und Form der Pflanzenorgane,” Gesammelte
Abhandlungen, 1892, ii., 1160. Arbeiten a. d. bot. Inst. Wurzburg,
1880-82.

2 Goebel, K., Einleitung in die experimentelle Morphologie der Pflanzen,
1908.

Regeneration 155

accepted this view and the writer made use of it in his
first experiments on regeneration and heteromorphosis
in animals.‘ At that time the idea of the existence of
such specific organ-forming substances was received
with some scepticism, but since then so many proofs
for their existence have been obtained that the idea
is no longer questioned. Such substances are known
now under the name of ‘‘internal secretions” or ‘‘hor-
mones’’; their connection with the theory of Sachs was
forgotten with the introduction of the new nomenclature.

It may be well to enumerate some of the cases in
which the influence of specific substances circulating
in the blood upon phenomena of growth has been
proven. One of the most striking observations in this
direction is the one made by Gudernatsch on the growth
of the legs of tadpoles of frogs and toads.?, The young
tadpoles have no legs, but the mesenchyme cells from
which the legs are to grow out later are present at an
early stage. From four months to a year or more may
elapse before the legs begin to grow. Gudernatsch
found that legs can be induced to grow in tadpoles at
any time, even in very young specimens, by feeding
them with the thyroid gland (no matter from what

tLoeb, J., Untersuchungen zur physiologischen Morphologie der
Tiere. I. Heteromorphose. Wurzburg, 1891. II. Organbildung und
Wachsthum. 1892. Reprinted in Studies in General Physiology.
Chicago, 1906.

2Gudernatsch, J. F., Zentralbl. f. Physiol., 1912, xxvi., 323; Arch. f.
Entwcklngsmech., 1912, Xxxv., 457; Am. Jour. Anat., 1914, Xv., 431.

156 Regeneration

animal). No other material seems to have such an
effect. The thyroid contains iodine, and Morse* states
that if instead of the gland, iodized amino acids are
fed to the tadpole the same result can be produced.
We must, therefore, draw the conclusion that the normal
outgrowth of legs in a tadpole is due to the presence
in the body of substances similar to the thyroid in their
action (it may possibly be thyroid substance) which
are either formed in the body or taken up in the food.
Thus we see that the mesenchyme cells giving rise to
legs may lie dormant for months or a year but will grow
out when a certain type of substances, e. g., thyroid,
circulates in the blood. There may exist an analogy
between the activating effect of the thyroid substance
and the activating effect of the spermatozo6n or butyric
acid (or other parthenogenetic agencies) upon the egg,
but we cannot state that the thyroid substance activates
the mesenchyme cells by altering their cortical layer.
The fact that the substance of the thyroid may
induce general growth in the human is too well known
to require more than an allusion in this connection.
When growth stops in children as a consequence of a
degeneration of the thyroid, feeding of the patient with
thyroid again induces growth. It may also suffice
merely to call attention to the connection between
acromegaly and the hypophysis.
It was formerly believed that the nervous system
t Morse, M., Jour. Biol. Chem., 1914, xix., 421.

Regeneration 157

acted as a regulator of the phenomena of metamor-
phosis in animals, but it was possible to show by simple
experiments that the central nervous system does not
play this r6le and that the regulator must be the blood
or substances contained therein. In the metamorphosis
of the Amblystoma larva the gills at the head and tail
undergo changes simultaneously, the gills being ab-
sorbed completely. The writer showed that in larve
in which the spinal cord was cut in two, no matter at
which level,—the sympathetic nerves were in all prob-
ability also cut—the two organs continued to undergo
metamorphosis simultaneously.* Uhlenhuth found that
if the eye of a salamander larva is transplanted into
another larva the transplanted eye undergoes its
metamorphosis into the typical eye of the adult form,
simultaneously with the normal eyes of the individual
into which it was transplanted. These and other
observations of a similar character leave no doubt that
substances circulating in the blood and not the central
nervous system are responsible for the phenomena of
growth and metamorphosis.

An interesting observation on the rdéle of internal
secretion in growth was made by Leo Loeb. When

t Loeb, J., Arch. f. Entwcklngsmech., 1897, iv., 502.

2 Uhlenhuth, E., 2bid., 1913, xxxvi., 211.

3 Loeb, Leo, Zentralbl. f. allg. Path. u. path. Anat., 1907, xviii., 563;
Zentralbl. f. Physiol., 1908, xxii., 498; 1909, xxiii., 73; 1910, xxiv., 2033
Arch. f. Entweklngsmech., 1909, xxvii., 89, 463; Jour. Am. Med. Assoc.,
1908, 1., 1897; 1909, liii., 1471.

158 Regeneration

the fertilized ovum comes in contact with the wall of
the uterus it calls forth a growth there, namely the
formation of the maternal placenta (decidua). This
author showed that the corpus luteum of the ovary gives
off a substance to the blood which alters the tissues in
the uterus in such a way that contact with any foreign
body induces this deciduoma formation. The case is of
interest since it indicates that the substance given off
by the corpus luteum does not induce growth directly,
but that it allows mechanical contact witha foreign body
to do so while without the intervention of the corpus
luteum substance no such effect of the mechanical stim-
ulus would be observable. The action of the substance
of the corpus luteum is independent of the nervous
system, since in a uterus which has been cut out and
retransplanted the same phenomenon can be observed.

Bouin and Ancel' have shown that the corpus luteum,
which in the case of pregnancy continues to exist for a
long time, is responsible for the changes in the mammary
gland in the first half of pregnancy, when an active
cell proliferation takes place in the gland. This process
can be interrupted by destroying the corpus luteum
artificially. During the second half of gravidity no
further cell proliferation takes place, but the cells begin
to secrete milk while during the period of cell prolifera-
tion such secretions do not occur.

* Quoted from M. Caullery, Les Problémes de la Sexualité, Paris, 1913,
p. 126.

Regeneration 159

Claude Bernard and Vitzou had shown that the
period of growth and moulting of the higher crustacea
is accompanied by a heaping up of glycogen in the liver
and subdermal connective tissue. Smith’ found that
during the period between two moultings, when there is
no growth, the storage cells are seen to be filled with
large and numerous fat globules instead of with glyco-
gen. He also found that in the Cladocera ‘‘the period
of active growth is accompanied by glycogen—as
opposed to fat—metabolism.”” He observed, moreover,
that if Cladocera are crowded at a low temperature the
fat metabolism (with inhibition to growth) is favoured,
while at high temperatures and with no crowding of
individuals the glycogen metabolism is favoured. In
the latter case a purely parthenogenetic mode of propa-
gation is observed, while in the former sexual reproduc-
tion takes place. The effect of crowding of individuals
is possibly due to products of excretion, which then act
on growth and reproduction indirectly by changing the
‘“slycogen metabolism” to ‘‘fat metabolism.”

All these cases agree in this, that apparently specific
substances induce or favour growth, not in the whole
body, but in special parts of the body. Sachs suggested
that there must be in each organism as many specific
organ-forming substances as there are organs in the
body.

We will now show that the assumption of the exist-

t Smith, Geoffrey, Proc. Roy. Soc., B. 1915, Ixxxviii., 418.

160 Regeneration

ence of such “‘organ-forming” substances (which may
or may not be specific) and of their flow in definite
channels explains the inhibitory influence of the whole
on the parts as well as the unbridled regeneration of the
isolated parts.

2. We have seen that the resting egg can be aroused
to development and growth by substances contained in
a spermatozo6n or by certain other substances men-
tioned in the preceding chapter. We will assume that
plants contain a large number of cells or buds which are
comparable to the resting egg cell, but which can be
aroused to action by certain substances circulating in
the sap; and that the same is effected for animal cells
by substances in the blood. In plants the cells which
can be aroused to new growth have very often a rather
definite location while in lower animals they are more
ubiquitous. For experimental purposes organisms
where these buds have a definite location are more
favourable, since we are better able to study the
mechanism underlying the process of activation and
inhibition (correlation). When a leaf of the plant
Bryophyllum calycinum is cut off and put on moist sand
or into water or even into air saturated with water
vapour, new plants will arise from notches of the leaf.
This is the usual way of propagating the plant and in no
other part of the leaf except the notches will new plants
arise. These notches therefore contain cells comparable
to seeds or to unfertilized eggs or to the mesenchyme

Regeneration 161

cells which give rise to legs in the tadpole of the frog.
The question arises: Why do notches in the leaf never
begin to grow while the leaf is attached to an intact
plant, and why do they grow when the leaf is isolated?
To this we are inclined to give an answer in the sense
of Bonnet, Sachs,
de Vries, and
Goebel, namely
that the flow of
(specific?) sub-
stances in the
plant determines
when and where
dormant buds or

anlagen shall be-

intogrow. Such Fis. 16. Growth of roots and shoots in a
Panes few notches of an isolated leaf
substances may of Bryophyllum ca'ycinum

originate or may

be present in the leaf; but as long as it is connected
with a normal plant they will be carried by the cir-
culation to the growing points of the stem and of the
roots and they cannot reach the notches; while when we
detach the leaf, either a new distribution or a new flow
of liquids will be established whereby the substances
reach some of the notches; and in these notches new
roots and a new shoot will be formed. When we
cut off a leaf and put it into moist air, not all but only
a few of the notches will, as a rule, grow out (Fig. 16);

162 Regeneration

but when we isolate each notch leaving as much
of the rest of the leaf as possible attached to it, each
notch will give rise to a new plant. (Fig. 17.) We
see, therefore, that it does not even require a whole
plant to cause inhibition but that we may observe the
tyranny of the whole over the parts in a single leaf.
The explanation
is as follows:
When we isolate
a leaf, some of
the notches will
commence to
grow into new
plants and this
growth will ar-
rest the develop-
ment of the

Fic. 17. If all the notches of a leaf are iso-
lated from each other each notch will give rise
to roots and a shoot, but the growth willbe other notches of
less rapid than in Fig. 16. Figs. 16 and 17 ;
were two leaves taken from the same node the leaf in the

of a plant. same way as
their development was suppressed by the whole plant.
The explanation is the same; those notches which
begin to grow first will attract the flow of substances
to themselves, thus preventing the other notches from
getting those substances. This idea is supported by
the fact that if all the notches are isolated from the
leaf each notch will give rise to a slowly growing

* Loeb, J., Bot. Gazette, 1915, Ix., 249.

Regeneration 163

plant, while if the leaf is not cut into pieces, and a few
notches only grow out, their growth is much more rapid.

In all these experiments the idea that the ‘‘isolation”
in itself is responsible for the growth still presents itself.
It can be disposed of by the following experiment which
never fails. Three leaves of Bryophyllum calycinum
are suspended in an atmosphere saturated with water
vapour but their tips are submersed in water (Figs.
18, 19, 20). The first leaf, Fig. 20, is entirely separated
from its stem, the second leaf, Fig. 19, remains connected
with the adjacent piece of stem, and the third leaf,
Fig. 18, remains also connected with this piece of stem
but the latter still possesses both leaves. The first leaf,
Fig. 20, produces new roots and shoots in the submerged
part in a few days; the second leaf, Fig. 19, produces
no roots or shoots for a long time. This might find its
explanation by the assumption that the first leaf, being
more isolated than the second, regenerates more quick-
ly. But this explanation becomes untenable owing to
the fact that the third leaf, Fig. 18, being less isolated
than both (possessing a second leaf in addition to
the stem), forms new roots and shoots also more
quickly than the second leaf. The phenomena become
intelligible in the following way. The fact that in the
second leaf shoots and roots are formed very late, if at
all, finds its explanation not in the lessened isolation
of this leaf, but in the fact that the formation of a
new shoot or of a callus in the piece of stem takes place

164 Regeneration

more quickly than the formation of roots and shoots
in the notches of a completely isolated leaf. The stem

Fic. 18 Fic. 19 Fic. 20

acts therefore as a centre of suction for the flow of
substances from the leaf and this prevents or retards
the formation of roots and shoots in the notches. In
the isolated leaf of Bryophyllum calycinum no callus
formation takes place and hence no flow of the sap

Regeneration 165

away from the leaf will occur. This will allow one or
more of the notch buds of this leaf to grow out and then
a flow will be established towards these growing buds.

In the third specimen, Fig. 18, the presence of two
leaves suppresses or, as a rule, retards the growth of a
shoot on the stem and possibly also the flow from one
leaf may block to some extent the flow from the op-
posite leaf if the piece of stem is very short. This puts
the leaves in a condition not as good as that in leaf Fig.
20, but better than in leaf Fig. 19.*

In the normal plant the buds in the notches of the
leaf remain dormant since the flow of the ‘‘stimulating”’
substances takes place towards the tips of the stem and
root, and because these substances are retained there in
excess. This is probably the real basis of the mysterious
dominance of the ‘‘whole”’ over its ‘‘parts” or of the
anlagen of the tip of the stem over those farther below.
When a piece of the stem of Bryophyllum is cut off and
its leaves are removed, the two apical buds will grow
out first. This ‘“‘dominance” finds its explanation
probably in the anatomical structure and the mechan-
ism of sap flow which tend to bring the “‘stimulating”’
substances first to the anlagen in the tip. In Laminaria
Setchell has been able to show directly that regenera-
tion always starts from that tissue which conducts the
nutritive material.

When we cut out a piece of a stem of Bryophyllum,

t With larger leaves the experiment may also succeed in moist air.

166 Regeneration

and remove all:the leaves, new shoots will be formed
from the two apical buds of the stem, and roots will
arise from the most basal nodes; provided that the stem
is suspended in air saturated with water vapour. The
growth in such a stem deprived of all leaves is slow. Ii
we remove all the leaves on such a piece of stem except
the two at the apical end, the stem will form only roots,
but these will develop much more rapidly than on a
stem without leaves. If we remove all the leaves
except the two at the basal end, the stem will only
form shoots (at the apical end) but these will develop
much more rapidly than in a leafless stem. Hence the
leaves accelerate the growth of roots towards the basal
end and inhibit it towards the apical end; and they
favour the growth of shoots towards the apical end and
inhibit it in the nodes located nearer the base.

We thus see that while the stem inhibits the growth
of the leaves connected with it, the latter accelerate the
growth in the stem. Both facts can probably be
explained on the same basis; namely, on the assumption
that it is the flow of substances from the leaf to the
stem which inhibits the growth of the notches and ac-
celerates the growth of the buds in the stem. On this
assumption it would also follow that the leaves send
root-forming substances towards the basal and shoot-
forming substances towards the apex of the stem. It
also seems to follow from recent as yet unpublished ex-
periments by the writer that the root-forming substances

Regeneration 167

are associated or identical with the substances which
cause geotropic curvature in the stem.

These observations show that
the phenomena of correlation or W
of the influence of the whole over
the parts is due to peculiarities of
circulation or the flow of sap; and

that the isolation prevents the sap
from flowing away to other parts

of the plant. There is no need for
assuming the existence of a mys- _
terious force which directs the piece :

Fic, 21

to grow into a whole.
3. Phenomena of inhibition or correlation such as
we have described in Bryophyllum are not lacking in
the regeneration of animals, as experiments on Tubu-
laria show.* Tubularia mesembryanthemum (Fig. 21)
is a hydroid consisting of a long stem terminating at
one end in a stolon

>= = which attaches it-
self to solid bodies

such as rocks, at
the other end in a polyp. The writer found that if
we cut a piece from a stolon and suspend it in an aquar-
ium it forms as a rule a polyp at either end (Fig. 22),

Fic. 22

tLoeb, J., Untersuchungen zur physiologischen Morphologie. I.
Heteromorphose. 1891. II. Organbildung und Wachsthum. Wiarz-

burg, 1892.

168 Regeneration

but the velocity with which the two polyps are
formed is not the same, the polyp at the oral end
of the piece being formed much more rapidly—a day
or one or two weeks sooner—than the aboral polyp.
The process of polyp regeneration at the aboral pole
could, however, be accelerated and its velocity made
equal to that of the regeneration of the oral polyp by
suppressing the formation of the latter. This was
accomplished by depriving the oral pole of the oxygen
necessary for regeneration, e. g., by merely putting the
oral end of the piece of stem into the sand. It was,
therefore, obvious that the formation of the oral polyp
retarded the formation of the aboral polyp. This
inhibition might have been due to the fact that a
specific organ-forming material needed for the forma-
tion of a polyp existed in sufficient quantity in the stem
for the formation of one polyp only at a time. This
idea, however, was found to be incorrect since when the
stem was cut into two or more pieces each piece formed
a polyp at once at its oral pole and regenerated the
aboral polyps also, but again with the usual delay. It
seemed more probable then that the cause of the
difference in the rapidity of polyp formation at both
ends lay in the fact that certain material flowed first
to the oral pole and induced polyp formation here but
that this flow was reversed as soon as the polyp at the
oral pole was formed or as soon as the formation of the
oral polyp was inhibited by lack of oxygen. The partial

Regeneration 169

or full completion of the formation of the oral polyp
acted as an inhibition to the further flow of material to
this pole. This idea was supported by an observation
made independently by Godlewski and the writer that
if a piece of stem be cut out of a Tubularia, and if the
piece be ligatured somewhere between the
two ends, the oral and the aboral polyps
are formed simultaneously. This would
be comprehensible on the assumption that
the retarding effect which the formation
of the oral has on the aboral polyp was
indeed of the nature of a flow of material
towards the oral pole.

Miss Bickford: found that the differ-
ence in time between the formation of
the two polyps disappears also when the
piece cut from the stem becomes so small
that it is of the order of magnitude of a
single polyp. In that case two incomplete polyps are
formed simultaneously at each end (Fig. 23). The
new head in the regeneration of Tubularia arises, as
Miss Bickford observed, from the tissue near the wound.
At some distance from the wound in the old tissue
two rows of tentacles arise, which are noticeable as
rows of longitudinal lines inside the stem before the
head is formed. Driesch noticed that the newly
formed head is the smaller the smaller the whole

t Bickford, E. E., Jour. Morphol., 1894, ix., 417.

Fic. 23

170 Regeneration

piece. (This is true, however, only in rather small
pieces.) There is, therefore, in small pieces a rough pro-
portionality between size of head and size of regenerat-
ing piece. Driesch’ uses this interesting fact to prove
the existence of an entelechy, while we are inclined to
see in it an analogue to the observation of Leo Loeb,
that the velocity of the process of healing in the case
of a deficiency of the epithelium. decreases when the
size of the uncovered area diminishes. While we do
not wish to offer any suggestion concerning the me-
chanism of these quantitative phenomena—they may
be related in some way with the velocity of certain
chemical reactions—we see no reason for assuming
that they cannot be explained on a purely physico-
chemical basis.

The writer noticed that certain pigmented cells
from the entoderm of the organism always gather at
that end where a new polyp is about to be formed.
These red or yellowish cells always collect first at the
oral end of a piece of stem. It may be that certain
substances given off by the pigmented cells at the cut
end are responsible for the polyp formation, but this is
only a surmise.

Another suggestion made by Child,? is that there
exists an axial gradient in the stem whereby the cells

* Driesch, H., Science and Philosophy of the Organism, i., 127.
2 Child, C. M., ‘Die physiologische Isolation von Teilen des Organ-
ismus,’’ Roux’s Vortrdge und Aufsdize, Leipzig, 1911.

Regeneration 171

regenerate the more quickly the nearer they are to the
oral pole. If this were correct, and we cut a long piece
from the stem of a Tubularia and bisect the piece, the
oral pole of the anterior half should regenerate more
quickly than the oral pole of the posterior half. Accord-
ing to the writer's observations on a Tubularian (T.
crocea) growing in the estuaries ae
near Oakland, California, both oral
ends regenerate equally fast in such \
cases.

4. The phenomena of regenera- 14
tion in Cerianthus membranaceus,
a sea anemone, can be easily under-
stood from the experiments on
Tubularians, if we imagine the

body wall of Cerianthus to consist ,

e d

of a series of longitudinal elements
running parallel to the axis of sym-
metry of the animal from the tenta-
cles to the foot. The number of these elements may
be supposed to correspond to the number of tentacles
in the outer row of the normal animal. Each such ele-
ment behaves like a Tubularian, with this difference,
however, that the elements in Cerianthus are more
strongly polarized than in Tubularia, and that each
one is able to form a tentacle at its oral pole only.
This fact can be nicely illustrated in the following way:
if a square or oblong piece (abcd, Fig. 24) be cut from

Fic. 24

172 Regeneration

the body wall of a Cerianthus in such a way that
one side, ac, of the oblong is parallel to the longitudinal
axis of the animal, tentacles will grow on one of the
four sides only; namely, on the side a b.* (Fig. 25.)
The other three free edges are not able to produce
tentacles. If an incision be made in the body wall of

Fic. 25 Fic. 26

a Cerianthus, tentacles will grow on the lower edge of
the incision (Fig. 26).

The writer tried whether or not by tying a ligature
around the middle of a piece of an Actinian this polarity
could be suppressed; but the experiments did not
succeed, inasmuch as the cells compressed by the liga-
ture died, and were liquefied through bacterial action so
that the pieces in front-and behind the ligature fell
apart. It is therefore impossible to decide whether or
not a current or a flow of substances in a certain direc-

«Loeb, J., ‘Untersuchungen zur physiologischen Morphologie der
Tiere.”’

Regeneration 173

tion through these elements is responsible for this
polarity, though this may be possible. The writer
found, however, that one condition is necessary for the
growth and regeneration of tentacles which also plays a
réle in the corresponding phenomena in plants, namely
turgidity. The tentacles of Cerianthus are hollow
cylinders closed at the tip, and by
liquid being pressed into them they
can be stretched and appear turgid. If,
however, an incision is made in the
body, the tentacles above the incision
can no longer be stretched out. In one
experiment the oral disk of a Cerian-
thus was cut off; very soon new tenta-
cles began to grow at the top, and after
having reached a certain size, an incision
was made in the animal. The tentacles
above the incision collapsed in conse-
quence and ceased to grow, while growth Fic. 27

of the others continued. On the lower

edge of the incision new tentacles began to grow.

It seems also possible that Morgan’s well-known ex-
periment on regeneration in Planaria can be explained by
a flow of substances. He? found that if a piece acd b
be cut out of a fresh-water Planarian at right angles
to the longitudinal axis (Fig. 27), at the front end anew
normal head, at the back end a new tail, will be regen-

t Morgan, T. H., Regeneration, New York, 1901.

174 Regeneration

erated (Fig. 28); but that if a piece acd bbe cut froma
Planarian obliquely (Fig. 29) instead of at right angles to
the longitudinal axis a tiny head is
a <>: formed at the foremost corner of the
a = piece a and a tiny tail at the hindmost
corner b (Fig. 30). Why isit that in the
oblique piece the head is formed in the
corner and not all along the
cut surface as is the case when ~ Qo
the cut is made at right angles NX
to the longitudinal axis? The |
writer is inclined to believe that io
the right answer to this ques- d

Fic. 28

tion has been given by Bar-
deen.' This author has pointed

out the apparent réle that the >

circulatory (or so-called di-
gestive) canals in Planarians
play in the localization of the
phenomena of regeneration, in- Fic. 29
as-much

as the new head always forms
symmetrically at the opening of
the circulatory vessel or branch
which is situated as much as
Fic. 30 possible at the foremost end of the

t Bardeen, C. R., Am. Jour. Physiol., 1901, v., 1; Arch. f. Entweklngs-
mech., 1993, Xvi., I.

Regeneration 175

regenerating piece of worm. He assumes that through
muscular action the liquids of the body are forced to
stream toward this end, and that this fact has some con-
nection with the formation of a new head. There can
be no doubt that the facts here mentioned agree with
Bardeen’s suggestion. The oblique pieces in Morgan’s
experiments which at first have the heads and tails

Fic. 31 cy Fic. 32

outside the line of symmetry of the middle piece,
gradually assume a normal position (Figs. 31, 32).
’ The writer is inclined to believe that this is due to
mechanical conditions. The head a ec of such an
oblique piece is asymmetrical, the one side a e being less
stretched than the other ec. The higher tension of the
piece e ¢ will have the effect of bringing e nearer c, since
we know that acid formation and hence energy pro-
duction increases in proportion to surface, 7. e., it must
be the greater the more it is stretched. The reverse is
true for the tail d f 6, and the effect here will be that f

176 Regeneration

will be pulled nearer d. In this way purely mechanical
conditions are responsible for the fact that the soft
tissues of the animal are gradually restored to their
true orientation.

As a final possible example of the influence of internal
secretion or substances contained in the blood may
be mentioned the following curious observation of
Przibram.t In a crustacean, Alpheus, the two chelz
(pincers) are not equal in size and form, one being
very much larger than the other. Przibram found that
when he cut off the larger pincer in such crustaceans
the remaining pincer assumes in the next moulting
the size and shape of the removed large pincer; while
in place of the removed pincer one of the small type
is produced. Hence a reversal of the two pincers is
thus brought about. If later on the large pincer is
again cut off the process is repeated and the original
dissymmetry is restored. Przibram was able to show
that the nervous system has no connection with this
phenomenon.

The elements which have entered into the discussion
thus far are, first, the flow of substances in preformed
channels; second, the existence of general or specific
substances required for the growing or regenerating
organ. A third element is to be added; namely the
“‘suction”’ effect upon these substances of a developing
organ. Thus we see that if one or a few of the notches

*Przibram, H., Arch. f. Entwcklngsmech., 1901, xi., 329.

Regeneration 177

in a leaf of Bryophyllum grow out the other notches-of
the leaf are inhibited from growing. There is enough
material present in the leaf for all the notches to grow
into shoots as is proved by the fact that all will grow
out if they are isolated from each other. This was
explained on the assumption that the notches of a
whole which happen to develop first, create a flow of
these substances from the rest of the leaf to themselves
and thus prevent any getting to the other notches. We
stated that this is supported by the fact that the few
notches growing out in an undivided leaf grow more
rapidly than the many shoots growing from each notch
of a divided leaf. But why should a growing'shoot or a
growing point in general produce such a suction? I
think this may be possible on the assumption that the
consumption of these substances by the growing organs
causes a low osmotic pressure of these substances in the
growing region and this fall of osmotic potential will
act as a cause for the further flow. This brings about
the apparent ‘‘suction”’ effect of the growing elements
upon the flow of substances.

5. We mentioned that when a piece is cut from a
Planaria between pharynx and head a new mouth is
formed in the middle. It should also be mentioned that
according to Child the piece after regeneration is
smaller than it was before.* This indicates that
material in the old cells has been digested or has under-

t Child, C. M., Senescence and Rejuvenescence. Chicago, 1915.
12

178 Regeneration

gone hydrolysis in order to furnish the nutritive material
for the new head and tail, since the piece cannot take
up any food from the outside before a mouth is formed.
These phenomena of autodigestion—the process itself
will be discussed in the last chapter—seem to occur in

many (if not all) phenomena of regeneration. It may
be that the collect-
ing of red cells at
the end in a Tubu-
larian where regen-
eration is about to
begin has to do with
the furnishing of
material by self-
digestion, since these
Fic. 33 cells are partly at
least destroyed in
the process. It is of interest to look for more ex-
amples of autodigestion accompanying phenomena
of regeneration.

The writer has observed more closely the transforma-
tion of an organ into more undifferentiated material in
Campanularia (Fig. 33), a hydroid.t This organism
shows a remarkable stereotropism. Its stolons attach
themselves to solid bodies, and the stems appear on
the side of the stolon exactly opposite the point or
area of contact with the solid body. The stems

t Loeb, J., Am. Jour. Physiol., 1900, iv., 60.

Regeneration 179

grow, moreover, exactly at right angles to the solid
surface element to which the stolon is attached. If such
a stem be cut and put into a watch glass with-
sea water, it can be observed that those polyps
which do not fall
off go through a
series of changes
2 which make it ap-
pear as if the dif-
ferentiated material of the polyp were
transformed into undifferentiated ma-
terial. The tentacles are first put to-
é gether like the hairs of a camel’s-hair
Fic. 34 brush (Fig. 34), and gradually the whole
fuses to a more or less shapeless mass
which flows back into the periderm (Fig. 35). It
follows from this that in this process certain solid
constituents of the
polyp, e. g., the cell
walls, must be
liquefied. This un-
differentiated mate-
rial formed from the Fic. 35
polyp may afterward
(low out again, giving rise to a stolon or a polyp; to the
former where it comes in contact with a solid body, to
the latter where it is surrounded by sea water. These
observations suggest the idea of reversibility of the

180 Regeneration

process of differentiation of organs and tissues, in cer-
tain forms at least. We have to imagine that some
of the cells or interstitial tissue is digested and that as
a consequence the organ loses its characteristic shape.

Giard and Caullery have found that a regressive
metamorphosis occurs in Synascidians, and that the
animals hibernate in this condition. The muscles of
the gills of these animals are decomposed into their
individual cells. The result is the formation of a
parenchyma which consists of single cells and of
cell aggregates resembling a morula.

Driesch,? experimenting on the regeneration of an
Ascidian, found that when he cut off the gills and
siphons of the animal the portion removed was able
to regenerate a whole animal. The gill-piece excised
contained no heart, no intestine, and no stolon, and all
these organs were regenerated from the gills. Ina.
number of cases the regeneration took place by bud
formation at the edge of the wound, but in other cases
the gills were transformed into an undifferentiated mass
of tissue from which the missing parts of the animals
arose by budding and new gills were formed.

It is probable that the two cases are only quantita-
tively different. In both, autodigestion of certain cell
constituents and possibly of whole cells must take
place in order to obtain material for the formation of the

t The writer quotes this after Driesch.
4 Driesch, H., Arch. f. Entwekingsmech., 1902, xiv., 247.

Regeneration 181

lost part of the Ascidian. If an interstitial tissue is
digested it becomes a question of how much of this tissue
undergoes hydrolysis. If there is little destroyed the
old shape of the gills remains, if too much is digested
the old gills become a shapeless mass in which a certain
number of the old cells are maintained and give rise to
the new animal by cell division. The material for the
new organs must of course be furnished from old cells
which have been digested.

If regeneration takes place in pieces which take up
no food the newly formed organs must originate from
material absorbed from cells of the animal which are
hydrolyzed and whose material serves as food for those
cells which grow. Very often this process of digestion
takes place without loss of the total form of the organ
and is overlooked by the pure morphologists. In
Campanularia also the process of collapse described
above is only apparent in a fraction of the cases as in
Driesch’s observations on Clavellina.* It is also possible
that the red and yellow entoderm cells which gather at
the end where the new polyp forms furnish the material
which is utilized for the process of growth of the cells
from which the tentacles arise (with or without giving
off specific ‘‘hormones’’ besides).

x One author, Miss Thatcher, in trying to repeat these observations,
did not notice the total collapse of the tissues and concluded that my
observations must have been wrong. The writer is fairly certain that
his observations were correct.

182 Regeneration

6. We have mentioned the ideas concerning a
design, or “‘entelechy,’’ acting as a guide to the
developing egg and have shown that this revival of
Platonic and Aristotelian philosophy in biology was
due toa misconception; namely, that the egg consisted
of homogeneous material which was to be differentiated
into an organism. For this supernatural task super-
natural agencies seemed required. But we have seen
that the unfertilized egg is already differentiated in a
way which makes the further differentiation a natural
affair. This idea of a quasi superhuman intelligence
presiding over the forces of the living is met with in the
field of regeneration, and here again it is based upon a
misconception. The lens of the eye is formed in the
embryo from the epithelium lying above the so-called
optic cup (the primitive retina). Where this retina
touches the epithelium the latter begins to grow into
the cup, the ingrowing piece of epithelium is cut off and
forms the lens, which probably under the influence of
substances secreted by the optic cup becomes trans-
parent. Certain animals like the salamander are able
to form a new lens when the old one has been removed
by operation, but the new lens is formed in an entirely
different way; namely, from the upper edge of the iris.
G. Wolf, who observed this regeneration used it to
endow the organism with a knowledge of its needs; the
idea of a Platonic preconceived plan or an Aristotelian
purpose suggested itself. But it can be shown that the

Regeneration 183

organism does in this case what it is compelled to do
by its physical and chemical structure.

Uhlenhuth* has shown by way of tissue culture that
the cells of the iris cannot grow and divide as long as
they are full of pigment granules as they normally are.
When the fine superficial membrane of the iris is torn
the pigment granules fall out and the cells can now grow
and multiply. If the lens is taken out of the eye of the
salamander the fine membrane of the iris is torn and
the pigment cells at the edge (especially the upper edge)
lose their pigment granules which fall down on account
of their specific gravity. As soon as this happens the
cells will proliferate. A spherical mass of cells is formed
which become transparent and which will cease to grow
as soon as they reach a certain size. The unanswered
question is: Why does the mass of cells become trans-
parent so that it can serveasalens? The answer is that
young cells when put into the optic cup always become
transparent no matter what their origin; it looks as if
this were due to a chemical influence exercised by the
optic cup or by the liquid it contains. Lewis has shown
that when the optic cup is transplanted into any other
place under the epithelium of a larva of a frog the
epithelium will always grow into the cup where the
latter comes in contact with the epithelium; and that
the ingrowing part will always become transparent.
This leaves us then with one puzzle still: Why is the

« Not yet published.

184 Regeneration

growth of the lens limited? The limitation in the
growth of organs is one of the most important problems
in growth and organ formation, though unfortunately
our knowledge of this topic is inadequate. ;

7. The botanist J. Sachs was the first to definitely
state that in each species the ultimate size of a cell is a
constant, and that two individuals of the same species
but of different size differ in regard to the number, but
not in regard to the size of their cells.* Amelung, a
pupil of Sachs, determined the correctness of Sachs’s
theory by actual counts. Sachs, in addition, recognized
that wherever there were large masses of protoplasm,
é. g., in siphonez and other cceloblasts, many nuclei
were scattered throughout the protoplasm. He inferred
from this that “each nucleus is only able to gather
around itself and control a limited mass of protoplasm.’’?
He points out that in the case of the animal egg the
reserve material—fat granules, proteins, and carbo-
hydrates—are partly transformed into the chromatin
substances of the nuclei, and that the cell division of the
egg results in the cells reaching a final size in which each
nucleus has gathered around itself that mass of proto-
plasm which it is able to control. Morgan’ and
Driesch* tested and confirmed the idea of Sachs for

tv. Sachs, J., “‘Physiologische Notizen,”’ vi., Flora, 1893.

2 Ibid., ix., 425, Flora, 1895.

3 Morgan, T. H., Arch. f. Entweklngsmech., 1895, ii., 81; 1901, xiii., 4163

1903, Xvi., I17.
4Driesch, H., Arch. f. Entwcklngsmech., 1898, vi., 198; 1900, x., 361.

Regeneration 185

the eggs of Echinoderms. We stated in the previous
chapter that Driesch produced artificially larvee of sea
urchins of one-eighth, one-fourth, and one-half their
normal size by isolating a single cleavage cell in one of
the first stages of segmentation of the fertilized sea-
urchin egg. He counted in each of the dwarf gastrule
resulting from these partial eggs the number of mesen-
chyme cells and found that the larve from a one-half
blastomere possessed only one-half, those from a one-
fourth blastomere only one-fourth, and those from a
one-eighth blastomere only one-eighth of the number of
cells which a normal larva developing from a whole egg
possessed. Moreover, he could show that when two
eggs were caused to fuse so as to produce a single larva
of double size, the gastrulz of such larve had twice the
number of mesenchyme cells. Driesch drew the con-
clusion from his observations that each morphogenetic
process in an egg reaches its natural end when the
cells formed in the process have reached their final
size.

Since each daughter nucleus of a dividing blastomere
has the same number of chromosomes as the original
nucleus of the egg, it is clear that in a normally fertilized
egg each nucleus has twice the mass of chromosomes
that is contained in the nucleus of a merogonic egg, 7. ¢.,
an enucleated fragment of protoplasm into which a
spermatozoén has entered and which is able to develop.
Such a fragment has only the sperm nucleus. This

186 Regeneration

phenomenon of merogony was discovered by Boveri
and was elaborated by Delage.* Boveri, in comparing
the final size of the cells in normal and merogonic eggs
after the cell divisions had come to a standstill, found
that this size is always in proportion to the original
mass of the chromatin contained in the egg; the cells of
the merogonic embryo, e. g., the mesenchyme cells, are
only half the size of the same cells in the normally
fertilized embryo. Driesch furnished a further proof
of Boveri’s law, that the final ratio of the mass of the
chromatin substance in a nucleus to the mass of proto-
plasm is a constant in a given species. Driesch com-
pared the size of the mesenchyme cells in a sea-urchin
embryo produced by artificial parthenogenesis with
those of a normally fertilized egg and found them half
of the size of the latter. When the fertilized eggs and
the parthenogenetic eggs are equal in size from the
start,—which is practically the case if eggs of the same
female are used,—the process of the formation of
mesenchyme cells comes to a standstill when their
number in the normally fertilized eggs is half as large
as the final number in the parthenogenetic egg.?
Boveri’s results as well as those of Driesch were ob-
tained by counting the cells formed by eggs of equal
size and not by simply measuring the size of the cells.
It is most remarkable that certain apparent exceptions

t Delage, Y., Arch. Zoél. expér., 1899, vii., 383.
2 Driesch, H., Arch. f. EntwckIngsmech., 1905, xix., 648.

Regeneration 187

to Boveri’s law which Driesch has actually found had
been predicted by Boveri.

These facts show that the growth of an organ comes
to a standstill when a certain size is reached or a certain
number of cells are formed. We cannot yet state why
this should be, but we are able to add that the formation
of a lens of normal size in the regeneration of the eye
is in harmony with the phenomena in the embryo.
There seems therefore no reason for stating that the
regeneration of the lens cannot be explained on a purely
physicochemical basis. The only justification for such
a statement on the part of Wolf is that he was not in
possession of the more complete set of facts now avail-
able through the work of Fischel and Uhlenhuth.

The healing of a wound is a process essentially simi-
lar to the regeneration of the lens. Normally the cells
which begin to proliferate after a wound is made in the
skin lie dormant, inasmuch as they neither grow nor
divide. When a wound is made certain layers of
epidermal cells undergo rapid cell division. Leo Loeb‘
has studied this case extensively. He found that if the
skin is removed anywhere, epidermis cells from the
wound edge creep upon the denuded spot and forma
covering. This may be a tropism (stereotropism) or it
may be a mere surface tension phenomenon. Next a
rapid process of cell division begins in the cells adjacent
to the wound these cells having been heretofore dormant.

« Loeb, Leo, Arch. f. Entwcklngsmech., 1898, vi., 297.

188 Regeneration

He is inclined to attribute this increase in the rate of
cell division to the stretching of the epithelial cells, and
he is supported in this reasoning by the observation that
the larger the wound the more rapid the process of
healing. During wound healing the mitoses first
increase markedly in the old epithelium. With the
closure of the wound a sudden fall in the mitoses takes
place. The closure of the wound causes an increase in
the number of epithelial rows over the defect. This
increase is therefore reached at an earlier period in the
larger wound since the process of mitosis is more rapid
here. Leo Loeb thinks that the pressure of the epithelial
cells upon each other leads to a rapid diminution in the
mitotic proliferation.?

t Spain, K. C., and Loeb, Leo, Jour. Exper. Med., 1916, xxiii., 107;
Loeb, L., and Addison, W.H. F., Arch. f. Entwckingsmech., 1911, Xxxii.,
44; 1913, XXXvii., 635.

2 The excessive formation of epithelial cells in the healing of wounds
has led the older pathologists to the genéralization that if something is
removed in the body an excessive compensation will take place. The
formation of antibodies has even been explained on this basis by Weiggert
and Ehrlich in their side-chain theory. Asa matter of fact, this generali-
zation is entirely incorrect and in regeneration of starfish, actinians,
flatworms, annelids, and possibly in all forms the reverse is true; e. g.,
if we cut off the anterior half of the body in Certanthus less is reproduced
than was cut away namely only tentacles and the mouth, but not the
missing piece of the body. Weiggert’s conception of regeneration was
probably based on the phenomenon of the healing of wounds, but the
excessive epithelium formation in this case is not the expression of a
general law of regeneration but of the peculiar mechanical conditions
which lead to mitoses. It would be a very strange coincidence indeed if
a theory of antibody formation based on such an erroneous generaliza-
tion should be correct.

Regeneration 189

Should it be possible that this is more generally the
case, é. g., also in the lens after it has reached a certain
size? The conditions limiting growth require further
investigation.

It is hardly necessary to point out that in these cases
we are seemingly dealing with cases of the inhibition of
growth which cannot be explained by the tyranny of
the whole over the parts, and that there must be condi-
tions at work other than the mere flow of substances
which can cause a cessation of growth. This can be
illustrated by certain observations on the egg.

8. The history of the egg shows a reversible condi-
tion of rest and of activity. The primordial egg cell
multiplies actively until a large number of eggs are
formed in the ovary which may reach into the millions
in the case of sea urchins or certain annelids. These
cell divisions then stop and the egg goes into the resting
stage in which it deposits the reserve material for the
development of the embryo. From this condition it
can only be called into activity again by the spermato-
zoon or the agencies of artificial parthenogenesis.

It seemed of interest to find out whether or not the
development of the egg may be reversed once more
after it has been activated. From all that has been said
in the chapter on artificial parthenogenesis, such a
reversal should take place in the cortical layer. The
result of these experiments seems to be that if a complete
destruction or change in the cortical layer has once

190 Regeneration

taken place —such as that caused by the entrance of
a spermatozoon into the egg—no reversal is possible;
although the development of the fertilized egg may be
suppressed for a long time by either low temperature
or lack of oxygen, or, in the case of seeds and spores, by
lack of water. But as soon as the conditions for the
chemical reactions in the egg are normal again, the
development may go on unless the egg has suffered by
the methods used to prevent development or by the
long duration of the suppression. With an incomplete
destruction of the cortical layer both development as
well as reversal of development are possible. Thus the
writer has shown that in the egg of Arbacia the effect
of the cortical alteration of the egg induced by the
butyric acid treatment or by the treatment with bases
can be reversed. When unfertilized eggs of Arbacia are
put for from two to five minutes into 50 c.c. sea water +
2.0 c.c. N/1o butyric acid they will all form a gelatinous,
somewhat atypical fertilization membrane; when put
back into normal sea water all will perish in a few hours
unless they are submitted to the short treatment with a
hypertonic solution mentioned in the previous chapter,
while if submitted to this treatment they will develop.
If, however, these eggs are transferred from the butyric
acid sea water not into normal sea water but into sea
water containing some NaCN (10 drops of ,4 per cent.
NaCN or KCN in 50 c.c. sea water), and if they remain
here for some time (e. g. overnight) they will not perish

Regeneration 191

when subsequently transferred back to normal sea water.
Such eggs will develop when fertilized with sperm.
The activating effect of the membrane formation has,
therefore, been reversed and the eggs have gone back
into the resting stage.t Wasteneys has found that the
rate of oxidation which was raised considerably by the
artificial membrane formation goes back to the value
characteristic for the resting eggs after the reversal
of their developmental tendency.? Similar results
were obtained in eggs activated with NH,OH. It
appears from this as though the change in the cortical
layer which leads to the development of the egg and
the increase in the rate of oxidations were reversible
in the egg of Arbacia.3

The writer had previously noticed that eggs of
Strongylocentrotus purpuratus, which had been treated
for two hours with hypertonic sea water, not infrequently
began to divide into two, four, or eight cells (and some-
times more) and then went back into the resting state
(except that they possessed the second factor required
for development as stated in Chapter V). It may be

t Loeb, Arch. f. Entwckingsmech., 1914, XXXviii., 277.

2 Wasteneys, H., Jour. Biol. Chem., 1916, xxiv., 281.

3 F. Lillie thinks that the KCN in this experiment merely inhibits the
change of the cortical layer necessary for development. This is con-
tradicted by two facts: first, the writer has shown in 1906 that KCN does
not inhibit the membrane formation, and, second, the eggs will not
return to the resting stage when put back into sea water too soon; in
that case they will disintegrate. This shows that in the KCN something
more happens than the mere block to disintegration.

192 Regeneration

remarked incidentally that such eggs at the time of cell
division contained the centrosomes and astrospheres,
and yet went back into a resting state, thus showing
that the centrosomes are only transitory organs or
organs which are only active under certain conditions.
It is quite possible that in these phenomena of reversal
not the whole of the cortical layer has undergone altera-
tion.

The writer must leave it undecided whether the
changes from the resting to the active state in body
cells can also be explained in analogy with these experi-
ments.

9. In the formation of the lens we have already
noticed an instance where the adjacent organ influences
growth inasmuch as the optic cup controlled the for-
mation of the lens. Such influences are quite commonly
observed. A piece of Tubularia when cut out from a
stem and suspended in water will regenerate at the
aboral pole not a stolon but a polyp, so that we have an
animal terminating at both ends of its body in a head.
The writer called such cases in which an organ is
replaced by an organ of a different kind hetero-
morphosis.

Contact with a solid body favours the formation of
stolons. Fig. 36 shows a piece of a stem of Pennaria
another hydroid, which was lying on the bottom of an
aquarium and which formed stolons at both ends a and
6b. In Margelis, another hydroid, the writer observed

Regeneration 193

that without any operation the apical ends of branches
which were in contact with solid bodies continued to
grow as stolons, while those surrounded by sea water
continued to grow as stems.

Herbst discovered a very interesting form of hetero-
morphosis in certain crustaceans; namely, that in the

Fic. 36

place of an eye which was cut off, an entirely different
organ could be formed, namely, an antenna. He
showed that the experimenter has it in his power to
determine whether the crustacean shall regenerate an
eye or an antenna in place of the eye. The latter will
take place when the optic ganglion is removed with the
eye, the former when it is not removed. These experi-
ments were carried out successfully on Palemon,
Palemonetes, Sicyonia, Palinurus, and other crus-
taceans.

The influence of gravitation is very familiar in plants;

194 Regeneration

‘in stems of Bryophyllum placed horizontally the roots
usually come out from the lower end of the callus.
Such phenomena are not often found in animals but
they exist here too as the following observation shows.
If we cut a piece ab (Fig. 37), from the stem ss of

Fic. 37

Antennularia antennina (Fig. 38), a hydroid, and put it
into the water in a horizontal position, new stems cd
(Fig. 37) may arise on its upper side. The small branches
on the under side of the old stem ad begin suddenly to
grow vertically downward.* In appearance and func-
tion these downward-growing elements are entirely
different from the branches of the normal Antennularia;
they are roots. In order to understand better the
transformation which thus occurs in these branches, it
may be stated that under normal conditions they have

tLoeb, J., Untersuchungen zur physiologischen Morphologie der
Tiere. II. Organbildung und Wachsthum. Wurzburg, 1892.

S Regeneration 195

4 a limited growth (see Fig. 38), are directed
4 upward, and have polyps on their upper
\N side. The parts which grow down (Fig.
f 37) have no polyps, but attach themselves
NA like true roots to solid bodies. Thus the
changed position of the stem alone, with-
out any operation, suffices to transform
the lateral branches, whose growth is
limited, into roots with unlimited growth.
ae The lateral branches on the upper side of
the stem do not undergo such a transfor-
mation into roots except in the immediate
surroundings of the place where a new stem
: arises. It seems that the formation of a
p new stem also causes an excessive growth
; of roots, possibly because the formation of
p new branches causes the removal of sub-
stances which naturally inhibit the forma-
tion of roots. Ifa piece from the stem be
put vertically into the water with top down-
ward, the uppermost point may continue
to grow as a stem, while the lowest point
may give rise to roots. In this case, there-
fore, a change in the orientation of organs
has the effect of changing the character
of organs.
There are only two ways by which we
NK can account for these influences of gravi-

196 Regeneration

tation. Either certain substances flow to the lowest
level and collecting there induce growth and possibly
changes in the character of growth (as in Aniennularia)
or if the cells have elements of different specific gravity
the relative position of these elements may possibly
change and influence in this way the conditions for
growth. The influence of gravitation as well as of con-
tact upon life phenomena are at present little under-
stood.

In all these cases of heteromorphosis the original
form is not restored. It is needless to say that they are
incompatible with the theory of natural selection.

The reader will have noticed that in this chapter one
term has not been mentioned which is commonly met
with in the literature, namely the ‘‘wound stimulus.”
As the writer had indicated in a former publication,
the word ‘‘stimulus”’ is generally used to disguise our
ignorance of (and also our lack of interest in) the causes
which underlie the phenomena which we investigate.
Regeneration very often does not take place near the
wound but at some distance from it. But even when
the regeneration takes place at the edge of the wound
the latter only serves to create conditions for regen-
eration, and these conditions cannot be expressed by
the word ‘‘stimulus.”’

While our knowledge of the réle of the whole in

*Loeb, J., Die chemische Entwicklungserregung des tiertschen Etes.
Berlin, 1909.

Regeneration 197

tegeneration is incomplete in a great many details it
seems that the known facts warrant the statement that
the phenomena of regeneration belong as much to the
domain of determinism as those of any of the partial
phenomena of physiology.

CHAPTER VIII

DETERMINATION OF SEX, SECONDARY SEXUAL “CHARAC-
TERS, AND SEXUAL INSTINCTS

I. The Cytological Basis of Sex Determination

1. It is a general fact that both sexes appear in
approximately equal numbers, provided a sufficiently
large number of cases are examined. This fact has
furnished the clue for the discovery of the mechanism
which determines the relative number of the two sexes.
The honour of having pointed the way to the solu-
tion of the problem belongs to McClung.* It has
been known that certain insects, e. g., Hemiptera and
Orthoptera, possess two kinds of spermatozoa but only
one kind of eggs. The two kinds of spermatozoa differ
in regard toa single chromosome, which is either lacking
or different in one-half of the spermatozoa.

The first one to recognize the existence of two kinds
of spermatozoa was Henking, who stated that in Pyr-
rhocoris (a Hemipteran) one-half of the spermatozoa

t McClung, C. E., ‘‘ The Accessory Chromosome—Sex Determinant?”
Biol. Bull., 1902, iii., 43.
198

Basis of Sex Determination 199

of each male possessed a nucleolus, while in the other
half it was lacking. Montgomery afterward showed
that Henking’s nucleolus was an accessory chromosome.
McClung was the first to recognize the importance of
this fact for the problem of sex determination. He
observed an accessory chrorhosome in one-half of the
spermatozoa of two forms of Orthoptera, Brachystola
and Hippiscus, and reached the following conclusion:

A most significant fact, and one upon which almost all
investigators are united in opinion, is that the element is
apportioned to but one-half of the spermatozoa. Assuming
it to be true that the chromatin is the important part of the
cell in the matter of heredity, then it follows that we have
two kinds of spermatozoa that differ from each other in a
vital matter. We expect, therefore, to find in the offspring
two sorts of individuals in approximately equal numbers,
under normal conditions, that exhibit marked differences
in structure. A careful consideration will suggest that
nothing but sexual characters thus divides the members
of a species into two well-defined groups, and we are logi-
cally forced to the conclusion that the peculiar chromosome
has some bearing upon the arrangement.

N. M. Stevens and E. B. Wilson’ have not only
proved the correctness of this idea for a number of
animals but have laid the foundation of our present
knowledge of the subject. Wilson showed that in those
cases where there are two types of spermatozoa, one

: Wilson, E. B., ‘Studies on Chromosomes,” Jour. Exper. Zoél., 1905,

ii., 371, 507; 1906, iii., I; 1909, vi., 69, 147; 1910, ix., 53; 1912, xiii., 345.
“Croonian Lecture," 1914, Proc. Roy. Soc., B. xxxviii., 333.

200 Basis of Sex Determination

with and one without an accessory or as it is now called
an X chromosome, all the cells of the female have one
chromosome more than the cells of the male. From
this he concludes correctly that in such species a female
is produced when the egg is fertilized by a spermato-
zoon containing an X chromosome, while a male is
produced when a spermatozo6n without an X chromo-
some enters the egg.

Such a form is Protenor,one of the Hemiptera. Wilson
made sure that all the eggs are alike in the number of
chromosomes, each egg containing an X chromosome in
addition to the six chromosomes characteristic of the
species Protenor. ‘There are two types of spermatozoa
in equal numbers in this species, each with six chromo-
somes, but one with, the other without, an X chromo-
some. The two possible chromosome combinations
between egg and spermatozoa are therefore as follows
(see the diagrammatic Fig. 39):

Egg Spermatozobn Result
(1) 6+ X + 6 = 12+ X= Male
(2) 64+X +6+X = 12+2X = Female

The egg which receives a spermatozo6én without an
X chromosome has after fertilization 12 +- X chromo-
somes and develops into a male; while the egg into
which a spermatozo6n with an X chromosome enters
gives rise to a female. Since all the body cells arise
from the fertilized egg by nuclear division and the

Basis of Sex Determination 201

chromosomes remain constant in number in all cells,
the consequence is that all the cells of a female Protenor
have two X chromosomes; while all the cells of a male
Protenor have only one X chromosome.

Before Fertilization

After Fertilization
Fic. 39

The chromosome situation in Protenor is a somewhat
extreme case, inasmuch as one X chromosome is
entirely lacking in the male. In other forms of Hemip-
tera, e. g., Lyge@us, there are also two types of spermato-
zoa appearing in equal numbers differing in regard to

202 Basis of Sex Determination

the X chromosome, but here it is only a difference in
size; one-half of the spermatozoa having a large X chro-
mosome, the other half instead a smaller chromosome.
Calling this latter the Y chromosome, the sex deter-
mination in this form is as follows: leaving aside the
chromosomes which are equal in both egg and sperma-
tozoén we may say that there is one type of egg con-
taining one large X chromosome; there are two types
of spermatozoa in equal numbers, one possessing a large
X chromosome, the other possessing a small Y chromo-
some. Wilson showed by a study of the chromosomes in
males and females that when one of the spermatozoa
containing a large X chromosome enters the egg, the
egg will dévelop into a female; while when one of the
spermatozoa containing a small Y chromosome enters
it will give rise to a male. Leaving aside the common
chromosomes of both sexes, a fertilized egg containing
XX gives rise to a female, while one containing XY
gives rise toa male. There is in this case as in that of
Protenor a preponderance of chromosome material in the
female, but this quantitative difference is not essential
for the determination of sex, since in some species the Y
chromosome may be as large as the X chromosome.

The main fact is that the female cells have the
chromatin composition XX, the male cells the composi-
tion XY, where Y is apparently qualitatively different
and often, but not necessarily, smaller than X, or
entirely lacking.

Basis of Sex Determination 203

It may be mentioned in passing that indirect evidence
exists indicating that in man there are also two kinds
of spermatozoa and one kind of egg, and that sex
depends on whether a male determining or a female
determining spermatozo6n enters the egg.

2. This mode of sex determination holds only for
those animals in which there is one type of egg and two
types of spermatozoa. Experimental evidence furnished
first by Doncaster in 1908 on a moth, Abraxas, indi-
cated that a number of other forms txists in which
matters are reversed, inasmuch as there are two types
of eggs and one type of spermatozoa. This condition of
affairs exists not only in the moth Abraxas, but also
in the fowl as shown by Pearl. In these forms it is
assumed that all the spermatozoa have one sex chromo-
some X, while there are two types of eggs, one possessing
the sex chromosome X, the other possessing Y. When
a spermatozo6n enters an egg with an X chromosome,
the egg will give rise to a male, while if it enters a Y
egg, a female will arise. The evidence pointing toward
this result is chiefly contained in experiments on sex-
limited or more correctly sex-linked heredity; 7.¢., a
form of heredity which follows the sex in a peculiar
way. Thus colour-blindness is a case of sex-linked
inheritance, since this abnormality appears over-
whelmingly in the male offspring of a colour-blind
person. Doncaster crossed two varieties of Abraxas dif-
fering in one character which was sex-linked, and the

204 Basis of Sex Determination

results of his crossings indicated that in this form there
are two types of eggs and one type of spermatozoa.*

These observations on sex-linked heredity confirm
the idea that the sex chromosomes determine the sex.
The most extensive and conclusive experiments along
this line are those by Morgan on the fruit fly Drosophila.
In this form there are two kinds of spermatozoa and
one kind of eggs; the egg has one X chromosome, while
one-half of the spermatozoa has an X the other a Y
chromosome; the entrance of the latter into an egg gives
rise to a male, of the former to a female.

While the eyes of the wild fruit fly Drosophila ampelo-
phila are red, Morgan? noticed in one of his cultures
a male that had white eyes. This white-eyed male was
mated to a red-eyed female. The offspring, the F,
generation, were all red eyed, males as well as females.
These were inbred and now gave in the F, generation
the following three types of offspring:

(I) 50 per cent. females, all with red eyes.
25 per cent. with red eyes.

2) 50 per cent. 1 5 3 :
(2) 50 per cent. male 25 per cent. with white eyes.

The character white eye was therefore transmitted
only to half the grandsons; it was a sex-linked charac-
ter. It is known from a study of the pedigrees of
colour-blind individuals that if the corresponding ex-

* Doncaster, L., The Determination of Sex. Cambridge, 1914.
1 Morgan, T. H., Heredity and Sex. New York, 1913.

Basis of Sex Determination 205

periment had been carried out with them, instead of
with white-eyed flies, the same proportions of normal
and colour-blind would have been found: namely, nor-
mal colour vision in the F, generation, in both males and
females, and half of the males of the F, generation
colour-blind, the other half and all the females with
normal vision. Of course, in man, intermarriage
between two different F, strains would have been re-
quired in place of the inbreeding of the F, ‘generation,
which took place in Morgan’s experiments. Morgan
interprets his experiments as follows. The normal red-
eyed Drosophila has one kind of eggs, each possessing
one X chromosome. This X chromosome has also the
factor for the development of red-eye pigment. The
white-eyed male has two kinds of spermatozoa, one
with an X chromosome, the other with a Y chromosome,
both lacking the factor for red-eye pigment. If we
designate the X chromosome with the factor for red-
eye pigment by X and the X and Y chromosomes
lacking the factor for redness with X and Y the follow-
ing combinations must result if we cross a normal red-
eyed female with a white-eyed male:

Eggs Sperm Result
x x XX _ red-eyed female
x Y XY _ red-eyed male

i
It is obvious that all the offspring of the first genera-

tion (the F, generation) must be red eyed, since all the

206 Basis of Sex Determination

eggs have one X chromosome with the factor for red.
According to the results obtained from cytological
studies which will be explained in the next chapter, the
females with the chromatin constitution XX will form
two types of eggs in equal numbers: namely, eggs with
an X and eggs with an X, 7.¢., all eggs have one X
chromosome, but in fifty per cent. of the eggs the X has
the factor for red, in fifty per cent. this factor is lack-
ing (X). The males having the chromosome constitu-
tion XY form two types of spermatozoa, one with an
X possessing the factor for red pigment and one, the Y
chromosomes, lacking this factor. If inbred the next
F, generation will give rise to the following four types
of offspring: (1) XX, (2) XX, (3) XY, (4) XY, all four
types in equal numbers.

(1) and (2) give females, both red eyed, since both
contain a red-factored KX chromosome. (3) and (4)
give males, (3) giving rise to red-eyed males, since it
contains a red-factored X chromosome, (4) producing
males with white eyes since this X chromosome is
lacking the factor for red eyes. Since all four combina-
tions must appear in equal numbers (provided the
experimental material is ample enough, which was the
case in these experiments), in the F, generation both
males and females should have red eyes and in the F,
generation all the females should have red eyes and
half of the males should have red, half white eyes,
These results were obtained.

Basis of Sex Determination 207

The experiments were carried further. No white-eyed
females had appeared thus far. On the same assump-
tions of the relation of the X, X, and Y chromosomes to
the heredity of sex as well as to eye colour it was
possible to predict under what conditions and in which
proportions white-eyed females should arise. Thus if
a red-eyed female of the F, generation (a cross between
white-eyed male and normal female) be mated with a
white-eyed male the result should be an equal number
of white-eyed males and white-eyed females if the
chromosome theory of sex determination were correct.
The reasoning would be as follows:

The red-eyed female, having the chromosome con-
stitution XX should form two kinds of eggs in equal
numbers with the constitution X and X; the white-
eyed male having the chromosome constitution XY
should form two kinds of spermatozoa X and Y. The
following four types of individuals must then be pro-
duced in equal numbers:

(1) KX, (2) XX, (3) KY, and (4) XY.

In this case (2) must give rise to white-eyed females
and (4) to white-eyed males, while (1) must give rise
to red-eyed females and (3) to red-eyed males. Hence
white-eyed males and females and red-eyed males and
females are to be expected in this case in equal num-
bers, and this was actually observed.

The numerical agreement in this and the other

208 Basis of Sex Determination

experiments between the expected and observed result
cannot well be an accident. The fact that the inheri-
tance of sex-linked characters in man follows the same
laws as in Drosophila is a strong argument in favour of
the assumption that in man, also, sex is determined by
two kinds of spermatozoa.

Morgan and his students discovered no less than
thirty-six sex-linked characters in Drosophila, and each
behaved in a similar way to the red and white eye
colour in regard to sex-linked inheritance, so that the
chromosome theory of sex determination rests on a safe
basis. That sex is merely determined by the number
of X chromosomes, not by the Y chromosome, is proved
by the facts that the Y chromosome may be completely
absent as in Protenor and that Bridges* has found a
type of female Drosophila with a chromosome formula
XXY whose sex was not affected by the supernu-
merary Y. ‘

3. On the basis of all these experiments and theories
it is comparatively easy to explain a number of pheno-
mena concerning sex ratios which before had been very
puzzling. In bees it had been shown many years ago
by Dzierzon that the males develop from unfertilized
eggs while the females, queens and workers, develop
from fertilized eggs. This is intelligible on the assump-
tion that the unfertilized egg contains only one X
chromosome while the spermatozoén carries into the

2 Bridges, C. B., Genetics, 1916, i., I.

Basis of Sex Determination 209

egg the second X chromosome. But if the male bee pro-
duces two types of spermatozoa we should expect that
only one-half of the fertilized eggs should be females,
the other half males. But it happens that of the two
types of spermatozoa only one is formed since in one of
the cell divisions which lead to the formation of sperma-
tozoa one viable spermatozoén only is formed while
the other one perishes. It is, therefore, quite pos-
sible that it is the female-producing spermatozoén
which survives while the male-producing spermatozo6n
dies.

It is occasionally observed that an insect shows one
sex on one side of its body and the opposite sex on the
other side. Boveri suggested that this phenomenon of
gynandromorphism is due to the fact that the spermato-
zoon for some unknown reason does not fuse with the
egg nucleus until after the egg has undergone its first
cell division. In this case it fuses with the nucleus of one
of the two cells into which the egg divides (or in some
cases even one of the later cells?). As a consequence
the one-half of the embryo which arises from the cell
which was not fertilized would have only one X chromo-
some and in a case like the bee would develop par-
thenogenetically, while the other half of the body,
developing from the cell into which a spermatozoén
has penetrated, would be fertilized. The latter half
of the body would be female, the former male. In his

last paper before his untimely death, Boveri has given
14

210 ‘Basis of Sex Determination

proof for the correctness of this interpretation as far as
gynandromorphism in the bee is concerned."

It seems to be generally true that where sexual
reproduction leads only to the formation of females
the case finds its explanation in the fact that the
male-producing spermatozoa perish and only the
female-producing spermatozoa survive. Such an ob-
servation was made by Morgan on a certain species
of phylloxerans.

The slight preponderance in the number of one sex
which is occasionally found—an excess of six per cent.
males over females in the human race—may well find
its explanation on the assumption of a slightly greater
mortality of the female-determining spermatozoa.

In certain forms parthenogenetic and sexual reproduc-
tion may alternate in a cycle, e. g., in plant lice, Dathnia,
and rotifers. In plant lice it has been observed for a
long time that when the plant is normal and the weather
warm the aphides remain wingless, reproduce par-
thenogenetically, and only females exist, and this may
last for years and for more than fifty generations; but
that when the plant is allowed to dry out both sexes
appear.

Here we are dealing with a limited determination of
sex inasmuch as the experimenter has it in his power to
prevent or allow the production of males. The facts
do not in all probability contradict the statements

1 Boveri, Th., Arch. f. Entwcklngsmech., 1915, xlii., 264.

Basis of Sex Determination 211

made concerning the réle of the X chromosomes in the
determination of sex. We have seen that where sex is
determined by two types of spermatozoa one type of
eggs is produced which possesses only one X chromo-
some. Such eggs might produce males if not fertilized
(as they do in bees), but they cannot produce females
because for that purpose they must have two X chromo-
somes. It has been shown for certain cases, and it may
be true generally, that if eggs of this type give rise to
parthenogenetic females they may do so because they
have for some reason two X chromosomes. Usually
such an egg loses one of the X chromosomes in a process
of nuclear division (the so-called reduction division)
which usually precedes fertilization. If this reduction
division is omitted the egg has two X chromosomes and
if such an egg develops parthenogenetically it gives rise
to a female. These cases do not, therefore, contradict
the connection between X chromosomes and sex deter-
mination established by cytological observations and
breeding experiments, on the contrary, they confirm it.
The question remains: How can external conditions
bring it about that the reduction division is omitted?
To this question no definite answer can be given at
present.

We may in passing mention the well-known observa-
tion that twins which originate from the same egg
always have the same sex; while twins arising from
different eggs show the usual variation as to sex. Twins

212 Basis of Sex Determination

|
coming from one egg have the same chorion and can

thereby be diagnosed as such. They can be produced
as we have stated in Chapter V by a separation of the
first two cleavage cells of the egg, each one giving rise
to a full embryo. It harmonizes with all that has been
said above that the sex of two such individuals must
be the same since they have the same number of X
chromosomes, the latter being determined in the human
trace by the nature of the spermatozoén which enters
the egg.

4. While thus far all the facts agree with the
dominating influence of certain chromosomes upon
sex determination, one group of facts has not yet been
explained: namely, hermaphroditism. By hermaphro-
ditism is meant the existence of complete and separate
sets of female and male gonads in the same individual.
This condition exists regularly not only in definite
groups of animals, e. g., certain snails, leeches, tape-
worms, but also, as everybody knows, in flowering
plants. While in some forms both kinds of sex cells,
male and female, are formed and mature simultaneously,
as, é. g., in the Ascidian Ciona (see Chapter IV), in others
they are formed successively, very often the sperma-
tozoa appearing first (protandric hermaphroditism).
In the long tapeworm Tenia each ring has testes
and ovaries, but the young rings are only male while
in the older rings the testes disappear and the ovaries
are formed. The same ring is in succession male and

Basis of Sex Determination 213

female. How can we reconcile the facts of hermaphro-
ditism with the chromosome theory of sex determina-
tion? Rhabdonema nigrovenosum, a parasite living in
the lungs of the frog, is hermaphroditic, but its eggs
produce not a hermaphroditic generation but one with
the two separate sexes; this generation. is not parasitic
and lives in the soil. The generation produced by these
separate males and females gives rise again to a her-
maphrodite which migrates into the lungs of the frogs.
According to Boveri and Schleip* the cells of the her-
maphrodite have twelve chromosomes. It produces two
types of spermatozoa with six and five chromosomes
respectively (one-half of the cells losing one chromosome
which is left at the line of cleavage between the two
cells); and one type with six chromosomes. In this way
separate males and females are produced by the her-
maphrodite, females with twelve and males with eleven
chromosomes.

The males produce again two kinds of spermatozoa,
male and female producing, but the male-producing
spermatozoa become functionless. This fusion of the
other spermatozo6n containing six chromosomes with
an egg having six chromosomes leads again to the for-
mation of the hermaphrodite with twelve chromosomes.
It is obvious that in this case the cause for the her-
maphroditism is not disclosed. " If chromosomes have

«Boveri, Th., Verhand. d. phys.-med. Gesellsch. Warzburg, 1911,
xli., 85. Schleip, W., Ber. d. naturf. Gesellsch., Freiburg i. Br., 1911, xix.

214 Basis of Sex Determination

anything to do with hermaphroditism there must be an
undiscovered element in the chromosomes which may
explain why the female as well as the hermaphrodite
have the same chromosome constitution; or we are
forced to look for another determinant outside the X
chromosomes or the chromosomes altogether. This
seems to be the only cytological work on the problem of
hermaphroditism. Experimental work has been begun
by Correns? and by Shull on the determination of
hermaphroditism in plants but lack of space forbids us
to give details.

II. The Physiological Basis of Sex Determination

5. As stated at the beginning of this chapter, the
chromosome theory of sex determination explained
only one feature of the problem, namely, the relative
numbers in which both sexes or only one sex, as the case
may be, are produced; and in this respect the evidence is
so complete that we must accept it. But with all this,
the problem of sex determination is not exhausted,
since a physiological solution of the problem of sex
determination demands an account of how the sex
chromosomes can induce the formation not only of
ovaries and testes but also of the other sex characters.
For the solution of this problem biology will have to
depend largely on experiments in which it is possible

« Correns, C., Biol. Centralbl., 1916, xxxvi., 12.

Basis of Sex Determination 218

to influence the formation of sex characters and of the
sex glands themselves.

The most striking observations in this direction
were made by Baltzer on a marine worm, Bonellia. In
this animal the two sexes are very different, the male
being a tiny parasite, a few millimetres in length, which
spends its life in the uterus of the female, whose size is
about five centimetres. A female carries as a rule
several and often a large number of the male parasites
in its uterus, which indicates that the males prevail
numerically. The fertilized eggs of the animals are
laid in the sea water where the larve hatch. At the
time of hatching all larve are alike. The differentiation
of the larve into the dwarf males and the giant females
can be determined at will. The larve have a tendency
to attach themselves to the proboscis of the female as
soon as they hatch. If given a chance to do so and if
they stick to the proboscis for more than three days they
will develop into males, which soon afterwards creep
into the female where they continue their parasitic
existence. If, however, no adult female Bonellia is put
into the aquarium in which the larve hatch, about
ninety per cent. of the larvee will, after a period of rest,
develop into females; the rest develop into males.
Those which develop into females will often show a
primary maleness which may manifest itself in the pro-
duction of sperm or of other secondary male sexual
characters. ‘This tendency is stronger the longer the

216 Basis of Sex Determination

period of rest lasts. If the larve are allowed to settle
on the proboscis of the adult female but are removed
too early hermaphrodites are produced having male and
female characters mixed.

Baltzer has suggested on the basis of some observa-
tions that the larve while on the proboscis of the female
absorb some substance secreted by the proboscis, and
this substance accelerates the further development
into a male and suppresses the female tendency. If
this substance from the proboscis does not reach the
larvee the tendency to become males is gradually
suppressed in the majority and only a few develop into
pure males or protandric hermaphrodites, while the
female characters are given a chance to develop.
Baltzer assumes, therefore,—as it seems to us correctly
—that in all larve the tendency for both sexual char-
acters is present, that they are, in other words, herma-
phrodites, but the chance for the suppression of one
and the development of the other group of characters
can be influenced by certain chemical substances which
the larva may take up.*

Giard has studied the effects of a curious form of cas-
tration brought about by parasites, which is followed
by a change in the sexual character of the castrated
animal. The phenomenon is very striking in certain
forms of crabs when they are attacked by a parasitic
crustacean, Sacculina. The two sexes differ in the crab

'Baltzer, F., Mutteil. d. zodlog. Station, Neapel, 1914, xxii.

Basis of Sex Determination 217

Carcinus menas by the form of the abdomen, but when
a male is attacked by the parasite its abdomen assumes
the female shape. Smith observed in another crab
that in such cases even the abdominal appendages of the
male may be transformed into those of a female. The
transformation is so complete that the older observers
had reached the conclusion that the parasite attacked
only the females, since they overlooked the fact that
the castration by the parasite transformed the sec-
ondary sexual characters of the male into those of a
female.

Giard observed that in a dicecious plant, Lychnis
dioica, a parasitic fungus brings about the transfor-
mation of the host into a hermaphrodite.

G. Smith has discovered a fact which shows that
chemical changes must underlie these morphological
transformations of primary or secondary sexual char-
acters. He noticed that in male crabs the presence of
the parasite Sacculina changes the contents of the
fatty constituents in the blood, making them equal
to that of the female. Vaney and Meignon had pre-
viously shown that during the chrysalid stage the female
silkworms have always more glycogen and less fat than
the males. The castration by parasites is paralleled
by what Caullery calls the castration by senility.*
In certain birds and also in mammals at the time when
the sexual glands cease to function certain secondary

*Caullery, M., Les Problémes de la Sexualité. Paris, 1913.

218 Basis of Sex Determination

sexual characters of the other sex make their appear-
ance. The most common case is that certain secondary
male characters appear in the old female (exceptionally
also in the young female with abnormal ovaries)
(arrhenoidy). Thus old female pheasants assume the
plumage of the male, and in the human female after
the menopause and especially among sterile women a
beard may begin to grow. The opposite phenomenon,
the old male assuming female characters, is not so
common. Very interesting observations on changes
in the plumage of castrated fowl have recently been
made by Goodale.*

It had long been observed by cattle breeders that in
the case of twins of different sex the female—the
so-called free-martin—is usually sterile. F. Lillie? has
recently discovered the cause of this interesting
phenomenon. Such twins originate from two different
eggs since the mother has two corpora lutea, one in
each ovary. In normal single pregnancies in cattle
there is never more than one corpus luteum present.
The two eggs begin to develop separately in each horn
of the uterus.

The rapidly elongating ova meet and fuse in the small
body of the uterus at some time between the 1o mm. and
the 20 mm. stage. The blood-vessels from each side then
‘anastomose in the connecting part of the chorion; a par-

ticularly wide arterial anastomosis develops, so that either

t Goodale, H. D., Biol. Bull., 1916, xxx., 286.
2 Lillie, F., Science, 1916, xliii., 611.

Basis of Sex Determination 219

fetus can be injected from the other. The arterial circula-
tion of each also overlaps the venous territory of the other,
so that a constant interchange of blood takes place. If
both are males or both are females no harm results from
this; but if one is male and the other female, the reproductive
system of the female is largely suppressed, and certain male
organs even develop in the female. This is unquestionably to
be interpreted as a case of hormone action.

The reproductive system of these sterile females is for
the most part of the female type, though greatly reduced.
The gonad is the part most affected; so much so that most
authors have interpreted it as testis.

It should be added, however, that this result cannot
at present be generalized, since in the hermaphrodites
the specific hormones of both sexes must circulate
without suppressing each other’s efficiency.

All these facts indicate that certain substances
secreted by the ovaries or testes may inhibit the de-
velopment of certain sexual characters of the opposite
sex. When these inhibitions are partly or entirely
removed the secondary sexual characters of the opposite
sex may appear. This fact may also be interpreted as
an indication of a latent hermaphroditism and if this
be correct the real and latent hermaphrodites differ
only by the degree of inhibition for one sex, this inhibi-
tion being lacking or less complete in the real than in
the latent hermaphrodite.

In the light of this conclusion the observations on
the regeneration of both ovaries and testicles which
Janda observed in a hermaphroditic worm, Criodrilus

220 Basis of Sex Determination

lacuum,* is no longer so mysterious. This worm nor-
mally possesses in the segments near the head a pair of
ovaries and several pairs of testes. Janda found that if
the anterior parts containing the gonads of these worms
are cut off a complete regeneration takes place, includ-
ing both types of gonads, ovaries as well as testes. As
a rule, more than one pair of ovaries appear in the
regenerated piece. This important experiment shows
that in a hermaphrodite both types of sex organs
can be produced from body cells or from latent buds
resembling body cells. This phenomenon would be
intelligible on the assumption that in the body of a
hermaphrodite substances circulate which favour the
development of both types of sex organs, while in a
dicecian animal probably only one type of sex organ
would be developed; the formation of the other being
inhibited.

Richard Goldschmidt has discovered in his breeding
experiments on the gipsy-moth (Lymantria dispar)
a phenomenon which will probably throw much light
on the physiology of sex determination. He found
that certain crosses between the Japanese and the
European gipsy-moth do not give pure sexes, males or
females, but mixtures of the sexual characters of both
sexes, and this mixture is a very definite one for defin-
ite crosses. These differences are such that it is
possible to grade the hybrids according to their mani-

Janda, V., Arch. f. Entwcklngsmech., 1912, Xxxili., 345; XXXiV., 557+

Basis of Sex Determination 221

festations of maleness or femaleness, both in mor-
phological characters and instincts. Goldschmidt calls
this peculiar phenomenon intersexualism, and its
essential feature is that the various degrees of inter-
sexualism can be produced at will by the right
combination of races.

Female intersexualism begins with animals which show
feathered antenne of medium size (feathered antennze
are a male character), but which are otherwise entirely
female in appearance except that they produce a smaller
number of eggs which are fertilized normally. In the next
stage patches of the brown male pigment appear on the
white female wings in steadily increasing quantity. The
instincts are still female, the males are attracted and
copulate. But the characteristic egg sponge laid by the
animal contains nothing but anal hairs in spite of the fact
that the abdomen is filled with ripe eggs. In the next stage
whole sections of the wings show male colouration, with
cuneiform female sectors between, the abdomen becomes
smaller, contains fewer ripe eggs, the instincts are only
slightly female, the males are attracted very little, and re-
production is impossible. In the next stage the male
pigment covers practically the whole wing, the abdomen
is almost male, but still contains ovaries with a few ripe
eggs, the instincts are intermediate between male and
female. Then follow very male-like animals which still,
show in different organs their female origin and have rudi-
mentary ovaries. . . . The end of the series is formed by
males, which show in some minor characters, such as
the shape of wings, still some traces of their female origin.

The series of the male intersexes starts with males show-
ing a few white female spots on their wings. These be-
come larger and larger, the amount of brown pigment

222 Basis of Sex Determination

correspondingly decreasing. . . . Hand in hand with this
the abdomen increases in size, reaching in the most extreme
cases two-thirds of the female size (without containing
eggs). The same is true for the instincts which become
more and more female.

(And also for the copulatory organs which also become
more and more female.)

As stated above, the main fact that every desired
degree of intersexualism can be produced at will by
properly combining the races for breeding, and the
intersexual potencies of the different races has been
worked out by Goldschmidt.*

6. The relation between chemical substances cir-
culating in the body—either derivatives of food taken
up from without or of chemical compounds formed
naturally inside the body—and the production of
sexual characters is best shown in the polymorphism
found among the social ants, bees, and wasps. Here we
have, as a rule, in addition to the two sexes a third
one, the workers, which are in reality rudimentary
and for that reason sterile females. They differ more or
less markedly from both the typical male and female
in their external form, and, as a rule cannot copulate
owing to their deficient structure. This third sex,
the sterile neuters, can be transformed at desire into
sexual females in certain species, as P. Marchal has
demonstrated. He worked with a form of social wasps

* Goldschmidt, R., Proc. Nat. Acad. Sc., 1916, ii., 53; Ztschr. induct.
Abstammungslehre, 1912, vii., and 1914, xi.

Basis of Sex Determination 223

in which the workers are sterile and smaller than the
real females. In such a society of wasps all the males
and workers die in the fall and only the fertilized
females survive, each one founding a new nest in the
following spring. From the first eggs laid, workers
arise, small in stature and sterile; these workers are
nourished by their mother. ‘Then these workers take
care of the feeding of all those larvae which arise from
the eggs which their mother continues to lay. Through-
out the spring only workers arise from the eggs. The
males appear in the summer, the real females towards
the end of the season when the sexes copulate.

Marchal isolated a number of the sterile workers,
providing them with food but giving them no larve
to raise. He found that the workers which thus far
had been sterile became fertile, producing, however,
only males. This latter fact is easily understood from
what has been said regarding the bees, namely, that
the female produces only one type of eggs, hence the
unfertilized egg can give rise only to males. The
astonishing or important point is that the ovaries of
the workers begin to develop as soon as they no longer
have a chance to nourish the larve, provided the food
which would have been given to the larve is now at
their disposal. In other words, the development of
their ovaries is the outcome of eating the food which
under normal conditions they would have given to the
larve. The food must, therefore, contain a substance

224 Basis of Sex Determination

which induces the development of eggs. The natural
sterility of the neuters or workers is, therefore, to use
P. Marchal’s expression, a case of “food castration,”
(“castration nutriciale’’).' The workers originate
from fertilized eggs and are therefore females, but for
the full development of the ovaries and the other
sexual characters something else besides the XX
chromosomes is needed and this is supplied in this case
by the quantity or quality of the food. May we not
conclude that the same thing may happen generally,
except that these substances are formed by the body
under the normal conditions of nutrition through the
influence of constituents of the second X chromosome?

It is known that the future queens among the bees
receive also a special type of food which the workers do
not receive. Again the idea of “food castration” of
the latter is suggested.

In rotifers Whitney? has shown that the cycle in the
production of males and females can be regulated by
the food. In some species a scanty supply of green
flagellates produced purely female offspring, while a
copious diet of the same green flagellates produced a
predominance of male grandchildren, sometimes as high
as ninety-five per cent. This was confirmed by Shull
and Ladoff.s

«This account of Marchal’s beautiful experiments is taken from
Caullery, M., Les Problémes de la Sexualité. Paris, 1913.

2 Whitney, D. D., Science, 1916, xliii., 176.

3 Shull, A. F., and Ladoff, S., Science, 1916, xliii., 177.

Basis of Sex Determination 225

7. The effects of the removal of the ovaries or
testes upon the development of secondary sexual
characters differ for different species. In insects the
secondary sexual characters are not altered by an
operative removal of the sexual glands as in the cater-
pillar, e.g., Ocneria dispar, according to Oudemans.
This result has been invariably confirmed by all subse-
quent workers, especially by Meisenheimer. Crampton
grafted the heads of pupz of butterflies upon the bodies
of other specimens of the opposite sex, but the sexual
characters of the head remained unaltered.

In vertebrates, however, there exists a distinct
influence of a secretion from the sexual glands upon the
development of certain of the secondary sexual char-
acters, which do not develop until sexual maturity.
In a way the observations on arrhenoidy and thelyidy
referred to above are indications of this influence.

Bouin and Ancel. had already suggested that the
sexual glands of mammals have two independent
constituents, the sexual cells and the interstitial tissue;
and that the latter tissue is responsible for the develop-
ment of the secondary sexual character. This has
been proved definitely by Steinach,* who showed that
when young rats are castrated certain secondary
sexual characters are not fully developed. The seminal
vesicles and the prostate remain rudimentary and

tSteinach, E., Zentralbl. f. Phystol., 1910, xxiv., 551; Arch. f. d. ges.
Physiol., 1912, cxliv., 72.

226 Basis of Sex Determination

the penis develops incompletely. Such animals when
adult recognize the female and seem to follow it, but
do not persist in their attention and neither erection
nor cohabitation occurs. When, however, the testes
are retransplanted into the muscles of the castrated
young animal (so that they are no longer connected
with their nerves) seminal vesicles, prostate, and penis
develop normally, and these animals show normal
sexual ardour and cohabitate with a female although the
female cannot become pregnant since the males cannot
ejaculate any sperm. When the retransplanted testes
were examined it was found that all the sperm cells
had perished, only the interstitial tissue of the testes
remaining. It was, therefore, proved that the develop-
ment of the seminal vesicles, the prostate, the penis,
and the normal sexual instincts and activities depends
upon the internal secretions from this interstitial tissue
and not upon the sex cells proper. This agrees with the
conclusions at which Bouin and Ancel had arrived by
ligaturing the vasa deferentia of male animals.
Steinach in another series of experiments castrated
young male rats and transplanted into them the ovaries
of young females. These ovaries did not disintegrate,
the eggs remaining, and corpora lutea were formed.
In such feminized individuals the seminal vesicles,
prostate, and penis did not reach their normal develop-
ment, and it was thereby proved that the internal secre-
tions from the ovary do not promote the growth of the

Basis of Sex Determination 227

secondary sexual male characters. On the contrary,
Steinach was able to show that the growth of the
penis was directly inhibited by the ovary, since in the
feminized males this organ remained smaller than in
the merely castrated animals. On the other hand the
infantile uterus and tube when transplanted into the
young male with the ovaries grow in a normal way,
and Steinach thinks that pregnancy in such feminized
males is possible if sperm be injected into the uterus.
In some regards the feminized males showed the mor-
phological habitus of females. Soon after the trans-
plantation of ovaries into a castrated male the nipples
of its mammary glands begin to grow to the large size
which they have in the female and by which the two
sexes can easily be discriminated. In addition the
stronger longitudinal growth of the body in the male
does not occur in the feminized specimens, the body
growth becomes that of a female; and likewise the fat
and hair of the feminized male resemble that of a real
female.

While the castrated males show an interest in the
females, the feminized males are absolutely indifferent
to females and behave like them when put together
with normal males; and, what is more interesting, they
are treated by normal males like normal females. The
sexual instincts have, therefore, also been reversed
in the feminized males by the substitution of ovaries
for testes.

228 Basis of Sex Determination

The inhibition of the growth of the penis by the ovary
is of importance; it supports the idea already expressed
that in hermaphrodites this inhibition of the growth
of the secondary organs of the other sex is only feeble
or does not exist at all.

We may finally ask whether there is any connection
between the cytological basis of sex determination by
special sex chromosomes and the physiological basis
of sex determination by specific substances or internal
secretions. It is possible that the sex chromosomes
determine or favour, in a way as yet unknown, the
formation of the specific internal secretion discussed
in the second part of this chapter. In this way all the
facts of sex determination might be harmonized, and it
may become clear that when it is possible to modify
secretions by outside conditions or to feed the body
with certain as yet unknown specific substances the
influence of the sex chromosomes upon the determi-
nation of sex may be overcome.

CHAPTER IX

MENDELIAN HEREDITY AND ITS MECHANISM*
I

1. The scientific era of the investigation of heredity
begins with Mendel’s paper on plant hybridization
which was not appreciated by his contemporaries.
Mendel invented a method for the quantitative study
of heredity which consisted essentially in crossing two
forms of peas differing only in one well-defined heredi-
tary character; and in following statistically and
separately the results of this crossing and that of the
inbreeding of the second and third generations of
hybrids. This led him to the recognition of one
essential feature of heredity; namely, that while the
hybrids of the first generation are all alike, each hy-
brid produces two types of sex cells in equal numbers,
one for each of the pure breeds which has been used
for the crossing. This takes place not only when the
forms used for the crossing differ in regard to one

tFor the literature on the subject the reader is referred to Morgan,
T. H., Sturtevant, A. H., Muller, H. J., and Bridges, C. B., The
Mechanism of Mendelian Heredity. New York, 1915.
229

230 Mechanism of Mendelian Heredity

character only but also if they differ for two or more
characters. The statement made is Mendel’s law of
heredity, or, more correctly, Mendel’s law of the
segregation of the hereditary characters of the parents
in the sex cells of the hybrids.t Mendel’s law allows
us to tabulate and calculate beforehand the relative
number of different forms which appear if the offspring
of a mating of two varieties are bred among themselves.

In order to do this it must be remembered also that
while in some cases the hybrid is an intermediate
between the two parent forms, in other cases it can-
not be discriminated from one of the two parent forms.
In such cases the character which appears in the
hybrid was called by Mendel the dominant character
and the one which disappeared the recessive character.
According to Bateson, who was the first to systematize
the phenomena of Mendelian heredity, recessiveness
means generally the absence of a character which is
present in the dominant type. When, e. g., the cross
between a tall and a dwarf form of pea gives in the
first generation only tall peas, on the basis of the
presence and absence theory the dominant form con-
tains a factor for growth which is lacking in the dwarf
form. While this theory fits many cases it meets with
difficulties in others. Thus the presence of a factor

* Mendel, G., “Experiment in Plant-Hybridization,” translated in
W. Bateson’s classical book on Mendel's Principles of Heredity.
Cambridge, 1909.

Mechanism of Mendelian Heredity 231

for pigment should be dominant over the absence of
such a factor, which is usually the case, inasmuch as
the cross of a coloured rat or rabbit with an albino is
black or coloured. There is, however, also a case where
whiteness is dominant over colour, as we shall see later.
This fact does not necessarily contradict the presence
and absence theory.*

When two pure breeds of parents differ in one char-
acter, é. g., two varieties of beans, one with a violet the
other with a white flower, the cross between the two
species (the F, generation) has pale violet flowers,
approximately intermediate between the two parents.
If these hybrids are bred among themselves the off-
spring is called the F, generation. According to
Mendel’s law the hybrids of the first F, generation all
have two kinds of eggs in equal numbers, one kind
representing the pure breed of the parents with violet,
the other of the pure breed with white flowers. The
same is true for the pollen cells. Hence the following
possible combinations must appear in the offspring
when the pale violet hybrids are inbred:

violet white...... eggs
violet white...... pollen

The four possible combinations are: (1) violet-—violet;

r The reader will find a critical discussion of the presence and absence
theory on page 220 of Morgan, Sturtevant, Muller, and Bridges, The
Mechanism of Mendelian Heredity. New York, 1915.

232 Mechanism of Mendelian Heredity

(2) violet-—white; (3) violet-—white; (4) white—white.
The first will result in pure violet flowers, the fourth
in pure white, and the second and third in pale violet
flowers. Since all four combinations will appear in
equal numbers when the number of crossings is suffi-
ciently large the numerical result will be:

violet: pale violet: white=1:2:1

Fifty per cent. of the F., generation will be pale
violet, 25 per cent. violet, and 25 per cent. white. The
violets and whites each will breed true when bred
among themselves since they are pure, and produce
only one type of eggs and pollen. The pale violets
are hybrids and will again produce the two types
of eggs and pollen, that is, if bred among themselves
will again give violets, pale violets, and whites in the
tratio1:2:1. This the experiment confirms.

As has been stated, it not infrequently happens that
all the hybrids of the first generation are alike. In such
cases the one character is ‘‘ recessive,’ i.e., overshadowed
or covered by the other the “dominant” character,
which alone appears in the hybrids. Thus when
Mendel crossed peas having round seeds with peas
having angular seeds all the hybrids had round seeds.
The round form is dominant, the angular recessive,
i. é., all the hybrids have round seeds. When these
hybrids were bred among themselves the next genera-

Mechanism of Mendelian Heredity 233

tion produced round and angular seeds in the ratio of
3:1 (5474 round to 1850 angular). The explanation
is as follows. Let R denote round, A angular character;
the pure breeds of parents have the gametic constitu-
tion RR and AA respectively. When crossed, all the
offsprings have the constitution RA and since A is
recessive this hybrid generation resembles the pure RR
parents. The F, generation produces two kinds of
eggs R and A and two kinds of pollen R and A in equal
numbers, and these if inbred give the following four
combinations in equal numbers:

RR, RA, AR, AA.

Since RA, AR, and RR all give round seeds the F,
generation produces round seeds to angular seeds in
the ratio of 3:1. The two organisms with the gametic
constitution RR and RA look alike, yet they are
different in regard to heredity. The gametically pure
form RR is called homozygous, the impure form RA
heterozygous.

2. W. S. Sutton' was the first to show that the
behaviour of the chromosomes furnishes an adequate
basis on which to account for Mendel’s law of the
segregation of the characters in the sex cells of the
hybrids. If we disregard the cases of parthenogenesis
and the X chromosomes, we may state that each

t Sutton, W. S., ‘‘ The Chromosomes in Heredity,” Biol. Bull., 1904,
iv., 231.

234 Mechanism of Mendelian Heredity

species is characterized by a definite number of
chromosomes, e. g.*

man (probably)........ BANCOTN seo teohe ances aos 20
MOUSE.........2------ 20 evening primrose....... 7
snail (Helix hortensis)... 22 nightshade............. 36
potato beetle.......... 18 tobacco............... 24
COULON . acura ence Ga xe 28 tomato................ 12
four o’clock............ 16 wheat...........+---55 8
garden pea............ 7

In the fertilization of the egg the number of chro-
mosomes is doubled (if we disregard for the moment
the complication caused by the X and Y chromosomes
which was considered in the previous chapter). It
was noticed by Montgomery that each chromosome
had a definite size and individuality, and he suggested
that homologous chromosomes existed in sperm and
egg and that in fertilization the homologous chromo-
somes of egg and sperm always joined and fused in the
special stage designated as synapsis, which will interest
us later. On the basis of this suggestion Sutton
developed the chromosome theory of the mechanism
of Mendelian heredity or segregation.

According to this theory, all the cells of an individual
(inclusive of the egg cells and sperm cells) have two
sets of homologous chromosomes, one from the father,
the other from the mother. Before the egg and sperm

«Morgan, T. H., Sturtevant, A. H., Muller, H. J., and Bridges, C.
B., Mechanism of Mendelian Heredity. New York, 1915, p. 26.

Mechanism of Mendelian Heredity 235

are ready for the production of a new individual, each
loses one set of homologous chromosomes in the so-
called reduction division, but the lost set is made up
indiscriminately of maternal as well as paternal chro-
mosomes, so that while one egg retains the maternal
chromosome A the other will retain the paternal one,
and soon. If before the reduction division all the eggs
had the chromosome constitution AA,, BB,, CC,, DD,
(where A B C D are the paternal and A, B, C, D, the
maternal chromosomes), after the reduction division
each daughter cell has a full set of four chromosomes,
but maternal and paternal mixed. Thus the one cell
may have AB,CD,, the other A,B,C.D, etc. This,
according to Sutton, is the basis of the Mendelian
heredity. Suppose the determiner of a certain char-
acter (violet colour of flower in the bean) is located
in a chromosome A of this species. The homologous
chromosome in beans with white colour may be desig-
nated as a. According to the chromosome theory of
Mendelian heredity a differs from A in one point,
though this difference is probably only of a chemical
character and not visible.

If an egg with A is fertilized by a pollen with a
(or vice versa), after fertilization the chromosome con-
stitution of the fertilized egg is Aa. All the other
homologous chromosomes are identical and therefore
need not be considered. All the nuclei of the F,
generation have the chromosome constitution Aa.

236 Mechanism of Mendelian Heredity

All will form eggs and pollen with nuclei of the same
chromosome constitution Aa, but all these sex cells
will go through the maturation division before they are
fertilized; and this reduction division leads to the
existence of two kinds of eggs in equal numbers, one
containing only the A, the other only the a chromosome;
and the same happens in the pollen. When therefore
the hybrids F, are mated among themselves, the
following four chromosome combinations will be pro-
duced:

es Pollen Possible combinations in fer-
a tilized eggs AA, Aa, aa, in

SS the ratio 1:2: 1.

Now this is exactly the ratio of Mendelian heredity
in the F, generation. The plant with the chromosome
constitution AA will form violet flowers, those with the
chromosome constitution Aa will form pale violet
flowers, and those with the chromosome constitution
aa will form white flowers.

To quote Sutton’s words:

The result would be expressed by the formula AA: Aa:
aa which is the same as that given for any character in a
Mendelian case. Thus the phenomena of germ cell divi-
sion and of heredity are seen to have the same essential
features viz., purity of units (chromosomes, characters)
and the independent transmission of the same; while as a
corollary it follows in each case that each of the two antago-

Mechanism of Mendelian Heredity 237

nistic units (chromosomes, characters) is contained by
exactly half the gametes produced.

It is obvious that Sutton by this idea did for heredity
in general what McClung had done for sex determi-
nation or sex heredity, that is, he showed that the
numerical results obtained in Mendelian heredity can
be accounted for on the basis that factors for hereditary
characters are carried by definite chromosomes. The
cytological basis of sex determination becomes only a
special case of the cytological basis of Mendelian
heredity. In the examples quoted the plants giving
rise to violet and to white flowers are homozygous
for the colour of flower having the chromosome constitu-
tion AA and aa respectively; while the plants with pale
violet flowers are heterozygous, having the chromosome
constitution Aa in their nuclei. The former give rise
to identical sex cells A and A or a and a; while the
heterozygous plants give rise to different sex cells A
anda.

From this point of view in Drosophila (and very
probably also in man) the female is homozygous for
sex having in all its cells the critical chromosome
constitution XX and giving rise to one type of eggs
only, each with one X chromosome; while the male in
these forms is heterozygous for sex having in all its
cells the chromosome constitution XY and forming
two different types of spermatozoa in equal numbers

238 Mechanism of Mendelian Heredity

X and Y. In Abraxas and in the fowl the female
is heterozygous for sex and the male homozygous.

3. If the chromosomes are the vehicle for Mendelian
heredity it should be possible to show that the various
hereditary characters which follow Mendel’s law must
be distributed over the various chromosomes; and it
should be possible to find out which characters are
contained in the same chromosome. It has already
been stated that sex-linked heredity is intelligible on
the ‘assumption that the X chromosome carries the sex-
linked characters. T. H. Morgan and his pupils have
shown with the greatest degree of probability that
corresponding linkages occur in the other chromosomes
and that there are in Drosophila exactly as many groups
of linkage as there are different chromosomes, namely
four.*

Mendel had found that when he crossed two species
of peas differing in regard to two pairs of characters,
he obtained in the F, generation results which he
calculated on the assumption that the segregation of the
two pairs of characters in the sex cells of the hybrids
took place independently of each other. To illustrate
by an example: When crossing a yellow round pea
with a green wrinkled variety in which the characters
round and yellow are dominant, green and wrinkled
recessive, all the hybrids of the F, generation had the

* Morgan, T. H., Sturtevant, A. H., Muller, H. J., and Bridges, C. B.,
The Mechanism of Mendelian Heredity. New York, 1915.

Mechanism of Mendelian Heredity 239

characters round and yellow. When these were inbred
the F, generation produced four types of seed in the
ratio 9 :3:3:1, namely:

(1) yellow round (315 seeds)
(2) yellow wrinkled (101 seeds)
(3) green round (108 seeds)
(4) green wrinkled (32 seeds)

The explanation according to Mendel’s theory is as
follows: Since the segregation of each pair of char-
acters occurs independently, there must be 3 yellow to I
green and also 3 round to I wrinkled in the F, genera-
tion. The yellow will, therefore, be round and wrinkled
in the ratio of 3:1, which will give 9 yellow round to 3
yellow wrinkled. The green will also be round and
wrinkled in the ratio of 3: 1, which will give 3 green round
to I green wrinkled, which is the ratio of 9: 3: 3: 1
found by Mendel.

On the basis of the chromosome theory the following
explanation could be given of this numerical relation.
The peas with yellow round seeds have sex cells with a
factor for both yellow and for round in two different
chromosomes; these two different chromosomes we will
designate with Y and R. The peas with green and
wrinkled seeds will have in their sex cells factors for
these characters in two homologous chromosomes g
and w, where g is the homologue of Y and w of R.
The cells of the hybrids of the F, generation will have

240 Mechanism of Mendelian Heredity

the chromosome constitution Yg Rw, where Y and g
and R and w are homologous chromosomes which will
lie alongside each other %%. In the formation of sex
cells a reduction of these four chromosomes to two
takes place whereby, according to the theory of Sutton,
the following two types of separation can take place:
YR and gw, or gR and Yw. (A separation into Yg
and Rw is impossible since the division takes place
only between homologous chromosomes.) Hence there
will be four types of eggs, YR, gw, gR, and Ywand the
same four types of pollen cells. The F, generation
will produce the sixteen possible combinations in equal
numbers: namely,

YRYR YRgw YReR YRYw
gwYR gwgew gweR gwYw
gRYR gRegew gRgR gRYw
YwYR Ywew YweR YwYw

Since wand g are recessives and therefore disappear
when in combination with their respective dominants
Y and R the result will be 9 YR (yellow round), 3
Yw (yellow wrinkled), 3 Rg (round green), and 1 gw
(green wrinkled) as Mendel actually observed and as
all investigators since have confirmed.

Bateson made the discovery that these Mendelian
ratios 9: 3: 3: 1 did not always occur when forms
differing in two characters were crossed. He found
typical and very constant deviations from this ratio

Mechanism of Mendelian Heredity 241

in definite cases and these cases he interpreted as being
due to ‘‘gametic coupling.”

These phenomena demonstrate the existence of a complex
interrelation between the factorial units. This interrela-
tion is such that certain combinations between factors may
be more frequent than others. The circumstances in which
this interrelation is developed and takes effect we cannot
as yet distinguish, still less can we offer with confidence
any positive conception as to the mode in which it is exerted.

Morgan has given an ingenious explanation of these
deviations on the basis of the chromosome theory of
Mendelian heredity. He assumes that they occur in
those cases where the two or more characters are con-
tained in the same chromosome. In that case the two
factors lying in the same chromosome should generally
be found together. Such was the case for instance in
the experiments with flies having red eyes and yellow
body colour versus white eyes and grey body colour, the
character for white eyes and yellow body being located
in the X chromosome (see preceding chapter), or in the
experiments on Abraxas. These phenomena are called
linkage, and the numerical results of linkage were given
in the preceding chapter in connection with the crossing
of sex-linked characters.

We have already mentioned that before the matura-
tion division occurs the homologous maternal and
paternal chromosomes fuse—the so-called synapsis

* Bateson, W., loc. cit., p. 157.
16

242 Mechanism of Mendelian Heredity

of the cytologists—and afterward separate again. It
had been observed by Janssens that in this stage of
fusion and subsequent separation a partial twisting
and a partial exchange between two chromosomes may
take place. Morgan assumes that this exchange
accounts for certain. deviations in the ratio of link-

\ @ YY
i} a
AW y ]
7 y
] |
Y J
j ]
|| a
BL 7 ¢
YY
ee Fic. 41

age. If in Fig. 4o the white and black signify two
homologous chromosomes I and I, containing the
two pairs of homologous factors AB and ab respectively,
the synapsis state would be as in Fig. 41. If the
separation were complete, either I or its homologue I,
might be lost in the maturation division of the egg.
If, however, the synapsis is slightly irregular, as in
Fig. 42, where the chromosomes are slightly twisted,
T and I, will not separate completely but an exchange

Mechanism of Mendelian Heredity 243

will take place, part of I, and I becoming exchanged.
This would result in the formation of two mixed chro-
mosomes Ab and aB (Fig. 42). This partial ex-
change of homologous chromosomes, which Morgan
calls “crossing over,” occurs, as he found in Drosophila,
in the egg only, not in the maturation division of the
sperm. He informs me that in the silkworm moth
Tanaka found that it occurs only in the male, while in
Primula it takes place both in the ovules and in the
pollen as shown by Gregory.

Morgan and his fellow-workers have put this theory
to numerous tests by breeding experiments and the
results have fully supported it. According to the.
chromosome theory linkage should occur only when
factors lie in the same chromosome. Hence it should be
possible, on the basis of this linkage theory, to foretell
how many linkage groups there may occur in a species;
namely, as many as there are chromosomes. In
Drosophila there are four pairs of chromosomes, and
Morgan and his fellow-workers found only four groups
of linked characters.* This agreement can be no mere
accident.

Carrying the assumption still farther, these authors
were able to show that each individual character has
in all probability a definite location in the chromosome,
so that it seems as if each individual chromosome

« The number of hereditary characters examined to test the theory was
over 130.

244 Mechanism of Mendelian Heredity

consisted of a series of smaller chromosomes, each of
which may be a factor in the determination of a heredi-
tary character which is transmitted according to Men-
del’s law of segregation. Biology has thus reached in the
chromosome theory of Mendelian heredity an atomistic
conception, according to which independent material
determiners for hereditary characters exist in a linear
arrangement in the chromosomes.

II
3

4. We are not concerned in this volume with the
many applications of the theory of heredity to the
breeding of plants, animals, and man; the reader
will find a discussion of these topics in the numerous
writings of the special workers on genetics.‘ We are,
however, interested in the bearing this work has on
the conception of the organism. Two questions present
themselves: Is the organism nothing but a mosaic
of hereditary characters determined essentially by de-
finite elements located in the chromosomes; and if
this be true, what makes a harmonious whole organism
out of this kaleidoscopic assortment? We call it a
kaleidoscopic assortment since a glance at the list of
hereditary characters found in one chromosome,
according to Morgan, shows that there is apparently

«Bateson, W., Mendel’s Principles of Heredity, 3d ed., 19133

Davenport, Chas. B., Heredity in Relation to Eugenics, 1911. Pearl, R.,
Modes of Research in_Genetics.

Mechanism of Mendelian Heredity 245

no physiological or chemical connection between
them, and second: How can a factor contained in the
chromosome determine a hereditary character of the
organism? To the first question we venture to offer
the answer which has been already suggested in various
chapters of this book, that the cytoplasm of the egg
is the future embryo in the rough; and that the factors
of heredity in the sperm only act by impressing the
details upon the rough block. This metaphor will
receive a more definite meaning by the answer to the
second question. The characters which follow Mende-
lian heredity are morphological features as well as
instincts. For the former we have already had occa-
sion to show in previous chapters to what extent they
depend upon the internal secretions or the existence of
specific compounds in the circulation, and the same is
true for the instincts (Chapters VIII and X). This
then leads us to the suggestion that these determiners
contained in the chromosomes give rise each to the
formation of one or more specific substances which
influence various parts of the body. We probably
do not notice all the effects in each case, but when a
special organ is affected in a conspicuous way, we con-
nect the factor with this organ or the special feature of
the organ which is altered, and speak of a determiner or
factor for that organ, or for one of its characters. We
also understand in this way why outside conditions
should be able to overcome the hereditary tendency

246 Mechanism of Mendelian Heredity

in certain cases, for instance why the influence of certain
hereditary factors for pigmentation should depend
tupon temperature as E. Baur observed.

The view, according to which the determiners in the
chromosomes only tend to give special characters to
the embryo or to the adult while the cytoplasm of
the egg may be considered the real embryo, receives
some support from the fact that the first development
of the egg is purely maternal, even if the egg nucleus
has been replaced by sperm of a different species.
If an egg of a sea urchin be cut into two pieces, one
with and one without a nucleus, and the enucleated
piece be fertilized with the sperm of a different species of
sea urchin, the blastula and gastrula stages are purely
maternal and only the skeleton of the pluteus stage
begins to betray the influence of the foreign sperm
inasmuch as this skeleton is purely paternal, according
to Boveri. In all experiments on hybridization it has
been found that the rate of cell division of the egg is a
purely maternal character. Thus when fish eggs of a
species, in which the rate of first segmentation of the
egg is about eight hours, are fertilized with sperm of a
species for which the same process requires about
thirty minutes or less at the same temperature, the
rate of segmentation is again about eight hours. There
is then no chromosome influence noticeable in the early
development.

When two forms of sea urchins, Strongylocentrotus

Mechanism of Mendelian Heredity 247

franciscanus and purpuratus,* are crossed, certain
features of the skeleton of the embryo, e. g., the so-called
cross-bars, are a dominant, inasmuch as they are found
in purpuratus and both the crosses, while they are
absent in franciscanus. The development prior to
the formation of the skeleton is purely maternal.
These observations again lend support to the idea that
the Mendelian factors of heredity must have the
embryo to work on and that the organism is not to be
considered a mere mosaic of Mendelian factors. This is
further supported by the idea that the species specificity
resides in the proteins of the unfertilized egg (see Chap-
ter III), and it is quite likely that this species specificity
decides which type of animal should arise from an egg.
The idea had been suggested that the factors which
determine the future character might be ferments or
enzymes, or substances from which such ferments de-
velop. A.R. Moore? pointed out that the cross-bars in
the skeleton of the hybrid between S. purpuratus and
franciscanus develop more slowly than in the pure breed
and that this should be expected if the determiners were
enzymes. Since the pure purpuratus has two deter-
miners for the development of the cross-bars (from
both egg and sperm), the hybrids only one (from either
t Loeb, J., King, W. O. R., and Moore, A. R., Arch. f. Entwcklngsmech..
1910, Xxix., 354. These experiments have been repeated at different
seasons of the year and in different years and have been found to be

constant.
2Moore, A. R., Arch. f. Entwcklngsmech., 1912, xxxiv., 168.

248 Mechanism of Mendelian Heredity

egg or sperm), the pure purpuratus should have twice
the enzyme mass of the hybrid. It is known that the
velocity of a chemical reaction increases in proportion
with the mass (or in some cases in proportion with
the square root of the mass) of the enzyme; the cross-
bars should therefore develop faster in the pure than
in the hybrid breeds, as was observed by Moore. It
was, however, not possible to obtain quantitative data.
On the other hand, it is obvious that this reasoning
would not hold for all cases. Thus when beans with
violet flowers are crossed with white-flowered beans
the hybrids are pale blue, which indicates that the
hybrids have less pigment than the pure violet. Now
we know that the mass of enzyme does not influence
the chemical equilibrium but only the velocity of the
reaction. The hybrids and pure violets differ, how-
ever, in the mass of violet pigment formed, that is to
say, in regard to the equilibrium. Hence the idea that
the determiners are enzymes or give rise to enzymes is
probably not applicable to cases of this type.
_ The experiments on the heredity of pigments are
at present almost the only ones which can be used for
an analysis of the chemical nature of the character
and its possible determiner. The important work of
G. Bertrand* and of Chodat? on the production of

«Bertrand, G., Ann. d. l’Inst. Pasteur, 1908, xxii., 381; Bull. Soc.
Chim., 1896, xv., 791.
2 Chodat, R., Arch. d. Sc. phys. et nat., 1915, xxxix., 327.

Mechanism of Mendelian Heredity 249

black pigment in the cells of animals and plants with
the aid of enzymes has paved the way for such work.
Bertrand has shown that tyrosine (p-oxyphenylamino-
propionic acid) is transformed into a black pigment
by an enzyme tyrosinase which occurs in numerous
organisms and is obviously the cause of pigment and
colouration in a great number of species. This discovery
was utilized in the study of the heredity of pigments
by Miss Durham, Gortner,* and very recently by On-
slow.?. The latter showed that from the skins of cer-
tain coloured rabbits and mice a peroxidase can be
extracted which behaves like a tryosinase toward
tyrosine in the presence of hydrogen peroxide. This
peroxidase was found in the skins of black agouti,
chocolate and blue rabbits, but not in yellow or orange
rabbits. The recessive whiteness in rabbits and mice
according to this author is due to the lack of the per-
oxydase. There exists a dominant whiteness in the
English rabbit which is due to a tyrosinase inhibitor
which destroys the activity of the tyrosinase ‘‘and the
dominant white bellies of yellow and agouti rabbits
are due to the same cause.” ‘“‘ Variations in coat colour
are probably due to a quantitative rather than to a
qualitative difference in the pigment present.”

One point might still be mentioned since it may help
to overcome a difficulty in visualizing the connection

tGortner, R. A., Trans. Chem. Soc., 1910, xcvii., 110.
2 Onslow, H., Proc. Roy. Soc., 1915, B. Ixxxix., 36.

250 Mechanism of Mendelian Heredity

between the localization of a factor in the chromosome
and the production of a comparatively large quantity of
a specific chemical compound, e. g., a chromogen or a
tyrosinase. We must remember that all the cells of
an organism have identical chromosomes, so that
a factor for an enzyme like tyrosinase is contained
in every cell throughout the whole body. It is
likely, however, that the same factor (which we may
conceive to be a definite chemical compound) will
find a different chemical substrate to work on in
the cells of different organs of the body, since the
different organs differ in their chemical composition.
Thus it is conceivable that in the production of tyro-
sinase or of tyrosine not a single chromomere of one
single cell is engaged, but the sum total of all these
individual chromomeres of all the cells in one or several
organs of the body. The writer has added this remark
especially in consideration of the fact that some authors
seem to feel that the chromosome conception of heredity
is incompatible with a physicochemical view of this
process.

Since we have mentioned this difficulty which some
writers seem to find in the chromosome theory of Men-
delian heredity, it may be added that a single factor
may suffice to determine a series of complicated reflexes.
Thus the heliotropic reactions of animals are due to the
presence of photosensitive substances, and it suffices
for the hereditary transmission of the complicated

Mechanism of Mendelian Heredity 251

purposeful reactions based on these tropisms that a
factor for the formation of the photosensitive substance
should exist.*

5. Another point should be emphasized, namely
that for Mendelian heredity it is immaterial whether
the character is introduced by the spermatozdon or
by the egg. This fact which Mendel himself already
recognized is in full harmony with the conclusion that
the chromosomes and not the cytoplasm are the bearers
of Mendelian heredity, since only in respect to the
chromosome constitution are egg and sperm alike,
while they differ enormously in regard to the mass of
protoplasm they carry. Wecan, therefore, be tolerably
sure that wherever we deal with a hereditary factor
which is determined by the egg alone the cytoplasm
of the latter is partly or exclusively responsible for the
result.

We have already mentioned the fact that the rate of
segmentation of the egg is such a character. Yet
this character is as definite as any Mendelian character,
and it would be as easy to discriminate two species
of eggs by the time required from insemination to the
beginning of cell division as it would be by any Men-
delian character of their parents.

The application of our modern knowledge of heredity
to human affairs has been discussed in a very original

« Loeb, J., ‘Egg Structure and the Heredity of Instincts,” The Monist,
1897, vii., 481.

252 Mechanism of Mendelian Heredity

way by Bateson in his address before the British
Association in Sydney to which the reader may be

referred.

«Bateson, W., Nature, 1916, xciii., 674.

CHAPTER X
ANIMAL INSTINCTS AND TROPISMS*

1. The idea that the organism as a whole cannot
be explained from a physicochemical viewpoint rests
most strongly on the existence of animal instincts
and will. Many of the instinctive actions are “pur-
poseful,”’ 2. e., assisting to preserve the individual and
the race. This again suggests ‘‘design” and a design-
ing “‘force,’’ which we do not find in the realm of
physics. We must remember, however, that there was
a time when the same ‘‘purposefulness” was believed
to exist in the cosmos where everything seemed to
turn literally and metaphorically around the earth,
the abode of man. In the latter case, the anthropo-
or geocentric view came to an end when it was shown
that the motions of the planets were regulated by
Newton’s law and that there was no room left for the

x Ideas similar to those expressed in this chapter may be found in the
writer’s former book Comparative Physiology of the Brain and Compara-
tive Psychology, New York, 1900, and in the books by George Bohn,
La Naissance de I'Intelligence, Paris, 1909, and La nouvelle Psychologie
animale, Paris, 1911.

253

254 Animal Instincts and Tropisms

activities of a guiding power. Likewise, in the realm
of instincts when it can be shown that these instincts
may be reduced to elementary physicochemical laws
the assumption of design becomes superfluous.

If we look at the animal instincts purely as observers
we might well get the impression that they cannot be
explained in mechanistic terms. We need only consider
what mysticism apparently surrounds all those instincts
by which the two sexes are brought together and by
which the entrance of the spermatozo6n into the egg
is secured; or the remarkable instincts which result in
providing food and shelter for the young generation.

We have already had occasion to record some cases
of instincts which suggest the possibility of physico-
chemical explanation; for example the curious experi-
ment of Steinach on the reversal of the sexual instincts
of the male whose testes had been exchanged for
ovaries. There is little doubt that in this case the
sexual activities of each sex are determined by specific
substances formed in the interstitial tissue of the
ovary and testes. The chemical isolation of the
active substances and an investigation of their action
upon the various parts of the body would seem to
promise further progress along this line.

Marchal’s observations on the laying of eggs by the
naturally sterile worker wasps are a similar case. The
fact that such workers lay eggs when the queen is
removed or when they are taken away from the larvze

Animal Instincts and Tropisms 255

may be considered as a manifestation of one of those
wonderful instincts which form the delight of readers
of Maeterlinck’s romances from insect life. Imagine
the social foresight of the sterile workers who when the
occasion demands it ‘‘raise’’ eggs to preserve the stock
from extinction! And yet what really happens is that
these workers, when there are no larve, can consume
the food which would otherwise have been devoured
by the larve; and some substance contained in this
food induces the development of eggs in the otherwise
dormant ovaries. What appeared at first sight as a
mysterious social instinct is revealed as an effect
comparable to that of thyroid substance upon the
growth of the legs of tadpoles in Gudernatsch’s
experiment (Chapter VII).

2. If we wish to show in an unmistakable way the
mechanistic character of instincts we must be able to
reduce them to laws which are also valid in physics.
That instinct, or rather that group of instincts, for
which this has been accomplished are the reactions
of organisms to light. The reader is familiar with the
tendency of many insects to fly into the flame. It
can be shown that many species of animals, from the
lowest forms up to the fishes, are at certain stages—
very often the larval stage—of their existence, slaves of
the light. When such animals, e. g., the larve of the
barnacle or certain winged plant lice or the cater-
pillars of certain butterflies, are put into a trough or

256 Animal Instincts and Tropisms

test-tube illuminated from one side only, they will rush
to the side from which the light comes and will continue
to do this whenever the orientation of the trough
or test-tube to the light is changed; while they will be
held at the window side of the vessel if the light or
the position of the vessel remains unchanged. This
instinct to get to the source of light is so strong that,
é. g., the caterpillars of Porthesia chrysorrhea die of
starvation on the window side of the vessel, with plenty
of food close behind. This powerful ‘‘instinct’’ is,
as we intend to show, in the last analysis, the expres-
sion of the Bunsen—Roscoe law of photochemical re-
actions. A large number of chemical reactions are
induced or accelerated by light, and the Bunsen—Roscoe
law shows that the chemical effect is in these cases,
within certain limits, equal to the product of the in-
tensity into the duration of illumination.

The “attraction” or “repulsion” of animals by the
light had been explained by the biologists in an anthro-
pomorphic way by ascribing to the animals a ‘“‘fond-
ness” for light or for darkness. Thus Graber, who had
made the most extensive experiments, gave as a result
the statement that animals which are fond of light
are also fond of blue while they hate the red, and
those which are fond of the “dark” are fond of red and
hate the blue.* In 1888 the writer published a paper

*Graber, V., Grundlinien zur Erforschung des Hielligheits- und
Farbensinnes der Tiere. Prag, 1884.

Animal Instincts and Tropisms 257

in which he pointed out that the so-called fondness of
animals for light and blue and for dark and red was
simply a case of an automatic orientation of animals
by the light comparable to the turning of the tips of a
plant towards the window of the room in which the
plant is raised.*

The phenomenon of a plant bending or growing to the
source of light is called positive heliotropism (while
we speak of negative heliotropism in all cases in which
the plant turns away from the light, as is observed
in many roots). The writer pointed out that animals
which go to the light are positively heliotropic (or
phototropic) and do so because they are compelled
automatically by the light to move in this direction,
while he called those animals which move away from
the light negatively heliotropic; they are automatically
compelled by the light to move away from it. What
the light does. is to direct the motions of the animals
and to explain this the following theory was proposed.
Animals possess photosensitive elements on the surface
of their bodies, in the eyes, or occasionally also in
epithelial cells of their skin. These photosensitive
elements are arranged symmetrically in the body and
through nerves are connected with symmetrical groups

of muscles. The light causes chemical changes in the

t Loeb, J., Sitzungsber. d. physik.—med. Gesellsch. Wurzburg, 1888.
Der Heliotropismus der Tiere und seine Ubereinstimmung mit dem Helio-
tropismus der Pflanzen. Warzburg, 1889. Arch. f. d. ges. Physiol.,
1897, Ixvi., 439.

258 Animal Instincts and Tropisms

eyes (or the photosensitive elements of the skin). The
mass of photochemical reaction products formed in
the retina (or its homologues) influences the central
nervous system and through this the tension or energy
production of the muscles. If the rate of photo-
chemical reaction is equal in both eyes this effect
on the symmetrical muscles is equal, and the muscles
of both sides of the body work with equal energy; as a
consequence the animal will not be deviated from the
direction in which it was moving. This happens when
the axis or plane of symmetry of the animal goes through
the source of light, provided only one source of light
be present. If, however, the light falls sidewise upon
the animal, the rate of photochemical reaction will be
unequal in both eyes and the rate at which the sym-
metrical muscles of both sides of the body work will .
no longer be equal; as a consequence the direction
in which the animal moves will change. This change
will take place in one of two ways, according as the
animal is either positively or negatively heliotropic; in
the positively heliotropic animal the resulting motion
will be toward, in the negatively heliotropic from, the
light. Where we have no central nervous system, as
in plants or lower animals, the tension of the contractile
or turgid organs is influenced in a different way, which
we need not discuss here.

The reader will perceive that according to the
writer’s theory two agencies are to be considered in

Animal Instincts and Tropisms _—259

these reactions: first, the symmetrical arrangement of
the photosensitive and the contractile organs, and sec-
ond, the relative masses of the photochemical reaction
products produced in both retine or photosensitive
organs at the same time. If a positively heliotropic
animal is struck by light from one side, the effect on
tension or energy production of muscles connected
with this eye will be such that an automatic turning
of the head and the whole animal towards the source of
light takes place; as soon as both eyes are illuminated
equally the photochemical reaction velocity will be
the same in both eyes, the symmetrical muscles of the
body will work equally, and the animal will continue
to move in this direction. In the case of the nega-
tively heliotropic animal the picture is the same except
that if only one eye is illuminated the muscles connected
with this eye will work less energetically. The theory
can be nicely tested for negatively heliotropic animals
in the larvee of the blowfly when they are fully grown,
and for positively heliotropic animals on the larve of
Balanus, and many other organisms.

. One of the difficulties in identifying the motions
of animals to or from the light with the positive and
negative heliotropism of plants consisted in the fact
that plants are mostly sessile (and respond to a one-
sided illumination with heliotropic curvatures to or
from the light), while most animals are free moving
and respond to the one-sided illumination by being

260 Animal Instincts and Tropisms

turned and compelled to move to or from the light.
This difficulty was overcome by the observation that

Fic. 43

sessile animals like the hydroid Eudendrium (Fig. 43)
or the tube worm Spirographis (Fig. 44) react to a one-

Fic. 44

sided illumination also with heliotropic curvatures like
sessile plants.* On the other hand, it had been found
before by Strassburger that free-swimming plant

* Loeb, J., Arch. f. d. ges. Physiol., 1890, xlvii,, 391; 1896, Ixiii., 273.

Animal Instincts and Tropisms — 261

organisms like the swarmspores of alge move to or
from the source of light as do free-swimming animals.

3. The writer suggested in 1897' that the light acts
chemically in the heliotropic reactions and in 1912 that
the heliotropic reactions probably follow the law of
Bunsen and Roscoe,? and it was possible to confirm
this idea by direct experiments.3 This law states
that the photochemical effect of light equals zt where 7
is the intensity of the light and ¢ the duration of illumi-
nation. The experiments were carried out on young
tegenerating polyps of Eudendrium by measuring the
time required to cause fifty per cent. of the polyps
to bend to the source of light. The intensity of light
was varied by altering the distance of the source of
light from the polyps. ‘Table VI gives the result.

TABLE VI
Time Required to Cause Fifty Per Cent. of the
Polyps to Bend towards the Source of Light
Distance between Polyps
and Source of Light
CALCULATED FROM
OBSERVED BunsEN-Roscor LAW
Metres Minutes Minutes
0.25 10
0.50 between 35 and 40 40
1.00 150 160
1.50 between 360 and 420 360

® Loeb, J., Arch. f. d. ges. Physiol., 1897, Ixvi., 439.
2 Loeb, J., The Mechanistic Conception of Life, Chicago, 1912, p. 27.
3 Loeb, J., and Ewald, W. F., Zentralbl. f. Physiol., 1914, xxvii., 1165.

262 Animal Instincts and Tropisms

We must therefore conclude that the heliotropic
curvature of the polyps is determined by a photochemi-
cal action of the light. The light brings about or
accelerates a chemical reaction which follows the Bunsen—
Roscoe law. As soon as the product of this reaction
on one side of the polyp exceeds that on the other by
a certain quantity, the bending occurs. When the
product if is the same for symmetrical spots of the
organism no bending can result. This is what our
theory suggested.

It is very difficult to prove directly the applicability
of the Bunsen—Roscoe law for free-moving animals,
but it can be shown that intermittent light is as effect-
ive as constant light of the same intensity, provided
that the total duration of the illumination by the
intermittent light is equal to that of the constant light,
and the duration of the intermission is sufficiently small
(Talbot’s law). Talbot’s law is in reality only a modi-
fication of the Bunsen—Roscoe law. Ewald has proved
in a very elegant way the applicability of Talbot’s
law to the orientation of the eyestalk of Daphnia.*
This makes it probable that the law of Bunsen—Roscoe
underlies generally the heliotropic reaction of animals.

It is of importance for the theory of the identity
of the heliotropism of animals and plants that in the
latter organisms the law of Bunsen and Roscoe is also
applicable. This had been shown previously by

t Ewald, W. F., Science, 1913, xxxviii., 236.

Animal Instincts and Tropisms 263

Fréschel? and by Blaauw.* In the following table
are given the results of Blaauw’s experiments on the
applicability of the Bunsen—Roscoe law for the helio-
tropic curvature of the seedlings of oats (Avena sativa).
The time required to cause heliotropic curvatures for
intensities of light varying from 0.00017 to 26520
metre-candles was measured. The product 7/, namely
metre-candles-seconds, varies very little (between 16
and 26).

TABLE VII

I II III I II, III
Duration of | Metre- | Metre- Duration of Metre- Metre-
Illumination | Candles \Candles-|| Illumination Candles |Candles-
Seconds Seconds

43 hours 0.00017 | 26.3 25 seconds 1.0998 | 27.5

13 a 0.000439] 20.6 8 . 3.02813] 24.2

10 “ 0.000609] 21.9 4 ts 5.456 21.8

6 id 0.000855] 18.6 2 a 8.453 16.9

" 0.001769] 19.1 I ‘ 18.94 18.9

roo minutes | 0.002706] 16.2 2/5 en 45.05 18.0

60 oa 0.004773| 17.2 2/25 ae 308.7 24.7

30 0.01018 | 18.3 1/25 ue 511.4 20.5

20 0.01640 | 19.7 1/55 es 1255 22.8

15 ae 0.0249 22.4 1/100 te 1902 19.0

8 oe 0.0498 23.9 1/400 ee 7905 19.8

4 ne 0.0898 21.6 1/800 ne 13094. 16.4

4oseconds | 0.6156 24.8 ||1/1000 =“ 20520 26.5

It is, therefore, obvious that the blind instinct
which forces animals to go to the light, e. g., in the case
of the moth, is identical with the instinct which makes

« Fréschel, P., Sitzwngsber. d. k. Akad. d. Wissensch., Wien, 1908, cxvii.
2 Blaauw, H. A., Rec. d. travaux botaniques Neérlandais, 1909, v., 209.

264 Animal Instincts and Tropisms

a plant bend to the light and is a special case of the
same law of Bunsen and Roscoe which also explains
the photochemical effects in inanimate nature; or in
other words, the will or tendency of an animal to move
towards the light can be expressed in terms of the
Bunsen-Roscoe law of photochemical reactions.

The writer had shown in his early publications on
light effects that aside from the heliotropic reaction of
animals, which as we now know depends upon the
product of the intensity and duration of illumination,
there is a second reaction which depends upon the
sudden changes in the intensity of illumination. ‘These
latter therefore obey a law of the form: Effect =f (#).'
Jennings has maintained that the heliotropic reactions
of unicellular organisms are all of this kind, but in-
vestigations by Torrey and by Bancroft? on Euglena
have shown that Jennings’s statements were based on
incomplete observations.

4. In these experiments only one source of light was
applied. “When two sources of light of equal intensity
and distance act simultaneously upon a _ heliotropic
animal, the latter puts its median plane at right angles
to the line connecting the two sources of light.’
This fact has been amply verified by Bohn, by Parker
and his pupils, and especially by Bradley Patten, who

* Loeb, J., Arch. f. d. ges. Physiol., 1893, liv., 81; Jour. Exper. Zodl.,
1907, iv., I51.

2 Bancroft, F. W., Jour. Exper. Zoél., 1913, xv., 383.

3 Loeb, J., Studies in General Physiology, Chicago, 1905, p. 2.

Animal Instincts and Tropisms 265

used it to compare the relative efficiency of two different
lights.

The behaviour of the animals under the influence of
two lights is a confirmation of our theory of heliotro-
pism inasmuch as the animal moves in such a direction
that the symmetrical elements of the surface of the
body are struck by light of the same intensity at the
same angle, so that as a consequence equal masses of
photosensitive substances are produced in symmetrical
elements of their eyes or skin in equal times. The
effect on the symmetrical muscles will be identical.
As soon as one of the lights is a little stronger the
animal will deviate towards this light, in case it is
positively heliotropic and towards the weaker light if it.
is negatively heliotropic. This deviation again is not
the product of chance but follows a definite law as
Patten’ has recently shown. He used the negatively
heliotropic larva of the blowfly. These larve were
made to record their trail while moving under the
influence of the two lights. The results of the measure-
ments of 2500 trails showing the progressive increase
in angular deviation of the larve (from the perpendicular
upon the line connecting the two lights), with increasing
differences between the lights, are given in the follow-
ing table. Since the deviation or angular deflection of
the larve is towards the weaker of the two lights it is
marked negative.

t Patten, Bradley M., Am. Jour. Physiol., 1915, xxxviii., 313.

266 Animal Instincts and Tropisms

TABLE VIII
Percentage Difference in the Average Angular Deflection of the
Intensity of the Two Lights Two Paths of the Larve towards

the Weaker Light

Per Cent. Degrees
° — 0.09
84 — 2.77
1624 — 5-75

25 — 8.86
334 — 11.92
50 —20.28
66% —30.90
8314 —46.81
100 —77.56

Let us assume that the negatively heliotropic animal
is at an equal distance from the two unequal lights and
placed so that at the beginning of the experiment its
median plane is at right angles to the line connecting
the two lights, but with its head turned away from
them. In that case the velocity of reaction in the
symmetrical photosensitive elements of the eyeless
larvee is greater on the side of the stronger light. Since
the animal is negatively heliotropic this will result in a
greater relaxation or a diminution of the energy pro-
duction of the muscles turning the head of the animal
towards the side of the stronger light. Hence the
animal will automatically deviate from the straight
line towards the side of the weaker light. By the
alteration of the position of its body the photosensitive
elements exposed to the stronger of the two lights

Animal Instincts and Tropisms —_267

will be put at a less efficient angle and hence the rate
of photochemical reaction on this side will be diminished.
The deviation from the perpendicular in which the
animal will ultimately move will be such that as a
consequence, the rate of photochemical reaction in
symmetrical elements is again equal. The ultimate
direction of motion will, according to our theory always
be such that the mass of chemical products formed
under the influence of light in symmetrical photo-
sensitive elements during the same time is equal.

_ Patten also investigated the question whether the
same difference of percentage between two lights would
give the same deviation, regardless of the absolute
intensities of the lights used. The absolute intensity
was varied by using in turn from one to five glowers.
The relative intensity between the two lights varied
in succession by 0, 814, 1634, 25, 3314, and 50 per
cent. Yet the angular deflections were within the
limits of error identical for each relative difference of
intensity of the two lights no matter whether, I, 2, 3,
4, or 5 glowers were used. The following table shows
the result.

268 Animal Instincts and Tropisms

TABLE IX

A TaBLeE BASED ON THE MEASUREMENTS OF 2700 TRAILS SHOWING
THE ANGULAR DEFLECTIONS AT FIvE DIFFERENT ABSOLUTE

INTENSITIES
Difference of Intensity between the Two Lights
Number
of
Glowers o 8g 1624 25 33% 50
percent. | per cent. | per cent. | per cent. | per cent. | per cent.
Deflectionin| Degrees
I —0.55 2.32 —5.27 —9.04 | —11.86 | —19.46
2 —0.10 —3.05 —6.12 —8.55 | —II.92 | —22.28
3 +0.45 —2.60 —5.65 —8.73 | —13.15 | —20.52
4 —0.025 | —2.98 —6.60 —9.66 | —11.76 | —19.88
5 —0.225 | —2.92 —5.125 | —8.30 | —10.92 | —19.28
Average | —0.09 —2.77 —5.75 —8.86 | —II.92 | —20.28

Such constancy of quantitative results is only
possible where we are dealing with purely physico-
chemical phenomena or where life phenomena are
unequivocally determined by purely physicochemical
conditions.

5. It seems difficult for some biologists, even with
the validity of the Bunsen—-Roscoe law proven, to
imagine that the movements of the animals under the
influence of light are not voluntary (or not dictated by
the mysterious ‘‘trial and error” method of Jennings).*

* According to this theory the animal is not directly oriented by the
outside force, e. g. the light, but selects among its random movements the

one which is most ‘‘suited’’ and keeps on moving in this direction. This
idea is untenable for most if not all the cases of tropisms and has been

Animal Instincts and Tropisms 269

But one wonders how it is possible on such an assump-
tion to account for the fact that the angle of deflection
of the larva of the fly when under the influence of two
lights of different intensities should be always the same
for a given difference in intensity; or why the time for
curvature in Eudendrium should vary inversely with
the intensity of illumination. It is, however, possible
to complete the case for the purely physicochemical
analysis of these instincts. John Hays Hammond, Jr.,
has succeeded in constructing heliotropic machines
which in the dark follow a lantern very much in the
manner of a positively heliotropic animal. The eyes
of this heliotropic machine consist of two lenses in
whose focus is situated the ‘‘retina” consisting of
selenium wire. The two eyes are separated from
each other by a projecting piece of wood which re-
presents the nose and allows one eye to receive light
while the other is shaded. The galvanic resistance of
selenium is altered by light; and when one selenium
wire is shaded while the other is illuminated, the elec-
tric energy (supplied by batteries inside the machine)
which makes the wheels turn (these take the place of

refuted by practically all the workers in this field, e. g., Parker and his
pupils, Bohn, H. B. Torrey, Holmes, Bancroft, Ewald, and others. It is
only upheld by Jennings and Mast; and is accepted among those to
whom the idea of a physicochemical explanation of life phenomena does
notappeal. Torrey and Bancroft (for the literature the reader is referred
to Bancroft’s paper, Jour. Exper. Zoél., 1913, xv., 383) have shown
directly that the theory of trial and error is not even correct for the
organism for which Jennings has developed this idea; namely Euglena.

270 Animal Instincts and Tropisms

the legs of the normal animal) no longer flows symmetri-
cally to the steering wheel, and the machine turns
towards the light. In this way the machine follows
a lantern in a dark room in a way similar to that of a
positively heliotropic animal. Here we have a model
of the heliotropic animal whose purely mechanistic
character is beyond suspicion, and we may be sure
that it is not “‘fondness’’ for light or for brightness
nor will-power nor a method of ‘‘trial and error” which
makes the machine follow the light.

6. It may also be of interest to know that in helio-
tropism the motions of the legs are automatically
controlled by the chemical changes taking place in
symmetrical elements of the retina. In order to prove
this point we will turn to the phenomenon of gal-
vanotropism. The galvanic current forces certain
animals to move in the direction of one of the two
electrodes just as the light forces the heliotropic animals
to move towards (or from) the source of light. The
change in the concentration of the ions at the
boundary of the various organs, especially the nerves,
determines the galvanotropic reactions. When the
shrimp Palemonetes is put into a trough with dilute
salt solution through which a current of a certain
intensity flows, the animal is compelled to move
towards the anode.’ It can walk forwards, back-
wards, or sidewise. Here we can observe directly

* Loeb, J., and Maxwell, S.S., Arch. f. d. ges. Physiol., 1896, Ixiii., 121,

Animal Instincts and Tropisms —271

that the effect of the current consists in altering the
tension of the muscles of the legs in such a way as to
make it easy for the animal to move toward the anode

and difficult to move toward the cathode. Thus if the
current be sent sidewise through the animal, say from
left to right (Fig. 45), the legs of the left side assume the
flexor position, those of the right the extensor position.
With this position of its legs the animal can easily move

272 +Animal Instincts and Tropisms

to the left, 7. e., the anode, and only with difficulty to
the right, z.e., the cathode. This change in the position
of the legs occurs when the animal is not moving at
all, thus showing that the galvanotropic movements
take place not because the animal intends to go to
the anode, but that the animal goes to the anode be-
cause its legs are practically prevented by the galvanic
current from working in any other way. This is
exactly what happens in the heliotropic motions of
animals, *

To understand what happens when the current goes
lengthwise through the body it should be stated that
Palemonetes uses the third, fourth, and fifth pairs of
legs for its locomotion. The third pair pulls in the
forward movement, and the fifth pair pushes. The
fourth pair generally acts like the fifth, and requires
no further attention. Ifa current be sent through the
animal longitudinally, from tail to head, and the strength
be increased gradually, a change soon takes place in
the position of the legs (Fig. 46). In the third pair the
tension of the flexors predominates, in the fifth the
tension of the extensors. The animal can thus move
easily with the pulling of the third and the pushing
of the fifth pairs of legs, that is to say, the current
changes the tension of the muscles in such a way that

*That the mechanisms by which heliotropic and galvanotropic
orientation is brought about are identical was shown by Bancroft
in Euglena (Bancroft, loc. cit.).

Animal Instincts and Tropisms 273

the forward motion is rendered easy, the backward
motion is difficult. Hence it can easily move toward

the anode, but only with difficulty toward the cathode.
If a current be sent through the animal in the opposite
direction, namely, from head to tail, the third pair
of legs is extended, the fifth pair bent; that is, the third

274. + Animal Instincts and Tropisms

pair can push, and the fifth pair pull. The animal
will thus move backward easily and forward with
difficulty, and it is thus driven to the anode again.

The explanation which Loeb and Maxwell proposed
for this influence of the current on the legs assumes
that there are three groups of ganglion cells in the
central nervous system of these animals which are
oriented according to the three main axes of the body;
(1) right-left and left-right, (2) backward, and (3) for-
ward. It depends upon whether the ganglion cells
or the nerve elements are in anelectrotonus, which
muscles are bent and which relaxed. It would lead
us too far to recapitulate the theory in this place, and
the reader who is interested in it is referred to Loeb
and Maxwell’s paper.‘ The importance of the ob-
servations lies in the fact that they show that any
element of will or choice on the part of the animal in
these motions is eliminated, that the animal moves
where its legs carry it, and not that the legs carry the
animal where the latter ‘‘wishes’’ to go.

7. This may be the place to dispel an error which has
sometimes crept into the discussion of the tropistic
reactions of animals. It has been stated occasionally
that it is the energy gradient and not the automatic
orientation of the animal by the light which makes
the positively heliotropic animal move towards the
source of light and the negatively heliotropic away

* Loeb, J., and Maxwell, S.S., Arch. f. d. ges. Physiol., 1896, Ixiii., 121.

Animal Instincts and Tropisms = 275

from it. Thus the positively heliotropic animal
would be compelled to move towards the source of
light as a consequence of the fact that the intensity
of the light increases the more the nearer the animal
apptoaches the source of light. If the source of light
be the reflected .

sky-light the dif- ‘ yy :
ference of intensity
at both ends of a
microscopic organ-
ism is so slight
that it is beneath

the limit capable LE
of influencing the LEED
i LLLP
motions. “ LEE oe
, a AE LLL LI
A simple experi Ge SAS
ment published by y Ze raga |
the writer in 1889 ™™™“¢"""b
suffices to dispel Rea

the idea that the

energy gradient determines the direction of the mo-
tion of an animal in tropistic reactions. Let direct
sunlight (S, Fig. 47) fall through the upper half of a
window (ww) upon a table, and diffused daylight (D)
through the lower half of the window on the same
table. A test-tube ac is placed on the table in sucha
way that its long axis is at right angles to the plane
of the window; and one half a 0 is in the direct sun-

276 Animal Instincts and Tropisms

light, the other half in the shade. If at the be-
ginning of the experiment the positively heliotropic
animals are in the direct sunlight at a, they promptly
move toward the window, gathering at the window
end c of the tube, although by so doing they go
from the sunshine into the shade.* This experi-
ment is in harmony with our idea that the effect
of light consists in turning the head of the animal
and subsequently its whole body toward the source of
light. By going from the strong light into the shade
the reaction velocity in both eyes is diminished equally
and hence there is no reason for the animal to change
its orientation, though its progressive motion may be
stopped for an instant by the change. But at the
boundary between sunlight and daylight a sudden
change from strong to weak light occurs. If the
energy gradient determined the direction of the posi-
tively heliotropic animal, the motion should stop at
the boundary from strong to weak light, which may
happen for an instant but which will not interfere with
the progressive motion of the animal.

8. Graber had found that when animals are put into
a trough covered half with blue and half with red glass,
those that are “‘fond”’ of light go under the blue, those
that are “fond” of darkness go under the red glass.
The writer pointed out that this result should be
expected on the basis of his theory of heliotropism, if

t Loeb, J., Dynamics of Living Matter, p. 126.

Animal Instincts and Tropisms 277

the assumption be correct that the red light is con-
siderably less efficient than light which goes through
blue glass (such glass also allows green rays to go
through). The botanists had already shown that red
glass is impermeable for the rays which cause helio-
tropic reactions of plants, and the writer was able to
show the same for the heliotropic reactions of animals.
Red glass acts, therefore, almost like an opaque body
for these animals.’

A closer examination of the most efficient rays for
the heliotropic reactions of different organisms has
revealed the fact that for some organisms a region in
the blue }=460—490 py, for others a region in the
yellowish-green, near about A= 520—530 py is the most
efficient.t For many plants and for some animals,
like Eudendrium and the larve of the worm Arenicola,
a region in the blue is most efficient; for certain, if not
most, animals a region in the yellow-green is most
efficient. Among unicellular green alge, Chlamydomo-
nas, has its maximal efficiency in the yellowish-green
and Euglena in the blue. According to observations
by Mast, some green unicellular organisms like Pan-
dorina, Eudorina, and Spondylomorum seem to behave
more like Chlamydomonas, while certain others behave
more like Euglena.? Wasteneys and the writer suggested

tLoeb, J., and Maxwell, S.S., Univ. Cal. Pub., 1910, Physiol., iii.,
195; Loeb and Wasteneys, Proc. Nat. Acad. Sc., 1915, i., 44; Science,
1915, xli., 328; Jour. Exper. Zodl., 1915, Xix., 23; 1916, XX., 217.

2 Mast, S. O., Proc. Nat. Acad. Sc., 1915, i., 622.

278 Animal Instincts and Tropisms

that there are two groups of heliotropic substances, one
with a maximum of photosensitiveness in the blue, the
other in the yellowish-green; and that the latter group
may or may not be related or identical with the visual
purple which is most rapidly bleached by light of a
wave length near }4=520—530 pu.

The ophthalmologist Hess? has utilized the helio-
tropic reactions of animals in an attempt to prove that
all animals from the lowest invertebrates up to the
fishes inclusive suffer from total colour-blindness. This
statement was based on the observation that for most
positively heliotropic animals the region in the yellowish-
green near A=520py seems the most efficient. Since
this region of the spectrum appears also as the brightest
to a totally colour-blind man, he concluded that all
these animals are totally colour-blind. There is no
reason why heliotropic reactions should be used as an
indicator for colour sensations; if totally colour-blind
human beings were possessed of an irresistible impulse
to run into a flame Hess’s assumption might be con-
sidered, but no such phenomenon exists in colour-
blind man. Moreover, v. Frisch? has shown by ex-
periments on the influence of the background on the
colouration of fish as well as by experiments on bees and

* Hess, C., “Gesichtssinn,”’ Winterstein's Handb. d. vergl. Physiol.,
I9Q13, iv.

2v. Frisch, K., “Der Farbensinn und Formensinn der Biene,”’ Zodl.

Jahrb. Abt. f. allg. Zoél. u. Physiol., 1914, xxxv. See also Ewald, W. F.,
Zischr. f. Sinnesphysiol., 1914, xlviii., 285.

Animal Instincts and Tropisms 279

on Daphnia that the reactions of these animals to light
of different wave-lengths indicate different effects
besides those of mere intensity. Thus v. Frisch could
train bees to go to a blue piece of cardboard distributed
among many cardboards of different shades of grey.
Bees thus trained would alight on any blue object
even if it contained no food. It would be impossible
to do this with totally colour-blind organisms.

9. Heliotropic reactions play a great réle in the
preservation of individuals as well as of species. In
order to understand this réle it must be stated that the
photosensitive substances appear often only under certain
conditions and that their effect is inhibited under other
conditions. Thus among ants the winged males and
females alone show positive heliotropism,’ while the
wingless workers are free from this reaction. This
positive heliotropism becomes violent at the time of the
nuptial flight and this phenomenon itself seems to be a
heliotropic phenomenon since it takes place in the
direction of the light. When the queen founds her nest
she loses her wings and becomes negatively heliotropic
again. Kellogg* has shown that the nuptial flight of
the bees is also a purely heliotropic phenomenon.
When a part of the hive remote from the entrance is
illuminated the bees rush to the light and can thus be
prevented from swarming. These phenomena suggest

t Loeb, J., Der Heliotropismus der Tiere, 1889.
2 Kellogg, V. L., Sctence, 1903, Xviii., 693.

280 Animal Instincts and Tropisms

that the presence of some substance secreted by the
sex glands may cause the intensification of the helio-
tropism which leads to the nuptial flight.

In certain species of Daphnia, fresh-water copepods,
and of Volvox, a trace of CO, suffices to make negatively
heliotropic or indifferent specimens violently positively
heliotropic.t Certain forms of marine copepods and
the larvee of Polygordius can be made positively helio-
tropic by lowering the temperature? and the larve of
the barnacle can be made negatively heliotropic by
strong light. It is quite possible that a change in the
sense of heliotropism by temperature and light is to
some extent at least responsible for the periodic depth
migrations of heliotropic animals. Many if not all
positively heliotropic animals can be made negatively
heliotropic by exposure to ultra-violet light. 4

A most interesting example of the réle of heliotropism
in the preservation of a species is shown in the cater-
pillars of Porthesia chrysorrhea. ‘The butterfly lays
its eggs upon a shrub. The larve hatch late in the fall
and hibernate in a nest on the shrub, as a rule not far
from the ground. As soon as the temperature reaches
a certain height, they leave the nest; under natural

* Loeb, J., Arch. f. d. ges. Physiol., 1906, cxv., 544.

2 [bid., 1893, liv., 81.

3Groom, Theo. T., and Loeb, J., Biol. Centralbl., 1890, x., 160;
Ewald, W. F., Jour. Exper. Zool., 1912, xiii., 591.

4Loeb, J., Arch. f. d. ges. Physiol., 1906, cxv., 564; Moore, A. R.,
Jour, Exper. Zoél., 1912, xiii., 573.

Animal Instincts and Tropisms = 281

conditions, this happens in the spring when the first
leaves have begun to form on the shrub. (The larve
can, however, be induced to leave the nest at any time
in the winter provided the temperature is raised suffi-
ciently.) After leaving the nest, they crawl directly
upward on the shrub where they find the leaves on
which they feed. Should the caterpillars move down
the shrub, they would starve, but this they never do,
always crawling upward to where they find their food.
What gives the caterpillar this never-failing certainty
which saves its life, and for which a human being
might envy the little larva? Is it a dim recollection of
experiences of former generations? It can be shown
that it is the light reflected from the sky which guides
the animal upward. When we put these animals into a
horizontal test-tube in a room, they all crawl toward
the window, or toward a lamp; the animal is positively
heliotropic. It is this positive heliotropism which
makes them move upward where they find their food,
when the mild air of the spring calls them forth from
their nest. At the top of the branch, they come in
contact with a leaf, and chemical or tactile influences
set the mandibles of the young caterpillar into activity.
If we put these larve into closed test-tubes which lie
with their longitudinal axes at right angles to the
window, they will all migrate to the window end,
where they stay and starve, even if their favourite leaves
are close behind them. They are slaves of the light.

282 Animal Instincts and Tropisms

The few young leaves on top of a twig are quickly
eaten by the caterpillar. The light, which saved its
life by making it creep upward where it finds food,
would cause it to starve could it not free itself from the
bondage of positive heliotropism. The animal, after
having eaten, is no longer a slave of the light, but can
and does creep downward. It can be shown that a
caterpillar, after having been fed, loses its positive
heliotropism almost completely and permanently. If
we submit unfed and fed caterpillars of the same nest
contained in two different test-tubes to the same
artificial or natural source of light, the unfed will
creep to the light and stay there until they die, while
those that have eaten will pay little or no attention
to the light. Their sensitiveness to light has dis-
appeared; after having eaten they become independent
of light and can creep in any direction. The restlessness
which accompanies the condition of hunger makes the
animal creep downward—which is the only direction
open to it—where it finds new young leaves on which
it can feed. The wonderful hereditary instinct, upon
which the life of the animal depends, is its positive
heliotropism in the unfed condition and its loss of this
heliotropism after having eaten. The latter pheno-
menon is in harmony with the experiments which show
that the heliotropism of certain species of Daphnia
disappears when the water becomes neutral.

And finally it may be pointed out that the majority

Animal Instincts and Tropisms 283

of green plants could not exist if their stems were not
positively, their roots negatively, heliotropic. It is
the positive heliotropism which makes the top grow
toward the light, which enables the leaves to get the
light necessary for assimilation, and the roots to grow
into the soil where they find the water and nutritive
salts.

10. While we do not wish to deal here with the
different tropisms it should be stated that aside from
heliotropism, chemotropism as well as stereotropism
play the most essential réle in the so-called instinctive
actions of animals. It is a problem of orientation by
the diffusion of molecules from a centre when a male
butterfly is deviated from its flight and alights on the
wooden box in which is enclosed a female of the same
species. We have already alluded to certain phenomena
of chemotropism in Chapter IV. Certain organisms
have a tendency to bring their bodies as much as
possible on all sides in contact with solid bodies; thus
the butterfly Amphipyra, which is a fast runner,
will come to rest under a glass plate when the plate
is put high enough above the ground so that it touches
the back of the butterfly. The animals which live
under stones or underground or in caves are as a rule
both negatively heliotropic and positively stereotropic.
Their tropisms predestine or force them into the life

they lead.
The sensitive area which forms the basis of tropisms

284 Animal Instincts and Tropisms

is as a rule developed not in the whole organism but
only in certain segments of the body. Thus the eyes
are located in the head. But when the action of
one segment becomes overpowering the whole or-
ganism follows the segment. It has been customary
among physiologists to speak of reflexes in such cases.
Thus, e. g., the arms of the male frog develop a powerful
positive stereotropism on their ventral surface during
the spawning season. It would avoid confusion to
realize that there is nothing gained in applying to this
tropism the meaningless term “‘reflex’’; it is better to
call them tropisms since the organism as a whole is
involved. If necessary we might speak of segmental
tropisms. The act of seeking the female as well as
that of cohabitation are in many cases combinations
of chemotropism and stereotropism. The development
of these tropisms depends upon the presence of certain
specific substances in the body, a fact emphasized
already in the case of heliotropism. In case of the
development of the segmental stereotropism of the male
frog at the time of spawning it has been shown that it
depends on an internal secretion from the testes.

It has been suggested by some authors that the
tropistic reactions are determined by some feeling or
emotion on the part of the organism. We have no
means of judging the emotions of lower animals (except
by “‘intuition”’). The writer suggested in 1899 in his
book on brain physiology that emotions may be deter-

Animal Instincts and Tropisms 285

mined by specific substances which also determine
the tropistic reaction (as well as phenomena of organ
formation, although this latter phenomenon has
nothing to do with the subject of instincts); and the
excellent work of Cannon’ has shown the réle of adre-
nalin in the expression of fear. It is, therefore, both
unwarranted and unnecessary to state that hypotheti-
cal emotions determine the tropistic reactions.

«Cannon, W. B., Bodily Changes in Pain, Hunger, Fear, and Rage,
New York, 1915.

CHAPTER XI
THE INFLUENCE OF ENVIRONMENT

1. The term environment in relation to an organism
may easily assume a mystic réle if we assume that it
can modify the organisms so that they become adapted
to its peculiarities. Such ideas are difficult to compre-
hend from a physicochemical viewpoint, according to
which environment cannot affect the living organism
and non-living matter in essentially different ways.
Of course we know that proteins will as a rule coagulate
at temperatures far below the boiling point of water
and that no life is conceivable for any length of time
at temperatures above 100° C., but heat coagulation of
proteins occurs as well in the test-tube as in the living
organism. If we substitute for the indefinite term
environment the’ individual physical and chemical
forces which constitute environment it is possible to
show that the influence of each of these forces upon the
organism finds its expression in simple physicochemical
laws and that there is no need to introduce any other

considerations.
286

The Influence of Environment 287

We select for our discussion first the most influential
of external conditions, namely temperature. The
reader knows that there is a lower as well as an upper
temperature limit for life. Setchell has ascertained
that in hot springs whose temperature is 43° C., or
above, no animals or green alge are found.t In hot
springs whose temperature is above 43° he found
only the Cyanophycee, whose structure is more closely
related to that of the bacteria than to that of the alge,
inasmuch as they have neither definitely differentiated
nuclei nor chromophores. The highest temperature
at which Cyanophycee occurred was 63°C. Not all the
Cyanophycee were able to stand temperatures above
43° C., but only a few species. The other Cyanophycee
were found at a temperature below 40° C., and were no
more able to stand higher temperatures than the real
alge or animals. The Cyanophycee of the hot springs
were as a rule killed by a temperature of 73°: From
this we must conclude that they contain proteins whose
coagulation temperature lies above that of animals
and green plants, and may be as high as 73°. Among
the fungi many forms can resist a temperature above
43° or 45°; the spores can generally stand a higher
temperature than the vegetative organs. Duclaux
found that certain bacilli (Tyrothrix) found in cheese
are killed in one minute at a temperature of from 80°

t Setchell, W. A., Science, 1903, XxVil., 934.

288 The Influence of Environment

to 90°; while for the spores of the same bacillus a
temperature of from 105° to 120° was required.*
Duclaux has called attention to a fact which is of
importance for the investigation of the upper tempera-
ture limit for the life of organisms.. According to this
author it is erroneous to speak of a definite temperature
as a fatal one; instead we must speak of a deadly
temperature zone. This is due to the fact that the
length of time which an organism is exposed to a higher
temperature is of importance. Duclaux quotes as an
example a series of experiments by Christen on the
spores of soil and hay bacilli. The spores were exposed
to a stream of steam and the time determined which
was required at the various temperatures to kill the

spores.

Tt took At 100" 0335065 geet eee Wiese over sixteen hours
BEE TOBATIOS aaa sey eeseg eas two to four hours

Goo MERE SEL Gr ae ons os ei hana thirty to sixty minutes
fo) RE, ESO aE BO. ties ied acetone five minutes or more
UALS (Nay reg sf does Sova wide decags one to five minutes

Pas ack |e agence rer eter eae opener one minute

In warm-blooded animals 45° is generally considered
a temperature at which death occurs in a few minutes;
but a temperature of 44°, 43°, or 42° is also to be
considered fatal with this difference only, that it takes

t Duclaux, E., Traité de microbiol., 1898, i., 280.

The Influence of Environment 289

a longer time to bring about death. This fact is to be
considered in the treatment of fever.

It is generally held that death in these cases is due
to an irreversible heat coagulation of proteins. Ac-
cording to Duclaux, it can be directly observed in
micro-organisms that in the fatal temperature zone
the normally homogeneous, or finely granulated, proto-
plasm is filled with thick, irregularly arranged bodies,
and this is the optical expression of coagulation. The
fact that the upper temperature limit differs so widely
in different forms is explained by Duclaux through
differences in the coagulation temperature of the various
proteins. It is, e. g. known that the coagulation
temperature varies with the amount of water of the
colloid. According to Cramer, the mycelium of Peni-
cillium contains 87.6 water to 12.4 dry matter, while
the spores have 38.9 water and 61.1 dry substance.
This may explain why the mycelium is killed at a
lower temperature than the spores. According to
Chevreul, with an increase in the amount of water,
the coagulation temperature of albuminoids decreases.
The reaction of the protoplasm influences the tempera-
ture of coagulation, inasmuch as it is lower when the
reaction is acid, higher when the reaction is alkaline.
The experiments of Pauli show also a marked influence
of salts upon the temperature of coagulation of colloids.

The process of heat coagulation of colloids is also a

function of time. If the exposure to high temperature
ro :

290 ~=3>6The Influence of Environment

is not sufficiently long, only part of the colloid coagulates;
in this case an organism may again recover.

Inside of these upper and lower temperature limits
we find that life phenomena are influenced by tempera-
ture in such a way that their rate is about doubled for
an increase of the temperature of 10° C., and that this
temperature coefficient for 10°, Q,., very often steadily
diminishes from the lower to the higher temperature;
so that near the lower temperature limit it becomes
often considerably greater than 2 and near the higher
temperature limit it becomes very often less than 2.*
This influence of temperature is so general that we are
bound to associate it with an equally general feature of
life phenomena; and such a feature would be most
likely the chemical reactions. It is known through the
work of Berthelot, van’t Hoff, and Arrhenius that the
temperature coefficient for the velocity of chemical
reactions is also generally of about the same order of
magnitude; namely 2 2 for a difference of 10°. In
chemical reactions there is also a tendency for Q,, to
become larger for lower temperature, and coefficients
of Q,, about 5 or 6 have repeatedly been found for
purely chemical reactions between 0° and 10°, e. g.,
for the inversion of cane sugar by the hydrogen ion.
The temperature coefficient for the reaction velocity
of ferments shows the same diminution of Q,, with

t A full discussion of the literature on temperature coefficients is given
in A. Kanitz’s book on Temperatur und Lebensvorgdnge, Berlin, 1915.

The Influence of Environment 291

rising temperature which is also noticed in most life
phenomena. Thus Van Slyke and Cullen" found that
the reaction rate of the enzyme urease ‘‘is nearly
doubled by every 10° rise in temperature between 10°
and 50°. Within this range the temperaturé coefficient
is nearly constant and averages 1.91. From 0° to 10° it
is 2.80, from 50° to 60° it is only 1.09. The optimum
is at about 55°.” The rapid fall of the temperature
coefficient for enzyme action at the upper temperature
limit has been ascribed by Tammann to a progressive
destruction of the active mass of enzyme by the higher
temperature (by hydrolysis). This will, however, not
account for the high value of the coefficient near the
lower limit. But is it not imaginable that at low
temperature an aggregation of the enzyme particles
exists which is also equivalent to a diminution of the
active mass of the enzyme and that this aggregation is
gradually dispersed by the rising temperature? This
would account for the fact that at a temperature near
o°C life phenomena stop because the enzymes are all
in a state of aggregation or gelation; that then more
and more are dissolved and the rate of chemical re-
action increases since the mass of enzyme particles
increases until all the enzyme molecules are dissolved
or rendered active. Under this assumption three
processes are superposed in the variation of the value

Van Slyke, D. D., and Cullen, G. E., Jour. Biol. Chem., 1914, xix.,
141.

292 ~The Influence of Environment

of Q,. with temperature: (1) the supposed increase in
the number of available ferment molecules with in-
creasing temperature near the lower temperature limit;
(2) the temperature coefficient of the reaction velo-
city which is nearly =2 for 10°C.; (3) the diminution
of the number of available ferment molecules by hydroly-
sis or some other action of the increasing temperature.
This latter is noticeable near the upper temperature
limit. The reason that 1 and 3 interfere more strongly
in life phenomena than in the chemical reactions of
crystalloid substances may possibly be accounted for
by the fact that the enzymes and most of the con-
stituents of living matter are colloidal, 7. e., consist of
particles of a considerably greater order of magnitude
than the molecules of crystalloids.*

We will now show the réle of the temperature
coefficient upon phenomena of development. F. R.
Lillie and Knowlton? first determined the influence of
temperature upon the development of the egg of the
frog and showed that it was of the same nature as that of
a chemical reaction. These experiments were repeated
a year later by O. Hertwig.:

t These considerations may meet the objections of Krogh to the
application of the van’t Hoff rule of temperature effect on reaction
velocity to life phenomena. See also the discussion of this subject in
Kanitz’s book.

2 Lillie, F. R., and Knowlton, E. P., Zodél. Bull., 1897, i.

3 Hertwig, O., Arch. mikrosk. Anat., 1898, li., 319. See also E.
Cohen, Vortrage fiir Aerate tiber physikalische Chemie. 2d ed. Leip-
zig, 1907.

The Influence of Environment 293

The time required for the eggs to reach definite
stages was measured for different temperatures and
it was found that the temperature coefficient Q,
between 2.5° and 6° was equal to 10 or more; between
6° and 15° it was between 2.6 and 4.5; between 10° and
20° it was 2.9 to 3.3, and between 20° and 24° it was
between 1.4 and 2.0. To anybody who has worked
on this problem it is obvious that no exact figures
can be obtained in this way, since the point when
a certain stage of development is reached is not
so sharply defined as to exclude a certain latitude of
arbitrariness. The writer found that very exact
figures can be obtained on the influence of temperature
upon development of the sea-urchin egg by measuring
the time from insemination to the first cell division.
Such experiments were carried out in a cold-water form
Strongylocentrotus purpuratus and a form living in
warmer water, Arbacia.t The figures on Arbacia
have been verified by different observers in different
years.

t Loeb, J., Arch. f. d. ges. Physiol., 1908, cxxiv., 411; Loeb J., and

Wasteneys, H., Biochem. Zischr., 1911, xxxvi., 345; Loeb J., and Cham-
berlain, M. M., Jour. Exper. Zo6l.,1915, Xix., 559+

294 The Influence of Environment

TABLE X

INFLUENCE OF TEMPERATURE UPON THE TIME (IN MINUTES) REQUIRED
: FROM INSEMINATION TO THE First CELL DIVISION

Arbacia
TEMPERA- Strongylocentrotus
TURE LoEB AND LoEB AND purpuratus
WASTENEYS CHAMBERLAIN
I9II 1915
°C, Minutes Minutes Minutes
3 532
4 469
5 352
6 275
7 498 291
8 410 41t 210
9 308 297-5 . 159
10 217 208 143
II 175 175
12 147 148 I31
13 129
14 116 121
15 100 100 100
16 85-5
17 79.5
18 68 68 87
19 65 78
20 56 56 75
21 53-3 78
22 47 46 75
23 45-5 Upper tempera-
24 42 ture limit
25 40 39-5
26 33-5
27-5 34
30 33
31 37

These figures permitted the determination of the
temperature coefficients Q,, with a sufficient degree of
accuracy (see next table). It seemed of importance

The Influence of Environment 295

to attempt to decide what the chemical reaction under-
lying these reaction velocities is (if it is a chemical
reaction). Loeb and Wasteneys' investigated the
temperature coefficient for the rate of oxidations in
the newly fertilized egg of Arbacta and found that the
temperature coefficient Q,, for that process does not
vary in the same way as the temperature coefficient
for cell division,

TABLE XI

TEMPERATURE COEFFICIENTS Qio FOR THE RATE OF SEGMENTATION
AND OXIDATIONS IN THE EGcGs oF Strongylocentrotus
AND Arbacia

Qro FOR RATE OF SEGMENTATION IN Qro for Rate of .
Oxidations in
‘TEMPERATURE Arbacia
Strongylocentrotus Arbacia
°C.
3-13 3-91 2.18
4-14 3.88
5-15 3.52 2.16
7-17 3.27 7.3 2.00
8-18 6.0
9-19 2.04 4-7
10-20 1.90 3.8 2.17
II-21 3.3
12-22 1.74 3.1
13-23 2.8 2.45
15-25 2.5 2.24
16-26 2.6
17.5-27.5 2.2 2.00
20-30 1.7 1.96

It is obvious that the temperature coefficient of
the rate of oxidations is remarkably constant, about
2 for 10°, for various temperatures and does not show

x Loc. cit.

296 The Influence of Environment

the variation from 7 or more to 2.2 for Q,, for the rate of
segmentation.

Kanitz? has shown that in a graph in which the
logarithms of the segmentation velocities are drawn
as ordinates and the temperatures as abscisse the
logarithms form two straight lines which are joined at
an angle. According to the law of van’t Hoff and
Arrhenius concerning the influence of temperature upon
velocities of chemical reactions the logarithms should
lie in a straight line. We are dealing therefore in these
cases with two exponential curves, one representing in
Arbacia the interval 7-13° and the second from 13-26°:
in Strongylocentrotus between 3-9° and 9-20°.

It was found in these experiments that if measure-
ments of the Q,, of later stages of development are
attempted the variations due to unavoidable difficulties
become too great to permit an equal degree of reliability
in the determinations.

The vast importance of this influence of temperature
upon the rate of development is seen in the fact that in
addition to the food supply the rate of the maturing
of plants and animals depends on this factor.

2. This influence of temperature upon develop-
ment has been used to find the conditions determining
fluctuating variation. The reader knows that by this
expression are understood the differences between in-
dividuals of a pure strain or breed. These variations

t Kanitz, A., loc. cit., p. 123.

The Influence of Environment 297

are not inherited, a fact contrary to the idea of Dar-
win, who assumed that by the selection of extreme
cases of fluctuating variation new varieties could de-
velop. What is the basis of this fluctuating variation?
The writer concluded that if fluctuating variations
were due to a slight variation in the quantity of a
specific substance—in some cases an enzyme—required
for the formation of a hereditary character, the tem-
perature coefficient might be used to test the idea. We
have just seen that the time required from insemina-
tion until the cell division of the first egg occurs is
very sharply defined for each temperature. If a large
number e.g. one hundred or more eggs are under obser-
vation simultaneously in a microscopic field it can be
seen that they do not all segment at the same time
but in succession; this is the expression of fluctuating
variation. Miss Chamberlain and the writer have
measured the time which elapses between the moment
the first egg of such a group segments and the moment
the last egg begins its segmentation, and found that this
latitude of variation is also very definite for each tem-
perature, and that its temperature coefficient is for
each interval of 10° practically identical with the
temperature coefficient of the segmentation for the
same interval.’ The slight deviations are practically
all in the same sense and accounted for by a slight
deficiency in the nature of the experiments. The

t Loeb, J., and Chamberlain, M. M., Jour. Exper. Zodl., 1915, XiX., 559+

298 The Influence of Environment

two following tables give the latitude of variations for
different temperatures for the first segmentation in
Arbacia and the temperature coefficient for this latitude

and the rate of segmentation.

efficients are practically identical.

These two latter co-

TABLE XII
Latitude Latitude
Temperature of Temperature of
Variation Varvation
°C; Minutes °C. Minutes
9 52.5 18 12.0
10 39-5 19 12.5
II 26.0 20 9.6
12 22.5 21 8.0
13 19.2 22 7.8
14 17.5 23 8.0
15 13.0 24 8.0
25 5-0
TABLE XIII
TEMPERATURE COEFFICIENT OF
Temperature
Interval Latitude
of Segmentation
Variation

°C.

9-19 4.2 4.7
10-20 3-9 3.8
II-21 3.2 3.3
12-22 2.8 3.1
13-23 2.4 2.8
14-24 2.3 2.8
15-25 2.6 2.5

The Influence of Environment 299

If we assume that the temperature coefficient for
the segmentation of the egg is that of a chemical re-
action (other than oxidation) underlying the process
of segmentation, the fluctuating variation in the time -
of the segmentations of the various eggs fertilized at the
same time is due to the fact that the mass of the enzyme
controlling that reaction varies within definite limits
in different eggs. The first egg segmenting at a given
temperature has the maximal, the last egg segmenting
has the minimal mass of enzyme. It should be added
that the time of the first segmentation is determined by
the cytoplasm and is not a Mendelian character, as
was stated in a previous chapter.

3. The point of importance to us is that the influ-
ence of temperature upon the organism is so constant
that if disturbing factors are removed it would be pos-
sible to use the time from insemination to the first
segmentation of an egg of Arbacia as a thermometer
on the basis of the table on page 295.

Facts of this character should dispose of the idea
that the organism as a whole does not react with that
degree of machine-like precision which we find in the
realm of physics and chemistry. Such an idea could
only arise from the fact that biologists have not been
in the habit of looking for quantitative laws, chiefly,
perhaps, because the difficulties due to disturbing
secondary factors were too great. The worker in
physics knows that in order to discover the laws of a

300 The Influence of Environment

phenomenon all the disturbing factors which might
influence the result must first be removed. When the
biologist works with an organism as a whole he is
rarely able to accomplish this since the various dis-
turbing influences, being inseparable from the life of
the organism, can often not be entirely removed. In
this case the biologist must look for an organism in
which by chance this elimination of secondary condi-
tions is possible. The following example may serve
as an illustration of this rather important point in
biological work. Although all normal human beings
have about the same temperature, yet if the heart-
beats of a large number of healthy human beings are
measured the rate is found to vary enormously. Thus
v. Korésy found among soldiers under the most favour-
able and most constant conditions of observations—
the soldiers were examined early in the morning before
rising—variations in the rate of heart-beat between
42 and 108. In view of this fact, those opposed to the
idea that the organism as a whole obeys purely physico-
chemical laws might find it preposterous to imagine that
the rate of heart-beat could be used asa thermometer.
Yet if we observe the influence of temperature on the
tate of the heart-beat of a large number of embryos of
the fish Fundulus, while the embryos are still in the
egg, we find that at the same temperature each heart
beats at the same rate, the deviations being only
slight and such as the fluctuating variations would

The Influence of Environment 301

demand.* This constancy is so great that the rate
of heart-beat of these embryos could in fact be used
as a rough thermometer. The influence of tempera-
ture upon the rate of heart-beat is completely reversible
so that when we measure the rate for increasing as well
as for decreasing temperatures we get approximately
the same values as the following table shows.

TABLE XIV
Time Required for Nineteen Heart-beats in
Temperature the Embryo of Fundulus
i Seconds
30 6.25
25 8.5
20 IL.5
15 19.0
10 32.5
5 61.0
10 33-5
15 18.8
20 12.0
25 10.0
30 6.0

Why does each embryo have the same rate of heart-
beat at the same temperature in contradistinction to
the enormous variability of the same rate in man?
-The answer is, on account of the elimination of all
secondary disturbing factors. In the embryo of Fun-
dulus the heart-beat is a function almost if not exclu-

* Loeb, J., and Ewald, W. F., Biochem. Zischr., 1913, lviii., 179.

302. The Influence of Environment

sively of two variables, the mass of enzymes for the
chemical reactions underlying the heart-beat and the
temperature. By inheritance the mass of enzymes is
approximately the same and in this way all the embryos
beat at the same rate (within the limits of the fluctuat-
ing variation) at the same temperature. This identity
exists, however, only as long as the embryo is relatively
quiet in the egg. As soon as the embryo begins to
move this equality disappears since the motion influ-
ences the heart-beat and the motility of different
embryos differs.

In man the number of disturbing factors is so
great that no equality of the rate for the same tem-
perature can be expected. Differences in emotions
or the internal secretions following the emotions,
differences in previous diseases and their after-effects,
differences in metabolism, differences in the use of
narcotics or drugs, and differences in activity are only
some of the number of variables which enter.

4. As stated above the temperature influences
practically all life phenomena in a similar characteristic
way, € g., the production of CO, in seeds and the
assimilation of CO, by green plants. The writer
would not be surprised if even the aberrations in the
colour of butterflies under the influence of temperature

* Clausen, H., Landwirtschaftl. Jahrb., 1890, xix., 893.
2 Matthaei, G. L.C., Trans. Philosoph. Soc., 1904, cxcvii., 47; Black-
man F. F., Ann. of Bot., 1905, xix., 281,

The Influence of Environment 303

turned out to be connected with the temperature co-
efficient. The experiments of Dorfmeister, Weismann,
Merrifield, Standfuss, and Fischer, on seasonal dimor-
phism and the aberration of colour in butterflies have
so often been discussed in biological literature that a
short reference to them will suffice. By seasonal
dimorphism is meant the fact that species may appear
at different seasons of the year in a somewhat different
form or colour. Vanessa prorsa is the summer form,
Vanessa levana the winter form of the same species.
By keeping the pupz of Vanessa prorsa several
weeks at a temperature of from o° to 1° Weismann
succeeded in obtaining from the summer chrysalids
specimens which resembled the winter variety, Vanessa
levana.

If we wish to get a clear understanding of the causes
of variation in the colour and pattern of butterflies,
we must direct our attention to the experiments of
Fischer, who worked with more extreme temperatures
than his predecessors, and found that almost identical
aberrations of colour could be produced by both ex-
tremely high and extremely low temperatures. This can
be seen clearly from the following tabulated results of
his observations. At the head of each column the
reader finds the temperature to which Fischer sub-
mitted the pup, and in the vertical column below are
found the varieties that were produced. In the vertical
column A are given the normal forms:

304 The Influence of Environment

TABLE XV
0° to 0° to A +35° to +36° to | +42° to
—20°C. | +10°C. | (Normal +37°C.| -+4r1°C. | +46°C.
Forms)

tchnusoides | polaris urtice ichnusa polaris ichnusoides

(nigrita) (nigrita)
antigone | fischeri to _ fischert antigone
(iokaste) (tokaste)
testudo dixeyt polychloros \erythromelas \dixeyt testudo
hygiea artemis antiopa epione artemis hygiea
elymi wiskottt |cardut _ wiskotti elymt
klymene __|merrifieldi \atalanta _ merrifieldt \klymene
weismannt |porima prorsa —_ porima weismannt

The reader will notice that the aberrations produced
at a very low temperature (from 0° to —20° C.) are
absolutely identical with the aberrations produced by
exposing the pupz to extremely high temperatures
(42° to 46° C.). Moreover, the aberrations produced
by a moderately low temperature (from 0° to 10° C.)
are identical with the aberrations produced by a moder-
ately high temperature (36° to 41° C.).

From these observations Fischer concludes that it is
erroneous to speak of a specific effect of high and of
low temperatures, but that there must be a common
cause for the aberration found at the high as well as
at the low temperature limits. This cause he seems
to find in the inhibiting effects of extreme temperatures
upon development.

If we try to analyse such results as Fischer’s from a

The Influence of Environment 305

physicochemical point of view, we must realize that
what we call life consists of a series of chemical reac-
tions, which are connected in a catenary way; inas-
much as one reaction or group of reactions (a) (e. g.,
hydrolyses) causes or furnishes the material for a
second reaction or group of reactions (0) (e. g., oxida-
tions). We know that the temperature coefficient for
physiological processes varies slightly at various parts
of the scale; as a rule it is higher near 0° and lower near
30°. But we know also that the temperature coefficients
do not vary equally for the various physiological pro-
cesses. It is, therefore, to be expected that the tem-
perature coefficients for the group of reactions of the
type (a) will not be identical through the whole scale
with the temperature coefficients for the reactions of
the type (8). If therefore a certain substance is formed
at the normal temperature of the animal in such quan-
tities as are needed for the catenary reaction (bd), it is
not to be expected that this same perfect balance will
be maintained for extremely high or extremely low
temperatures; it is more probable that one group of
reactions will exceed the other and thus produce aber-
rant chemical effects, which may underlie the colour
aberrations observed by Fischer and other experi-
menters.

It is important to notice that Fischer was also able
to produce aberrations through the: application of
narcotics. Wolfgang Ostwald has produced experi-

306 The Influence of Environment

mentally, through variation of temperature, dimor-
phism of form in Daphnia.

5. Next or equal in importance with the tempera-
ture is the nature of the medium in which the cells
are living.

It has often been pointed out that the marine animals
and the cells of the body of metazoic animals are
surrounded by a medium of similar constitution, the
sea water and the blood or lymph, both media be-
ing salt solutions differing in concentration but con-
taining the three salts NaCl, KCl, and CaCl, in about
the same relative concentration, namely 100 mole-
cules NaCl: 2.2 molecules of KCl: 1.5 molecules of
CaCl, This has suggested to some authors the poetical
dream that our home was once the ocean, but we can-
not test the idea since unfortunately we cannot experi-
ment with the past. Plants, unicellular fresh-water
alge, and bacteria do not demand such a medium for
their existence.

Herbst had shown that when sea-urchin larve were
raised in a medium in which only one of the constitu-
ents of the sea water was lacking (not only NaCl, KCl,
or CaCl,, but also Na.SO,, NaHCO,, or Na.HPO,),
the eggs could not develop into plutei; from which he
concluded that every constituent of the sea water was
necessary. This would indicate a case of extreme
adaptation to all the minutie of the external medium.

Experiments on a much more favourable animal

The Influence of Environment 307

for this purpose, namely, the eggs of the marine fish
Fundulus, gave altogether different results. The eggs
of this marine fish develop naturally in sea water but
they develop just as well in fresh or in distilled water,
and the young fish when they are made to hatch in
distilled water will continue to live in this medium.
This proves that these eggs require none of the salts
of the sea water for their development. When these
eggs are put immediately after fertilization into a pure
solution of NaCl of that concentration in which this
salt exists in the sea water practically all the eggs die
without forming an embryo; but if a small quantity
of CaCl, is added every egg is able to form one,
and these embryos will develop into fish and the latter
will hatch. This led the writer to the conclusion that
these fish (and perhaps marine animals in general)
need the Ca of the sea water only to counteract the
injurious effects which a pure NaCl solution has if it
is present in too high a concentration." When we raise
the eggs in a pure NaCl solution of a concentration
=m/8 practically every egg will develop; and even in
a m/4 or 3/8 m many or some eggs will form embryos
without adding Ca; it may be that a trace of Ca present
in the membrane of the egg may suffice to counter-
balance the injurious action of a weak salt solution.

Loeb, J., “The Poisonous Character of a Pure NaCl Solution,”
Am. Jour. Physiol., 1900, iii., 329; Arch. f. d. ges. Phystol., 1901, Ixxxviii.,
68; Am. Jour. Physiol., 1902, vi., 411; Biochem. Ztschr., 1906, ii., 81.

308 The Influence of Environment

The concentration of the NaCl in the sea water at.
Woods Hole (where these experiments were made)
is about m/2, and as soon as this concentration of NaCl
is reached the eggs are all killed as a rule before they
can form an embryo, unless a small but definite amount
of Ca is added. It was found that the eggs can be
raised in much higher concentrations of NaCl, but in
that case more Ca must be added. The following
table gives the minimal amount of CaCl, which must
be added in order to allow fifty per cent. of the eggs
to form embryos. (The eggs were put into the solu-
tion an hour or two after fertilization.)

TABLE XVI
Concentration Cc. m/16 CaClz
of Required for 50 ¢.c.

NaCl NaCl Solution
m.

3/8 0.1

4/8 0.3

5/8 0.5

2 3 0.6

7 0.9

8/8 1.2-1.4

9/8 1.8-2.0
10/8 2.0-2.5
ue 2.0
12/8 3-0-3.5
13/8 oO

This indicates that the quantity of CaCl, required
to counteract the injurious effects of a pure solution
of NaCl increases approximately in proportion to the

The Influence of Environment 309

square of the concentration of the NaCl solution.?
The reader will notice that the eggs can survive and
develop in a solution of three times the concentration
of sea water, provided enough Ca is added.

It was found also that not only Ca but a large num-
ber of other bivalent metals were able to counteract
the injurious action of an excessive NaCl solution;
namely Mg, Sr, Ba, Mn, Co, Zn, Pb, and Fe;? only Hg
and Cu could not be used since they are themselves
too toxic. The antagonistic efficiency of the bivalent
cations other than Ca was, however, smaller than
that of Ca. The following table gives the high-
est concentration of NaCl solution in which the
newly fertilized eggs of Fundulus can still form an
embryo.’

50 c.c. 10/8 m NaCl+4 c.c. m/1 MgCl,
50 c.c. 14/8 m NaCl+1 c.c. m/1 CaCl,
50 c.c. 11/8 m NaCl+1 cc. m/1 SrCl,

50 cc. 7/8 m NaCl+1c.c. m/1 BaCl,

On the other hand it was seen that in all the chlorides
with a univalent cation, LiCl, KCl, RbCl, CsCl,
NH,Cl, the eggs could form embryos up to a certain
concentration of the salt; but that this concentration
could be raised by the addition of Ca.

t Loeb, J., Jour. Biol. Chem., 1915, xxiii., 423.

2 Loeb, J., “On the Physiological Effects of the Valency and Possibly

the Electrical Charges of Ions,” Am. Jour. Physiol., 1902, vi., 411.
3 Loeb, J., Jour. Biol. Chem.,'1914, xix., 431.

310 The Influence of Environment

TABLE XVII

CONCENTRATIONS AT WHICH THE EGGS NO LONGER ARE ABLE TO
Form EmsBryos

In the Same Salts with the Addition
In the Pure Salts of r¢e.c.m CaClz to 50 c.c.
Solution

LiCl gore s agen s cae about 6/32 m >5/8m
NaChivs sod eos.sace s m/2 >14/8m
KiGliss coe ececeatees >11/16m > 8/8m

<6/8 m
RbCh ss secs ss >8/8m >9/8m

<7/8 m
CsChie inne stetineas >3/8 m >8/8 m

<4/8 m

In short it turned out that the injurious action of
the pure solution of any chloride (or any other anion)
with a univalent metal could be counteracted to a
considerable extent by the addition of small quantities
of a salt with a bivalent metal. It was also found
in the early experiments of the writer that the bivalent
or polyvalent anions had no such antagonistic effect upon
the injurious action of the salts with a univalent cation.

We therefore see that what at first sight appeared in
the experiments of Herbst a necessity, namely, the
presence of each constituent of the sea water, turns
out as a special case of a more general law; the salts
with univalent ions are injurious if their concentration
exceeds a certain limit and this injurious action is
diminished by a trace of a salt with a bivalent cation.

Why was it not possible to prove this fact for the

The Influence of Environment 311

eggs of the sea urchin? Before we answer this ques-
tion, we wish to enter upon the discussion of the nature
of the injurious action of a pure NaCl solution of a
certain concentration and of the annihilation of this
action by the addition of a small quantity of Ca. The
writer suggested in 1905 that the injurious action of a
pure NaCl solution consisted in rendering the membrane
of the egg permeable for NaCl, whereby the germ
inside the membrane is killed; while the addition of a
small amount of Ca (or any other bivalent metal)
prevents the diffusion of Na into the egg,* possibly, as
T. B. Robertson? suggested, by forming a precipitate
with some constituent of the membrane, whereby the
latter becomes more impermeable. The correctness
of this idea can be demonstrated in the following way.
When eggs of Fundulus, which are three or four days
old and contain an embryo, are put into a test-tube
containing 3 m NaCl they will float on this solution for
about three or four hours; after that they will sink to
the bottom. Before this happens the egg will shrink
and when it ceases to float the embryo is usually dead.
This is intelligible on the assumption that the NaCl
solution entered the egg, increased its specific gravity
so that it could not float any longer and killed the
embryo. When we add, however, I c. c. 10/8 m
CaCl, to 50 c.c. 3 m NaCl the eggs will float, the

t Loeb, J., Arch. f. d. ges. Physiol., 1905, cvii., 252.
2 Robertson, T. B., Ergeb. d. Physiol., 1910, x., 216.

312. ~The Influence of Environment

heart will continue to beat normally and the em-
bryo will continue to develop for three days or more,
because the calcium prevents the NaCl from entering
into the egg. For if we put a newly hatched embryo
into 50 c.c. 3 m NaCl+1 c.c. 10/8 m CaCl, it will die
almost instantly; hence the membrane must have
acted for three or more days as a shield which pre-
vented the NaCl from diffusing into the egg in the
presence of CaCl,.

The same experiments cannot be demonstrated in
the sea-urchin egg, first, because it can live neither in
distilled water nor in very dilute nor very concentrated
solutions; and second, because it is not separated as is
the germ of the Fundulus egg from the surrounding
solution by a membrane which is under proper condi-
tions practically impermeable for water and salts.

Nevertheless it can be shown that the results at
which we arrived in our experiments on Fundulus
are of a general application. Osterhout? has shown
that plants which grow in the soil or in fresh water
are readily killed by a pure NaCl solution of a certain
concentration, while they can resist the same concen-
tration of NaCl if some CaCl, is added. Wo. Ostwald?
has shown the same for a species of Daphnia. We,
therefore, come to the conclusion that the injurious

t Loeb, J., Biochem. Zischr., 1912, xlvii., 127.

2Osterhout, W. J. V., Bot. Gazette, 1906, xlii., 127; 1907, xliv., 257;
Jour. Biol., Chem., 1906, i., 363.

3 Ostwald, Wo., Arch. f. d. ges. Physiol., 1905, cvi., 568.

The Influence of Environment 313

action following an alteration in the constitution of the
sea water is in some of the cases due to an increase in
the permeability of the membranes of the cell, whereby
substances can diffuse into the cell which when the
proper balance prevails cannot diffuse. For this
balance the ratio of the concentration of the salts with
univalent cation Na and K over those with bivalent

CNa+K salts
CCa+Mg salts

cation Ca and Mg is of the greatest

importance.

6. The importance of this quotient appears in
the so-called ‘behaviour’ of marine animals. We
have mentioned the newly hatched larve of the
barnacle in connection with heliotropism. These larvee
swim in a trough of normal sea water at the surface,
being either strongly positively or negatively helio-
tropic. They collect as a rule in two dense clusters,
one at the window and one at the room side of the
dish. If such animals are put into a solution of NaCl+-
KCI (in the proportion in which these salts exist in
the sea water), they will fall to the bottom unable to
rise to the surface. They will, however, rise to the
surface and swim energetically to or from the window
if a certain quantity of any of the chlorides of a biva-
lent metal, Mg, Ca, or Sr, is added, but these movements
will last only a few minutes when only one of these
three salts is added; and then the animals will fall to
the bottom again. If, however, two salts, e. g., MgCl,

314 The Influence of Environment

and CaCl,, are added the animals will stay permanently
at the surface and react to light as they would have
done in normal sea water. These animals also can
resist comparatively large changes in the concentration
of the sea water, and it seemed of interest to find out
CNaCl+KCl
CMgCl,+CaCl.
allowed all the animals to swim at the surface, had
a constant value. The MgCl.+CaCl, solution was
3/8 m and contained the two metals in the proportion
in which they exist in the sea water; namely, 11.8 mole-
cules MgCl, to 1.5 molecules CaCl,. The next table
gives the result.t Since these experiments lasted a
day or more each, usually two different concentrations
of NaCl+KCl of the ratio 1:2 or 1:4 were compared
in one experiment.

whether the quotient , which just

TABLE XVIII

Numb i Cae. Value
er ea Las 3/8 m CaCla+ Ore
Experiment NaCi+KCl Reset CMg+Ca

1 m/16 0.3 27.8

m/8 0.4-0.5 37.0

7 | m/8 0.5 33.3

m/4 0.9-1.0 35-1

3 3/16 m 0.7 35-7

3/8 m 1.3 38.5

* Loeb, J., Jour. Biol. Chem., 1915, xxiii., 423.

The Influence of Environment 315

TABLE XVIII—Continued

Numb : Cre. Value o,
we er ge” Saad 3/8 m Ca Ch re CNa4+ 2
& 2Cla pelea A Se ae
Experiment Nacl+KCh Required CMg+Ca
m/8 0.5 36.0
4 m/2 1.8-1.9 39.2
m/4 0.8-0.9 39.2
5 | m/2 1.6-1.7 40.3
5/16 m 0.9 46.3
6 5/8m 1.7 49.0
3/16 m 0.6 41.7
7 6/8 m 2.4 41.7

CNa+K
CCa+Mg
remains very nearly constant with varying concentra-
tions of CNa+K.

In former experiments on jellyfish the writer had
shown that there exists an antagonism between Mg
and Ca’, and this observation was subsequently con-
firmed by Meltzer and Auer? for mammals. It was
observed that in a solution of NaCl+KCl+MgCl,
the larvee of the barnacle were also not able to remain
at the surface for more than a few minutes, while an
addition of some CaCl, made them swim permanently
at the surface. Various quantities of MgCl, were
added to a mixture of m/4 or m/2 NaCl+-KCl, to find

«Loeb, J., Jour. Biol. Chem., 1905-06, 1., 427.
4 Meltzer, S. J., and Auer, J., Am. Jour. Physiol., 1908, xxi., 400.

These experiments indicate that the ratio of

316 The Influence of Environment

out how much CaCl., was required to allow them to
swim permanently at the surface.

TABLE XIX
C.c. of m/16 CaCl, Neces-
sary to Induce the Ma-
jority of the Larve to
Swim in
m/2(Na-+K)|m/4(Na+K) -
50 ac. NaCl+KCl+0.75 c.c. 3/8 m MgClz 0.2
50c.c. NaCl+KCl+ 1.5 c.c. 3/8m MgCl, 0.4 0.3
50c.c. NaCl+KCl+ 2.5 c.c. 3/8m MgCl. 0.4 0.4
50 c.c. NaCl+KCil+ 5.0 c.c. 3/8m MgCl, 0.7-0.8 0.7-0.8
50 c.c. NaCl+KCl+10.0 c.c. 3/8 m MgCl, 1.6 1.6
50c.c. NaCl+KCl+15.0 c.c. 3/8m MgCl. 1.8
50c.c. NaCl+KCl-+20.0 c.c. 3/8m MgCl, 1.8

In order to interpret these figures correctly we must
remember that we are dealing with two different an-
tagonisms, one between the salts with univalent and
bivalent metals and the other between Mg and Ca.
The former antagonism is satisfied by the addition of
Mg, inasmuch as enough Mg was present for this
purpose in all solutions. What was lacking was the
balance between Mg and Ca. The experiments in
Table XIX therefore answer the question of the ratio
between Mg and Ca. If we consider only the concen-
trations of Mg between 2.5 and 10.0 c. c. 34 m MgCl,
—which are those closest to the normal concentration
of Mg in the sea water—we notice that CCa must
vary in proportion to CMg. If we now combine the
results of this and the previous paragraph we may

The Influence of Environment 317

express them in the form of the theory of physiologically
balanced salt solutions, by which we mean that in the ocean
(and in the blood or lymph) the salts exist in such ratio
that they mutually antagonize the injurious action which
one or several of them would have if they were alone in
solution. This law of physiologically balanced solu-
tions seems to be the general expression of the effect
of changes in the constitution of the salt solutions for
marine or all aquatic organisms.

This chapter would not be complete without an
intimation of the réle of buffers in the sea water and
the blood, by which the reaction of these media is pre-
vented from changing in a way injurious to the organ-
ism. These buffers are the carbonates and phosphates.
Instead of saying that the organisms are adapted to
the medium, L. Henderson has pointed out the fitness
of the environment for the development of organisms
and one of these elements of fitness are the buffers against
alterations of the hydrogen ion concentration.? The
ratio in which the salts of the different metals exist in
the sea water is another. It is obvious that the quan-
titative laws prevailing in the effect of environment
upon organisms leave no more room for the interfer-
ence of a ‘directing force”’ of the vitalist than do the
laws of the motion of the solar system.

t This theory was first expressed by the writer in Am. Jour. Physiol.,

1900, iii., 434.
2 Henderson, L., The Fitness of the Environment. See also Michaelis,

L., Die Wasserstoffionenconzentration. Berlin, 1914.

CHAPTER XII

ADAPTATION TO ENVIRONMENT
J

1. Itisassumed by certain biologists that the envir-
onment influences the organism in such a way as to
increase its adaptation. Were this correct it would
not contradict a purely physicochemical conception of
life; it would only call for an explanation of the me-
chanism by which the adaptation is brought about.
There are striking cases on record which warn us
against the universal correctness of the view that
the environment causes an adaptive modification of
the organism. Thus the writer pointed out in 1889
that positive heliotropism occurs in organisms which
have no opportunity to make use of it,’ e. g., Cuma
rathkii, a crustacean living in the mud, and the
caterpillars of the willow borer living under the bark
of the trees. We understand today why this should
be so, since heliotropism depends upon the presence of
photosensitive substances, and it can readily be seen

1 Loeb, J., Der Heliotropismus der Tiere und seine Ubereinstimmung
mit dem Heliotropismus der Pflanzen. Wurzburg, 1890 (appeared in
1889).

318

Adaptation to Environment 319

that the question of use or disuse has nothing to do
with the production of certain harmless chemical com-
pounds in the body. A much more striking example
is offered in the case of galvanotropism. Many or-
ganisms show the phenomenon of galvanotropism,
yet, as the writer pointed out years ago, galvano-
tropism is purely a laboratory product and no animal
has ever had a chance or will ever have a chance to
be exposed to a constant current except in the labora-
tory of a scientist. This fact is as much of a puzzle
to the selectionist and to the Lamarckian (who would
be at a loss to explain how outside conditions could
have developed this tropism) as to the vitalist who
would have to admit that the genes and supergenes
indulge occasionally in queer freaks and lapses. The
only consistent attitude is that of the physicist who
assumes that the reactions and structures of animals
are consequences of the chemical and physical forces,
which no more serve a purpose than those forces re-
sponsible for the solar systems. From this viewpoint
it is comprehensible why utterly useless tropisms or
structures should occur in organisms.

2. A famous case for the apparent adaptation of
animals to environment has been the blind cave ani-
mals. It is known that in caves blind salamanders,
blind fishes, and blind insects are common, while such
forms are comparatively rare in the open. This fact
has suggested the idea that the darkness of the cave

320 Adaptation to Environment

was the cause of the degeneration of the eyes. A closer
investigation leads, however, to a different explanation.
Eigenmann has shown that of the species of salamanders
living habitually in North American caves, two have
apparently quite normal eyes. They are Sphelerpes
maculicauda and Spelerpes stejnegeri. ‘Two others liv-
ing in caves have quite degenerate eyes, Typhlotriton
speleus and Typhlomolge rathbuni. If disuse is the
direct cause of blindness we must inquire why Spelerpes
is not blind.

Another difficulty arises from the fact that a blind
fish Typhlogobius is found in the open (on the coast of
southern California) in shallow water, where it lives
under rocks in holes occupied by shrimps. The
question must again be raised: How can it happen
that in spite of exposure to light Typhlogobius is
blind?

The most important fact is perhaps the one found
by Eigenmann in the fishes of the family of Amblyop-
side. Six species of this group live permanently in
caves, are not found in the open, and have abnormal ?
eyes, while one lives permanently in the open, is never
found in caves, and one comes from subterranean
springs. The one form which is found only in the
open, Chologaster cornutus, has a simplified retina as
well as a comparatively small eye, in other words, its
eye is not normal. This indicates the possibility that
the other representatives which are found only in

Adaptation to Environment 321

' caves also might have abnormal eyes even if they had
never lived in caves.

Through these facts the old idea becomes question-
able, namely, that the cave animals had originally
been animals with normal eyes which owing to disuse
had undergone a gradual hereditary degeneration.

Recent experiments made on the embryos of the fish
Fundulus have yielded the result that it is possible to
produce blindness in fish by various means other than
lack of light. Thus the writer found that by crossing
the egg of Fundulus with the sperm of a widely different
species, namely, Menidia, blind embryos were produced
very frequently; that is to say such embryos had the
degenerate eyes characteristic of blind cave fishes.
Very often no other external trace of an eye, except a
gathering of pigment, could be found, while a close
histological examination would possibly have resulted
in the demonstration of rudiments of a lens and other
tissues of the eye.

Another method of producing blind fish embryos
consists in exposing the egg immediately, or soon after
fertilization, to a temperature between 0° and 2° C.
for a number of hours. Many embryos are killed by
this treatment, but those which survive behave very
much like the hybrids between Fundulus and Menidia,
4. e.,a number of them have quite degenerated eyes.
If the eggs have once formed an embryo they can be

t Loeb, J., Biol. Bull, 1915, Xxix., 50.

322 Adaptation to Environment

kept at the temperature of o° for a month or more
without giving rise to blind animals. Occasionally
such rudimentary eyes were also observed when eggs
were kept in a solution containing a trace of KCN.
Stockard has succeeded in producing cyclopean eyes
in Fundulus by adding an excess of magnesium salt
to the sea water in which the eggs developed or by
adding alcohol, and McClendon has confirmed and
added to these results.

The writer tried repeatedly, but in vain, to produce
Fundulus with deficient eyes by keeping the embryos
in the dark. Sperm and egg were not allowed to be
exposed to the light yet the embryos without exception
had normal eyes.

F. Payne raised sixty-nine successive generations of
a fly Drosophila in the dark, but the eyes and the re-
action of the insects to light remained perfectly normal.

Uhlenhuth has recently demonstrated in a very
striking way that the development of the eyes does
not depend upon the influence of light or upon the
eyes functioning. He transplanted the eyes of young
salamanders into different parts of their bodies where
they were no longer connected with the optic nerves.
The eyes after transplantation underwent a degenera-
tion which was followed by a complete regeneration.
He showed that this regeneration took place in com-
plete darkness and that the transplanted eyes remained
normal in salamanders kept in the dark for fifteen

Adaptation to Environment 323

months. Hence the eyes which were no longer in
connection with the central nervous system, which
had received no light, and could not have functioned,
regenerated and remained normal. The degeneration
which took place in the eyes immediately after being
transplanted was apparently due to the interruption
of the circulation in the eye, and the regeneration
commenced in all probability with the re-establishment
of the circulation in the transplanted organ.

In our own experiments it can be shown that the
circulation in the embryo was deficient in all cases
where the eyes degenerated. The hybrids between
Fundulus and Menidia have often a beating heart but
rarely a circulation (although they form blood); and
the same phenomenon occurred in the embryos which
were exposed to a low temperature at an early period
of their lives. Hence all the facts agree that conditions
which lead to an abnormal circulation (and conse-
quently also to an abnormal or inadequate nutrition
of the embryonic eye) may prevent development and
lead to the formation of blind fishes. Eigenmann
states that no blood-vessels enter the eye of the blind
cave salamander Typhlotriton. The presence or ab-
sence of light does not usually interfere with the circu-
lation or nutrition of the embryonic eye, and hence
does not as a rule lead to the formation of degenerated
eyes.

This would lead us to the assumption that the blind

324 Adaptation to Environment

fish owe their deficiency not to lack of light but to a
condition which interferes with the circulation in the
embryonic eye. Such a condition might be brought
about by an anomaly in the germ plasm or in one
chromosome, the nature and cause of which we are not
able to determine at present; but which, since it occurs
in the germ plasm or the chromosomes, must be heredi-
tary. This would explain why it is, that animals
with perfect eyes may occur in caves and why perfectly
blind animals may occur in the open. It leaves, how-
ever, one point unexplained; namely, the greater fre-
quency of blind species in caves or in the dark and the
relative scarcity of such forms in the open.

Eigenmann has shown that all those forms which
live in caves were adapted to life in the dark before
they entered the cave." These animals are all nega-
tively heliotropic and positively stereotropic, and with
these tropisms they would be forced to enter a cave
whenever they are put at the entrance. Even those
among the Amblyopside which live in the open have
the tropisms of the cave dweller. This eliminates the
idea that the cave adapted the animals for the life in
the dark.

Only those animals can thrive in caves which for their
feeding and mating do not depend upon visual mechan-

tCuénot has proposed the term preadaptation for such cases and
this term expresses the situation correctly. Cuénot, L., La Génése des
Espéces animales, Paris, 1911,

Adaptation to Environment 325

isms; and conversely, animals which are not provided
with visual mechanisms can hold their own in the open,
where they meet the competition of animals which
can see, only under exceptional conditions. Thisseems
to account for the fact that in caves blind species are
comparatively more prevalent than in the open.

In other words, the adaptation of blind animals to
the cave is only apparent; they were adapted to cave
life before they entered the cave. Many animals are
obviously burdened with a germinal abnormality
giving rise to imperfection and smallness of the eye—
the hereditary factor involved may have to do with
the development of the blood-vessels and lymphatics
of the eye. Such mutants can survive more easily in
the cave, where they do not have to meet the competi-
tion of seeing forms, thanin the open. Inman also an
hereditary form of blindness is known, the so-called
hereditary glaucoma. It has nothing to do with light,
but the disease seems to be due to an hereditary
anomaly of the circulation in the eye.

Kammerer’ has recently reported that by keeping
the blind European cave salamander Proteus anguinus
under certain conditions of illumination he succeeded
in producing two specimens with larger eyes. Accord-
ing to him the eyes of Proteus may develop to a
certain point and then retrogress again. He states
that by keeping young salamanders alternately for a

t Kammerer, P., Arch. f. Entwcklngsmech., 1912, xxxiii., 349.

326 Adaptation to Environment

week or two in sunlight and in a dark room where
they were exposed to red incandescent light, two males
formed somewhat larger eyes. The first year no altera-
tion was visible. In the second year a slight increase
in the size of the eyes was noticeable under the skin.
In the third year the eye protruded slightly and this
increased somewhat in the fourth year.

There is thus far only one case on record in animal
biology in which the light influences the formation of
organs. The writer found that the regeneration of the
polyps of the hydroid Eudendrium does not take place
if the animals are kept in the dark, while the polyps
will regenerate if exposed to the light;" and the time of
exposure may be rather short according to Goldfarb. ?
It is possible that Proteus resembles in this respect
Eudendrium; it should be stated, however, that of
many different forms tried by the writer over a number
of years, Eudendrium was the only one which gave evid-
ence of such an influence of light. Of course it is not
impossible that the light might influence reflexly the
development of blood-vessels in the eyes of certain
animals, e. g., Proteus, and thus allow the eyes of Proteus
to grow a little larger.

We therefore come to the conclusion that it is not
the cave that made animals blind but that animals with
a hereditary tendency towards a degeneration of the

* Loeb, J., Arch. d. f. ges. Physiol., 1896, 1xiii., 273.
2Goldfarb, A. J., Jour. Exper. Zoél., 1906, iii., 129; 1910, viii., 133-

Adaptation to Environment 327

eyes can survive in a cave while they can only excep-
tionally survive in the open. The cause of the de-
generation is a disturbance in the circulation and
nutrition of the eye, which is as a rule independent of
the presence or absence of light.

We may by way of a digression stop for a moment to
consider the most astonishing and uncanny case of
adaptation; namely, the formation of the transparent
refractive media, especially the lens in front of the
retina. It is due to these media that the rays which
are sent out by a luminous point can be united to an
image point on the retina. One part of this process is
understood; namely, the formation of alens. Wherever
the optic cup of the embryo is transplanted under the
epithelium the latter will be transformed into a trans-
parent lens. When the upper edge of the iris is in-
jured in the salamander so that the cells can multiply,
the mass of newly formed cells also becomes transparent
and a lens is formed. ‘This indicates the existence of
a substance in the optic cup which makes the epithelial
cells transparent; and which also limits the size of the
lens which is formed. The lens is not always a perfect
optical instrument, on the contrary, it is as a rule
somewhat defective. Of course, a great many details
concerning the process of lens regeneration have still
to be worked out.

3. It is well known that most marine animals die if
put into fresh water and vice versa; and in salt lakes or

328 Adaptation to Environment

ponds with a concentration of salt so high that most
marine animals would succumb if suddenly transferred
to such a solution we have a limited fauna and flora.
The common idea is that marine animals become
adapted to fresh water or vice versa; or to the condi-
tions in salt lakes; especially if the changes take place
gradually. Yet it can be shown that the existence of
these different faunas can be explained without the
assumption of an adaptive effect of the environment.’
The writer has worked with a marine fish Fundulus
whose eggs develop naturally in sea water which, how-
ever, will develop just as well in distilled water; and
the young fish hatching in distilled water live and grow
in this medium. Most of the adult fish die after several
days, when put suddenly into distilled water, but they
can live in fresh water which contains only a trace of
salt. They can also live in very concentrated sea
water, e. g., twice the normal concentration. Suppose
that a bay of the ocean containing such fish should
suddenly become landlocked and the concentration
of the sea water be thus raised to twice its natural
amount; the majority of forms would die and only
Fundulus and possibly a few other species with the
same degree of resistance would survive. An investi-
gator examining the salinity of the water and not know-
ing the natural resistance of Fundulus to changes in
concentration would be inclined to assume that he had
before him an instance of a gradual adaptation of the

Adaptation to Environment 329

fish to a higher concentration of the sea water; whereas
the fish was already immune to this high concentration
before coming in contact with it.

This fish seemed a favourable object from which to
find out how far an adaptation to the environment
really existed; and the result was surprising. By
changing the concentration of the sea water gradually
it is possible to raise the natural resistance of the fish
only a trifle, not much over ten per cent. The con-
centration of the natural sea water is a little over that
of a m/2 solution of NaCl+KCl+CaCl, in the pro-
portion in which these three salts exist in the sea water.
When adult Fundulus are put into a 10/8 m solution
of NaCl+KCl+CaCl, in the proportion in which these
salts occur in sea water they die in less than a day, but
when put from sea water directly into a 8/8 m or
9/8 m solution they can live indefinitely. It was found"
that if the concentration of the sea water was raised
gradually (by m/8 a day) the fish on the fifth day could
resist a 10/8 m solution of NaCl+KCl+CaCl, for a
month (or possibly indefinitely; the experiment was
discontinued after that period). When a 10/8 m solu-
tion was allowed to become more concentrated slowly
by evaporation (at room temperature) all the fish died
rapidly when the concentration was 12/8 m or even
below. In higher concentrations they can live only
a day or two. These experiments show that while the

t Loeb, J., Biochem. Ztschr., 1913, liii., 391.

330 Adaptation to Environment

fish is naturally immune to a 9/8 m NaCl+KCl+CaCl,
solution, by the method of slowly raising the concen-
tration it may be made to tolerate a 10/8 m or 11/8 m
solution, but not more. These fish when once adapted
to a 10/8 m solution can be put suddenly into a very
weak solution, e. g.,a m/80 NaCl, without suffering and
when brought back into a 10/8 m solution of NaCl+
KCl+CaCl, they will continue to live. If they remain
for several days in the weak solution their power of
resistance to 10/8 m NaCl+KCl+CaCl, solution is
weakened.

What change takes place when the fish is made more
resistant and why is its normal resistance so great?
The answer based on the writer’s experiments seems
to be as follows: Fundulus is comparatively resistant
to sudden changes in the concentration of the sea water
between m/80 and 9/8 m because it possesses a com-
paratively impermeable skin whose permeability is
not seriously altered by sudden changes within these
limits of concentration; while if these limits are ex-
ceeded and the fish are brought suddenly into too high
a concentration the skin becomes permeable and the
fish dies, the gills becoming unfit for use or nerves
being injured by the salt which diffuses into the fish.

The fact, that by slowly raising the concentration
to 10/8 m the fish may resist this limit, is in reality
no adaptation. There is no sharp limit between the
injurious and non-injurious concentration. We have

Adaptation to Environment 331

seen that the fish is naturally immune to a 9/8 m solu-
tion. It is also naturally immune to a 10/8 m or 11/8 m
solution if we give it time to compensate the injurious
effects of a 10/8 m solution by the repairing action of
its blood or kidneys. Beyond this no rise is possible.
In reality adaptation does not exist in this case.

In former experiments the writer had shown that a
pure NaCl solution of that concentration in which this
fish naturally lives kills it very rapidly, while it lives
in such a solution indefinitely if a little CaCl, is added.
The explanation of this fact is that the pure NaCl solu-
tion is able to diffuse into the tissues of the animal
while the addition of a trace of CaCl. renders the mem-
brane practically impermeable to NaCl. The question
then arose whether it was possible to make the fish more
resistant to a pure NaCl solution of sufficiently high
concentration and how this could be done. On the basis
of the idea of an adaptive effect of the environment we
should expect that by gradually raising the concentra-
tion of a pure NaCl solution the latter would gradually
alter the animal and ‘make it more resistant. The
method of procedure suggested was therefore to put
the fish first in low and gradually into increasing con-
centrations of NaCl. This method was tried and found
futile for the purpose. Fundulus when put from sea
water (after having been washed) into a 6/8 m NaCl
solution die in about four hours. When kept previ-
ously in a weaker NaCl solution they die if anything

332 Adaptation to Environment

more quickly. But it is possible to make them live
longer in a 6/8 m solution of NaCl; we have to proceed,
however, by a method which is in contrast with the
ideas of the adaptive influence of the environment.
When the fish are first treated with sea water (cr with
a mixture of NaCl+KCl+CaCl.) of a higher concen-
tration so that they become adapted to a 10/8 m solu-
tion of NaCl+KCl+CaCl, or to 10/8 m sea water,
they become also more resistant to an otherwise toxic
solution of NaCl. Fish taken directly from sea water
were killed in less than four hours when put into a
6/8 m NaCl solution, while fish of the same lot previously
adapted to 10/8 m sea water in the manner described
above lived two or three days in a 6/8 m NaCl solution.

It is not impossible that it was the high concentration
of calcium in the 10/8 m sea water which rendered the
fish more immune to a subsequent treatment with NaCl.
We know why a pure NaCl solution kills them and we
also know why the addition of CaCl, protects them
against this pernicious effect. It is rather strange that
where the conditions of the experiments are clear we
find nothing to indicate an adaptive effect of the
environment.

4. Ehrlich’s work on trypanosomes seems to indicate
a remarkable power of adaptation on the part of or-
ganisms to certain poisons. If the writer understands
these experiments correctly they consisted in infecting

1 Loeb, J., Biochem. Zitschr., 1913, liii., 391.

Adaptation to Environment 333

a mouse with a certain strain of trypanosomes, and
treating it with a certain arsenic compound, which
inhibited somewhat the propagation of the parasites
but did not kill them all. Four or five days later
trypanosomes from this mouse were transmitted to
another mouse and after twenty-four hours this mouse
was treated with a stronger dose of the same arsenic
compound; and this process was repeated. After the
third transmission or later, the trypanosomes can resist
considerably higher doses of the same poison than at
first and this resistance is retained for years. Ehrlich
seems to have taken it for granted that he had succeeded
in transforming the surviving trypanosomes into a
type which is permanently more resistant to the arsenic
compound than was the original strain.

The writer is not entirely convinced that in these
experiments a possibility was sufficiently considered
which is suggested by Johannsen’s experiments on the
importance of pure lines in work on heredity. Ac-
cording to this author a strain of trypanosomes taken
at random should, in all likelihood, contain a population
consisting of strains with different degrees of resistance.
If a high but not the maximal concentration of an
arsenic compound is repeatedly injected into the in-
fected mice the weaker populations of trypanosomes
are killed and only the more resistant survive. These
of course continue to retain their resistance if trans-
planted to hosts of the same species. According to this

334 Adaptation to Environment

interpretation the arsenic-fast strain may possibly
have existed before the experiments were made, and
Ehrlich’s treatment consisted only in eliminating the
less resistant strains.

On the other hand, it has been shown that if an
arsenic-fast strain of trypanosomes is carried through
a tetse fly it loses its arsenic-fastness. This fact may
possibly eliminate the applicability of the pure line
theory to a discussion of the nature of the arsenic-
fastness, but it seems that further experiments are
desirable.

5. Dallinger stated that he succeeded in adapting
certain protozoans to a temperature of 70° C. by
gradually raising their temperature during several
years. It is desirable that this statement be verified;
until this is done doubts are justified. Schottelius
found that colonies of Micrococcus prodigiosus when
ransferred from a temperature of 22° to that of 38°
no longer formed pigment and trimethylamine. After
the cocci had been cultivated for ten or fifteen gen-
erations at 38° they failed to form pigment even
when transferred back to 22° C. Dieudonné™ used
Bacillus fluorescens for similar purposes. At 22° it
forms a fluorescing pigment and trimethylamine, but
not at 35°. By constantly cultivating this bacillus
at 35° Dieudonné found that after the fifteenth genera-
tion had been cultivated at 35° the bacillus produced

* Dieudonné, A., Arb. a. d. kais. Gesndhtsmt., 1894, ix., 492.

Adaptation to Environment 335

pigment and trimethylamine at 35°. Davenport and
Castle* found that tadpoles of a frog kept at 15° went
into heat rigour at 40.3° C., while those kept for twenty-
eight days at 25° were not affected by this temperature
but we1t into heat rigour at 43.5°.. When the latter
tadpoles were put back for seventeen days to a tem-
perature of 15° they had lost their resistance to high
temperature partially, but not completely, since they
went into heat rigour at 41.6°. The authors suggest
that this adaptation to a higher temperature is due to
a loss of water on the part of protoplasm, whereby
the latter becomes more resistant to an increase in
temperature. This idea was put to a test by Kryz?,
who found that the coagulation temperature of their
muscle plasm is not altered by keeping cold-blooded
animals at different temperatures.

Loeb and Wasteneys? found that Fundulus taken
from a low temperature of 10° C. die in less than two
hours when suddenly transferred to sea water of 29° C.;
and in a few minutes if suddenly transferred to a tem-
perature of 35° C. If, however, the fish were trans-
ferred to a temperature of 27° C. for forty hours they
could live indefinitely in sea water of 35°. By exposing
the fish each day two hours to a gradually rising tem-

t Davenport, C. B., and Castle, W. E., Arch. f. Entwcklngsmech.,
1896, ii., 227.

2 Kryz, F., Arch. f. Entwcklngsmech., 1907, xxiii., 560.

3 Loeb, J., and Wasteneys, H., Jour. Exper. Zodl., 1912, xii., 543-

336 Adaptation to Environment

perature they could render them resistant to a tem-
perature of 39°. The remarkable fact was that fish
if once made resistant to a high temperature (35°) did
not lose this resistance when kept for four weeks at
from 10° to 14° C. Control fish taken from the same
temperature died in from two to four minutes; im-.
munized fish taken from 10° and put directly to 35° C.
lived for many hours or indefinitely. They will even
retain this immunity when kept for two weeks at a
temperature of 0.4° C.

Why is it that an animal can in general resist a high
temperature better if the latter is raised gradually
than when it is raised suddenly? Physics offers us
an analogy to this phenomenon in the experience that
glass vessels which burst easily when their temperature
is raised suddenly, remain intact when the temperature
is raised gradually. Glass is a poor conductor of heat
and when the temperature is raised suddenly inside a
glass cylinder the inner layer of the cylinder expands
while the outer layer on account of the slowness of
conduction of heat does not expand equally and the
cylinder may burst. We might assume that the sud-
den increase in temperature brings about certain changes
in the cells (e. g., an increase in permeability or destruc-
tion of the surface layer?). If the rise of temperature
occurs gradually the blood or lymph or the cell sap
may have time to repair the damage, and this repair
seems to be irreversible, at least for some time, as the

Adaptation to Environment 337

experiments on Fundulus seem to indicate. If the
temperature rises too rapidly the damage cannot be
repaired quickly enough by the cell or body liquids.

It is also to be considered that substances might
-be formed in the body at a higher temperature which
do not exist at a lower temperature, and vice versa,
and this might explain results like those of Schottelius
or Dieudonné and many others.

6. The theory of an adapting effect of the environ-
ment has often been linked with the assumption of the
inheritance of acquired characters. The older claims
of the hereditary transmission of acquired characters,
such as Brown-Séquard’s epilepsy in guinea pigs after
the cutting of the sciatic nerve, have been shown to be
unjustified or have found a different and more rational
explanation. Recently P. Kammerer has claimed to
have proven by new experiments that by environmental
changes, hereditary changes can be produced.

It has been mentioned already that the mature male
frogs and toads possess during the breeding season lumps
on the thumbs or arms which are pigmented and which
bear numerous minute horny black spines; these secon-
dary sexual characters serve the male frog in holding
the females in the water during copulation. There is
one species which does not possess this sexual character,
namely the male of the so-called midwife toad (Alytes
obstetricans). In this species the animals copulate on
land, and it is natural to connect the lack of this secon-

338 Adaptation to Environment

dary sexual character in the male with its different
breeding habit. Kammerer now forced such toads to
copulate in water instead of on land (by keeping the
animals in a terrarium with a high temperature). He
makes the statement that by forcing the parents to
lay their eggs during successive spawning periods in
water he finally obtained offspring which under normal
temperature conditions lay their eggs naturally in
water; in other words, they have changed their habits.
We will not discuss this part of his statement since
the breeding habits of animals in captivity are liable
to be abnormal. But Kammerer makes the further
important statement? that the male offspring of such
couples will in the third generation produce the swell-
ing on the thumb and the usual roughness, and in
the fourth generation black pads and hypertrophy
of the muscles of the forearm will appear. In other
words, he reports having succeeded in producing an
inheritance of an acquired morphological character
which has never been known to occur in this species.
Bateson, on account of the importance of the case,
wished to examine it more closely and I will quote his
report.

The systematists who have made a special study of
Batrachia appear to be agreed that Alytes in nature does
not have these structures; and when individuals possessing
them can be produced for inspection it will, I think, be time

«Kammerer, P., Arch. f. Entwckingsmech., 1909, xxviii., 448.

Adaptation to Environment 339

to examine the evidence for the inheritance of acquired
characters more seriously. I wrote to Dr. Kammerer in
July, 1910, asking him for the loan of such a specimen and
on visiting the Biologische Versuchsanstalt in September
of the same year I made the same request, but hitherto
none has been produced. In matters of this kind much
generally depends on interpretations made at the time of
observation; here, however, is an example which could
readily be attested by preserved material.*

More recently the same author has reported another
hereditary morphological change brought about by
outside conditions.2. A certain salamander (Salaman-
dra maculosa) has yellow spots on a generally dark
skin. Kammerer states that if such salamanders are
kept on a yellow ground they become more yellow,
not by an extension of the chromatophores (which would
not be surprising) but by actual multiplication and
growth of the yellow pigment cells; while the black
skin is inhibited in its growth. The reverse is true if
these salamanders are kept on black soil; in this case
according to Kammerer the growth of the yellow cells
of the skin is inhibited while the black part of the skin
grows. Curiously enough, according to him, these in-
duced changes are hereditary. Here again we are deal-
ing with the inheritance of an acquired morphological
character.

«Bateson, W., Problems of Genetics, pp. 201-202. Yale University

Press, 1913.
2Kammerer, P., Arch. f. Entwekingsmech., 1913, XXxvi., 4.

340 Adaptation to Environment

Megusar* has repeated Kammerer’s experiments on
salamanders but contradicts him by stating that the
colour of the soil has no influence on the colouration
of salamanders. Of course, we know the phenomenon
of colour adaptation in which the animal changes its
colour pattern according to the environment. This
is an effect of the retina image on the skin and has been
interpreted by the writer as a case of colour tele-
photography, for which no physical explanation has
yet been found.? This phenomenon, however, does not
lead to any hereditary change of colour.

Kammerer makes many statements on the heredity
of acquired modifications of instinct; indeed he claims
that an interest in music on the part of parents pro-
duces offspring with musical talent. In such claims
much depends upon the subjective interpretation of
the observer.

The writer is not aware that there is at present on
record a single adequate proof of the heredity of an
acquired character. We have records of changes in
the offspring by poisoning the germ plasm by alcohol
given to parents—as in Stockard’s well-known experi-
ments—or by. exposing butterflies to extreme tempera-
tures, but in these cases the germ cells were poisoned
or altered by the alcohol or by chemical compounds
produced at very low or very high temperatures. This

1 Werner, F., Biol. Centralbl., 1915, xxxv., 176.
2 Loeb, J., The Mechanistic Conception of Life. Chicago, 1912.

Adaptation to Environment 341

is of course an entirely different thing from stating
that by inducing the midwife toad to lay its eggs in
the water the male offspring acquires the pads and
horns of other species of frogs on its thumb; or that
by keeping black salamanders on yellow paper the off-
spring is more yellow. Yet if there is an inheritance of
acquired characters which can in any way throw light
on the so-called phenomena of adaptation it must
consist in results such as Kammerer claims to have
obtained.

While the writer does not decline to accept Ehrlich’s
interpretation of the arsenic-fast strains of trypano-
somes or Kammerer’s statements in regard to the inheri-
tance of acquired character, he feels that more work
should be done before they can be used for our problem.

7. This attitude leaves us in a quandary. The
whole animated world is seemingly a symphony of
adaptation. We have mentioned already the eye
with its refractive media so well curved and placed
that a more or less perfect image of the outside objects
is focussed exactly on the retina; and this in spite of
the fact that lens and retina develop independently;
we have mentioned and discussed the cases of instincts
or automatic arrangements which are required to per-
petuate life—the attraction of the two sexes and the
automatic mechanisms by which sperm and egg are
brought together; the maternal instincts by which the
young are taken care of; and all those adaptations by

342 Adaptation to Environment

which animals get their food and the suitable conditions
of preservation. Can we understand all these adapta-
tions without a belief in the heredity of acquired char-
acters? As a matter of fact the tenacity with which
some authors cling to such a belief is dictated by the
idea that this is the only alternative to the supra-
naturalistic or vitalistic ideas. The writer is of the
opinion that we do not need to depend upon the as-
sumption of the heredity of acquired characters, but that
physiological chemistry is adequate for this purpose.

The earlier writers explained the growth of the legs
in the tadpole of the frog or toad as a case of an adapta-
tion to life on land. We know through Gudernatsch
that the growth of the legs can be produced at any
time even in the youngest tadpole, which is unable to
live on the land, by feeding the animal with the thyroid
gland. As we have stated in Chapter VII, it is quite
possible that in nature the legs of the tadpole begin to
grow when enough of the thyroid or a similar compound
has been formed or is circulating in the animal.

It might justly be claimed as a case of adaptation
that the egg attaches itself to the wall of the uterus
and calls forth the formation of the decidua. We
have mentioned the observation of Leo Loeb that the
corpus luteum of the ovary gives off a substance to the
blood which alters the tissues in the uterus in such a
way that contact with any foreign body (e. g., the egg)
induces this decidua formation. Again what appeared

Adaptation to Environment 343

as adaptation when unknown turns out to be a result
of the action of a definite chemical substance circulating
in the body.

It appears as a case,of adaptation that the eggs of
the majority of animals cannot develop without a
spermatozoén, and yet we can imitate the activating
effect of a spermatozoén on the egg by definite chemical
compounds, which leads to the suggestion that the
activating effect of the spermatozo6n on the egg might
be due to the fact that it carries such a compound. |

The wonderful adaptations exhibited in the mating
instincts seem to be due to definite substances secreted
by the sex glands, as was shown by Steinach (Chapter
VII). Here, again, the process as popularly conceived,
is the reverse of the truth; those survive that have the
equipment,—they did not acquire the equipment under
the influence of environment.

It is absolutely imperative for green plants that their
stems and leaves be exposed to the light since only in
this way are they able to form carbohydrates; and it is
equally essential that the roots should grow into the soil
so that the plant may get the nitrates and phosphates
required to build up its proteins and nucleins. This
result is, in the language of adaptationists, brought
about by an adaptive response of the plant to the light.
In reality this adaptive responsé is due (Chapter X)
to the presence of a photosensitive substance present
in almost all green plants.

344 Adaptation to Environment

Lewis has shown that if the optic cup is transplanted
under the skin of a young larva into any part of the
body the skin in contact with the optic cup will form
a lens; it looks as if a chemical substance from the
optic cup were responsible for the formation of the lens.

These examples might be multiplied indefinitely.
They all indicate that apparent morphological and
instinctive adaptations are merely caused by chemical
substances formed in the organism and that there is
no reason for postulating the inheritance of acquired
characters. We must not forget that there are just
as many cases where chemical substances circulating
in the body lead to indifferent or harmful results. As
an example of the first type, we may mention the exist-
ence of heliotropism in animals living in the dark, of
the latter type, the inheritance of deficiencies like
colour-blindness or glaucoma.

While it is possible for forms with moderate dis-
harmonies to survive, those with gross disharmonies
cannot exist and we are not reminded of their possible .
existence. As a consequence the cases of apparent
adaptation prevail in nature.

The following observation may serve to give an idea
how small is the number of existing or durable forms
compared with the number of forms incapable of exist-
ence. We have mentioned the fact observed by Moenk-
house, the writer, and Newman, that it is possible to
fertilize the eggs of each marine bony fish with the

Adaptation to Environment 345

sperm of practically every other marine bony fish.
The number of teleosts at present in existence is about
ten thousand. If we accomplish all possible hybridiza-
tions, one hundred million different crosses will result.
Of these only a small fraction of one per cent. can live
(see Chapter I), and it is generally the lack of a
proper circulation which inhibits them from reaching
maturity. It is, therefore, no exaggeration to state
that the number of species existing today is only an
extremely small fraction of those which can and pos-
sibly do originate, but which escape our notice and
disappear because they cannot live or reproduce. If
we consider these facts we realize that the mere laws
of chance are adequate to account for the fact of the
apparently purposeful adaptations; as they are ade-
quate to account for the Mendelian numbers.

CHAPTER XIII
EVOLUTION

Darwin’s work has been compared to that of Coper-
nicus and Galileo inasmuch as all these men freed the
mind from the incubus of Aristotelian philosophy which,
with the efficient co-operation of the church and the
predatory system of economics, caused the stag-
nation, squalor, immorality, and misery of the
Middle Ages. Copernicus and Galileo were the
first to deliver the intellect from the idea of a uni-
verse created for the purpose of man; and Darwin
rendered a similar service by his insistence that
‘accidental and not purposeful variations gave rise
to the variety of organisms. In this struggle for
intellectual freedom the names of Huxley and Haeckel
must be gratefully remembered, since without them
Darwin’s idea would not have conquered hu-
manity.

Darwin assumed that the small fluctuating variations
could accumulate to larger variations and thus cause

new forms to originate.
346

Evolution 347

It was the merit of de Vries" to have pointed out that
fluctuating variations are not hereditary and hence
could not have played the réle assigned to them by
Darwin, while discontinuous variations as they appear
in the so-called ‘‘sports” or mutations are inherited.
This was an important step in the history of the theory
of evolution. It did not touch the foundation of Darwin’s
work, namely the substitution of the idea of an acci-
dental evolution for that of a purposeful creation; it
only modified the conception of the possible mechanism
of evolution. According to de Vries, there are special
species or groups of species which are in a state of muta-
tion. He considers the evening primrose on which he
made his observations as one of these forms. Morgan
and his pupils have observed over 130 mutations in a
fly Drosophila. From our present limited knowledge we
must admit the possibility that the tendency toward the:
production of mutants is not equally strong in different
forms. Whether this part of de Vries’s idea is or is not
correct there can be no doubt that variations occur which
consist in the loss and apparently, though in rarer cases,
in the gain ora modification of a Mendelianfactor. Ifwe
wish to visualize the basis of such a change we may do so
by imagining well-defined chemical constituents in one or
more of the chromomeres undergoing a chemical change.

tde Vries, H., The Mutation Theory, translated by Farmer, J. B.,

and Darbishire, A. D., Chicago, 1909. Species and Varieties. Chicago,
1906. Gruppenweise Artbildung. Berlin, 1913.

348 Evolution

This way of looking at the origin of variation has
had the effect of putting an end to the vague specula-
tions concerning the evolution of one form from another.
We demand today the experimental test when such a
statement is made and as a consequence the amount
of mere speculation in this field has diminished
considerably.

It is possible that any further progress concerning
evolution must come by experimental attempts to
bring about at will definite mutations. Such attempts
have been reported but they are not all beyond the
possibility of error.t The most remarkable among
them are those by Tower who by a very complicated
combination of effects of temperature and moisture
claims to have produced definite mutations in the
potato beetle. The conditions for these experiments
are so expensive and complicated that a repetition by
other investigators has not yet been possible.

It is, however, still uncertain whether the mere addi-
tion or loss of Mendelian characters can lead to the
origin of new species. Species specificity is determined
by specific proteins (Chapter III.), while some Mende-
lian characters at least seem to be determined by hor-
mones or substances which need neither be proteins nor
specific for the species.

«For a critical discussion of the details, see Bateson, W., Problems
of Genetics, New Haven, 1913, Chapter X.

CHAPTER XIV
DEATH AND DISSOLUTION OF THE ORGANISM

I. It is an old saying that we cannot understand
life unless we understand death. The dead body, if
its temperature is not too low and if it contains enough
water, undergoes rapid disintegration. It was natural
to argue that life is that which resists this tendency to
disintegration. The older observers thought that the
forces of nature determined the decay, while the vital
force resisted it. This idea found its tersest expres-
sion in the definition of Bichat, that ‘‘life is the sum
total of the forces which resist death.”” Science is not
the field of definitions, but of prediction and control.
The problem is: first, how does it happen that as soon
as respiration has ceased only for a few minutes the
human body is dead, that is to say, will commence to
undergo disintegration, and second, what protects the
body against this decay while the respiration goes on,
although temperature and moisture are such as to
favour decay?

The earlier biologists had already raised the question
349

350 Death and Dissolution of the Organism

why it was that the stomach and intestine did not
digest themselves. The hydrochloric acid and the
pepsin in the stomach and the trypsin in the intestine
digest proteins taken in in the form of food; why do
they not digest the proteins of the cells of the stomach
and the intestine? They will promptly digest the
stomach as soon as the individual is dead, but not during
life. A self-digestion may also be caused if the arteries
of the stomach are ligatured. Claude Bernard and
others suggested that the layer of mucus protected
the cells of the stomach and of the intestine from the
digestive enzymes; or that the epithelial layer had a
protective effect. Pavy suggested that the alkali of
the blood had a protective action. All these theories
became untenable when Fermi showed that all kinds
of living organisms, protozoans, worms, arthropods,
are not digested in solutions of trypsin as long as they
are alive, while they are promptly digested in the same
solution when dead.* This is in harmony with the
fact that many parasites live in the intestine without
being digested as long as they are alive. Fermi con-
cluded that the living cell cannot be attacked by the
digestive ferments, while with death a change occurs
by which they can be attacked. But what is this
change? Fermi seems to be inclined to think that the
“living molecule” of protein is not hydrolysable (per-
haps because the enzyme cannot attach itself to it?),
t Fermi, C., Centralbl. f. Bacteriologie, Abt. 1, 1910, Ivi., 55.

Death and Dissolution of the Organism 351

while a change in the constitution or configuration of
the proteins takes place after respiration has ceased,
The fact that the living cell resists the digestive action
of trypsin and pepsin has found two other modes of
explanation, first, that the cells are surrounded by a
membrane or envelope through which the enzyme can-
not diffuse, and second, that the living cells possess
antiferments. But the so-called antiferments are
also said to exist after the death of the cell, whereas
after death the cell is promptly digested. Frederica,
as well as Klug, has shown that worms which are not
attacked by trypsin are digested by this enzyme when
they are cut into small pieces; although the pieces
of course contain the antienzyme. The other sugges-
tion that a membrane impermeable for trypsin protects
the cells would explain why living protozoa are not
digested by trypsin, but it leaves another fact unex-
plained, namely, the autodigestion of all the cells after
death by enzymes contained in the cells themselves.

2. The disintegration of the body after death is not
caused exclusively or even chiefly by the digestive en-
zymes of the intestinal tract or the micro-organisms enter-
ing the dead body from the outside, but by the enzymes
contained in the cells themselves. This phenomenon of
autolysis’ was first characterized by Hoppe-Seyler.?

t Levene, P. A., Autolysis. The Harvey Lectures, 1905-1906, p. 73,
gives a full account of the work on this subject up to 1905.
2 Hoppe-Seyler, F., Tabinger med.-chem. Untersuchungen, 1871, p. 499.

352 Death and Dissolution of the Organism

All organs suffering death within the organism, in the
absence of oxygen, undergo softening and dissolution in a
manner resembling that of putrefaction. In the course of
that process, albuminous matter gives rise to leucin and
tyrosin, fat to free acids andsoaps. This maceration, iden-
tical with the pathological conception of softening, is ac-
complished without giving rise to ill odour and is a process
similar to the one resulting from the action of water, acids,
and digestive enzymes.

In work of this kind, rigid asepsis is required to
exclude the possibility of bacterial infection and this
was first done by Salkowski, who showed that in
aseptically kept tissues like liver and muscle the amount
of substances that can be extracted with hot water
increases considerably. By the work of others, especi-
ally Martin Jacoby and Levene, it was established that
the power of self-digestion is shared by all organs.
Analysis of the products of the autodigestion of tissues
shows that, e. g., the amino acids, which constitute the
proteins, are produced. Dakin, Jones, and Levene
demonstrated the hydrolytic products of the nucleins,
in the case of the self-digestion of tissues.'

Again the question arises: Why do the tissues not
undergo autolysis during lifetime and what protects
them, and the answer is that self-digestion is a conse-
quence of the lack of oxidations. The presence of
antiferments must continue after death and cannot
be the cause which prevents the self-digestion during

* Levene, P. A., Am. Jour. Physiol., 1904, xii., 276.

Death and Dissolution of the Organism 353

life, since nothing indicates the destruction of the
hypothetical antidigestive enzymes through lack of
oxygen. The recent work of Bradley and Morse*
and of Bradley? has thrown some light on the problem.
These authors found that proteins of the liver which
are indigestible can be made digestible by the liver
enzymes if an acid salt or a trace of acid is added to
the mixture. A m/200 HCl solution gives marked
acceleration of the autodigestion of the liver. This
would explain why autodigestion takes place after
oxidations cease. In many if not all the cells, acids
are constantly formed during lifetime, e. g., lactic acid,
which through oxidation are turned to CO., and this
diffuses into the blood so that the H ion concentration
in the cells does not rise materially. If, however, the
oxidations cease, as is the case after death, the forma-
tion of lactic acid continues, but the acid is not oxidized
to CO, and thus removed, and as a consequence the
H ion concentration increases in the cells and the self-
digestion of proteins, which the digestive enzymes con-
tained in the cells themselves could not attack formerly,
becomes possible. Acid increases the digestibility
of a protein, probably by salt formation. Theoreti-
cally we should not be surprised that while in the liver
an increase in the CH favours autolysis in other tissues
the same result is produced by the reverse effect. We
t Bradley H. C., and Morse, M., Jour. Biol. Chem., 1915, xxi., 209.

2 Bradley, H. C., ibid., 1915, xxii., 113.
23

354 Death and Dissolution of the Organism

might say that the preservation of a certain CH prob-
ably at or near the point of neutrality during life pre-
vents self-digestion, while the gross alteration of the
Cu in either direction after death (or after the cessa-
tion of oxidations in the tissues) induces autolysis.
Bradley indeed suggests that many of the phenomena
of autolysis during lifetime, such as atrophy, necrosis,
involution, might be due to an increase in the Cu in
the tissues.

These facts agree with the suggestion of Fermi that
in the living cell the proteins cannot be attacked by
the digestive enzymes but relieves us of the necessity
of making the monstrous assumption of a “living
molecule” of proteins as distinct from a ‘‘dead”’ mole-
cule. The difference between life and death is not one
between living and dead molecules, but more -likely
between the excess of synthetic over hydrolytic
processes.

In the second chapter we mentioned the interesting
idea of Armstrong that when a synthesis is brought
about by a digestive enzyme (e. g., maltase) not the
original substrate is formed (e. g., maltose) but an
isomer, in this case isomaltose; and this isomer is not
attacked by the enzyme maltase. We thus get a
rational understanding of the statement which Claude
Bernard used to make but which remained at his time
mysterious: Ja vie, cest la création. During life,
when nutritive material is abundant, through the

Death and Dissolution of the Organism 355

reversible action of certain enzymes, synthetic com-
pounds are formed from the building stones furnished
by the blood. These synthetic isomers cannot be
hydrolyzed by the enzymes by which they are formed
and hence on account of the isomeric structure are
immune against destruction. It is not impossible that
the increase of the concentration of acid in the cells
after death transforms the isomers into that form in
which they can be digested by the enzymes contained
in the cell. Another possibility is that the increase in
digestibility brought about by an increase in CH in
the cell is due to the hydrating effect of acids on proteins
with a subsequent increase in digestibility. Whatever
the answer may be, the work done since Claude Bernard
has removed that cloud of obscurity which in his days
surrounded the prevalence of synthetic action in the
living and of disintegration in the dead tissues.

3. We have already referred to the connection
between the lack of oxygen and the onset of autolysis
and disintegration of tissues in the body. It is of
interest that there are cells in which the disintegration
under the influence of lack of oxygen is so rapid that
it can be followed under the microscope. The writer
has observed that certain cells undergo complete irre-
versible dissolution in a very short time under the
influence of lack of oxygen, e. g., the first segmentation
cells of the egg of a teleost fish Ctenolabrus.*

* Loeb, J., Arch. f. d. ges. Physiol., 1895, Ixii., 249.

356 Death and Dissolution of the Organism

When these eggs are deprived of oxygen at the time they
reach the eight- or sixteen-cell stage, it can be noticed that
the membranes of the blastomeres are transformed into
small droplets within half an hour or more, according to
the temperature. These droplets begin to flow together,
forming larger drops. [Figures 48 to 51 show the successive

6 ae?
A

ory,

ID
BE eS

FIG. 50 Fic. 51

stages of this process.] When the eggs are exposed to the
air in time, segmentation can begin again; but if a slightly
longer time is allowed to elapse, the process becomes irre-
versible and life becomes extinct. Such clear structural
changes cannot often be observed in the eggs of other
animals under the same conditions. Are these changes of
structure (apparently liquefaction of solid elements) respon-
sible for death under such conditions? In order to obtain
an answer to this question, the writer investigated the

Death and Dissolution of the Organism 357

effect of the lack of oxygen upon the heart-beat of the em-
bryo of the same fish Cienolabrus. This egg is perfectly
transparent and the heart-beat can easily be watched.
When these eggs are put into an Engelmann gas chamber
and a current of pure hydrogen is sent through, the heart
may cease to beat in fifteen or twenty minutes; it stops
beating suddenly, before the number of heart-beats has
diminished noticeably, and ceases beating before all the
free oxygen can have had time to diffuse from the egg.
In one case the heart beat ninety times per minute before
the hydrogen was sent through; four minutes after the
current of hydrogen had passed through the gas chamber,
the rate of the heart-beat was eighty-seven per minute,
three minutes later it was seventy-seven, and then the
. beats stopped suddenly. It is hard to believe that this
cessation could have been caused by lack of energy.
Hydrolytic processes alone could furnish sufficient energy to
maintain the heart-beat for some time, even if all the oxygen
had been used up. The suddenness of the standstill at a
time when the rate had hardly diminished seems to be
more easily explained by a sudden collapse of the machine;
it might be that liquefaction or some other change of
structure occurs in the heart or its ganglion cells, compar-
able to that which we mentioned before. In another fish
Fundulus, where the cleavage cells undergo no visible
changes in the case of lack of oxygen, the heart of the em-
bryo can continue to beat for about twelve hours in a cur-
rent of hydrogen. In this case the rate of the heart-beat
sinks during the first hour in the hydrogen current from
about one hundred to twenty or ten per minute; then it
continues to beat at this rate for ten hours or more. In
this case one might believe that during the period of steady
diminution of the tension of oxygen in the heart (during
the first hour), the heart-beat sinks steadily while it keeps
up at a low but steady rate as long as the energy for the

358 Death and Dissolution of the Organism

beat is supplied solely by hydrolytic processes; but there
is certainly no change in the physical structure of the cells
noticeable in Fundulus, and consequently there is no sudden
standstill of the heart.

Budgett: has observed that in many infusorians visible
changes of structure occur in the case of lack of oxygen’;
as a rule the membrane of the infusorian bursts or breaks
at one point, whereby the liquid contents flow out. Har-
desty and the writer found that Paramcecium becomes more
strongly vacuolized when deprived of oxygen, and at last
bursts. Amoebe likewise become vacuolized and burst
under these conditions. Budgett found that a number of
poisons, such as potassium cyanide, morphine, quinine,
antipyrine, nicotine, and atropine, produce structural
changes of the same character as those described for lack
of oxygen. As far as KCN is concerned, Schoenbein had
already observed that it retards the oxidation in the tissues,
and Claude Bernard and Geppert confirmed this observa-
tion. For the alkaloids, W. S. Young has shown that they
are capable of retarding certain processes of autoxidation.
This accounts for the fact that the above-mentioned poisons
produce changes similar to those observed in the case of lack
of oxygen.?

The phenomenon of rapid disintegration when de-
prived of oxygen (or in the presence of KCN) seems to
be general as Child? has shown in extensive experiments.
Child has used it to show that younger animals disin-
tegrate more rapidly than older or larger ones, and he
uses this fact for a theory of senescence. He connects

t Budgett, S. P., Am. Jour. Physiol., 1898, i., 210."

2Loeb, J., The Dynamics of Living Matter, New York, 1906, pp.
19-21.

3 Child, C. M., Senescence and Rejuvenescence. Chicago, 1915.

Death and Dissolution of the Organism 359

the more rapid disintegration of the young animal with
a greater metabolism. Without wishing to doubt
Child’s interesting observations the writer is not
quite certain whether the more rapid disintegration
of the younger forms is not a result of the fact that the
walls of membranes in the young are softer than those
of the older animals, and hence are more readily lique-
fied. Such a difference could be due to mere chemical
constitution, e. g., the increase in Ca in the membrane
with the increase in age. In old age in man the deposit
of Ca in the blood-vessels is a frequent occurrence.
These facts may help us to understand the nature of
death and dissolution of the body in higher animals.
Death in these animals is due to cessation of oxidations,
but the surprising fact is that if the oxidations have
been interrupted but a few minutes life cannot be
restored even by artificial respiration. This suggests
that the respiratory ganglia in the medulla oblongata
suffer an irreparable injury or an irreversible change
(comparable to that just described in the cells of Cteno-
labrus) even when deprived of oxygen for only a short
time. As a consequence of the irreversible injury to
the medulla the respirations cease permanently, the

tIt is a fact that in the early cells of Ctenolabrus the dissolution of
the cell walls through lack of O precedes death, since when oxygen is
admitted early enough the cells recover again. In infusorians the
bursting of the animal due to lack of O occurs suddenly, while the animal
is still moving, and this bursting is the cause of death, and not the
reverse.

360 Death and Dissolution of the Organism

heart-beat must also cease, and gradually the different
tissues must undergo the dissolution characteristic
of death. While all the cells may be immortal they
are only so in the presence of oxygen and the nutritive.
solution which the circulating blood furnishes. With
the proper supply of oxygen cut off they can no longer
live.

4. It 1s an unquestionable fact that each form has
a quite definite duration of life. Unicellular organisms
are immortal; but for the higher organisms with sexual
reproduction the duration of life is almost as character-
istic as any morphological peculiarity of a species.
No species can exist unless the natural life of its in-
dividuals outlasts the period of sexual maturity; and
unless the average duration of life is long enough to
allow as many offspring to be brought into the world
as will compensate for loss by death. The male bee
dies before it is a year old, while the queen may live
several years. In a certain species of butterflies, the
Psychide, the parthenogenetic female lays its eggs
while still in the cocoon and then dies without ever
leaving the cocoon. The imago of the ephemera
leaves the water in the evening, copulates, lets its eggs
fall into the water, and is dead the next morning. The
imperfect condition of their mandibles and alimentary
canal makes them unfit for a long duration of life. The
males of the rotifers which are devoid of organs of
digestion live but a few days.

Death and Dissolution of the Organism 361

In the Zodlogical Station at Naples in 1906, an
actinian, Actinia equina, was alive after having been
in captivity fifteen years, and another one, Cerianthus,
had been observed for twenty-four years. Korschelt
kept earthworms for as long as ten years. The fresh-
water mussel may reach the age of sixty years or more
and crayfish may live for over twenty years. The
differences in the duration of life of mammals are too
well known to need discussion. If the cells and tissues
are immortal, how does it happen that the duration
of life is so characteristic for each species?

Metchnikoff* has recently investigated the cause of
“natural”? death in the butterfly of the silkworm. The
butterfly in this species lacks the organs necessary for
taking up food, like the male rotifer or the ephemeridz
and hence is already, by this fact, condemned to a
short life. Metchnikoff observed that these butterflies
could live twenty-three days, but the average duration
of life was 15.6 for the males and 16.6 days for the
females; and that seventy-five per cent. of them con-
tained no parasitic fauna or flora in their intestine.
They lose considerably in weight during their lives,
but the males still contain the fat body at the time of
death. None of the changes accompanying ‘‘old age”’
in man are found in the tissues of these butterflies
before death. Metchnikoff is inclined to believe that
the animal is poisoned by some excretion retained in

t Metchnikoff, E., Ann. d. l’Inst. Pasteur, 1915, Xxix., 477-

362 Death and Dissolution of the Organism

the body; namely, the urine, and that this poison also
causes the symptoms of weakness which characterize
the animal. He could prove the toxic character of their
urine on other animals. This combined with starvation
could sufficiently account for the short duration of
life. The facts of the case show that it is due to an
imperfection in the construction of the organism such
as one would expect to find more or less in each animal
if one discards the idea of purposefulness and divine wis-
dom in nature. Only a slight, perhaps an infinitesimal,
fraction, of those species which are theoretically possible
and which at one time or another arise can survive.
Those which are durable show all transitions from
the grossest disharmonies to an apparent lack of such
shortcomings.

5. Minot had tried to prove that the death of meta-
zoa is due to the greater differentiation and special-
ization of their tissues. Admitting the immortality of
the unicellular organisms he argues that death is the
price metazoa pay for the higher differentiation of their
cells. This is of course purely metaphorical, but we
may put it into a form in which it is capable of discus-
sion in physicochemical terms, by assuming that death
is a necessary stage in the development of a species.
We are inclined, however, to follow Metchnikoff and
suspect some poison accidentally or unavoidably formed
in the body or some structural shortcoming as the cause
of “natural” death.

Death and Dissolution of the Organism 363

An unusually favourable object for the study of
natural death is the animal egg. The egg of the starfish
Asterias forbesii when taken out of the body is usually
immature, but in the spawning season it ripens in sea
‘water. The writer* observed that eggs which ripen
disintegrate very rapidly when not fertilized. This
disintegration may be due to a process of autolysis,
whith sets in only after the egg has extruded the two
polar bodies. The writer found that by preventing
the maturation of the egg either by withdrawing the
oxygen or by replacing the alkaline sea water by a
neutral solution or by exposing the eggs for some time
to acidulated sea water, the disintegration could also
be prevented.

Further experiments showed that even in the mature
egg rapid disintegration could be prevented by lack of
oxygen, and similar results were obtained by Mathews.
When the egg is fertilized it does not disintegrate
in the presence of oxygen but it gradually dies in
the absence of oxygen. One is almost tempted to
say that while the fertilized egg is a strict aérobe the
mature unfertilized egg is an anaérobe. This latter
statement, however, becomes doubtful since the pre-
sence of oxygen may help the disintegration only in-
directly by allowing certain changes to go on in the
egg. The important points for us are that duration of
life in the mature unfertilized egg is comparatively

t Loeb, J., Biol. Bull., 1902, iii., 295.

364 Death and Dissolution of the Organism

short and that the entrance of a spermatozoén or the
process of artificial parthenogenesis saves the life of
the egg. Loeb and Lewis found that the life of the
unfertilized sea-urchin egg (which is usually mature
when removed from the ovaries) can also be prolonged -
when its oxidations are suppressed. The decay of the
unfertilized egg seems to be due to the fact that those
alterations in the cortical layer which underlie the
membrane formation and which are responsible for
the starting of development gradually take place.
In such a condition the egg will die quickly unless
deprived of oxygen. This view is supported by the
observation of Wasteneys that unfertilized eggs of
Arbacia show an increased rate of oxidations when
allowed to remain for some time in sea water; we have
seen in Chapter V that such an increase also accom-
panies artificial membrane formation.

6. If the limited duration of life of an organism is
determined by one or more definite harmful chemical
processes, we should expect to find a temperature co-
efficient for the duration of life or at least be able to
show that if all other conditions are the same the dura-
tion of life is for a given organism a function of
temperature. The writer* investigated the dura-
tion of life of fertilized and unfertilized eggs of
Strongylocentrotus purpuratus for the upper temper-
ature limits.

* Loeb, J., Arch. f. d. ges. Physiol., 1908, cxxiv., 411.

Death and Dissolution of the Organism 365

TABLE XX

Duration of life of the eggs of S. purpuratus

Temperature
Unfertilized Fertilized
°C: Minutes ; Minutes
2 >i%
: (zy v4
31 | >2%
<3
30 >3 >4
<5 <5
29 eo
us ie 8 | > II
<10 <13
27 about 18 | is .
> 35 >
26 | < 40 | 2 a
> 76
25 { < 81
> 168 > 192
24 { < 200 | < 209
Hours
22 10%
21 24
20 7

These observations show a very high temperature
coefficient near the upper temperature limit, and this

366 Death and Dissolution of the Organism

may account at least partly for the fact that in tropical
seas the pelagic fauna is so much more limited than in
polar seas.t It is quite probable that the high tem-
perature coefficients at the utmost limits are only an
expression of the coagulation time of certain proteins.

P. and N. Rau state that in the cold certain butter-
flies live longer, and similar statements exist for the
silkworm, but these statements are not based on exact
experiments, which are difficult. Dr. Northrop and
the writer have started experiments on the influence
of temperature on the duration of life of the fly Droso-
phila. Newly hatched flies were kept first without
food except water and air at 34°, 28°, 24°, 19°, 14°,
and 10°; and second with cane sugar. The average
duration of life was as follows:

With water days With cane sugar days
BAe ace Saat cesar ee eee Dalaid antaaus uuche bead hee ug raha ac 6.2
BB sted hemes es ae BD Aracm ey dea ga ae 7.2
QA e-ea wieteceat nanan es BA cei see a Se eee eee 9.4
TO (ics shh Oe setae? 1 ee Cree party eer eae 12.3
i ee 8.3
TO? sectiiningre gaat 11.9

«K. Brandt (“Uber den Nitratgehalt des Ozeanwassers und seine
biologische Bedeutung,’ Abh. d. kais. Leop. Carol. deutsch. Akad. d.
Naturfoscher.,1915)accounts for this fact by the assumption that through
the greater activity of the denitrifying bacteria in the tropical waters
the amount of available nitrates is here comparatively smaller than
in the polar oceans. The writer fully appreciates the importance of this
fact but nevertheless is inclined also to see a limiting factor in the
enormously rapid decline of the duration of life at the upper temperature
limits.

Death and Dissolution of the Organism 367

These experiments show that there is a definite
temperature coefficient for the duration of life and that
this coefficient is of the order of magnitude of that of
a chemical reaction. We are continuing these experi-
ments with animals in the presence of food. It should,
however, be remembered that the fly carries with it
a good deal of reserve material from the larval period.
We have carried on simultaneously determinations of
the temperature coefficients of the duration of the
larval and pupa stage of these organisms at the same
temperatures and found ratios similar to those given
above for the duration of life with water only.

7. Metchnikofft has furnished the scientific facts
for our understanding of senescence. He has demon-
strated that the changes in tissue which give rise to
phenomena of senility are due to the action of phago-
cytes. Thus the ganglion cells are altered (digested?)
and destroyed by ‘‘neuronophags” and this is the
main cause of mental senility. Definite phagocytic
cells, the osteoclasts, slowly dissolve the bones (by
the excretion of an acid?) and this leads to the known
fragility of the bones in old age. The whiteness of
the hair is due to the action of phagocytes; in the
muscles in old age the contractile elements are destroyed
by the sarcoplasm, and so on. It agrees with these
facts that where organs are absorbed in the embryonic
development of an animal, as e. g., the tail of the tad-

t Metchnikoff, E., The Prolongation of Life. New York, 1907.

368 Death and Dissolution of the Organism

pole in metamorphosis, the phenomenon is due to a
process of phagocytosis (and autolysis). We have
mentioned the fact that in the larva of the Amblystoma
the absorption of the gills and of the tail occurs simul-
taneously and that both must be caused by a constituent
of the blood. Such a constituent may be responsi-
ble for phagocytosis and autolysis in the organs under-
going absorption. Metchnikoff calls attention to the
fact that certain infectious diseases, e. g., syphilis, may
bring about precocious senility; and he mentions also
the senile appearance of young cretins which is due to
the diseased thyroid. “It is no mere analogy to sup-
pose that human senescence is the result of a slow but
chronic poisoning of the organism.” He assumes that’
in man this poisoning is caused by the products of
fermentation in the large intestine and that the micro-
organisms responsible for these fermentations may
therefore be regarded as the real cause of senility in
man. Parrots which are long-lived birds have a limited
flora of microbes in their intestine, while cows and horses
which are short-lived in comparison with man possess
an extraordinary richness of the intestinal flora. But,
needless to say, it is not the quantity of microbes alone
which is to be considered, the nature of the microbes is
of much greater importance.

Certain plants like the Californian Sequoia gigantea
may be considered as practically immortal since they
live several thousands of years; other plants, the an-

Death and Dissolution of the Organism 369

nuals, die after fructification. Metchnikoff quotes
from a letter by de Vries that this author prolonged
the life of Ginotheras by cutting the flowers before
fertilization.

Under ordinary conditions the stem dies after producing
from forty to fifty flowers, but if cutting be practised new
flowers are produced until the winter cold intervenes. By
cutting the stem sufficiently early the plants are induced
to develop new buds at the base and these buds survive
winter and resume growth in the following spring.

Metchnikoff suggests that it is a poison formed in the
plant (in connection with fructification?) which kills
the annuals, while it is not formed or is less harmful
in the perennials. He compares the situation to the
death of the lactic acid bacilli if the lactic acid is allowed
to accumulate. This hypothesis is certainly worthy
of consideration, and it is quite possible that in addi-
tion to structural shortcomings poisons formed by
certain organs of the body as well as poisons formed
by bacteria account for the phenomenon of death in
metazoa.

INDEX

Abraxas, 203, 238, 241

Acquired characters, inheritance
of, 337 ff.

Actinia equina, 361

Adaptation, 12, 318 ff.; to life in
caves, 319 ff.; fresh and salt
water, 327 ff.; poisons, 332 ff.;
temperature, 334 ff.; caused by
hormones, 342

Addison, W. H. F., 188

Agglutination, of corpuscles by
sera, 67 ff.; of sperm, 78, 82 ff.

Allolobophora terrestris, 46

Alpheus, 176

Alytes obstetricans, 337, 338

Amanita phalloides, 63

Amblystoma, 157, 368

Amelung, 184

Amphipyra, 283

Analogies between living and
dead matter, 14 ff.

Anaphylaxis reaction, 61 ff.

Ancel, 158, 225 ff.

Antagonistic salt action.
Balanced salt solutions.

Ant laria antennina, 194, 196

Apes, blood relationship to man,
54, 56 ff.

Apolant, 45

Arbacia, 75 ff., 96, 99, 101, III,
114, 150, 190 ff., 293 ff., 298,
299, 364

Arenicola, 277

Armstrong, E. F., 26, 28, 354

Arrhenius, S., 33 ff., 88, 290, 296

Arrhenoidy, 218, 225

Artificial parthenogenesis, 95 ff.;
in sea urchins, 95 ff.; new
method of, 98, 99; by blood,
101 ff.; by sperm extract, 103;

See

by acids, 105; by mechanical
agitation, 107; in starfish, 110;
role of hypertonic solution, 112,
115, 116; and oxidation, 116,
117, 118; and permeability,
119 ff.; in frogs, 124; and de-
termination of sex, 125

Artificial production of life, 38-39

Assimilation of COz without
chlorophyll, 17 ff.

Astertas, 49, 81, 110, 363; ochracea,
73 .; capitaia, 74

Asterina, 75, 81, 110

Astrospheres, 115 ff., 192

Auer, J., 315

Autolysis, 351 ff.

Avena, 263

B. coli communis, 36; typhosus, 363
fluorescens, 334

Bacteria, growth of, 15 ff., 29, 71
ff.; specificity in, 41 ff.

“ Bacterio-purpurin,” 41

Balanced salt solutions, 307-3173
ot of, 317; and adaptation,
331 O.

Balanus, 259

Baltzer, F., 215 ff.

Bancroft, F. W., 70, 125, 127, 264,
269 ff.

Bang, 63

Bardeen, C. R., 174 ff.

Barnacle, larve of, 313 ff.

Bataillon, 124

Bateson, W., 230, 240 ff., 338, 348

Batrachia, 338

Baur, E., 48, 246

Bayliss, 63

Becquerel, P., 36 ff.

Beggiatoa, 19

371

372

Beijerinck, M., 20

Berkeley, Lord, 111

Bernard, Claude, 2 ff., 26, 159,
350, 354) 3551 358

Berthelot, 290

Bertrand, G., 248 ff.

Beutner, R., 140

Bichat, 2, 349

Bickford, E. E., 169

Blaauw, H. A., 263

Blackman, F. F., 302

Blastomeres, 141 ff.

Blind animals, 319 ff.

Blood, transfusion of, 53 ff.

Blood relationship, established by
transfusion, 53, 54 ff.; precipitin
reaction, 55 ff.; anaphylaxis re-
action, 61 ff.; hemoglobin crys-
tals, 64 ff.

Blood serum, precipitin reaction
of, 54 ff.; effect of, on unfer-
tilized eggs, 101 ff., 124

Blowfly, heliotropism of larve of,
265 ff.

Bohn, G., 253, 264, 269

Bombinator igneus, 46

Bonellia, 215

Bonnet, 154, 161

Bordet, 54 ff., 60

Bouin, 158, 225 ff.

Boveri, Th., 8, 126, 128 ff., 134,
138 ff., 150 ff., 186 ff., 209 ff.,

246
Brachystola, 199
Bradley, H. C., 27, 64, 353, 354
Brandt, 366
Braus, H., 147
Bridges, C. B., 208, 229, 231 ff.
Brown, A. P., 64 ff.
Bruchmann, H., 93
Bryophyllum calycinum, 153, 160

261, 263, 264
Burrows, 31

Campanularia, 178, 181
Cannon, W. B., 285
Carcinus menas, 217

Index

Cardamine pratensis, 90

Carrel, 31

Cassia bigapsularis, 37

Castle, W. E., 89 ff., 335

Caullery, M., 158, 180, 217

Cave animals, 319 ff.

Cell division, 15, 29, 129 f.;
suppression of, 113 ff.

Cells, nutritive media of, 15 ff;
immortality of, 30 ff.; mi-
grating, 44; mesenchyme, 51 ff.,
130 ff., 147, 155 ff.

Certanthus membranaceus, 171 ff.,
188, 361

Chetopterus, 78 ff. :

Chamberlain, M. M., 293, 297

Chapman, H. G., 60

Chemotropism of spermatozoa,

92 ff.

Chevreul, 289

Child, C. M., 7, 170, 177, 358

Chlamydomonas, 277

Chodat, R., 248

Chologaster, 320

Christen, 288

Chromosomes, réle of, in sex
determination, 198 ff.; theory of
Mendelian heredity, 233

Chun, 142

Ciona intestinalis, 89 ff., 212

Cladocera, 159

Clausen, H., 302

Clavellina, 181

Cohen, E., 292

Cohn, 41 ff.

Compton, 90

Conklin, E. G., 129, 134, 143,
145 ff.

Constancy of species, 40-43

Copernicus, 346

Corpus luteum, action of, 157-158

Correlation, 154, 167

Correns, C., 90 ff., 214

Cramer, 289

Crampton, H. E., 143, 225

Criodrilus lacuum, 219-220

oe over of chromosomes,
241 ff.

Crystals, differences between living
organisms and, 14 ff.

Cienolabrus, 355, 357, 359

Ctenophores, 142

Index

Cuénot, L., 12, 324

Cullen, G. E., 24, 291

Cuma rathkii, 318

Cyanophycee, 287

Cytisus biflorus, 37

ot of eggs as future
embryo, 8, 9, 70, 126, 151 ff.

Dakin, 352

Dallinger, 334

Daphnia, 210, 262, 279, 280, 282,
306, 312

Darbishire, A. D., 347

Darwin, 90, 297, 346 ff.

Darwinian theory, 5 ff.

Davenport, C. B., 244, 335

Death, 349 ff.; natural, cause of,
364,369

Decidua formation induced by
corpus luteum, 157-158

Delage, Y., 107, I10, 111, 123, 126,

de la Rive, 24

de Meyer, J., 127

Dendrostoma, 101

Dentalium, 144

Design, 4, 5

Determination of sex, in bees, 208
ff.; in phylloxerans, 210; in
Bonellia, 215

Development of egg, 127 ff.

de Vries, H., 6, 42, 154, 161, 347,
369

Dewitz, 93

Dieudonné, C., 334, 337

“Directive force,” 2

Disharmonies, 7

Divisibility of living matter, limits
of, 148-151

Dominance, 230

Doncaster, L., 203

Dorfmeister, 303

Driesch, H., 4 ff., 128, 133, 136,
138 En 147, 150, 169 ff., 180 ff.,
18.

Drosophila ampelophila, 204 ff.,
237, 243, 322, 347, 366

Duclaux, E., 288, 289

v. Dungern, 80

Duration of life, 360 ff.

Durham, 249 ,

373

Dutrochet, 154
Dzierzon, 208

Ectoderm formation, 130 ff.

Egg, as the future embryo, 8, 9,
70, 126, 151; artificial partheno-

enesis of, 95 ff.; organisms
rom, 128 ff.; determining unity
of organism, 151-152; chromo-
somes in,198 ff.

Egg structure, 129 ff.; influence of
centrifugal force on, 135; and
regulation, 139, 140, 141; and
fluidity of protoplasm, 141

Ehrlich, 45, 322, 332 ff., 3413
side-chain theory of, 88, 188

Eigenmann, 320, 323 ff.

Electromotive forces, origin in
living organs, 140

Engelmann, 357

Engler, 24

Entelechy, 4, 170, 182

Environment, influence of, 286 ff.;
temperature, 288 ff., 344 ff.;
salinity, 306; adaptation to, 319

Enzyme action, 23 ff., 297, 302

Ernst, A., 21 :

Eternity of life, 34 ff., 360

Eudendrium, 260, 261, 269, 277,
278, 326

Eudorina, 277

Euglena, 264, 269, 272, 277

Euler, H., 21

Evolution, 346 ff.; and mutation,

348
Ewald, W.F., 261 ff., 269, 280, 301

Farmer, J. B., 347

Fermi, 350, 354

Fertilization, heterogeneous, 48 ff.,
51, 73 ff.; specificity in, 71 ff.;
and oxidation, 117 ff.; and per-
meability, 119 ff.

“Fertilizin,”’ 84, 87 ff., 93

Fischel, 187

Fischer, 303 ff.

Fish, 55

Fitness of environment, 317

Fitzgerald, J. G., 63

Flow of substances and regenera

_ tion in Bryophyllum, 161 ff.

374

Fluctuating variations, 6, 297 ff.,
346 ff.

Folin, 22

Food, influence on polymorphism
in wasps, 222 ff.

Food castration, 224; influence on
sexual cycle in rotifers, 224; on
metamorphosis in tadpoles, 155

Ford, 63

Forssmann, 63

Frédéricq, 351

Free-martin, cause of sterility,
218-219

Friedenthal, H., 53 ff., 60

Frisch, K., 278, 279

Fréschel, P., 263

Fuchs, H. M., 90

Fucus, 123

Fundulus heteroclitus, 51, 116,
147, 300, 301, 302, 307 ff., 321
ff., 328 ff., 335, 337, 357 ff.

pao 346 ‘
alvanotropism, I1, 270 ff., 319
Gay, F. P., 62 f. :
Generation, spontaneous, 14 ff.,

Genes, 4 ff., 152, 319

Genus and species, chemical basis
of, 40 ff.

Geppert, 358

Germination in seeds, 35 ff.

Giard, 180, 216 ff.

Godlewski, E., 48, 75, 78, 120,
126, 169

Godlewski, E., Sr., 18

Goebel, K., 154, 161

Goldfarb, A. J., 326

Goldschmidt, R., 220 ff.

Goodale, H. D., 218

Gortner, R., 249

Graber, V., 256, 276

Grafting, heteroplastic, in animals,
46; in plants, 47

Gravitation, influence on organ
formation in Antennularia, 194
ff.; on the egg of the frog, 141

Gray, J., 122

Gregory, 243

Groom, T. T., 280

Growth, termination of, 184; in-
fluence of cell size, 187

Index

Gudernatsch, J. F., 155, 255, 342
Guyer, 124
Gynandromorphism, 209

Haeckel, 346

Half-embryos and whole embryos,
I4l, 142

Hammond, J. H., Jr., 269

Harden, 16

Hardesty, 358

Harmonious character of organism,
5, 6, 318 ff., 341 ff.

Harrison, 31

Hartley, 111

Healing of wound, 187

Hektoen, 66

Heliotropism, 11 ff., 257 ff., 318;
heredity of, 250 ff.; change of,
279, 280 ff.; and adaptation,
318

Helmholtz, 34

Hemoglobins, crystallographic
measurements of, 64 ff.

Henderson, L., 317

Henking, 198 ff.

Herbst, C., 97, 147, 193, 306, 310

Heredity, of genus and species,
40 ff., 70, 151, 152; Mendelian,
70, 151 ff., 229 ff., 348; of sex,
198; sex-linked, 203 ff., 238 ff.;
and evolution, 348

Herlant, M., 78 ff., 115 ff.

Hermaphroditism, 89 ff., 212 ff.,
216, 219 ff. See also Inhibition
and Regeneration.

Hertwig, O., 97, 123, 292

Hertwig, R., 95, 97

278

Hess, C.,

Heterogeneous hybrids, purely
maternal, 49, 50

Heterogeneous transplantation,

Murphy's experiments on, 44 ff.;
limitation of, 46

Heteromorphosis, 155, 193-196

Hill, C., 25

Hippiscus, 199

Holmes, S. J., 269

Hoppe-Seyler, 351

Hormones, 145, 155, 181, 219; and
Mendelian heredity, 245 ff.,
348; and adaptation, 342. See
also Organ-forming substances.

Index

Huxley, 346

Hybridization, heterogeneous, in
sea urchins, 48 ff., 73 ff.;in
Se 51; in plants (Mendel’s),

230 ff.
Hydrolytic enzymes, action of,
24; reversible action of, 24 ff.
Hypertonic solution, 99, 111 ff.

Imitation of cell structures by
colloids, 39

Immortality, of cancer cells, 30;

, of somatic cells, 30 ff.; of life in
general, 34 ff.

Inheritance, of colour-blindness,
203, 204, 205; of eye pigment in
Drosophila, 204 ff.; of pigments,
248 ff.; of acquired characters,

337 UH.

Inhibition of regeneration in Bryo-
phyllum, 162 ff.

Inhibition of sexual characters of
opposite sex, in pheasants, 218;
lack of in hermaphrodites, 219;
in Bonellia, 226

Instincts, 10 ff., 253 ff.; sexual,
198 ff.

Intersexualism, 221

Intestine, formation of, 130 ff.

Isoagglutinins, 66 ff., 92

Isolation of blastomeres, 136 ff.

facebys 352
anda, V., 219 ff.
Jansky, 67
Janssens, 242
Jennings, H. S., 264 ff.
ensen, 45
oest, 46
ohannsen, W., 42, 333
ones, 352
ost, 90

Kammerer, P., 325, 337 ff.
Kanitz, A., 290, 292, 296
Kastle, J. H., 26 ff.
Kellogg, V. L., 279
Kelvin, 34

King, W. O. R., 50, 247
Klug, 351

v. Knafil, E., 106
Knowlton, E. P., 292

375

Kofoid, C. A., 143
v. Korésy, 300
Korschelt, 361
Krakatau, 21
Kraus, 54 ff.
Krogh, 292

Kryz, F., 335
Kupelwieser, H., 75

Lack of oxygen, influence on dis-
integration of tissue, 355 ff.

Ladoff, S., 224

Lamarck, 6

Laminaria, 165

Landois, L., 53

Landsteiner, 66

Lanice, 143 ff.

Lankester, E. R., 41

Leathes, } . B., 63

Leucena leucocephala, 37

Levene, 351, 352

Lewis, 183, 344, 364

Light, influence on organ forma-
tion, in cave animals, 319 ff.;
in Proteus, 325; in Eudendrium,
326. See also Heliotropism.

Lillie, F. R., 80, 82 ff., 87 ff., 93,
134, I9I, 218, 292

Lillie, R. S., 101, 107, 110, 120 ff.

Lipase, synthetic action of, 26

Living and dead matter, specific
differences between, 14 ff.

Lloyd, D. J., 111

Localization of Mendelian charac-
ters in individual chromosomes,

243, 244
Loeb, Leo, 30 ff., 45, 157, 170,
187 ff., 342
Loevenhart, A. S., 26 ff.
Lumbricus rubellus, 46
Lychnis dioica, 217
Lycopodium, 93
Lygeus, 201
Lymantria dispar, 220
Lymneus, 142
Lymphocytes, réle of, 45 ff.
Lyon, E. P., 134 ff.

Macfadyen, A., 36

Maeterlinck, 255

Magnus, W., 60

Maltase, synthetic action of, 25

376

Marchal, P., 222 ff., 254

Margelis, 192

Mass of chromatin and of cyto-
plasm, 186

Mast, 269, 277

Mathews, A. P., 107, 363

Matthaei, G. L. C., 302

Maxwell, S.S., 270, 274, 277

McClendon, J. F., 122, 322

McClung, C. E., 68, 198 ff., 237

Megusar, 340

Meignon, 217

Meisenheimer, 225

Meltzer, S. J., 315

Membrane formation, 86 ff.; arti-
ficial, 98 ff.

Mendel, G., 23, 229 ff.

Mendelian characters, and evolu-
tion, 70, 348; and internal se-
cretions, 243, 348; and enzymes,
247, 248, 249 ;

Mendelian, factors of heredity,
4 ff., 68, 151 ff.; mutation, 66;
dominant, 90; segregation,
229ff. See also Non-Mendelian
inheritance.

Mendelian heredity, mechanism
of, 229 ff.; and chromosomes,
233 ff.; and hormones, 245 ff.,
348; and enzymes, 247 ff.

Menidia, 51, 321, 323

Merogony, 120, 126, 186

Merrifield, 303

Mesenchyme formation, 130 ff.

Metamorphosis of tadpoles in-
duced by thyroid, 155, 156

Metchnikoff, 361 ff., 367 ff.

Michaelis, L., 62, 317

Micrococcus prodigtosus, 334

Micromeres, 132 ff.

Minot, 362

Moenkhaus, W. J., 51, 344

Molisch, 20

Montgomery, 199, 234

Moore, A. R., 50, 247 ff., 280

Morgan, T. H., 46, 68, 89 ff., 95,
116, 126, 134, 141 ff., 173, 175,
184, 204 ff., 229 ff., 241 ff., 244,
347

Morse, M., 156, 353

Morton, J. J., 44

Moss, W. L., 67

Index

Muller, H. J., 229, 237 ff.

Murphy, J. B., 44 ff.

Mutation, 6, 42, 2433; and evolu-
tion, 347, 348

Myers, 55

Nathanson, 19

Natural death, 361 ff.

Neilson, 110

Newman, 344

Newton's Law, 253

Nitrifying bacteria, 16 ff.

Non-Mendelian inheritance, genus
and species characters, 70, 151,
251; rate of segmentation,
246; first development, 247

Northrop, 366

Nostocace@, 21

Nussbaum, M., 149

Nuttall, G. H. F., 56 ff

Ocneria dispar, 225

Cnotherus, 369

Onslow, H., 249

Organ-forming substances or
hormonesin regeneration, 154 ff.;
causing metamorphosis in tad-
poles, 155-157; decidua forma-
tion, 158; development of milk
glands, 158; Sachs’s theory of,
159

Organisms from eggs, 128 ff.

Origin of life, 14 ff., 33 ff.

Osborne, 23

Osterhout, W. J. V., 312

Ostwald, Wo., 29, 305, 312

Oudemans, 225

Overton, 123

Palemon, 193

Palemonetas, 193; geotropism of,
270

Palinurus, 193

Pandorina, 277

Parker, G. H., 264, 269

Parthenogenesis, artificial, 95 ff.;
“spontaneous, ’’ 107

Pasteur, 14 ff., 24, 33, 38

Patten, B., 264

Pauli, W., 289

Pavy, 350

Payne, F., 322

Index

Pearl, R., 203, 244

Penicillium, 289

Pennaria, 192

i Siig synthetic action of, 28,

Pfeffer, 92 ff.

Phagocytosis, 367

Planarta, 173 ff., 177

Planorbis, 142 ;

Plants, heteroplastic grafting in,
47 ff.; regeneration in, 160 ff.

Polygordius, 280

Polymorphism, 222

Porthesia, 256, 280 ff.

Preadaptation, 12, 324

Precipitin reaction, 54 ff.

Preformation of organism in egg,
128 ff., 142-145

Presence and absence theory, 230ff.

Primula, 243

Proteins, specific reactions of,
54 ff.; and species specificity, 68;
and evolution, 70, 348

Protenor, 200 ff., 208

Proteus, 325 ff.

Przibram, H., 176

Pure lines, 333, 334

Pycnopodia spuria, 74

Pyrrhocoris, 198

Radiation pressure, r6le in trans-
mission of spores through inter-
stellar space, 34 ff.

Rana, esculenta, 46; palustris, 46;
virescens, 46

Rate of segmentation, a non-
Mendelian. hereditary charac-
ter, 246

Rau, 366

Reaction, tropistic, 11 ff., 92 ff.,
147, 178, 187, 255 ff.; precipi-
tin, 54 ff.; anaphylaxis, 61 ff.

Regeneration, 9 ff., 153 ff.; in
plants, 160 ff.; in Bryophyllum,
161-167; in animals, 167 ff.;
in Tubularia, 167-170; in
Cerianthus, 171 ff.; in Planar-
ians, 173-176; in Alpheus, 176;
and autolysis, 178-181; of lens,
182, 183; external influences
on, 192 ff.; of gonads in her-
maphrodites, 219

377

Regulation, 139, 140, I41; in
regeneration, see Regeneration.

| Reichert, E. T., 64 ff.

Reseda, 90
i a of spores, 36; seeds, 36

Reversibility of development, in
Campanularia, 178 ff.; in As-
cidians, 180; in egg, 189 ff.; in
Antennularia, 194

Rhabdonema nigrovenosum, 213

Richet, C., 61

Richter, 34 .

Ringer solution, 99

Robertson, T. B., 28 ff., 62 ff.,
104, 311

Roentgen rays, 45

Roscoe, see Bunsen

Rotifers, determination of sexual
cycle by food, 224

Roux, W., 141 ff.

Saccharomyces, 36; cerevisie, 60

Sacculina, 216 ff.

Sachs, 88,

v. ed J., 145, 154 &., 159, 161,
I

Salamandra maculosa, 339
Salkowski, 352

Salts required for life, 306 ff.
Sansum, W. D., 64
Schizophycee, 21

Schleip, W., 213
Schoenbein, 358
Schottelius, 334, 337
Schroeder, 14, 33
Schultze, O., 141
Schtitze, 55

Schwann, 33

| Schwarzschild, 34

Secretions, internal, 145, 155, 157

Self-digestion, 350 ff.

Self-sterility, 89 ff.

Senescence, 367

Sequoia, 31, 368

Setchell, W. A., 165, 287

Sex, of parthenogenetic frogs, 1253
of twins, 211

Sex chromosome, 199 ff.

Sex determination, cytological
basis of, 198 ff.; physiological
basis of, 214 ff.

378

Sexual characters, 198 ff.

Shibata, 93

Shull, A. F., 214, 224

Sicyonia, 193

Side-chain theory, 88, 188

Smith, Geoffrey, 159, 217

Smith, Graham, 58

Spain, K. C., 188

Spallanzani, 33

Species, chemical basis of, 4o ff.;
specificity of, 41 ff.; incompati-
bility of, not closely related,

44 ff.

Species specificity, determined by
proteins, 63, 68, 348; apparently
not by nucleins, 69

Specificity, of grafted tissues, 47;
of spermatozoa, 48; of blood
sera, 53 ff.; in fertilization, 71 ff.;
of activation of sperm by eggs,
80 ff.

Spelerpes, 320

Spermatozoa, fertilization of eggs
by, 72 ff.; activation by eggs
of, 80 ff.; agglutination of, 82
ff.; cluster formation of, 83;
chemotropism of, 92 ff.; cul-
tivating of, 126 ff.; chromosomes
of, 198 ff.

Spirographis, 260

Spondylomorum, 277

Spontaneous generation, 33, 38

Spooner, G. B., 134

Standfuss, 303

Staphylococcus pyogenes aureus, 36

Steffenhagen, K., 55

Steinach, E., 225 ff., 254, 343

Stereotropism, 178, 187, 283

Stevens, Miss, 68, 199

Stimulus, 196

Stockard, 322, 340

Strassburger, 260

Streaming as means of egg differ-
entiation, 145, 146

Strongylocentrotus franciscanus, 50,
52, 75, 81 ff., 103, 247

Strongylocentrotus lividus, 129

Strongylocentrotus purpuratus, 52,
73 ff., 81 ff., 94, 98 ff., 103, 108,
109, 111 ff., 137, 191, 246 ff.,
293 ff., 364; larve of, 49 ff.

Sturtevant, A. H., 229 ff.

Index

Styela, 146

Sulphur bacteria, 19 ff.

Supergenes, 5, 9, 136, 319

Sutton, W. 5S., 68, 233 ff.

Synthesis of living matter, by
micro-organisms, 15 ff.; by
enzymes, 24 ff.

Synthetic action of enzymes, 23
ff., 38

Tenia, 212

Talbot, 262

Tammann, 291

Tanaka, 243

Taylor, A. E., 27, 69 ff.

Tchistowitch, 54 ff.

Teleost fish, crosses of, 6 ff., 345

Temperature, effect on heliotrop-
ism, 280; upper limit for organ-
isms, 287 ff.; effect on life, 288
ff.; on butterflies, 303 ff.; adapta~
tion to, 334 ff.

Temperature coefficient, 290 ff.,
305; for enzyme, 291; for de-
velopment, 292 ff.; for oxida-
tions, 295; and fluctuating var-
jation, 296 ff.; for fheart-beat,
300 ff.; for duration of life, 366

Thatcher, Miss, 181

Thyroid inducing metamorphosis
in tadpoles, 155, 156

Tichomiroff, 95

Tissue culture of spermatozoa, 127

Tissues, transplantation of, 30 ff.,
44 ff.; cultivation of, 31 ff.; spe-
Cificity of, 44 ff.

Torrey, H. B., 264, 269

Tower, 348

Transfusion of blood, 53

Transplantation, of tissues, 30 ff.,
44 ff.; of cancers, 45; of anlagen,
x of eye of salamander, 157;
of testes, 226; of ovaries, 227

Traube, 28

Treub, 21

Trial and error, 268, 270

Trifolium arvense, 37

Tropisms, 11 ff., 92 ff., 147, 178,
187, 253 ff.; and instincts, 253;
theory of, 257 ff.

Tropisms, in embryonic develop-
ment, 147; of cave animals, 324

Index

Trypanosomes, 332 ff.

Trypsin, synthetic action of, 27

Tuber brumale, 60

Tubularia crocea, 171

Tubularia mesembryanthemum,167,
169, 192

Twins, origin of, 136 ff.; sex of,
211

Tyndall, 33

Typhlogobius, 320

Typhlomolge, 320

Typhlotriton, 320, 323

Tyrosinase, 249, 250

Tyrosine, 249, 250

v. Uexktll, J., 4 ff., 128, 139
Uhlenhuth, E., 157, 183, 187
Uhlenhuth, P., Py > 66, 322
Underhill, F.

Vanessa, prorsa, 303; Jevana,
303,
Vaney, 217

Van Slyke, D. D., 22, 24, 291
van’t Hoff, 24 ff., 290, 292, 296
Variation, 6, 297 ff., 346-348

379

Vitzou, 159
Volvox, 280

Walcott, 42, 61

Warburg, O., 117 ff.

Warming, 4r

Wasps, polymorphism in, 222-
224; sex determination, 255 ff.

Wassermann, 55

Wasteneys, H., 29, 82, 87, 112,
113, ne 191, 277, 293, 295,
335» 364

Weiggert, 188

Weismann, 7, 30, 393

Wells, H. G., 62, 69

Welsh, D. A, 60

Werner, F., 340

Wheeler, W.M. +43

White, Te 36

Whitney, D. D., 224

Wilson, E. B. , 68, 143, 199 ff.

Winkler, 47

Winogradsky, S., 16 ff., 42

Wolf, G., 182, 187

Yeast cells, cultivation of, 15 ff.
Young, 16, 358

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