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Title: The Organism as a Whole
       From a Physicochemical Viewpoint


Author: Jacques Loeb



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THE ORGANISM AS A WHOLE

From a Physicochemical Viewpoint

by

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

Copyright, 1916
by
Jacques Loeb

The Knickerbocker Press, New York




                                   To

                             THE MEMORY OF

                             DENIS DIDEROT

          Of the _Encyclopedie_ and the _Systeme 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."

                                                     JOHN MORLEY.

    (Article Diderot, _Encyclopaedia Britannica_.)




PREFACE


It is generally admitted that the individual physiological 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 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 spermatozoon, but apparently we cannot make a spermatozoon
develop into an organism without the cytoplasm of an egg, although
sperm and egg nucleus transmit equally the Mendelian characters. The
conception 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 hereditary
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 conception 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. Schaefer's masterly presidential address on "Life" or the
addresses of Correns and Goldschmidt on the determination of sex.
Credit should also have been given to Professor Raymond Pearl for the
discrimination between 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 Institut of Berlin, Dr. P. A. Levene of the
Rockefeller Institute, Professor T. H. Morgan of Columbia University,
and Professor Hardolph Wasteneys of the University 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 mechanistic 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.

                                                            J. L.

   The Rockefeller Institute
     for Medical Research,
        _August, 1916_




CONTENTS


                                                                  PAGE

  CHAPTER I

    Introductory Remarks                                             1


  CHAPTER II

    The Specific Difference between Living and
    Dead Matter and the Question of the
    Origin of Life                                                  14


  CHAPTER III

    The Chemical Basis of Genus and Species:                        40

       I.--The Incompatibility of Species not Closely Related       44

      II.--The Chemical Basis of Genus and Species and of
           Species Specificity                                      53


  CHAPTER IV

    Specificity in Fertilization                                    71


  CHAPTER V

    Artificial Parthenogenesis                                      95


  CHAPTER VI

    Determinism in the Formation of an Organism from an Egg        128


  CHAPTER VII

    Regeneration                                                   153


  CHAPTER VIII

    Determination of Sex, Secondary Sexual Characters,
    and Sexual Instincts:

       I.--The Cytological Basis of Sex Determination              198

      II.--The Physiological Basis of Sex Determination            214


  CHAPTER IX

    Mendelian Heredity and its Mechanism                           229


  CHAPTER X

    Animal Instincts and Tropisms                                  253


  CHAPTER XI

    The Influence of Environment                                   286


  CHAPTER XII

    Adaptation to Environment                                      318


  CHAPTER XIII

    Evolution                                                      346


  CHAPTER XIV

    Death and Dissolution of the Organism                          349

  Index                                                            371




The Organism as a Whole

CHAPTER I

INTRODUCTORY REMARKS


1. 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 even a scientific vitalist, would think of treating the process
of digestion, metabolism, production of heat, and electricity or even
secretion or muscular contraction in any other than a purely chemical
or physicochemical 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 concerned, we find
a totally different situation. The same physiologists who in the
explanation of the individual processes would follow the strictly
physicochemical viewpoint and method would consider the reactions of
the organism as a whole as the expression of non-physical agencies.
Thus Claude Bernard,[1] who in the investigation of the individual
life processes was a strict mechanist, declares that the making of a
harmonious 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 phenomena
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
respiration and circulation or the activity of glands, and so on. 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.

[1] Bernard C., _Lecons sur les Phenomenes de la Vie_. Paris, 1885, i.,
22-64.

    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 physicochemical
    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 advance) and arouses the admirable
    subordination and harmonious 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 inaccessible to us, and if we believe
    anything else we commit an error and become the dupes of metaphors
    and take figurative 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 physicochemical basis--but the harmonious
character of the 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 development
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[2] and v. Uexkuell,[3] both brilliant biologists,
occupy today a standpoint not very different from that of Claude
Bernard. Driesch assumes that there is an Aristotelian "entelechy"
acting as directing guide in each organism; and v. Uexkuell suggests
a kind of Platonic "idea" as a peculiar characteristic of life which
accounts for the purposeful character of the organism.

v. Uexkuell supposes as did Claude Bernard and as does Driesch
that in an organism or an egg the ultimate 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 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."[4] v. Uexkuell's ideas concerning the nature of a
Mendelian factor and of the "supergenes" 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 homogeneous, 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. Uexkuell and Driesch that the egg has no structure, the
development of structure becomes a difficult problem--but this is not
the real situation.

[2] Driesch, H., _The Science and Philosophy of the Organism_. 2 vols.
The Gifford Lectures, 1907 and 1908.

[3] v. Uexkuell, J., _Bausteine zu einer biologischen Weltanschauung_.
Muenchen, 1913.

[4] v. Uexkuell, J., _Bausteine zu einer biologischen Weltanschauung_.
Muenchen, 1913, p. 216.

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. Uexkuell consider the Darwinian
theory a failure. We may admit that the theory of a formation 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,[5] Darwin's theory still serves the purpose of
explaining how without any pre-established 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.

[5] de Vries, H., _Die Mutationstheorie_. Leipzig, 1901.

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 organisms
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, _i. 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 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 99-99/100 per cent. of possible teleosts
a process 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 evolution or if there had never been any evolution.

3. v. Uexkuell 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 understand how they can force each other into a
harmonious whole[6]; even if we make ample allowance for the law of
chance and the corresponding wastefulness in the world of the living.
But it is doubtful whether this idea of the role 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) characters 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 experiments 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.

[6] 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
Weismann 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 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 acceptance of the chromosome theory of
heredity is compatible with a consistent physicochemical conception of
the organism as a whole.

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 nowhere shown more
strikingly than in the field of regeneration. 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 substances in the circulation can collect
there. The mysterious influence of the whole on these parts consists
often merely of the fact that the circulating specific 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 parts 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 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 duration 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
role ascribed to the adaptations of organisms. 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 throws a different light upon the
seemingly purposeful character of animal reactions. Heliotropism
depends primarily upon the presence of photosensitive substances
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 environment 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.
Cuenot speaks of "preadaptation" 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 organizing 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.

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. However,
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 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 division
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 products. 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, 10 gm., ammonium tartrate, 0.2 gm. to 0.5 gm.,
and fused ash from yeast, 0.1 gm.[7] 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.

[7] Pasteur, L., _Annal. d. Chim. et d. Physique_, 1862, 3 ser., lxiv.,
1.

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 role in the alcoholic fermentation of sugar.

Chlorophyll, under the influence of the red rays of light, manufactures
the sugars from the CO{2} 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
prerequisite for the synthesis of proteins in living organisms?

The investigations of Winogradsky on nitrifying,[8] sulphur and perhaps
also on iron bacteria have to all appearances pointed a way out of this
difficulty. It seemed probable that there were specific micro-organisms
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
ammonia to nitrites were grown on the following medium; 1 gm. ammonium
sulphate, 1 gm. potassium phosphate, 1 gm. magnesium carbonate, to
1 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 compounds
takes place. "Since this bound carbon in the cultures can have no
other source than the CO{2} and since the process itself can have no
other cause than the activity of the nitrifying organism, no other
alternative was left but to ascribe to it the power of assimilating
CO{2}."[9] "Since the oxidation of NH{3} is the only source of chemical
energy which the nitrifying organism can use it was clear _a priori_
that the yield in assimilation must correspond to the quantity of
oxidized nitrogen. It turned out that an approximately constant ratio
exists between the values of assimilated carbon and those of oxidized
nitrogen." This is illustrated by the results of various experiments as
shown in Table I.

[8] Winogradsky, S., "Die Nitrification," _Handb. d. tech. Mykol._,
1904-06, iii., 132.

[9] Winogradsky, _loc. cit._, p. 163 and ff.

TABLE I

    --------------+---------+---------+---------+---------
                  | _No. 5_ | _No. 6_ | _No. 7_ | _No. 8_
                  +---------+---------+---------+---------
                  |  _mg._  |  _mg._  |  _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.[10]

[10] Godlewski, E., _Anz. d. Akad. d. Wissensch. in Krakau_, 1892, 408;
1895, 178.

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 the hydrogen
sulphide which is formed as a reduction product from CaSO{4} 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{2}S + O{2} = 2H{2}O + S{2}

    (2) S{2} + 3O{2} + 2H{2}O = 2H{2}SO{4}

The sulphuric acid is at once neutralized by carbonates.

Winogradsky assumes that the oxidation of H{2}S by the sulphur bacteria
is the source of energy which plays the same role as the oxidation
of NH{3} 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
accomplished with a purely mineral culture medium, like that of the
nitrite bacteria. On the basis of this assumption they should also be
able to form sugars from the CO{2} of the air.

Nathanson[11] discovered in the sea water the existence of bacteria
which oxidize thiosulphate to sulphuric acid. They will develop if some
Na{2}S{2}O{3}, is added to sea water. These bacteria can only develop
if CO{2} from the air is admitted or when carbonates are present. For
these organisms the CO{2} cannot be replaced by glucose, urea, or
other organic substances. Such bacteria must therefore possess the
power of producing sugar and starch from CO{2} without the aid of
chlorophyll. Similar observations were made by Beijerinck on a species
of fresh-water bacteria.[12]

[11] Nathanson, _Mitteil. d. zool. Station_, Neapel, 1902.

[12] Beijerinck, M., _Folia Microbiologica_, 1914, iii., 91.

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{4},
CO{2}, and O{2} besides NH{3}, or SH{2}, existed. This would lead us
to consider it possible that the first organisms 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 development.

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."[13] Of course all life on the islands
was extinct. When Treub in 1886 first visited the island, he found
that blue-green algae 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 algae referred to were according to Euler of the nostoc
type. Nostoc does not require sugar, since it can produce that compound
from the CO{2} 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 _Nostocaceae_ generally
appear as the first settlers on sand the conclusion has been drawn that
they or the group of _Schizophyceae_ to which they belong formed the
first settlers of our planet.[14] This conclusion is not quite safe
since in the settlement of Krakatau as well as in the first colonizing
of sand areas the nature of the first settler is determined chiefly by
the carrying power of wind (or waves and birds).

[13] Ernst, A., _The New Flora of the Volcanic Island of Krakatau_,
Cambridge, 1908.

[14] Euler, H., _Pflanzenchemie_, 1909, ii. and iii., 140.

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 compounds of plants or animals, these
substances undergo a digestion, _i. e._, 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 proteins
required for the formation of the material of a growing animal.[15]

[15] This fact was thoroughly established by Mendel and Osborne. A
summary of their work is given in Underhill, F. P., _Physiology of the
Amino Acids_, 1916.

3. The essential difference between living and non-living matter
consists then in this: the living cell synthetizes 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 complicated 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 isolating the enzyme of
the yeast cell which causes the alcoholic fermentation of sugar; and
this gave rise to the 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 hydrolyzing 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 substrate is disrupted
in the "strain" of the new combination 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 enzyme 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.[16]

[16] Van Slyke, D. D., and Cullen, G. E., _Jour. Biol. Chem._, 1914,
xix., 141.

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 expressed the idea that the hydrolytic enzymes
should 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 products 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 possibility that in the transitory combination between
enzyme molecule and substrate a change in the molecular configuration
of the substrate or in the distribution of intramolecular strain may
take place. The first apparently complete confirmation of van't Hoff's
suggestion appeared in the form of the synthesis of maltose from grape
sugar by the enzyme maltase, which decomposes maltose into grape sugar.
By adding the enzyme maltase from yeast to a forty per cent. solution
of glucose Croft Hill[17] obtained a good yield of maltose. It turned
out, however, that what he took for maltose was not this compound
but an isomer, namely isomaltose, which has a different molecular
configuration and cannot be hydrolyzed by the enzyme maltase.

[17] Hill, C., _Jour. Chem. Soc._, 1898, lxxiii., 634.

Lactose is hydrolyzed from kephyr by an enzyme lactase into galactose
and glucose; by adding this enzyme to galactose and glucose a synthesis
was 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 assuming "that the enzyme has a specific
influence in promoting the formation of the biose which it cannot
hydrolyze."[18] 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.

[18] Armstrong, E. F., _Proc. Royal Soc._, 1905, B. lxxvi., 592.

The principle enunciated by Armstrong, that in the synthetic action of
hydrolytic enzymes not the original 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 phenomena. 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 contradicted 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 was added to the products
of the hydrolysis of ethylbutyrate, ethyl alcohol, and butyric acid
by the same enzyme.[19] Taylor[20] 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.

[19] Kastle, J. H., and Loevenhart, A. S., _Am. Chem. Jour._, 1900,
xxiv., 491.

[20] Taylor, A. E., _Univ. Cal. Pub._, 1904, _Pathology_, i., 33;
_Jour. Biol. Chem._, 1906, ii., 87.

This suggests possibly a specific action of the enzyme. If this slight
reversible action had any biological significance (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.[21]

[21] Bradley, H. C., _Jour. Biol. Chem._, 1913, xiii., 407.

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 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 confronted 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 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, _i. e._, the formation 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 medium 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 harmful
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,[22] as otherwise the synthesis of specific
proteins would have to come to a standstill.

[22] This would lead to the idea that the enzymes in the cell also
synthetize molecules of their own kind, or that, in other words, the
synthetic processes in the cell are of the nature of autocatalysis.
Loeb, _Der chemische Character des Befruchtungsvorgangs_, Leipzig,
1908. Robertson, 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. Entwicklngsmech._, 1913,
xxxvii., 485. Ostwald, Wo., _Ueber die zeitlichen Eigenschaften der
Entwicklungsvorgaenge_, Leipzig, 1908.

This problem of synthesis leads to the assumption of immortality of the
living cell, since there is no _a priori_ reason why this synthesis
should ever come to a standstill of its own accord as long as enough
food is available 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 necessity of death for the body cells of the latter. Leo
Loeb was led by his investigations on the transplantation 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 apparently
indefinitely so that it was obvious that the transplanted 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 as we speak of
the immortality of the germ cells of such animals.[23]

[23] Loeb, Leo, _Jour. Med. Res._, 1901, vi., 28; _Arch. f.
Entwicklngsmech._, 1907, xxiv., 655.

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,[24] 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 ganglionic 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.[25] 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; and every two days a few cells from the
margin of the piece have to be transferred to a new culture medium.

[24] Loeb, Leo, _Ueber die Entstehung von Bindegewebe, Leucocyten und
rothen Blutkoerperchen aus Epithel und ueber eine Methode isolierte
Gewebsteile zu zuechten_. Chicago, 1897.

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

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 comparatively 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 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 physicochemical 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,
Pasteur, Tyndall, and all those who have worked with pure cultures of
micro-organisms, have proved that no spontaneous generation of living
from non-living matter can be demonstrated; and the statements to the
contrary 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_,[26] Arrhenius discusses the possibility 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 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 organisms
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 incandescent all over its surface and
any seeds on it would therefore be deprived of their germinating power."

[26] Arrhenius, S., _Worlds in the Making_, London and New York, 1908,
p. 212.

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 diameter 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 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 transference 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 deg. 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 warrant 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 grain found in
the Egyptian tombs was still able to germinate has long been recognized
as a myth. Miss White[27] 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 seventeen years. In a drier climate
they last longer than in a moist climate. It is of importance that
the hydrolyzing 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
treatment. Macfadyen[28] 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 deg. has no appreciable effect on the
    vitality of micro-organisms. To judge by the results there appeared
    no reason to doubt that the experiment might have been successfully
    prolonged for a still longer period.

[27] White, J., _Proc. Roy. Soc._, 1909, B, lxxxi., 417.

[28] Macfadyen, A., _Proc. Roy. Soc._, 1903, lxxi., 76.

Paul Becquerel[29] found that seeds which possess a very thick
integument may live longer than the grain in Miss White's experiments.
The thickness of the integument prevents the exchange of gases between
air and seed. Thus seeds of leguminoses (_Cassia bicapsularis_,
_Cytisus biflorus_, _Leucaena leucocephala_, and _Trifolium 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.

[29] Becquerel, P., _Revue generale des Sciences_, 1914, xxv., 559.

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 germination 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 far from a sun. 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 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 potential 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 capable of forming molecules of their own kind from a simple
nutritive solution. With such synthetic enzymes 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 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 combination 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{2}, and N--but also
on the physical side if the necessity 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 organism 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 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 endowed, as Warming put it, with an "unlimited plasticity,"
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 covering putrefying animal matter
in a vessel and forming a continuous membrane along its wall. These
red bacteria were of very different shape, size, and grouping, 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 character 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-purpurin_" seemed justly
to indicate the existence of 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, in spite of their common colour and
their association. Later observations showed that Cohn was right.
Winogradsky[30] 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.

[30] Winogradsky, S., _Beitraege zur Morphologie und Physiologie der
Bacterien_. Leipzig, 1888.

The method of pure line breeding inaugurated by Johannsen[31] 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.

[31] Johannsen, W., _Elemente der exacten Erblichkeitslehre_. 2d ed.,
1913.

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 mutation the old species may persevere. Walcott has found
fossils of annelids, snails, crustaceans, and algae in a precambrian
formation in British Columbia whose age (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, _i. e._, the permanence of specificity
may therefore be considered as established as far back as two or
possibly two hundred millions of years. The definiteness and constancy
of each species must be determined 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 separated 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.


_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 another, 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.[32] 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, the
so-called lymphocytes, which develop in the lymphatic 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 this idea. 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
immunity to a transplantation of cancer produced by Jensen, Leo Loeb,
and Ehrlich and Apolant through the previous transplantation of tissue
in such an animal was due to the fact that this previous tissue
transplantation 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.

[32] Murphy, J. B., _Jour. Exper. Med._, 1913, xvii., 482; 1914, xix.,
181; xix., 513; Murphy and Morton, J. J., _Jour. Exper. Med._, 1915,
xxii., 204.

A lesser though still marked degree of incompatibility exists also in
lower animals for grafts from a different species.[33] 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 permanent union
between the anterior and posterior ends of two species of earthworms,
_Lumbricus rubellus_ and _Allolobophora terrestris_. Born and later
Harrison 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 characteristic 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 specificity was not yet fully recognized. The role of
lymphocytes in these cases has never been investigated. The grafted
piece always retained the characteristics of the species from which it
was taken.

[33] The reader is referred to Morgan's book on _Regeneration_ (New
York, 1901), for the literature on this subject.

Plants possess no leucocytes and we therefore see that they tolerate
a graft of foreign tissues better than is the case in animals. As
a 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 furnish
only nutritive sap to the graft and in this respect does not behave
very differently from an artificial nutritive 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 occurred 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 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.[34] 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.

[34] Baur, E., _Einfuehrung in die experimentelle Vererbungslehre_.
Berlin, 1911, p. 232.

3. Another manifestation of the incompatibility of distant species
is found in the domain of fertilization. The eggs of the majority of
animals cannot develop unless a spermatozoon enters. The entrance
of a spermatozoon 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[35]
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 Californian 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.

[35] Literature on this subject in Chapter IV.

[Illustration: FIG. 1. Five-days-old larvae from a sea urchin
(Strongylocentrotus purpuratus) <F> and a starfish (Asterias) <M>.
(Front view.)]

[Illustration: FIG. 2. Five-days-old larvae of Strongylocentrotus
purpuratus produced by artificial parthenogenesis. (Side view.) The
larvae in Figs. 1 and 2 are identical in appearance, proving that
heterogeneous hybridization leads to a larva with purely maternal
characters.]

If such heterogeneous hybridizations are carried out, two striking
results are obtained. The one is that the resulting larva has only
maternal characteristics (Figs. 1 and 2), as if the sperm had
contributed no hereditary material to the developing embryo. This
result could not have been predicted, for if we fertilize the egg of
the same Californian sea urchin, _Strongylocentrotus purpuratus_, with
the sperm of a very closely related sea urchin, _S. franciscanus_,
the hereditary effect of the spermatozoon is seen very distinctly in
the primitive skeleton formed by the larva.[36] (Fig. 3.) In the case
of the heterogeneous hybridization the spermatozoon acts practically
only as an activating agency upon the egg and not as a transmitter of
paternal qualities.

[36] Loeb, J., King, W. O. R., and Moore, A. R., _Arch. f.
Entwicklngsmech._, 1910, xxix., 354.

[Illustration: FIG. 3. Five-days-old larvae of two closely related forms
of sea urchins (S. purpuratus <F> and S. franciscanus <M>). In this case
the larva has also paternal characters as shown by the skeleton.]

The second striking fact is that while the sea-urchin 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
heterogeneous hybrids begins or assumes a severe character 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 larvae.

He investigated the effects of heterogeneous hybridization also in
fishes, which are a much more favourable object. The egg of the marine
fish _Fundulus heteroclitus_ can be fertilized with the sperm of almost
any other teleost fish, as Moenkhaus[37] 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,[38] and found the same two striking facts which he had already
observed in the heterogeneous cross between sea urchin and starfish:
first, practically no transmission of paternal characters, and second,
a sickly condition of the embryo which begins early and which increases
with further development. The heterogeneous fish hybrids between,
_e. g._, _Fundulus heteroclitus_ <F> and _Menidia_ <M> 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. In exceptional cases circulation may be
established and in these a normal embryo may result, but such an embryo
is chiefly maternal.

[37] Moenkhaus, W. J., _Am. Jour. Anat._, 1904, iii., 29.

[38] Loeb, J., _Jour. Morphol._, 1912, xxiii., 1.

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_
<F> and _S. franciscanus_ <M> is very sturdy and shows no abnormal
mortality as far as the writer's observations go. If, however, the
reciprocal crossing is carried out, namely that of _S. franciscanus_
<F> and _S. purpuratus_ <M>, the development is at first normal, but
beginning with the time of mesenchyme formation the majority of larvae
become sickly and die; and again the question may be raised whether
or not the beginning 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 protoplasm of the egg, since otherwise we could not understand 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 incompatibility is not yet understood. It is quite
possible that this mechanism is not the same in all the cases mentioned
here, and that it may be different when two 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[39] showed by experiment that if blood of a foreign species
was introduced into an animal the blood corpuscles of the transfused
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.[40] This was the beginning
of the investigations in the field of serum specificity which were
destined to play such a prominent role in the development of medicine.
Friedenthal was able to show later that if to 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 deg.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
incompatible, and this discovery was justly considered by Friedenthal
as a confirmation of the idea of the evolutionists that the anthropoid
apes and the human are blood relations.[41]

[39] Landois, L., _Zur Lehre von der Bluttransfusion_. Leipzig, 1875.

[40] This is probably true only within the limits of exactness used in
these experiments.

[41] Friedenthal, H., "Experimenteller Nachweis der
Blutverwandtschaft." _Arch. f. Physiol._, 1900, 494.

This line of investigation had in the meanwhile entered upon a new
stage when Kraus, Tchistowitch, and Bordet discovered and developed
the precipitin reaction, 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, _i. 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 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 (_e. 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 rabbits 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 reaction 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 Schuetze 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[42] found, by trying the white
of egg of different species of birds, that the precipitin reaction
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.

[42] Uhlenhuth, P., and Steffenhagen, K., Kolle-Wassermann, _Handb. d.
pathol. Mikroorg._, 2nd Ed., 1913, iii., 257.

To Nuttall[43] 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 precipitate 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 quantitative specificity of blood
serum. This statement may 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.

[43] Nuttall, George H. F., _Blood Immunity and Blood Relationship_,
Cambridge Univ. Press, 1904.

TABLE II

QUANTITATIVE TESTS WITH ANTI-PRIMATE SERA

_Tests with Antihuman Serum_

  ---------------------------+--------------+-------------------------
        Blood of             | _Precipitum_ |      _Percentage_
                             |   _Amount_   |
  ---------------------------+--------------+-------------------------
  _Primates_                 |              |
    Man                      |     .031     | 100
    Chimpanzee               |     .04      | 130 (loose precipitum)
    Gorilla                  |     .021     |  64
    Ourang                   |     .013     |  42
    Cynocephalus mormon      |     .013     |  42
    Cynocephalus sphinx      |     .009     |  29
    Ateles geoffroyi         |     .009     |  29
                             |              |
  _Insectivora_              |              |
    Centetes ecaudatus       |     .0       |   0
                             |              |
  _Carnivora_                |              |
    Canis aureus             |     .003     |  10 (loose precipitum)
    Canis familiaris         |     .001     |   3
    Lutra vulgaris           |     .003     |  10 (concentrated serum)
    Ursus tibetanus          |     .0025    |   8
    Genetta tigrina          |     .001     |   3
    Felis domesticus         |     .001     |   3
    Felis caracal            |     .0008    |   3
    Felis tigris             |     .0005    |   2
                             |              |
  _Ungulata_                 |              |
    Ox                       |     .003     |  10
    Sheep                    |     .003     |  10
    Cobus unctuosus          |     .002     |   7
    Cervus porcinus          |     .002     |   7
    Rangifer tarandus        |     .002     |   7
    Capra megaceros          |     .0005    |   2
    Equus caballus           |     .0005    |   2
    Sus scrofa               |     .0       |   0
                             |              |
  _Rodentia_                 |              |
    Dasyprocta cristata      |     .002     |   7 (concentrated
                             |              |        serum clots)
    Guinea-pig               |     .0       |   0
    Rabbit                   |     .0       |   0
                             |              |
  _Marsupialia_              |              |
    Petrogale xanthopus    } |              |
    Petrogale penicillata  } |              |
    Onychogale frenata     } |              |
    Onychogale unguifera   } |     .0       |   0
    Onychogale unguifera   } |              |
    Macropus bennetti      } |              |
    Thylacinus cynocephalus} |              |
  ---------------------------+--------------+-------------------------

    Among the Primate bloods that of the Chimpanzee gave too high a
    figure, owing to the precipitum being flocculent 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
    Cynocephalus 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 Carnivore bloods can be explained by the fact that one
    gave a loose precipitum, and the other was a somewhat concentrated
    serum.[44]

[44] Nuttall, _Blood Immunity and Blood Relationship_, pp. 319 and 320.

We have mentioned that even the proteins of the egg are specific
according to Uhlenhuth. Graham Smith, one of Nuttall's collaborators,
applied the latter's quantitative method to this problem and confirmed
the results of Nuttall. A few examples may serve as an illustration.

TABLE III

TESTS WITH ANTI-DUCK'S-EGG SERUM

    --------------------------------+--------------+--------------
            _Material tested_       |  _Amount of  | _Percentage_
                                    |  precipitum_ |
    --------------------------------+--------------+--------------
    Duck's          egg-albumin     |    .0384     |     100
    Pheasant's           "          |    .0328     |      85
    Fowl's               "          |    .0234     |      61
    Silver Pheasant's    "          |    .0140     |      36
    Blackbird's          "          |    .0065     |      15
    Crane's              "          |    .0051     |      14
    Moorhen's            "          |    .0046     |      12
    Thrush's             "          |    .0046     |      12
    Emu's                "          |    .0018     |       5
    Hedge-Sparrow's      "          |    trace     |       ?
    Chaffinch's          "          |      .       |       0
    Tortoise serum                  |    trace     |       ?
    Turtle serum                    |      "       |       ?
    Alligator serum                 |      .       |       0
    --------------------------------+--------------+--------------

    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-EGG SERUM

    --------------------------------+--------------+--------------
            _Material tested_       |  _Amount of  | _Percentage_
                                    |  precipitum_ |
    --------------------------------+--------------+--------------
    Fowl's       egg-albumin (old)  |    .0159     |     100
    Fowl's             "    (fresh) |    .0140     |      88
    Silver Pheasant's  "            |    .0075     |      47
    Pheasant's         "            |    .0075     |      47
    Crane's            "            |    .0046     |      29
    Blackbird's        "            |    .0046     |      29
    Duck's             "            |    .0037     |      23
    Moorhen's          "            |    .0028     |      18
    --------------------------------+--------------+--------------

    Thrush, Emu, Greenfinch, and Hedge-sparrow egg-albumins were tested
    and gave traces of precipita, as also did Tortoise and Turtle sera.
    The egg-albumins of the Tortoise, Frog, Skate, and two species of
    Dogfish did not react. Alligator, Frog, Amphiuma, and Dogfish sera
    also yielded no results.[45]

[45] Nuttall, pp. 345 and 346.

By improving the quantitative method in various ways, Welsh and
Chapman[46] 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 (precipitin) together with the specific
antisubstance."

[46] Welsh, D. A., and Chapman, H. G., _Jour. Hygiene_, 1910, x., 177.

The Bordet reaction was not only useful in indicating the specificity
and blood relationship for animals but also among plants. Thus Magnus
and Friedenthal[47] were able to demonstrate with Bordet's method the
relationship between yeast (_Saccharomyces cerevisiae_) and truffle
(_Tuber brumale_).

[47] Magnus, W., and Friedenthal, H., _Ber. d. deutsch. bot.
Gesellsch._, 1906, xxiv., 601.

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 specificity of living organisms. It is only logical to
conclude that the fossil forms of invertebrate animals and of algae
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,[48] 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 anaphylaxis, was able
to produce this phenomenon, and later he showed that it was generally
impossible to produce anything resembling anaphylaxis by non-protein
substances, _e. g._, cocain or apomorphin.[49] Wells isolated from
egg-white four different proteins (three coagulable proteins and one
non-coagulable) which can be distinguished from each other by the
anaphylaxis reaction, although all come from the same biological
object.[50] 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.

[48] Richet, C., _L'anaphylaxie_. Paris, 1912.

[49] Quoted from Wells, H. G., _Jour. Infect. Diseases_, 1908, v., 449.

[50] _Ibid._, 1911, ix., 147.

A pretty experiment by Gay and Robertson[51] 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 hydrolysis of an
    approximately four per cent. neutral solution of potassium
    caseinate by the action of pure pepsin at 36 deg.C.

[51] Gay, F. P., and Robertson, T. B., _Jour. Biol. Chem._, 1912, xii.,
233.

He considered this a case of a real synthesis of proteins from
the products of its hydrolytic cleavage. This 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 products of complete peptic
digestion of casein had no such effect, but the synthetic product of
this digestion obtained by Robertson's method has the same specific
antigenic properties as paranuclein, thus making it appear that
Robertson had indeed succeeded 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 confirmed, and Fitzgerald
and Leathes[52] reached the conclusion 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 are therefore the bearers of the specificity of
living organisms.

[52] Fitzgerald, J. G., and Leathes, J. B., _Univ. Cal. Pub._, 1912,
"Pathology," ii., 39.

Bradley and Sansum[53] found that guinea-pigs sensitized to beef or dog
hemoglobin fail to react or react but slightly to hemoglobin of other
origin. The hemoglobins tried were dog, beef, cat, rabbit, rat, turtle,
pig, horse, calf, goat, sheep, pigeon, chicken, and man.

[53] Bradley, H. C., and Sansum, W. D., _Jour. Biol. Chem._, 1914,
xviii., 497.

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 hemoglobins of the blood by Reichert and Brown,[54] 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).

[54] Reichert, E. T., and Brown, A. P., "The Differentiation and
Specificity of Corresponding Proteins and other Vital Substances in
Relation to Biological Classification and Organic Evolution." Carnegie
Institution Publication No. 116, Washington, 1909.

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 hemoglobin 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 isomorphous 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 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
Mendelian mutation except the morphological differences. It is not
impossible that while species differ by the constitution 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 discriminated from each other by the precipitin
reaction.[55]

[55] Uhlenhuth, _Das biologische Verfahren zur Erkennung und
Unterscheidung von Menschen und Tierblut_, Jena, 1905, p. 102.

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 "isoagglutinins" 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 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 systematic
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,[56] 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 1, 2.
             Corpuscles agglutinated by sera of Groups 2, 4.

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

[56] Moss, W. L., _Johns Hopkins Hospital Bulletin_, 1910, xxi., 62.

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 1.
    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 overwhelming numbers, which
indicates that, as a rule, the sera agglutinate the blood corpuscles
of individuals of the other groups, but not those of individuals
belonging to the same group. The phenomenon that a serum agglutinates
no corpuscles (Group 1), or that the corpuscles 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 constitution and hence may give rise to the specific
biological 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 protamine or histone, the latter a non-coagulable protein,
more resembling a split product of one of the larger coagulable
proteins.

A. E. Taylor[57] found that if the spermatozoa of the salmon are
injected into a rabbit, the blood of the animal acquires the power of
causing cytolysis of salmon spermatozoa. 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 testicles a protein resembling albumin was separated by
precipitation.[58]

    The albumin behaved like ordinary serum albumin or egg albumin,
    producing typical and fatal anaphylactic reactions and being
    specific when tried against mammalian sera. The nucleinate did not
    produce any reactions when guinea-pigs were given small sensitizing
    and larger intoxicating 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.

[57] Taylor, A. E., _Jour. Biol. Chem._, 1908, v., 311.

[58] Wells, H. G., _Jour. Infect. Diseases_, 1911, ix., 166.

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 constituents 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 spermatozoon,
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 spermatozoon to develop into an embryo,[59] while we can
induce the egg to develop into an embryo without a spermatozoon. 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.

[59] Loeb, J., and Bancroft, F. W., _Jour. Exper. Zool._, 1912, xii.,
381.




CHAPTER IV

SPECIFICITY IN FERTILIZATION


1. We have become acquainted with two characteristics 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, namely, 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 fertilization 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 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 majority 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 spermatozoon 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
spermatozoon--enters the female gamete--the egg--and this starts the
development. In the case of most animals the egg cannot develop unless
the spermatozoon enters. The question arises: How does the spermatozoon
activate the egg? And also how does it happen that the spermatozoon
enters the egg? We will first consider the latter question. These
problems can be answered best from experiments 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 connection is again a
phenomenon of specificity. The spermatozoon can, as a rule, only enter
an egg of the same or a closely related species, but not that of one
more distantly related. What is the character of this specificity?
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 fertilized with a distant species for which this egg is naturally
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 tetraethylammoniumhydroxide 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 fertilization takes
place as a rule, while with the same sperm and eggs the fertilization
is successful again if both are mixed in the hyperalkaline solution.
From 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 _Asterias_ sperm
into the egg of _purpuratus_; and that if C{Ca} was increased it was
not necessary to add as much NaOH.

The spermatozoon enters the egg through the so-called fertilization
cone, _i. e._, a protoplasmic process comparable to the pseudopodium of
an amoeboid cell. The analogy of the process of phagocytosis--_i. e._,
the taking up of particles by an amoeboid cell--and that of the
engulfing of the spermatozoon 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 spermatozoon
which in the case of _S. purpuratus_ <F> and _Asterias_ <M> can be
supplied by a slight increase in the C{OH} or C{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 sperm of _Asterina_ only one per cent. could be fertilized.[60]
Godlewski succeeded by the same method in fertilizing the eggs of a
Naples starfish with the sperm of a crinoid.[61] The writer did not
succeed in bringing about the fertilization 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_.

[60] Loeb, J., _Arch. f. d. ges. Physiol._, 1903, xcix., 323; 1904,
civ., 325; _Arch. f. Entwcklngsmech._, 1910, xxx., II., 44; 1914, xl.,
310; _Science_, 1914, xl., 316.

[61] Godlewski, E., _Arch. f. Entwcklngsmech._, 1906, xx., 579.

Kupelwieser[62] 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 spermatozoon can enter the eggs of widely different species but
with rare exceptions all the embryos will die in an early stage of
development.[63]

[62] Kupelwieser, H., _Arch. f. Entwcklngsmech._, 1909, xxvii., 434;
_Arch. f. Zellforsch._, 1912, viii., 352.

[63] See Chapter II.

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 might block the entrance of the sperm of sea urchin into
eggs of their own species. This was found to be correct; 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/2
NaCl 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 mixture of m/2 NaCl and 3/8 m MgCl{2} 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 spermatozoon enters the eggs
can be shown by the fact that the eggs do not divide (although 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{2}, containing in
addition one drop of a N/100 solution of NaOH (or NH{3} or benzylamine
or butylamine) or eight drops of m/100 NaHCO{3}, most, and often
practically all of the eggs at once form fertilization membranes
and segment at 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{2}+CaCl{2} (in the
proportion in which these salts exist in the sea water) or into a
neutral mixture of NaCl+MgCl{2}+KCl+CaCl{2}. In such neutral mixtures
the eggs form fertilization membranes and begin to segment. The eggs
are not fertilized in a neutral solution of NaCl or of NaCl+KCl.[64]

[64] Loeb, J., _Science_, 1914, xl., 316; _Am. Naturalist_, 1915,
xlix., 257.

It is, therefore, obvious that if we diminish the alkalinity of the
solution surrounding the egg and deprive this solution of CaCl{2} we
establish the same block to the entrance of the spermatozoon 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 specificity which allows
the sperm to enter an egg is a surface 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 jelly which surrounds the egg of
_purpuratus_.[65] It is thus not impossible that the specificity which
favours the entrance of a spermatozoon into an egg of its own species
may consist in an agglutination between spermatozoon and egg protoplasm
(or its fertilization cone); and that this agglutination is favoured if
the C{OH} or C{Ca} or both are increased within certain limits.

[65] Loeb, _Arch. f. Entwcklngsmech._, 1914, xl., 310.

Godlewski discovered a very interesting form of block to the entrance
of the spermatozoon into the egg which takes place if two different
types of sperm are mixed. He had found that the sperm of the annelid
_Chaetopterus_ 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 _Chaetopterus_ and the sperm of
sea urchins are mixed the mixture is not able to induce development or
membrane formation, since now neither spermatozoon can enter; blood
has the same inhibiting effect as the foreign sperm. The mixture does
not interfere with the development of the eggs if they are previously
fertilized.[66]

[66] Godlewski, E., _Arch. f. Entwcklngsmech._, 1911, xxxiii., 196.

The phenomenon was further investigated by Herlant[67] who found that
if the sperm of a sea urchin is mixed with the sperm of certain
annelids (_Chaetopterus_) 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 remained
unfertilized will now fertilize the egg. From these and similar
observations Herlant draws the conclusion 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 surface 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 agglutination after
some time if mixed with the sperm of a sea urchin or a starfish.[68]
We can imagine that the precipitate forms a film around the egg and
acts as a block for the agglutination between egg and spermatozoon. The
block can be removed mechanically by washing.

[67] Herlant, M., _Anat. Anzeiger_, 1912, xlii., 563.

[68] Loeb, J., _Jour. Exper. Zool._, 1914, xvii., 123.

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 C{OH} or C{Ca}) are not fulfilled. On the other hand, living but
immobile sperm cannot enter the egg under any conditions. If we add a
trace of KCN to the sperm of _Arbacia_ so that the spermatozoon 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 spermatozoon 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 spermatozoon to make the latter stick
to the surface of the egg and agglutinate and that later forces of a
different character bring the spermatozoon into the egg. The fact that
under normal conditions a very slight degree of motility on the part of
the spermatozoon 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. Lillie[69] has pointed out that
this may be a specific effect. The writer tested this idea on the sperm
and eggs of two species of starfish 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 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 only rendered motile by
the proximity of eggs. The following table gives the result.[70]

[69] Lillie, F. R., _Jour. Exper. Zool._, 1914, xvi., 523.

[70] Loeb, J., _Am. Naturalist_, 1915, xlix., 257.

TABLE V

SPECIFICITY OF ACTIVATION OF SPERM BY EGGS

 --------------+------------+-------------+---------------+-------------
               |_Asterias_  | _Asterina_  |_Franciscanus_ |_Purpuratus_
               |    <M>     |     <M>     |     <M>       |     <M>
 --------------+------------+-------------+---------------+-------------
 _Asterias_    |_Immediately|No           |Moderately     |Slight effect
<F> (immature) | very       | activation. | active.       | in immediate
               | motile._   |             |               | contact with
               |            |             |               | egg.
               |            |             |               |
 _Asterina_    | Not motile.|_Violent     |_Violent       |Slight effect
<F> (immature) |            | activity._  | activity._    | only  near
               |            |             |               | the egg.
               |            |             |               |
 _Franciscanus_|Slightly    |No motility. |_Immediately   |_Immediately
<F> (mature)   | motile.    |             | active._      | active._
               |            |             |               |
               |            |             |               |
 _Purpuratus_  |Slightly    |Slight effect|_Immediately   |_Immediately
<F> (mature)   | motile     | in immediate| active._      | active._
               | after some | contact     |               |
               | time.      | with eggs.  |               |
 --------------+------------+-------------+---------------+-------------

The spermatozoa of starfish show a marked specificity inasmuch as they
are strongly activated only by the eggs of their own species, although
in this experiment 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 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 therefore 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 activating effect of the eggs of various
marine teleosts upon sperm of the same species.

4. F. R. Lillie[71] has studied the very striking phenomenon 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 Californian 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 seconds and the result is a homogeneous sperm
suspension. 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 species, 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 1 or 2 mm. 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.

[71] Lillie, F. R., _Science_, 1913, xxxviii., 524; _Jour. Exper.
Zool._, 1914, xvi., 523; _Biol. Bull._, 1915, xxviii., 18.

This phenomenon seems to occur in sea urchins and annelids. The writer
has vainly looked for it in different 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 clusters 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 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 substance which forms a precipitate with a
substance on the surface of the spermatozoon 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 impact 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."[72]
But this assumption seems to go beyond the facts inasmuch as the
existence of such an agglutinating substance can only 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
agglutinate, _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 hundred per cent. of such
eggs can be fertilized by sperm.[73]

[72] Lillie, F. R., _loc. cit._

[73] Loeb, J., _Jour. Exper. Zool_., 1914, xvii., 123; _Am.
Naturalist_, 1915, xlix., 257.

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
_Strongylocentrotus 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 _purpuratus_ are put for two hours into hypertonic sea water
(50 c.c. of sea water+8 c.c. 2-1/2 m NaCl) and then transferred 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 fertilization membrane and now begin to
develop into larvae; and if the spermatozoon gets into a blastomere of
the 2- or 4-cell stage normal plutei will result. When the sperm is
added while the eggs are in active parthenogenetic cell division the
individual blastomeres into which a spermatozoon enters will also form
a fertilization 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 development whether the spermatozoon enters into
a blastomere 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
spermatozoon into an egg which is already fertilized.[74]

[74] Loeb, J., _Arch. f. Entwcklngsmech._, 1907, xxii., 479;
_Artificial Parthenogenesis and Fertilization_, Chicago, 1913, p. 240.

When the spermatozoon 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 spermatozoon. 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 acid.
This membrane is so tough in the egg of _Strongylocentrotus_ that
no spermatozoon can get through it; in the egg of _Arbacia_ the
membrane is occasionally replaced by a soft gelatinous film. If no
second treatment 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 characteristic of fertilized eggs.[75]
When the membrane is too tough to allow the spermatozoon to enter the
egg it can be shown that if the membrane is torn mechanically the egg
can still be fertilized by sperm.

[75] Loeb, J., _Science_, 1913, xxxviii., 749; _Arch. f.
Entwcklngsmech._, 1914, xxxviii., 277; Wasteneys, H., _Jour. Biol.
Chem._, 1916, xxiv., 281.

Should it be possible that the spermatozoon can no longer agglutinate
with the fertilized egg or that those phagocytotic reactions which we
suppose to play a role in the entrance of the spermatozoon into the egg
are no longer possible after a spermatozoon has entered? The mere fact
of development is apparently not the cause which bars a spermatozoon
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 membrane formation does not create such a
block although 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 spermatozoon--we should assent to such an
assumption--but that the "fertilizin" acts like an "amboceptor" between
egg and spermatozoon, the latter being the complement, the former the
antigen. The pathologist would probably object to this interpretation
since no "amboceptor" 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 spermatozoon into the egg.[76]

[76] 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. Arrhenius showed that the same phenomenon can be obtained when
a weak base like NH{4}OH is neutralized by a weak acid (_e. g._, boric
acid); hence we should assume that NH{4}OH consists of ten different
forms of ammonia. Both cases, the saturation of toxin with antitoxin
and ammonia with boric acid are equilibrium phenomena. (Arrhenius, S.,
_Quantitative Laws in Biological Chemistry_, London, 1915.)

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 role in the theories of evolution. We are here only
concerned with the mechanism which determines the block to the entrance
of a spermatozoon into an egg of the same hermaphroditic individual.

Castle[77] 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[78] found that the eggs
of various females differ in their power of being fertilized by sperm
of the same individual while one hundred per cent. could usually be
fertilized with sperm of a different individual. He 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[79] has reported results similar to those of
Castle and Morgan.

[77] Castle, W. E., _Bull. Mus. Comp. Zool._, Harvard, 1896, xxvii.,
203.

[78] Morgan, T. H., _Jour. Exper. Zool._, 1904, i., 135; _Arch. f.
Entwcklngsmech._, 1910, xxx., 206.

[79] Fuchs, H. M., _Jour. Genet._, 1915, iv., 215.

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 monoecious (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.[80]

[80] Quoted from Fuchs.

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, "inhibitory" substances,
and finds that they can apparently be transmitted to the offspring. He
made experiments on _Cardamine pratensis_ which is self-sterile.[81] He
fertilized two individuals of _Cardamine_ crosswise and raised sixty
plants of the first generation. He compared the fertility of these
F{1} plants toward (_a_) their parents, and (_b_) 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
    (_C_) sterile with both parents. Type BG

[81] Correns, C., _Biol. Centralbl._, 1913, xxxiii., 389.

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 children 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 spermatozoon is important. Should it be possible
that the block created by the entrance of a spermatozoon into the egg
is also due to an inhibitory substance carried by a spermatozoon into
the egg; and furthermore that the effect of the inhibitory substance
should be the prevention of further agglutination of the spermatozoon
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 hermaphrodite and a spermatozoon of the same
individual. The experiments on the agglutinins have shown that while
isoagglutinins (_i. e._, agglutinins for other individuals of the same
species) are common auto-agglutinins (_i. e._, agglutinins for cells of
the same individual) rarely if ever occur.

7. A positive chemotropism of the spermatozoa toward an egg of the same
species has been demonstrated in a few cases, but it seems that this
phenomenon 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.

    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 accumulate in the tube. The malic acid acts as well
    in the form of a free acid as in the form of salts.[82]

[82] Pfeffer, _Untersuchungen aus dem botanischen Institut zu
Tuebingen_, 1881-1885, i., 363.

These experiments were continued and amplified by Shibata.
Bruchmann[83] 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.

[83] Bruchmann, H., _Flora_, 1909, ic., 193.

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 simultaneously by the two
sexes. This can be easily observed 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 spermatozoon enters into the egg. The
question arises: How does the spermatozoon cause the egg to develop
into a new organism? The spermatozoon 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 spermatozoon. No
progress was possible in this field until ways were found to replace
the action of the living spermatozoon by well-known physicochemical
agencies.[84] 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 disintegrated in
their experiments without giving rise to larvae. In 1899 the writer
succeeded in causing the unfertilized eggs of the sea urchin _Arbacia_
to develop into swimming larvae, blastulae, gastrulae, 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 larvae, blastulae, gastrulae, and plutei.[85] 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 spermatozoon enters. These experiments furnished proof that the
activating effect of the spermatozoon upon the egg can be replaced by a
purely physicochemical agency.[86]

[84] The substitution of well-known physicochemical agencies
for the mysterious action of the spermatozoon 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.

[85] Loeb, J., _Am. Jour. Physiol._, 1899, iii., 135; 1900, iii., 434.

[86] Loeb, J., _Artificial Parthenogenesis and Fertilization_, Chicago,
1913. The reader is referred to this book for the literature on the
subject.

The first method used in the production of larvae from the unfertilized
eggs did not lend itself to an analysis of the activating effect of the
spermatozoon 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 spermatozoon causes
the egg to lose water. No further progress was possible until another
method of artificial parthenogenesis was found. When a spermatozoon
enters the egg of a sea urchin or starfish 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 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 generally
assumed, moreover, that the process of membrane formation was of no
significance in the phenomenon of fertilization, except perhaps that
the fertilization membrane 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.

[Illustration:

FIG. 4. Unfertilized egg surrounded by spermatozoa (whose flagellum is
omitted in the drawing).

FIG. 5. The same egg after a spermatozoon has entered. The
fertilization membrane is separated from the egg by a clear space.]

In 1905 the writer found that membrane formation, or rather the change
of the surface of the egg underlying the membrane formation, is the
essential feature in the activation of the egg by a spermatozoon. 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/10
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 deg. C. or less--develop into swimming blastulae[87]; 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 spermatozoon and nuclear division.

[87] The reader will find a description of the development of this egg
in the next chapter.

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 treatment with hypertonic
sea water or with lack of oxygen or with KCN they developed into normal
larvae. This new or improved method of artificial parthenogenesis 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/10 butyric acid (2.6 c.c.
in the case of S. _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. 2-1/2 m NaCl
or Ringer solution or cane sugar) in which they remain, at 15 deg. C.
from thirty-five to sixty minutes in the case of _purpuratus_, and from
17-1/2 minutes to 22-1/2 minutes at 23 deg. in the case of _Arbacia_ at
Woods Hole. If the eggs are then transferred 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 deg. C.) and that a slight overexposure to the
hypertonic sea water injures the eggs so that development is abnormal. By
this method it is possible to imitate the activating effect of the living
spermatozoon upon the egg in every detail and eggs treated in this way
will develop in large numbers into perfectly normal larvae. 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 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
larvae, 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 ascertain
what substances or agencies caused membrane formation in the egg,
since it now became clear that the spermatozoon 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 hypotonic
solutions; (5) rise in temperature, and (6) certain salts, _e. g._,
BaCl{2} and SrCl{2} in the case of the egg of _purpuratus_, and
according to R. Lillie, NaI or NaCNS in the egg of _Arbacia_. Whenever
we submit an unfertilized 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.[88]

[88] The reader is referred for details to the writer's book on the
subject.

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, _Dendrostoma_, was able to
cause membrane formation in the egg of the sea urchin. When added in a
dilution of 1 c.c. of serum to 500 or 1000 c.c. of sea water to eggs of
_purpuratus_ a certain number formed fertilization membranes. It was
found later that the serum and tissue 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, however,
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{2}
for from five to ten minutes (or possibly a little longer) before
exposing them to the foreign blood serum. BaCl{2} acts similarly. The
fact that SrCl{2} alone can cause membrane formation 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{2} 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 susceptibility for the sperm of
foreign species. Thus the eggs of _Strongylocentrotus purpuratus_
require a higher concentration of sperm extract than the eggs of _S.
franciscanus_. 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 spermatozoon produces this
effect; and as a rule no previous sensitizing action of SrCl{2} is
required. When we bring the unfertilized eggs of _S. franciscanus_ into
contact with the living sperm of starfish or shark or even of fowl,
the eggs form a fertilization membrane without previous sensitization.
A specific substance from the foreign spermatozoon causes membrane
formation before the spermatozoon has time to enter the egg. The effect
is the same as if artificial membrane formation had been called forth
with butyric acid, _i. 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 spermatozoon, only the living spermatozoon
of the same species can induce membrane formation. This might find
its explanation on the assumption that the active substance contained
in the foreign sperm or serum is water-soluble and a protein, while
the activating or membrane-forming substance in the spermatozoon is
insoluble in water but soluble in the egg (or in lipoids). If this
assumption is correct the two substances are essentially different.

Robertson[89] has succeeded in extracting a substance from the sperm
of the sea urchin which causes membrane formation of the sea-urchin
egg after the latter has been sensitized by a treatment with SrCl{2}.
It seems to the writer that if the substance extracted by Robertson
were the real fertilizing agent contained in the spermatozoon it should
fertilize the egg without a previous sensitization of the egg with
SrCl{2} being required.

[89] Robertson, T. B., _Arch. f. Entwcklngsmech._, 1912, xxxv., 64.

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 _in_ the acid sea water and soon afterward will
cytolyze 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, _e. g._, butyric, are added to sea water the mineral acid
acts as if it were not present, _i. e._, the eggs form membranes when
transferred back to sea water if the concentration 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 solubility is comparatively high,
can only bring about a cytolysis 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 assumption that the specific membrane-inducing
substance in the spermatozoon 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 development might be an alteration
of the surface of the egg, in all probability an alteration of the
superficial layer probably of the nature of a superficial cytolysis.
The question remains: What could be the physicochemical nature of
this cytolysis? The writer had suggested in former papers that in the
cytolysis underlying 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.[90] 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 mechanism 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.[91]

[90] Loeb, J., _Ueber den chemischen Charakter des
Befruchtungsvorgangs_, etc., Leipzig, 1908.

[91] v. Knaffl, E., _Arch. f. d. ges. Physiol._, 1908, cxxiii., 279.

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 observed that unfertilized starfish eggs may begin to develop
apparently without any outside "stimulus," and A. P. Mathews found
that slight mechanical agitation 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 urchin 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.[92]

[92] Loeb, J., _Artificial Parthenogenesis and Fertilization_, p. 255.

The durability of emulsions varies, and where an emulsion is very
durable shaking has no effect, while where it is at the critical point
of separating into two continuous 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 fertilization
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 separated 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 destruction 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 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.[93] 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.

[93] It has been stated by several writers that the eggs of the sea
urchin can no longer form the fertilization membrane when the jelly
surrounding the egg is dissolved. The writer has found that if the
jelly surrounding the eggs of _Strongylocentrotus purpuratus_ is
dissolved by acid the eggs still form a fertilization membrane upon the
entrance of a spermatozoon.

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 concerning 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. Thus in 1901 Loeb[94] and Neilson
showed that a short treatment with HCl and HNO{3} sufficed to cause
some eggs of _Asterias_ in Woods Hole to develop into larvae without
a second treatment being needed, and Delage[95] showed the same for
CO{2}; and in 1905 the writer found that the eggs of the Californian
starfish _Asterina_ can be induced to form a membrane by butyric acid
treatment and that ten per cent. of these eggs developed into normal
larvae. Quite recently R. S. Lillie observed that the eggs of _Asterias_
at Woods Hole can be caused to form membranes and develop into larvae by
a treatment with butyric acid and that the time of exposure required to
get a maximal number of larvae 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.[96]

[94] Loeb, J., _Artificial Parthenogenesis and Fertilization_, 1913, p.
250 and ff.

[95] Delage, Y., _Arch. d. Zool. exper. et gen._, 1902, x., 213; 1904,
ii., 27; 1905, iii., 104.

[96] Lillie, R. S., _Jour. Biol. Chem._, 1916, xxiv., 233.

All this leads us to the conclusion that the main effect of the
spermatozoon 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 alteration without endangering the rest of the egg may induce
its development. The spermatozoon, therefore, causes 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 disintegrate 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 deg. C. for _purpuratus_
and 17-1/2 to 22-1/2 minutes for _Arbacia_ at 23 deg. C.) in a solution
which is isosmotic with 50 c.c. sea water +8 c.c. 2-1/2 m NaCl,[97] they
will develop into larvae, many of which may be normal. Any hypertonic
solution 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 diminishes in certain
limits with the concentration of OH ions in the solution.

[97] 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.
Entwcklngsmech._, 1914, xxxviii., 402.

It is strange that in the eggs of _purpuratus_ the corrective 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 retarded 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 artificial membrane formation alone starts the
development 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 corrective 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 following artificial membrane formation is prevented for
a day at least. The same result can be obtained by adding ten drops
of 1/10 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 concentration
required do not suppress or even lower the oxidations in the egg to
any considerable extent,[98] but they prevent the processes of cell
division. Hence it seems that the egg disintegrates so rapidly after
artificial membrane formation because it is killed by those processes
leading to nuclear division or cell division which are induced by
the artificial membrane formation. 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 hypertonic solution supplies the corrective
factor required, so that the egg can then undergo cell division at room
temperature without disintegrating.

[98] Loeb, J., and Wasteneys, H., _Jour. Biol. Chem._, 1913, xiv., 517;
_Biochem. Ztschr._, 1913, lvi., 295.

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 observers 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 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{2} formed by the eggs--since we know that CO{2} 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
presumable explanation for this is that the lack of oxygen prevents
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 unfertilized egg can live for days in such solutions.[99] In
a series of papers, beginning in 1905, he showed that the fertilized
egg will live longer in hypertonic, hypotonic, 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.[100] It
is thus obvious that coincident with the changes underlying nuclear
division or cell division alterations occur in the sensitiveness of
the egg to salt solutions of abnormal concentration or constitution,
_e. g._, NaCl+CaCl{2} isotonic with sea water, hypertonic, or hypotonic
solutions.

[99] Loeb, J., _Biochem. Ztschr._, 1906, ii., 81.

[100] Loeb, J., _Arch. f. d. ges. Physiol._, 1906, cxiii., 487;
_Biochem. Ztschr._, 1910, xxvi., 279, 289; xxvii., 304; xxix., 80;
_Arch. f. Entwcklngsmech._, 1914, xl., 322.

We must, therefore, conclude that artificial membrane 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 destruction; that if it is given time it can recover from this
condition and that the treatment with the hypertonic solution also
brings about this recovery rapidly and reliably.

Herlant[101] suggested that the corrective effect of the hypertonic
solution consisted in the proper development 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 impossible.[102] 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 formation 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 suggestion of
Herlant deserves to be taken into serious consideration.

[101] Herlant, M., _Arch. de Biol._, 1913, xxviii., 505.

[102] 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.

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.[103] In 1906 he found
that the rapid disintegration of the eggs of the sea urchin which
follows artificial 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 formation alone.[104] 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 spermatozoon 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, however, in an unfertilized egg without
membrane formation, 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.[105] According to Warburg it
is probable that the oxidations 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{4}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.[106] 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.

[103] The writer found that the eggs of _Fundulus_ will segment a
number of times even if all the oxygen has apparently been removed.

[104] Loeb, J., _Biochem. Ztschr._, 1906, ii., 183.

[105] Thus the treatment of an unfertilized egg without membrane with a
hypertonic solution combines two effects, first the general cytolytic
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.

[106] Warburg, O., _Sitzungsber. d. Heidelberger Akad. d. Wissnsch._,
B. 1914.

The question remains then: How can membrane formation or the alteration
of the cortical layer underlying membrane formation cause an increase
in the rate of oxidations? 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.

Another possibility is that the act of fertilization increases the
permeability of the egg. This idea, which seems attractive, was first
suggested and discussed by the writer in 1906.[107] He had found that
when fertilized and unfertilized eggs were put into abnormal salt
solutions, _e. g._, pure solutions of NaCl, the fertilized eggs died
more rapidly than the unfertilized 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 obviously 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 solutions 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.[108]

[107] Loeb, J., _Biochem. Ztschr._, 1906, ii., 87.

[108] Unless the egg is left so long in the pure NaCl solution that its
permeability is increased.

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
develop because it contains too much CO{2} but that the CO{2} can
escape from the egg as soon as its permeability is increased through
the destruction of the cortical layer of the egg.[109] After the
CO{2} has escaped, the excessive permeability must be restored to its
normal value and this is the role of the hypertonic treatment. It is,
however, difficult to harmonize the assumption of an impermeability of
the unfertilized egg for CO{2} 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 spermatozoon enters.
The cortical layer is removed along the cut surface and there is no
reason why the CO{2} 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{2}
since the latter causes membrane formation if contained in the sea
water in sufficiently high concentration.

[109] Lillie, R. S., _Jour. Morphol._, 1911, xxii., 695; _Am. Jour.
Physiol._, 1911, xxvii., 289.

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 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 develop 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 larvae without further
treatment. It is impossible to apply Lillie's theory to these facts,
for the simple reason that the treatment with hypertonic sea water was
just long enough to induce development 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 treatment of
the eggs with a hypertonic solution diminishes their permeability; the
reverse would be much more probable.

Lillie's theory also fails to explain that mere treatment of the eggs
with a hypertonic solution can bring about their development into
larvae. 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[110] has shown that the electrical conductivity of the egg
is increased after fertilization, and J. Gray[111] 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 spermatozoon 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 exclusively by an increase in the permeability of the
egg.[112]

[110] McClendon, J. F., Publications of the Carnegie Institution, No.
183, 125; _Am. Jour. Physiol._, 1910, xxvii., 240.

[111] Gray, J., _Proc. Cambridge Philosophical Society_, 1913, xvii., 1.

[112] R. Lillie has recently shown that in a hypotonic solution water
diffuses more rapidly into a fertilized than into an unfertilized
egg. 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 understand how this observation can throw any light on the
mechanism of development, since water diffuses rapidly enough into the
unfertilized egg.

7. When the experiments on artificial parthenogenesis 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 spermatozoon
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 echinoderms
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[113] was courageous
enough to make an attempt to raise parthenogenetic larvae 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.

[113] Delage, Y., _Compt. rend. Acad. Sc._, 1909, cxlviii., 453.

Better opportunities were offered when a method was discovered which
induced the development of the unfertilized eggs of the frog. In
1907, Guyer made the surprising 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 the frog. The writer has tried
it in vain on the eggs of many other forms. He has at present seven
parthenogenetic 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 spermatozoon.
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 parthenogenetic 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 spermatozoa of normal appearance, and no eggs.[114] (Figs. 7
and 8.) This would indicate that the frog belongs to those animals in
which the male is heterozygous for sex.

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

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
spermatozoon--although normally the egg of this form does not develop
unless a spermatozoon enters--corroborates the idea expressed in
previous chapters that the egg is the future embryo and animal; and
that the spermatozoon, aside from its activating effect, only transmits
Mendelian characters to the egg. The question arises: Is it possible
to cause a spermatozoon to develop into an embryo? The idea has
been expressed that the egg was only the nutritive medium on which
the spermatozoon developed into an embryo, but this idea has been
rendered untenable by the experiments on artificial parthenogenesis.
Nevertheless the question whether or not the spermatozoon 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 spermatozoon 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. Incidentally these experiments on merogony also prove that
the mere mechanical tearing of the cortical layer,--which must happen
in the separation of the unfertilized 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
spermatozoon underwent transformation into a nucleus, but no mitosis
or aster formation was observed.[115] These experiments should be
continued.

[115] Loeb, J., _Artificial Parthenogenesis and Fertilization_,
Chicago, 1913.




CHAPTER VI

DETERMINISM IN THE FORMATION OF AN ORGANISM FROM AN EGG


1. The writer in a former book (_Dynamics of Living Matter_, 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. Uexkuell seem
to find it impossible to account for the development of such machines
from an undifferentiated egg on a purely physicochemical basis. A
study of Driesch's very interesting and important book[116] 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 differentiated organisms from such
undifferentiated material, a problem which does not seem capable of
a solution by physicochemical agencies alone. But the supposition
of a structureless egg is wrong, since Boveri has 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.

[116] Driesch, H., _Science and Philosophy of the Organism_. London,
1908 and 1909.

In this chapter we shall attempt the task among others of showing
how, on the basis of the simple physicochemical structure of the
unfertilized egg, the main organ of self-preservation of the organism,
the intestine, is formed through the mere process 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 structure 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 constituents and these, probably
on account of their chemical difference, do not all begin to grow or
divide simultaneously and equally.

Boveri[117] 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 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 unpigmented _C_. Observation
has shown that each one of these regions gives rise to a definite
constituent of the egg: _A_ furnishes the mesenchyme from which the
skeleton and the connective tissue originate; _B_ is the material for
the formation of the intestine, and _C_ gives rise to the ectoderm.

[117] Boveri, Th., _Verhandl. d. physik.-med. Gesellsch._, Wuerzburg,
1901, xxxiv., 145.

[Illustration: FIG. 9]

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
concentration and probably the more so the greater the distance from
_B_; and the same may probably be said for the substances determining
mesenchyme and ectoderm formation. Hence the unfertilized egg contains
already a rough preformation of the embryo inasmuch as the main axis of
the embryo and the arrangement of its first organs are determined.

[Illustration: FIG. 10]

[Illustration: FIG. 11]

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). The next divisions bring about an
unequal division of the material. Four cells will be formed of ectoderm
material _C_ and only little intestine material _B_, the other four
cells containing _B_ and _A_. These latter form at the next division
four very small colourless cells, the so-called micromeres, _A_ (Fig.
12), from which the mesenchyme, skeleton, 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 separation
of the three groups of substances is probably not as complete as our
purely diagrammatic drawing (Fig. 12) indicates.

[Illustration: FIG. 12]

The cell 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 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 divisions,
when the number of cells is theoretically 1024, in reality not quite
so many (between 800 and 900). The next step consists in the cells
derived from the material _A_ (mesenchyme and micromeres) gliding into
the hollow sphere, where they form a ring, the physicochemical process
responsible for this gliding being yet unknown. At the opening of this
ring an active 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 skeleton by the formation of crystals consisting of the CaCO{3} by
the mesenchyme cells surrounding the intestine. For the establishment
of the principle in which we are interested the description of
morphogenesis need not be carried farther.

[Illustration: FIG. 13]

[Illustration: FIG. 14]

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 assumption but show, as
Boveri has pointed out, a prearrangement 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 demonstrated is only conceivable if
the material of the egg has not too high a degree of fluidity; we may
consider it as consisting essentially of a semi-solid gel which is not
homogeneous throughout the egg but divided into three strata.

2. Lyon[118] tried to ascertain whether by centrifuging the sea-urchin
egg it was possible to modify its structure and thereby affect the
later embryo. He and subsequent experimenters found that it only is
possible 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 investigators 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 hydrolyzing enzymes, _e. g._, trypsin into the digestive
tract.

[118] Lyon, E. P., _Arch. f. Entwcklngsmech._, 1907, xxiii., 151;
Morgan, T. H., and Spooner, G. B., _ibid._, 1909, xxviii., 104; Morgan,
_Jour. Exper. Zool._, 1910, ix., 594; Conklin, E. G., _ibid._, 1910,
ix., 417; Lillie, F. R., _Biol. Bull._, 1909, xvi., 54.

When the surrounding solution is altered in constitution 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 larvae 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 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[119] 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
organism 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 gastrulae formed are often if not always symmetrical to each
other (Fig. 15), as the writer had a chance to observe in the egg of
_Strongylocentrotus purpuratus_[120] in the following experiment. The
eggs of the sea urchin _Strongylocentrotus 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{2} 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 embryos 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.

[119] Driesch, H., _Ztschr. f. wissnsch. Zool._, 1891, liii., 160.

[120] Loeb, J., _Arch. f. Entwcklngsmech._, 1909, xxvii., 119.

[Illustration: FIG. 15]

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 blastulae 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.[121] 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.

[121] Driesch, H., _Arch. f. Entwcklngsmech._, 1900, x., 361.

When the contents of the egg are displaced by pressure the result
will be determined by the location of 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[122] 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--_i. e._, a division into three strata, one forming the ectoderm,
the second the entoderm, and the third the mesoderm--was sufficient
to explain the various phenomena 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 development from a purely
physicochemical viewpoint. It is, therefore, only fair to point out
that Boveri[123] has furnished the facts for a simpler explanation,
which seems to have escaped the notice of antimechanists.[124]

[122] Driesch, H., _Arch. f. Entswcklngsmech._, 1902, xiv., 500.

[123] Boveri, Th., _Verhandl. d. physik. med. Gesellsch._, Wuerzburg,
N.F., 1901, xxxiv., 145.

[124] v. Uexkuell makes in his last book (_Bausteine zu einer
biologischen Weltanschauung_, Muenchen, 1913, p. 24) the following
statement: "Driesch succeeded in showing that the germ cell has no
trace of a machine-like structure but consists entirely of equivalent
parts." This is not correct.

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 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
electromotive forces which are observed in living tissues originate
at the boundaries between a watery and a water-immiscible phase, like
oleic acid or lecithin.[125] In his earlier writings[126] 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.

[125] Loeb, J., and Beutner, R., _Biochem. Ztschr._, 1912, xli., 1;
xliv., 303; 1913, li., 288; li., 300; 1914, lix., 195.

[126] Loeb, J., _The Dynamics of Living Matter_. New York, 1906.
Introductory Remarks.

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 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 symmetry 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.[127] The frog's egg consists
of two substances, a lighter one which is on top and a heavier one
below. Although viscous, the two substances 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 develop
independently into two whole embryos instead of into two half-embryos.
In Roux's experiment this flow of protoplasm was avoided. Morgan showed
that 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.[128]
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 redistribution 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.

[127] Roux, W., _Virchow's Archiv_, 1888, cxiv., 113.

[128] Morgan, T. H., _Embryology of the Frog_. New York.

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 embryo in 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 _Lymnaeus_ turn to the right,
those of _Planorbis_ to the left. It was observed by Crampton[129],
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 cleavage. The asymmetry of the body in snails is therefore
already preformed in the egg.[130]

[129] Crampton, H. E., _New York Academy of Sciences_, 1894; Kofoid, C.
A., _Proc. Am. Acad. Arts and Sciences_, 1894, xxix.

[130] Conklin, E. G., _Anat. Anzeig._, 1903, xxiii., 577; _Heredity and
Environment in the Development of Man_. Princeton, 1915, p. 171.

E. B. Wilson[131] 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 Annelid. 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
    remaining cell segments as if it still formed a part of an entire
    embryo.[132] 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 normal development. The posterior cell
    develops into a segmented larva with a prototroch, an asymmetrical
    pre-trochal or head region, and 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 prototroch and a pre-trochal
    region, with an apical organ, but produces no post-trochal region,
    develops no trunk or setae, 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 development 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 structure. 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 possibility 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.

[131] Wilson, E. B., _Science_, 1904, xx., 748; _Jour. Exper.
Zool._, 1904, i., 1, 197.

[132] The reader will notice the absence of "regulation."

Conklin has established the existence of a definite structure in the
unfertilized eggs of Ascidians, Amphioxus, 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 differentiation may come about. Sachs suggested that
all differentiation and the formation of every organ presupposes
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 exists
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 differentiation. Such streaming begins,
according to Conklin,[133] in the egg just before fertilization when
the surface layer of the egg protoplasm

    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.

    [133] 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
    introductions to modern biology in the English literature.

    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 posterior 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 in a 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 nervous 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.[134]

    [134] Conklin, E. G., _loc. cit._, p. 117.

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 reactions. 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.[135] Driesch[136] 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 formation of organs has been discussed by Herbst.[137]

[135] Loeb, J., _Jour. Morphol._, 1893, xiii., 161; _The Mechanistic
Conception of Life_. Chicago, 1912, p. 106.

[136] Driesch, H., _Science and Philosophy of the Organism_, i., p. 104.

[137] Herbst, C., _Formative Reize in der tierischen Ontogenese_.
Leipzig, 1901.

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 hindleg, will develop. Braus[138] has proved this
by transplanting 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 transplanted into the spot from which the hindlegs grow
out under natural conditions. There is therefore nothing to indicate
"regulation."

[138] Braus, H., _Muenchener Med. Wochnschr._, 1903, 1 (II.), No. 47, p.
2076.

The same is true for the formation of the eye and probably in general.
We have to consider the formation 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 functions; yet we
know that such pulps can still perform some of the characteristic
chemical processes of the organ; _e. g._, the alcoholic fermentation
characteristic 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 without 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,[139]
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 characteristic 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.[140]

[139] Nussbaum, M., _Arch. f. mikroscop. Anat._, 1886, xxvi., 485.

[140] It must not be overlooked that in bacteria and the blue algae no
distinct differentiation into nucleus and protoplasm can be shown.
To these organisms, therefore, the experiments of Nussbaum cannot be
applied.

The observations of Nussbaum and those who repeated his experiments
showed that although two different structures are required, not the
whole mass of an 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.[141] 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 smallest
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 results disagree with the statement of Boveri and
support the observation of the writer.

[141] Loeb, J., _Arch. d. f. ges. Physiol._, 1893, lv., 525.

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 algae, 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 suggested 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 adds only
the finer details to the rough block. Such a 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 organism 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 individuality; and this could be accomplished by substances
circulating in the liquids of the body as we shall see in later
chapters.




CHAPTER VII

REGENERATION


1. 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 regeneration. The leaf of the Bermuda
"life plant"--_Bryophyllum 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. When a 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,[142] 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[143] and
de Vries have accepted this view and the writer made use of it in his
first experiments on regeneration and heteromorphosis in animals.[144]
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 "hormones"; their connection with the theory
of Sachs was forgotten with the introduction of the new nomenclature.

[142] v. Sachs, J., "Stoff und Form der Pflanzenorgane," _Gesammelte
Abhandlungen_, 1892, ii., 1160. Arbeiten a. d. bot. Inst. Wuerzburg,
1880-82.

[143] Goebel, K., _Einleitung in die experimentelle Morphologie der
Pflanzen_, 1908.

[144] Loeb, J., Untersuchungen zur physiologischen Morphologie der
Tiere. I. Heteromorphose. Wuerzburg, 1891. II. Organbildung und
Wachsthum. 1892. Reprinted in _Studies in General Physiology_. Chicago,
1906.

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.[145] 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 animal). No other material seems to have such an effect. The
thyroid contains iodine, and Morse[146] 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.

[145] Gudernatsch, J. F., _Zentralbl. f. Physiol._, 1912, xxvi., 323;
_Arch. f. Entwcklngsmech._, 1912, xxxv., 457; _Am. Jour. Anat._, 1914,
xv., 431.

[146] Morse, M., _Jour. Biol. Chem._, 1914, xix., 421.

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 spermatozoon 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 acted as a regulator
of the phenomena of metamorphosis in animals, but it was possible to
show by simple experiments that the central nervous system does not
play this role 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 absorbed completely. The writer showed that in larvae in which the
spinal cord was cut in two, no matter at which level,--the sympathetic
nerves were in all probability also cut--the two organs continued to
undergo metamorphosis simultaneously.[147] 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.[148] 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.

[147] Loeb, J., _Arch. f. Entwcklngsmech._, 1897, iv., 502.

[148] Uhlenhuth, E., _ibid._, 1913, xxxvi., 211.

An interesting observation on the role of internal secretion in growth
was made by Leo Loeb.[149] When 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
with a foreign body to do so while without the intervention of the
corpus luteum substance no such effect of the mechanical stimulus 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.

[149] Loeb, Leo, _Zentralbl. f. allg. Path. u. path. Anat._, 1907,
xviii., 563; _Zentralbl. f. Physiol._, 1908, xxii., 498; 1909, xxiii.,
73; 1910, xxiv., 203; _Arch. f. Entwcklngsmech._, 1909, xxvii., 89,
463; _Jour. Am. Med. Assoc._, 1908, l., 1897; 1909, liii., 1471.

Bouin and Ancel[150] 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 proliferation such secretions do not occur.

[150] Quoted from M. Caullery, _Les Problemes de la Sexualite_, Paris,
1913, p. 126.

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[151]
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 glycogen. 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 propagation is observed, while
in the former sexual reproduction 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 "glycogen
metabolism" to "fat metabolism."

[151] Smith, Geoffrey, _Proc. Roy. Soc._, B. 1915, lxxxviii., 418.

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 existence 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 spermatozoon or by certain other
substances mentioned 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
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?) substances in the plant determines when and where dormant
buds or anlagen shall begin to grow. Such substances may 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 circulation 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); 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.[152] (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 arrest the development of the other notches
of the leaf in the same way as their development was suppressed by the
whole plant.

[152] Loeb, J., _Bot. Gazette_, 1915, lx., 249.

[Illustration: FIG. 16. Growth of roots and shoots in a few notches of
an isolated leaf of _Bryophyllum calycinum_]

[Illustration: FIG. 17. If all the notches of a leaf are isolated from
each other each notch will give rise to roots and a shoot, but the
growth will be less rapid than in Fig. 16. Figs. 16 and 17 were two
leaves taken from the same node of a 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 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
quickly. 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 more
quickly than the formation of roots and shoots in the notches of a
completely isolated leaf. The stem 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 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.

[Illustration: FIG. 18  FIG. 19  FIG. 20]

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
opposite 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.[153]

[153] With larger leaves the experiment may also succeed in moist air.

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 mechanism 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
regeneration always starts from that tissue which conducts nutritive
material.

When we cut out a piece of a stem of _Bryophyllum_, 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. If 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 accelerates 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 experiments by the writer
that the root-forming substances are associated or identical with the
substances which cause geotropic curvature in the stem.

These observations show that the phenomena of correlation or 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 mysterious force which directs the piece to
grow into a whole.

[Illustration: FIG. 21]

3. Phenomena of inhibition or correlation such as we have described
in _Bryophyllum_ are not lacking in the regeneration of animals, as
experiments on _Tubularia_ show.[154] _Tubularia mesembryanthemum_
(Fig. 21) is a hydroid consisting of a long stem terminating at one
end in a stolon which attaches itself 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 aquarium it forms as a rule a polyp
at either end (Fig. 22), 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 formation 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 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.

[154] Loeb, J., Untersuchungen zur physiologischen Morphologie. I.
Heteromorphose. 1891. II. Organbildung und Wachsthum. Wuerzburg, 1892.

[Illustration: FIG. 22]

[Illustration: FIG. 23]

Miss Bickford[155] found that the difference 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 piece. (This is
true, however, only in rather small pieces.) There is, therefore, in
small pieces a rough proportionality between size of head and size
of regenerating piece. Driesch[156] 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 mechanism 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 physicochemical basis.

[155] Bickford, E. E., _Jour. Morphol._, 1894, ix., 417.

[156] Driesch, H., _Science and Philosophy of the Organism_, i., 127.

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,[157] is that there exists an axial
gradient in the stem whereby the cells 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. According to the writer's observations
on a Tubularian (_T. crocea_) growing in the estuaries near Oakland,
California, both oral ends regenerate equally fast in such cases.

[157] Child, C. M., "Die physiologische Isolation von Teilen des
Organismus," Roux's _Vortraege und Aufsaetze_, Leipzig, 1911.

4. The phenomena of regeneration in _Cerianthus membranaceus_, a sea
anemone, can be easily understood from the experiments on Tubularians,
if we imagine the body wall of _Cerianthus_ to consist of a series of
longitudinal elements running parallel to the axis of symmetry of the
animal from the tentacles 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 element 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
(_a b c d_, Fig. 24) be cut from the body wall of a _Cerianthus_ in
such a way that one side, _a c_, 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_.[158] (Fig. 25.) The other three
free edges are not able to produce tentacles. If an incision be made in
the body wall of a _Cerianthus_, tentacles will grow on the lower edge
of the incision (Fig. 26).

[158] Loeb, J., "Untersuchungen zur physiologischen Morphologie der
Tiere."

[Illustration: FIG. 24]

[Illustration: FIG. 25]

[Illustration: 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
ligature 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 direction 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 role 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 _Cerianthus_ was cut off; very
soon new tentacles 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 consequence and ceased to grow, while
growth of the others continued. On the lower edge of the incision new
tentacles began to grow.

[Illustration: FIG. 27]

It seems also possible that Morgan's well-known experiment on
regeneration in _Planaria_ can be explained by a flow of substances.
He[159] found that if a piece _a c d b_ be cut out of a fresh-water
Planarian at right angles to the longitudinal axis (Fig. 27), at the
front end a new normal head, at the back end a new tail, will be
regenerated (Fig. 28); but that if a piece _a c d b_ be cut from
a Planarian obliquely (Fig. 29) instead of at right angles to the
longitudinal axis a tiny head is formed at the foremost corner of the
piece _a_ and a tiny tail at the hindmost corner _b_ (Fig. 30). Why is
it that in the oblique piece the head is formed in the corner and not
all along the cut surface as is the case when the cut is made at right
angles to the longitudinal axis? The writer is inclined to believe
that the right answer to this question has been given by Bardeen.[160]
This author has pointed out the apparent role that the circulatory (or
so-called digestive) canals in Planarians play in the localization of
the phenomena of regeneration, inasmuch as the new head always forms
symmetrically at the opening of the circulatory vessel or branch which
is situated as much as possible at the foremost end of the 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 connection 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 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 e c_ of such an
oblique piece is asymmetrical, the one side _a e_ being less stretched
than the other _e c_. The higher tension of the piece _e c_ will have
the effect of bringing _e_ nearer _c_, since we know that acid formation
and hence energy production increases in proportion to surface, _i. e._,
it must be the greater the more it is stretched. The reverse is true for
the tail _d f b_, and the effect here will be that _f_ 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.

[159] Morgan, T. H., _Regeneration_, New York, 1901.

[160] Bardeen, C. R., _Am. Jour. Physiol._, 1901, v., 1; _Arch. f.
Entwcklngsmech._, 1903, xvi., 1.

[Illustration: FIG. 28]

[Illustration: FIG. 29]

[Illustration: FIG. 30]

[Illustration: FIG. 31]

[Illustration: FIG. 32]

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.[161] In a crustacean, _Alpheus_, the
two chelae (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.

[161] Przibram, H., _Arch. f. Entwcklngsmech._, 1901, xi., 329.

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 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.[162] This indicates that material in the
old cells has been digested or has undergone 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 collecting of red cells
at the end in a Tubularian where regeneration is about to begin has
to do with the furnishing of material by self-digestion, since these
cells are partly at least destroyed in the process. It is of interest
to look for more examples of autodigestion accompanying phenomena of
regeneration.

[162] Child, C. M., _Senescence and Rejuvenescence_. Chicago, 1915.

[Illustration: FIG. 33]

The writer has observed more closely the transformation of an organ
into more undifferentiated material in _Campanularia_ (Fig. 33), a
hydroid.[163] 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 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 which make it appear as if the differentiated material of the
polyp were transformed into undifferentiated material. The tentacles
are first put together like the hairs of a camel's-hair 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 undifferentiated material formed
from the polyp may afterward flow 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 process of differentiation
of organs and tissues, in certain 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.

[163] Loeb, J., _Am. Jour. Physiol._, 1900, iv., 60.

[Illustration: FIG. 34]

[Illustration: FIG. 35]

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.[164]

[164] The writer quotes this after Driesch.

Driesch,[165] 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. In a 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.

[165] Driesch, H., _Arch. f. Entwcklngsmech._, 1902, xiv., 247.

It is probable that the two cases are only quantitatively 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 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_.[166] 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).

[166] 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.

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 to a
misconception; namely, that the egg consisted of homogeneous material
which was to be differentiated into an organism. For this supernatural
task supernatural 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 transparent. 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 organism does in this case what
it is compelled to do by its physical and chemical structure.

Uhlenhuth[167] 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 transparent so that it can serve as a lens? 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 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.

[167] Not yet published.

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.[168]
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, _e. g._, in siphoneae and other
coeloblasts, 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."[169] He points
out that in the case of the animal egg the reserve material--fat
granules, proteins, and carbohydrates--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 protoplasm which it is able
to control. Morgan[170] and Driesch[171] tested and confirmed the
idea of Sachs for the eggs of Echinoderms. We stated in the previous
chapter that Driesch produced artificially larvae 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 gastrulae
resulting from these partial eggs the number of mesenchyme cells
and found that the larvae 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 gastrulae of such larvae had twice
the number of mesenchyme cells. Driesch drew the conclusion 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.

[168] v. Sachs, J., "Physiologische Notizen," vi., _Flora_, 1893.

[169] _Ibid._, ix., 425, _Flora_, 1895.

[170] Morgan, T. H., _Arch. f. Entwcklngsmech._, 1895, ii., 81; 1901,
xiii., 416; 1903, xvi., 117.

[171] Driesch, H., _Arch. f. Entwcklngsmech._, 1898, vi., 198; 1900,
x., 361.

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,
_i. e._, an enucleated fragment of protoplasm into which a spermatozoon
has entered and which is able to develop. Such a fragment has only
the sperm nucleus. This phenomenon of merogony was discovered by
Boveri and was elaborated by Delage.[172] 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 protoplasm is a constant in a given species. Driesch compared
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.[173] Boveri's results as well as
those of Driesch were obtained 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 to Boveri's law which
Driesch has actually found had been predicted by Boveri.

[172] Delage, Y., _Arch. Zool. exper._, 1899, vii., 383.

[173] Driesch, H., _Arch. f. Entwcklngsmech._, 1905, xix., 648.

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 available through the work of
Fischel and Uhlenhuth.

The healing of a wound is a process essentially similar 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[174] 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 form a
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. 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.[175] 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.[176]

[174] Loeb, Leo, _Arch. f. Entwcklngsmech._, 1898, vi., 297.

[175] Spain, K. C., and Loeb, Leo, _Jour. Exper. Med._, 1916, xxiii.,
107; Loeb, L., and Addison, W. H. F., _Arch. f. Entwcklngsmech._, 1911,
xxxii., 44; 1913, xxxvii., 635.

[176] The excessive formation of epithelial cells in the healing of
wounds has led the older pathologists to the generalization 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. As a matter
of fact, this generalization 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 _Cerianthus_ 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 generalization should be
correct.

Should it be possible that this is more generally the case, _e. 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 conditions 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 condition 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
spermatozoon 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 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/10 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 1/10 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 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.[177] 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.[178] Similar results were
obtained in eggs activated with NH{4}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_.[179]

[177] Loeb, _Arch. f. Entwcklngsmech._, 1914, xxxviii., 277.

[178] Wasteneys, H., _Jour. Biol. Chem._, 1916, xxiv., 281.

[179] F. Lillie thinks that the KCN in this experiment merely inhibits
the change of the cortical layer necessary for development. This is
contradicted 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.

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 sometimes 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 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
alteration.

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 experiments.

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 formation 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 heteromorphosis.

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 _b_. In _Margelis_, another hydroid, the writer observed 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.

[Illustration: FIG. 36]

Herbst discovered a very interesting form of heteromorphosis in certain
crustaceans; namely, that in the 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 experiments were carried
out successfully on _Palaemon_, _Palaemonetes_, _Sicyonia_, _Palinurus_,
and other crustaceans.

The influence of gravitation is very familiar in plants; 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.

[Illustration: FIG. 37]

If we cut a piece _a b_ (Fig. 37), from the stem _s s_ of _Antennularia
antennina_ (Fig. 38), a hydroid, and put it into the water in a
horizontal position, new stems _c d_ (Fig. 37) may arise on its upper
side. The small branches on the under side of the old stem _a b_
begin suddenly to grow vertically downward.[180] In appearance and
function 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 a
limited growth (see Fig. 38), are directed upward, and have polyps on
their upper side. The parts which grow down (Fig. 37) have no polyps,
but attach themselves like true roots to solid bodies. Thus the
changed position of the stem alone, without any operation, suffices
to transform the lateral branches, whose growth is limited, into
roots with unlimited growth. The lateral branches on the upper side
of the stem do not undergo such a transformation into roots except in
the immediate surroundings of the place where a new stem arises. It
seems that the formation of a new stem also causes an excessive growth
of roots, possibly because the formation of new branches causes the
removal of substances which naturally inhibit the formation of roots.
If a piece from the stem be put vertically into the water with top
downward, the uppermost point may continue to grow as a stem, while the
lowest point may give rise to roots. In this case, therefore, a change
in the orientation of organs has the effect of changing the character
of organs.

[180] Loeb, J., Untersuchungen zur physiologischen Morphologie der
Tiere. II. Organbildung und Wachsthum. Wuerzburg, 1892.

[Illustration: FIG. 38]

There are only two ways by which we can account for these influences of
gravitation. Either certain substances flow to the lowest level and
collecting there induce growth and possibly changes in the character
of growth (as in _Antennularia_) 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 contact upon life phenomena
are at present little understood.

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,[181] 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 regeneration, and these
conditions cannot be expressed by the word "stimulus."

[181] Loeb, J., _Die chemische Entwicklungserregung des tierischen
Eies_. Berlin, 1909.

While our knowledge of the role of the whole in regeneration 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 CHARACTERS, 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 solution of the problem belongs
to McClung.[182] 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 to a single
chromosome, which is either lacking or different in one-half of the
spermatozoa.

[182] McClung, C. E., "The Accessory Chromosome--Sex Determinant?"
_Biol. Bull._, 1902, iii., 43.

The first one to recognize the existence of two kinds of spermatozoa
was Henking, who stated that in _Pyrrhocoris_ (a Hemipteran) one-half
of the spermatozoa 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 chromosome 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 logically forced to the conclusion that the peculiar
    chromosome has some bearing upon the arrangement.

N. M. Stevens and E. B. Wilson[183] 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 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 spermatozoon containing an X
chromosome, while a male is produced when a spermatozoon without an X
chromosome enters the egg.

[183] Wilson, E. B., "Studies on Chromosomes," _Jour. Exper. Zool._,
1905, ii., 371, 507; 1906, iii., 1; 1909, vi., 69, 147; 1910, ix.,
53; 1912, xiii., 345. "Croonian Lecture," 1914, _Proc. Roy. Soc._, B.
lxxxviii., 333.

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
chromosomes, but one with, the other without, an X chromosome. The
two possible chromosome combinations between egg and spermatozoa are
therefore as follows (see the diagrammatic Fig. 39):

      _Egg_      _Spermatozoon_        _Result_

    (1) 6 + X        + 6         =   12 + X = Male

    (2) 6 + X        + 6 + X     =   12 + 2X = Female

The egg which receives a spermatozoon without an X chromosome has after
fertilization 12+X chromosomes and develops into a male; while the egg
into which a spermatozoon 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 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.

[Illustration: FIG. 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 Hemiptera, _e. g._, _Lygaeus_, there are also two types of
spermatozoa appearing in equal numbers differing in regard to the
X chromosome, but here it is only a difference in size; one-half of
the spermatozoa having a large X chromosome, the other half instead
a smaller chromosome. Calling this latter the Y chromosome, the sex
determination in this form is as follows: leaving aside the chromosomes
which are equal in both egg and spermatozoon we may say that there is
one type of egg containing 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 chromosome. 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 develop
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 to a 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 composition XY, where Y is apparently
qualitatively different and often, but not necessarily, smaller than X,
or entirely lacking.

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 spermatozoon 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_,
indicated that a number of other forms exists 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 chromosome X, while
there are two types of eggs, one possessing the sex chromosome X,
the other possessing Y. When a spermatozoon 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; _i. e._, 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 overwhelmingly in the male
offspring of a colour-blind person. Doncaster crossed two varieties of
_Abraxas_ differing in one character which was sex-linked, and the
results of his crossings indicated that in this form there are two
types of eggs and one type of spermatozoa.[184]

[184] Doncaster, L., _The Determination of Sex_. Cambridge, 1914.

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 ampelophila_ are red,
Morgan[185] 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{1} generation, were all red eyed, males as well as females. These
were inbred and now gave in the F{2} generation the following three
types of offspring:

[185] Morgan, T. H., _Heredity and Sex_. New York, 1913.

    (1) 50 per cent. females, all with red eyes.

    (2) 50 per cent. males { 25 per cent. with red eyes.
                           { 25 per cent. with white eyes.

The character white eye was therefore transmitted only to half the
grandsons; it was a sex-linked character. It is known from a study of
the pedigrees of colour-blind individuals that if the corresponding
experiment 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, normal colour vision in the F{1} generation, in
both males and females, and half of the males of the F{2} generation
colour-blind, the other half and all the females with normal vision. Of
course, in man, intermarriage between two different F{1} strains would
have been required in place of the inbreeding of the F{1} 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 following combinations must result if we cross a normal red-eyed
female with a white-eyed male:

    _Eggs_   _Sperm_       _Result_

     =X=        X      =X=X red-eyed female

     =X=        Y      =X=Y red-eyed male

It is obvious that all the offspring of the first generation (the
F{1} generation) must be red eyed, since all the 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 =X=X will form two types
of eggs in equal numbers: namely, eggs with an =X= and eggs with an X,
_i. e._, 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
lacking (X). The males having the chromosome constitution =X=Y 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{2} generation will give rise to the following four types of
offspring: (1) =X==X=, (2) =X=X, (3) =X=Y, (4) XY, all four types in
equal numbers.

(1) and (2) give females, both red eyed, since both contain a
red-factored =X= 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 combinations must appear in equal
numbers (provided the experimental material is ample enough, which was
the case in these experiments), in the F{2} generation both males and
females should have red eyes and in the F{2} generation all the females
should have red eyes and half of the males should have red, half white
eyes. These results were obtained.

The experiments were carried further. No white-eyed females had
appeared thus far. On the same assumptions 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{2}
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 constitution =X=X 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 produced in equal numbers:

(1) =X=X, (2) XX, (3) =X=Y, 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 numbers, and
this was actually observed.

The numerical agreement in this and the other experiments between the
expected and observed result cannot well be an accident. The fact that
the inheritance 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[186] has found a
type of female _Drosophila_ with a chromosome formula XXY whose sex was
not affected by the supernumerary Y.

[186] Bridges, C. B., _Genetics_, 1916, i., 1.

3. On the basis of all these experiments and theories it is
comparatively easy to explain a number of phenomena 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 assumption that the unfertilized egg
contains only one X chromosome while the spermatozoon carries into the
egg the second X chromosome. But if the male bee produces 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 spermatozoa one viable
spermatozoon only is formed while the other one perishes. It is,
therefore, quite possible that it is the female-producing spermatozoon
which survives while the male-producing spermatozoon 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
spermatozoon 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 chromosome and in a case
like the bee would develop parthenogenetically, while the other half
of the body, developing from the cell into which a spermatozoon 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 proof for the correctness of this interpretation as
far as gynandromorphism in the bee is concerned.[187]

[187] Boveri, Th., _Arch. f. Entwcklngsmech._, 1915, xlii., 264.

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 observation 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 reproduction may alternate
in a cycle, _e. g._, in plant lice, _Daphnia_, 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
parthenogenetically, 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
made concerning the role 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
chromosome. 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 chromosomes. 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 determination 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 observation 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
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 race by the nature of the spermatozoon 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 hermaphroditism 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, _e. g._, in the Ascidian _Ciona_ (see
Chapter IV), in others they are formed successively, very often the
spermatozoa appearing first (protandric hermaphroditism). In the long
tapeworm _Taenia_ 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 female.
How can we reconcile the facts of hermaphroditism with the chromosome
theory of sex determination? _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 hermaphrodite which migrates into the lungs of the frogs.
According to Boveri and Schleip[188] the cells of the hermaphrodite
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 hermaphrodite, females with twelve and
males with eleven chromosomes.

[188] Boveri, Th., _Verhand. d. phys.-med. Gesellsch._ Wuerzburg, 1911,
xli., 85. Schleip, W., _Ber. d. naturf. Gesellsch._, Freiburg i. Br.,
1911, xix.

The males produce again two kinds of spermatozoa, male and female
producing, but the male-producing spermatozoa become functionless.
This fusion of the other spermatozoon containing six chromosomes with
an egg having six chromosomes leads again to the formation of the
hermaphrodite with twelve chromosomes. It is obvious that in this case
the cause for the hermaphroditism is not disclosed. If chromosomes
have 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[189] and by Shull on the determination of hermaphroditism in
plants but lack of space forbids us to give details.

[189] Correns, C., _Biol. Centralbl._, 1916, xxxvi., 12.


_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 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 larvae hatch. At the time of hatching
all larvae are alike. The differentiation of the larvae into the dwarf
males and the giant females can be determined at will. The larvae 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 larvae hatch, about ninety per cent. of
the larvae 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 production of sperm
or of other secondary male sexual characters. This tendency is stronger
the longer the period of rest lasts. If the larvae 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 observations that the larvae
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 larvae 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 larvae the tendency for both sexual characters
is present, that they are, in other words, hermaphrodites, 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.[190]

[190] Baltzer, F., _Mitteil. d. zoolog. Station_, Neapel, 1914, xxii.

Giard has studied the effects of a curious form of castration 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 _Carcinus
maenas_ 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 secondary sexual
characters of the male into those of a female.

Giard observed that in a dioecious plant, _Lychnis dioica_, a parasitic
fungus brings about the transformation 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 characters. 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 previously 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.[191] In certain birds and also in mammals at
the time when the sexual glands cease to function certain secondary
sexual characters of the other sex make their appearance. 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.[192]

[191] Caullery, M., _Les Problemes de la Sexualite_. Paris, 1913.

[192] Goodale, H. D., _Biol. Bull._, 1916, xxx., 286.

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[193] 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.

[193] Lillie, F., _Science_, 1916, xliii., 611.

    The rapidly elongating ova meet and fuse in the small body of the
    uterus at some time between the 10 mm. and the 20 mm. stage. The
    blood-vessels from each side then anastomose in the connecting part
    of the chorion; a particularly wide arterial anastomosis develops,
    so that either fetus can be injected from the other. The arterial
    circulation 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 development 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 inhibition 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 lacuum_,[194] is no longer so mysterious. This
worm normally 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, including 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 dioecian animal probably only one type of sex organ
would be developed; the formation of the other being inhibited.

[194] Janda, V., _Arch. f. Entwcklngsmech._, 1912, xxxiii., 345;
xxxiv., 557.

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
definite crosses. These differences are such that it is possible to
grade the hybrids according to their manifestations of maleness or
femaleness, both in morphological characters and instincts. Goldschmidt
calls this peculiar phenomenon intersexualism, and its essential
feature is that the various degrees of intersexualism can be produced
at will by the right combination of races.

    Female intersexualism begins with animals which show feathered
    antennae of medium size (feathered antennae 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 reproduction 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 rudimentary 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 showing a few
    white female spots on their wings. These become larger and larger,
    the amount of brown pigment 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.[195]

[195] Goldschmidt, R., _Proc. Nat. Acad. Sc._, 1916, ii., 53; _Ztschr.
induct. Abstammungslehre_, 1912, vii., and 1914, xi.

6. The relation between chemical substances circulating 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 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. Throughout
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 larvae 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 larvae, provided the food which would have been given to
the larvae 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 larvae. The food must,
therefore, contain a substance 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").[196] 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?

[196] This account of Marchal's beautiful experiments is taken from
Caullery, M., _Les Problemes de la Sexualite_. Paris, 1913.

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[197] 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.[198]

[197] Whitney, D. D., _Science_, 1916, xliii., 176.

[198] Shull, A. F., and Ladoff, S., _Science_, 1916, xliii., 177.

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 caterpillar,
_e. g._, _Ocneria dispar_, according to Oudemans. This result has
been invariably confirmed by all subsequent workers, especially by
Meisenheimer. Crampton grafted the heads of pupae 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 characters, 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
development of the secondary sexual character. This has been proved
definitely by Steinach,[199] 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 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 development 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.

[199] Steinach, E., _Zentralbl. f. Physiol._, 1910, xxiv., 551; _Arch.
f. d. ges. Physiol._, 1912, cxliv., 72.

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 development, and it was thereby proved that
the internal secretions from the ovary do not promote the growth of
the 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 morphological habitus of females. Soon
after the transplantation 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.

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
determination of sex may be overcome.




CHAPTER IX

MENDELIAN HEREDITY AND ITS MECHANISM[200]


_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 hereditary 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 hybrid 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 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.[201] 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.

[200] For 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.

[201] Mendel, G., "Experiment in Plant-Hybridization," translated in
W. Bateson's classical book on Mendel's _Principles of Heredity_.
Cambridge, 1909.

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 cannot 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 contains 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 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.[202]

[202] 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.

When two pure breeds of parents differ in one character, _e. g._, two
varieties of beans, one with a violet the other with a white flower,
the cross between the two species (the F{2} generation) has pale
violet flowers, approximately intermediate between the two parents. If
these hybrids are bred among themselves the offspring is called the
F{2} generation. According to Mendel's law the hybrids of the first
F{1} 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
         |\ /|
         | X |
         |/ \|
    violet   white .... pollen

The four possible combinations are: (1) violet--violet; (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 sufficiently
large the numerical result will be:

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

Fifty per cent. of the F{2} 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
ratio 1:2:1. 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. e._, all the hybrids have round seeds. When
these hybrids were bred among themselves the next generation 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 constitution
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{1} 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{2} 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[203] 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 species is characterized by a
definite number of chromosomes, _e. g._[204]

[203] Sutton, W. S., "The Chromosomes in Heredity," _Biol. Bull._,
1904, iv., 231.

[204] Morgan, T. H., Sturtevant, A. H., Muller, H. J., and Bridges, C.
B., _Mechanism of Mendelian Heredity_. New York, 1915, p. 26.

    man (probably)             24    corn                      20
    mouse                      20    evening primrose           7
    snail (_Helix hortensis_)  22    nightshade                36
    potato beetle              18    tobacco                   24
    cotton                     28    tomato                    12
    four o'clock               16    wheat                      8
    garden pea                  7

In the fertilization of the egg the number of chromosomes 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 chromosomes 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 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 chromosomes, so that while one egg retains the maternal
chromosome _A_ the other will retain the paternal one, and so on.
If before the reduction division all the eggs had the chromosome
constitution _AA{1}_, _BB{1}, _CC{1}_, _DD{1}_ (where _A_ _B_ _C_ _D_
are the paternal and _A{1}_ _B{1}_ _C{1}_ _D{1}_ 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{1}__CD{1}_, the other _A{1}__B{1}__C{1}__D{1}_,
etc. This, according to Sutton, is the basis of the Mendelian heredity.
Suppose the determiner of a certain character (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 designated
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 constitution 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{1} generation have the chromosome constitution _Aa_. 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{1} are mated among themselves, the following
four chromosome combinations will be produced:

    Eggs        Pollen

    A   a       A    a

    [Transcriber's note: Diagram illustrating the four possible pairings
    of chromosomal characters]

    Possible combinations in fertilized eggs
    _AA_, _Aa_, _aa_, in the ratio 1:2:1.

Now this is exactly the ratio of Mendelian heredity in the F{2}
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 division 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 antagonistic 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 determination 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 constitution _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_ and _a_.

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 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.[205]

[205] Morgan, T. H., Sturtevant, A. H., Muller, H. J., and Bridges, C.
B., _The Mechanism of Mendelian Heredity_. New York, 1915.

Mendel had found that when he crossed two species of peas differing in
regard to two pairs of characters, he obtained in the F{2} 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{1} generation had the characters round and
yellow. When these were inbred the F{2} 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 characters occurs independently, there
must be 3 yellow to 1 green and also 3 round to 1 wrinkled in the F{2}
generation. 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 1 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{1} generation will have the
chromosome constitution Yg Rw, where Y and g and R and w are homologous
chromosomes which will lie alongside each other YRgw[Transcriber's
note: displayed in the text as YR over gw, i.e. like a fraction but
without a dividing bar]. 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 Yw and the
same four types of pollen cells. The F{2} generation will produce the
sixteen possible combinations in equal numbers: namely,

    YRYR   YRgw  YRgR  YRYw
    gwYR   gwgw  gwgR  gwYw
    gRYR   gRgw  gRgR  gRYw
    YwYR   Ywgw  YwgR  YwYw

Since w and 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 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 interrelation 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.[206]

[206] Bateson, W., _loc. cit._, p. 157.

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
contained 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 maturation division occurs
the homologous maternal and paternal chromosomes fuse--the so-called
synapsis 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 linkage. If in Fig. 40 the white
and black signify two homologous chromosomes I and I{1} 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{1} 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, I and I{1} will not
separate completely but an exchange will take place, part of I{1} and
I becoming exchanged. This would result in the formation of two mixed
chromosomes Ab and aB (Fig. 42). This partial exchange 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.

[Illustration: FIG. 40]

[Illustration: FIG. 41]

[Illustration: FIG. 42]

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.[207] This agreement can be
no mere accident.

[207] The number of hereditary characters examined to test the theory
was over 130.

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 consisted of a series of smaller chromosomes, each of which
may be a factor in the determination of a hereditary character which is
transmitted according to Mendel'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_

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.[208] 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 definite
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 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 Mendelian heredity are
morphological features as well as instincts. For the former we have
already had occasion 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
connect 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
in certain cases, for instance why the influence of certain hereditary
factors for pigmentation should depend upon temperature as E. Baur
observed.

[208] Bateson, W., _Mendel's Principles of Heredity_, 3d ed., 1913;
Davenport, Chas. B., _Heredity in Relation to Eugenics_, 1911. Pearl,
R., _Modes of Research in Genetics_.

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_ _franciscanus_ and
_purpuratus_,[209] 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 Chapter III), and it is quite likely that this species specificity
decides which type of animal should arise from an egg.

[209] 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.

The idea had been suggested that the factors which determine the future
character might be ferments or enzymes, or substances from which such
ferments develop. A. R. Moore[210] 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 determiners for the development of the cross-bars (from both
egg and sperm), the hybrids only one (from either 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.

[210] Moore, A. R., _Arch. f. Entwcklngsmech._, 1912, xxxiv., 168.

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,
however, 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[211] and of Chodat[212] on the production of 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_-oxyphenylaminopropionic 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,[213] and very recently by
Onslow.[214] The latter showed that from the skins of certain coloured
rabbits and mice a peroxidase can be extracted which behaves like a
tyrosinase 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
peroxydase. 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."

[211] Bertrand, G., _Ann. d. l'Inst. Pasteur_, 1908, xxii., 381; _Bull.
Soc. Chim._, 1896, xv., 791.

[212] Chodat, R., _Arch. d. Sc. phys. et nat._, 1915, xxxix., 327.

[213] Gortner, R. A., _Trans. Chem. Soc._, 1910, xcvii., 110.

[214] Onslow, H., _Proc. Roy. Soc._, 1915, B. lxxxix., 36.

One point might still be mentioned since it may help to overcome a
difficulty in visualizing the connection 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 tyrosinase 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 Mendelian 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 purposeful reactions based
on these tropisms that a factor for the formation of the photosensitive
substance should exist.[215]

[215] Loeb, J., "Egg Structure and the Heredity of Instincts," _The
Monist_, 1897, vii., 481.

5. Another point should be emphasized, namely that for Mendelian
heredity it is immaterial whether the character is introduced by the
spermatozoon 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. We can, 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 Mendelian character of their
parents.

The application of our modern knowledge of heredity to human affairs
has been discussed in a very original way by Bateson in his address
before the British Association in Sydney to which the reader may be
referred.[216]

[216] Bateson, W., _Nature_, 1916, xciii., 674.




CHAPTER X

ANIMAL INSTINCTS AND TROPISMS[217]


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
"purposeful," _i. e._, assisting to preserve the individual and the
race. This again suggests "design" and a designing "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 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.

[217] Ideas similar to those expressed in this chapter may be found
in the writer's former book _Comparative Physiology of the Brain and
Comparative Psychology_, New York, 1900, and in the books by George
Bohn, _La Naissance de l'Intelligence_, Paris, 1909, and _La nouvelle
Psychologie animale_, Paris, 1911.

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 spermatozoon 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 physicochemical explanation; for example the
curious experiment 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
larvae 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 larvae, can consume the food which would otherwise
have been devoured by the larvae; 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 larvae of the barnacle or certain winged plant
lice or the caterpillars of certain butterflies, are put into a trough
or 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, _e. g._, the caterpillars of
_Porthesia chrysorrhoea_ 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 expression of
the Bunsen-Roscoe law of photochemical reactions. 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 intensity into the
duration of illumination.

The "attraction" or "repulsion" of animals by the light had been
explained by the biologists in an anthropomorphic way by ascribing to
the animals a "fondness" 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.[218] In 1888 the writer published a paper 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.[219]

[218] Graber, V., _Grundlinien zur Erforschung des Helligkeits- und
Farbensinnes der Tiere_. Prag, 1884.

[219] Loeb, J., _Sitzungsber. d. physik.-med. Gesellsch_. Wuerzburg,
1888. _Der Heliotropismus der Tiere und seine Uebereinstimmung mit
dem Heliotropismus der Pflanzen._ Wuerzburg, 1889. _Arch. f. d. ges.
Physiol._, 1897, lxvi., 439.

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 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 photochemical 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 symmetrical 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 these reactions: first, the
symmetrical arrangement of the photosensitive and the contractile
organs, and second, the relative masses of the photochemical reaction
products produced in both retinae 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 negatively 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 larvae of the blowfly
when they are fully grown, and for positively heliotropic animals on
the larvae 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 turned and compelled to move to or from the
light. This difficulty was overcome by the observation that sessile
animals like the hydroid _Eudendrium_ (Fig. 43) or the tube worm
_Spirographis_ (Fig. 44) react to a one-sided illumination also with
heliotropic curvatures like sessile plants.[220] On the other hand,
it had been found before by Strassburger that free-swimming plant
organisms like the swarmspores of algae move to or from the source of
light as do free-swimming animals.

[220] Loeb, J., _Arch. f. d. ges. Physiol._, 1890, xlvii., 391; 1896,
lxiii., 273.

[Illustration: FIG. 43]

[Illustration: FIG. 44]

3. The writer suggested in 1897[221] that the light acts chemically in
the heliotropic reactions and in 1912 that the heliotropic reactions
probably follow the law of Bunsen and Roscoe,[222] and it was possible
to confirm this idea by direct experiments.[223] This law states
that the photochemical effect of light equals _i t_ where _i_ is
the intensity of the light and _t_ the duration of illumination.
The experiments were carried out on young regenerating 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.

[221] Loeb, J., _Arch. f. d. ges. Physiol._, 1897, lxvi., 439.

[222] Loeb, J., _The Mechanistic Conception of Life_, Chicago, 1912, p.
27.

[223] Loeb, J., and Ewald, W. F., _Zentralbl. f. Physiol._, 1914,
xxvii., 1165.

TABLE VI

    ------------+----------------------------------------------
    _Distance   |_Time Required to Cause Fifty Per Cent. of the
     between    | Polyps to Bend towards the Source of Light_
     Polyps and +----------------------+-----------------------
     Source of  |       Observed       |    Calculated from
     Light_     |                      |   Bunsen-Roscoe 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
    ------------+----------------------+-----------------------

We must therefore conclude that the heliotropic curvature of the
polyps is determined by a photochemical 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 _it_ 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 effective 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 modification 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_.[224] This
makes it probable that the law of Bunsen-Roscoe underlies generally the
heliotropic reaction of animals.

[224] Ewald, W. F., _Science_, 1913, xxxviii., 236.

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 Froeschel[225] and by Blaauw.[226] In the following table are
given the results of Blaauw's experiments on the applicability
of the Bunsen-Roscoe law for the heliotropic 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 _i t_, namely
metre-candles-seconds, varies very little (between 16 and 26).

[225] Froeschel, P., _Sitzungsber. d. k. Akad. d. Wissensch._, Wien,
1908, cxvii.

[226] Blaauw, H. A., _Rec. d. travaux botaniques Neerlandais_, 1909,
v., 209.

TABLE VII

    ---------------+--------------+----------
           I       |      II      |   III
    _Duration of   |    _Metre-   | _Metre-
     Illumination_ |    Candles_  | Candles-
                   |              | Seconds_
    ---------------+--------------+----------
        43 hours   |     0.00017  |   26.3
        13   "     |     0.000439 |   20.6
        10   "     |     0.000609 |   21.9
         6   "     |     0.000855 |   18.6
         3   "     |     0.001769 |   19.1
       100 minutes |     0.002706 |   16.2
        60    "    |     0.004773 |   17.2
        30    "    |     0.01018  |   18.3
        20    "    |     0.01640  |   19.7
        15    "    |     0.0249   |   22.4
         8    "    |     0.0498   |   23.9
         4    "    |     0.0898   |   21.6
        40 seconds |     0.6156   |   24.8
        25 seconds |     1.0998   |   27.5
         8    "    |     3.02813  |   24.2
         4    "    |     5.456    |   21.8
         2    "    |     8.453    |   16.9
         1    "    |    18.94     |   18.9
       2/5    "    |    45.05     |   18.0
      2/25    "    |   308.7      |   24.7
      1/25    "    |   511.4      |   20.5
      1/55    "    |  1255        |   22.8
     1/100    "    |  1902        |   19.0
     1/400    "    |  7905        |   19.8
     1/800    "    | 13094        |   16.4
    1/1000    "    | 26520        |   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 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 (di/dt).[227] Jennings has maintained that the
heliotropic reactions of unicellular organisms are all of this kind,
but investigations by Torrey and by Bancroft[228] on _Euglena_ have
shown that Jennings's statements were based on incomplete observations.

[227] Loeb, J., _Arch. f. d. ges. Physiol._, 1893, liv., 81; _Jour.
Exper. Zool._, 1907, iv., 151.

[228] Bancroft, F. W., _Jour. Exper. Zool._, 1913, xv., 383.

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."[229] This
fact has been amply verified by Bohn, by Parker and his pupils, and
especially by Bradley Patten, who used it to compare the relative
efficiency of two different lights.

[229] Loeb, J., _Studies in General Physiology_, Chicago, 1905, p. 2.

The behaviour of the animals under the influence of two lights is a
confirmation of our theory of heliotropism 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[230]
has recently shown. He used the negatively heliotropic larvae of the
blowfly. These larvae were made to record their trail while moving under
the influence of the two lights. The results of the measurements of
2500 trails showing the progressive increase in angular deviation of
the larvae (from the perpendicular upon the line connecting the two
lights), with increasing differences between the lights, are given in
the following table. Since the deviation or angular deflection of the
larvae is towards the weaker of the two lights it is marked negative.

[230] Patten, Bradley M., _Am. Jour. Physiol._, 1915, xxxviii., 313.

TABLE VIII

    -----------------------+-------------------------------
    _Percentage Difference | _Average Angular Deflection of
       in the Intensity    |  the Two Paths of the Larvae
       of the Two Lights_  |   towards the Weaker Light_
    -----------------------+-------------------------------
          _Per Cent._      |           _Degrees_
              0            |             -0.09
              8-1/3        |             -2.77
             16-2/3        |             -5.75
             25            |             -8.86
             33-1/3        |            -11.92
             50            |            -20.28
             66-2/3        |            -30.90
             83-1/3        |            -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 larvae 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
production 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 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 photosensitive 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, 8-1/3,
16-2/3, 25, 33-1/3, 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, 1, 2, 3, 4, or 5 glowers
were used. The following table shows the result.

TABLE IX

A TABLE 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    |    0    |  8-1/3  | 16-2/3  |   25    | 33-1/3  |   50
  Glowers_|per cent.|per cent.|per cent.|per cent.|per cent.|per cent.
 ---------+---------+---------+---------+---------+---------+---------
          |                  _Deflection in Degrees_
     1    |  -0.55  |  -2.32  |  -5.27  |  -9.04  | -11.86  | -19.46
     2    |  -0.10  |  -3.05  |  -6.12  |  -8.55  | -11.92  | -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  | -11.92  | -20.28
 ---------+---------+---------+---------+---------+---------+---------

Such constancy of quantitative results is only possible where we are
dealing with purely physicochemical 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).[231] But
one wonders how it is possible on such an assumption 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 represents 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 electric energy (supplied by batteries inside the
machine) which makes the wheels turn (these take the place of the legs
of the normal animal) no longer flows symmetrically 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.

[231] 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 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 not appeal. Torrey and Bancroft
(for the literature the reader is referred to Bancroft's paper, _Jour.
Exper. Zool._, 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_.

6. It may also be of interest to know that in heliotropism 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 galvanotropism. 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
_Palaemonetes_ 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.[232] It can walk forwards, backwards, or
sidewise. Here we can observe directly 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 to the left, _i. e._, the anode, and only with difficulty
to the right, _i. 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 because 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.[233]

[232] Loeb, J., and Maxwell, S. S., _Arch. f. d. ges. Physiol._, 1896,
lxiii., 121.

[233] That the mechanisms by which heliotropic and galvanotropic
orientation is brought about are identical was shown by Bancroft in
_Euglena_ (Bancroft, _loc. cit._).

[Illustration: FIG. 45]

To understand what happens when the current goes lengthwise through the
body it should be stated that _Palaemonetes_ 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. If a
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 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 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.

[Illustration: FIG. 46]

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) forward. 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.[234] The importance
of the observations 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.

[234] Loeb, J., and Maxwell, S. S., _Arch. f. d. ges. Physiol._, 1896,
lxiii., 121.

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 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 approaches the source of light. If the
source of light be the reflected sky-light the difference of intensity
at both ends of a microscopic organism is so slight that it is beneath
the limit capable of influencing the motions.

[Illustration: FIG. 47]

A simple experiment published by the writer in 1889 suffices to dispel
the idea that the energy gradient determines the direction of the
motion of an animal in tropistic reactions. Let direct sunlight (_S_,
Fig. 47) fall through the upper half of a window (_w w_) upon a table,
and diffused daylight (_D_) through the lower half of the window on
the same table. A test-tube _a c_ is placed on the table in such a
way that its long axis is at right angles to the plane of the window;
and one half _a b_ is in the direct sunlight, the other half in the
shade. If at the beginning 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.[235] This experiment
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 positively 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.

[235] Loeb, J., _Dynamics of Living Matter_, p. 126.

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 the assumption be correct
that the red light is considerably 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 heliotropic 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 {gamma} = 460-490 {nm}, for others a
region in the yellowish-green, near about {gamma} = 520-530 {nm} is
the most efficient.[236] For many plants and for some animals, like
_Eudendrium_ and the larvae 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 algae,
_Chlamydomonas_, has its maximal efficiency in the yellowish-green and
_Euglena_ in the blue. According to observations by Mast, some green
unicellular organisms like _Pandorina_, _Eudorina_, and _Spondylomorum_
seem to behave more like _Chlamydomonas_, while certain others behave
more like _Euglena_.[237] Wasteneys and the writer suggested 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 {gamma} = 520-530 {nm}.

[236] Loeb, 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. Zool._, 1915,
xix., 23; 1916, xx., 217.

[237] Mast, S. O., _Proc. Nat. Acad. Sc._, 1915, i., 622.

The ophthalmologist Hess[238] has utilized the heliotropic 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 {gamma} = 520 {nm} 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 considered, but no such phenomenon exists in colour-blind man.
Moreover, v. Frisch[239] has shown by experiments on the influence of
the background on the colouration of fish as well as by experiments on
bees and 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.

[238] Hess, C., "Gesichtssinn," _Winterstein's Handb. d. vergl.
Physiol._, 1913, iv.

[239] v. Frisch, K., "Der Farbensinn und Formensinn der Biene," _Zool.
Jahrb. Abt. f. allg. Zool. u. Physiol._, 1914, xxxv. See also Ewald, W.
F., _Ztschr. f. Sinnesphysiol._, 1914, xlviii., 285.

9. Heliotropic reactions play a great role in the preservation of
individuals as well as of species. In order to understand this role 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,[240] 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[241] 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 that the presence of some substance secreted by the sex glands
may cause the intensification of the heliotropism which leads to the
nuptial flight.

[240] Loeb, J., _Der Heliotropismus der Tiere_, 1889.

[241] Kellogg, V. L., _Science_, 1903, xviii., 693.

In certain species of _Daphnia_, fresh-water copepods, and of _Volvox_,
a trace of CO{2} suffices to make negatively heliotropic or indifferent
specimens violently positively heliotropic.[242] Certain forms of
marine copepods and the larvae of _Polygordius_ can be made positively
heliotropic by lowering the temperature[243] and the larvae of the
barnacle can be made negatively heliotropic by strong light.[244]
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 ultraviolet light.[245]

[242] Loeb, J., _Arch. f. d. ges. Physiol._, 1906, cxv., 564.

[243] _Ibid._, 1893, liv., 81.

[244] Groom, Theo. T., and Loeb, J., _Biol. Centralbl._, 1890, x., 160;
Ewald, W. F., _Jour. Exper. Zool._, 1912, xiii., 591.

[245] Loeb, J., _Arch. f. d. ges. Physiol._, 1906, cxv., 564; Moore, A.
R., _Jour. Exper. Zool._, 1912, xiii., 573.

A most interesting example of the role of heliotropism in the
preservation of a species is shown in the caterpillars of _Porthesia
chrysorrhoea_. The butterfly lays its eggs upon a shrub. The larvae
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 conditions, this happens
in the spring when the first leaves have begun to form on the shrub.
(The larvae can, however, be induced to leave the nest at any time in
the winter provided the temperature is raised sufficiently.) 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 larvae
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.

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 disappeared; 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 phenomenon 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 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 role 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 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 organism 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 determined 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[246]
has shown the role of adrenalin in the expression of fear. It is,
therefore, both unwarranted and unnecessary to state that hypothetical
emotions determine the tropistic reactions.

[246] 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 role if we assume that it can modify the organisms so that
they become adapted to its peculiarities. Such ideas are difficult
to comprehend 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 deg. 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.

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 deg. C.,
or above, no animals or green alga are found.[247] In hot springs
whose temperature is above 43 deg. he found only the _Cyanophyceae_,
whose structure is more closely related to that of the bacteria
than to that of the algae, inasmuch as they have neither definitely
differentiated nuclei nor chromophores. The highest temperature at
which _Cyanophyceae_ occurred was 63 deg. C. Not all the _Cyanophyceae_
were able to stand temperatures above 43 deg. C., but only a few species.
The other _Cyanophyceae_ were found at a temperature below 40 deg. C.,
and were no more able to stand higher temperatures than the real algae
or animals. The _Cyanophyceae_ of the hot springs were as a rule killed
by a temperature of 73 deg. 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 deg. Among the fungi many forms
can resist a temperature above 43 deg. or 45 deg.; 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 deg. to 90 deg.; while for the spores
of the same bacillus a temperature of from 105 deg. to 120 deg. was
required.[248]

[247] Setchell, W. A., _Science_, 1903, xxvii., 934.

[248] Duclaux, E., _Traite de microbiol._, 1898, i., 280.

Duclaux has called attention to a fact which is of importance for
the investigation of the upper temperature 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.

    It took at 100 deg.         over sixteen hours
    "   "   "  105-110 deg.     two to four hours
    "   "   "  115 deg.         thirty to sixty minutes
    "   "   "  125-130 deg.     five minutes or more
    "   "   "  135 deg.         one to five minutes
    "   "   "  140 deg.         one minute

In warm-blooded animals 45 deg. is generally considered a temperature at
which death occurs in a few minutes; but a temperature of 44 deg., 43
deg., or 42 deg. is also to be considered fatal with this difference
only, that it takes 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. According to Duclaux, it can
be directly observed in micro-organisms that in the fatal temperature
zone the normally homogeneous, or finely granulated, protoplasm 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
_Penicillium_ 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 temperature 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 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 temperature in such a way that their rate
is about doubled for an increase of the temperature of 10 deg. C., and
that this temperature coefficient for 10 deg., Q10, 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.[249] 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 greater
than or equal to 2 for a difference of 10 deg. In chemical reactions
there is also a tendency for Q{10} to become larger for lower
temperature, and coefficients of Q{10} about 5 or 6 have repeatedly
been found for purely chemical reactions between 0 deg. and 10 deg.,
_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{10} with rising temperature which is also noticed
in most life phenomena. Thus Van Slyke and Cullen[250] found that the
reaction rate of the enzyme urease "is nearly doubled by every 10 deg.
rise in temperature between 10 deg. and 50 deg. Within this range the
temperature coefficient is nearly constant and averages 1.91. From O deg.
to 10 deg. it is 2.80, from 50 deg. to 60 deg. it is only 1.09. The
optimum is at about 55 deg." 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 0 deg. 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 reaction 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 of Q{10} with temperature: (1) the supposed
increase in the number of available ferment molecules with increasing
temperature near the lower temperature limit; (2) the temperature
coefficient of the reaction velocity which is nearly =2 for 10 deg.C.;
(3) the diminution of the number of available ferment molecules by
hydrolysis 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 constituents of living matter
are colloidal, _i. e._, consist of particles of a considerably greater
order of magnitude than the molecules of crystalloids.[251]

[249] A full discussion of the literature on temperature coefficients
is given in A. Kanitz's book on _Temperatur and Lebensvorgaenge_,
Berlin, 1915.

[250] Van Slyke, D. D., and Cullen, G. E., _Jour. Biol. Chem._, 1914,
xix., 141.

[251] 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.

We will now show the role of the temperature coefficient upon phenomena
of development. F. R. Lillie and Knowlton[252] 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.[253]

[252] Lillie, F. R., and Knowlton, E. P., _Zool. Bull._, 1897, i.

[253] Hertwig, O., _Arch. mikrosk. Anat._, 1898, li., 319. See also E.
Cohen, _Vortraege fuer Aerste ueber physikalische Chemie._ 2d ed. Leipzig,
1907.

The time required for the eggs to reach definite stages was measured
for different temperatures and it was found that the temperature
coefficient Q{10} between 2.5 deg. and 6 deg. was equal to 10 or more;
between 6 deg. and 15 deg. it was between 2.6 and 4.5; between 10 deg.
and 20 deg. it was 2.9 to 3.3, and between 20 deg. and 24 deg. 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_.[254] The figures on _Arbacia_
have been verified by different observers in different years.

[254] Loeb, J., _Arch. f. d. ges. Physiol._, 1908, cxxiv., 411; Loeb
J., and Wasteneys, H., _Biochem. Ztschr._, 1911, xxxvi., 345; Loeb J.,
and Chamberlain, M. M., _Jour. Exper. Zool._, 1915, xix., 559.

TABLE X

INFLUENCE OF TEMPERATURE UPON THE TIME (IN MINUTES) REQUIRED FROM
INSEMINATION TO THE FIRST CELL DIVISION

    -----------+-----------+----------------------------------
               |        _Arbacia_        |
    Temperature+-----------+-------------+_Strongylocentrotus_
               | Loeb and  |  Loeb and   |    _purpuratus_
               | Wasteneys | Chamberlain |
               |   1911    |    1915     |
    -----------+-----------+-------------+--------------------
      deg. C.  | _Minutes_ |  _Minutes_  |     _Minutes_
         3     |           |             |        532
         4     |           |             |        469
         5     |           |             |        352
         6     |           |             |        275
         7     |   498     |             |        291
         8     |   410     |    411      |        210
         9     |   308     |    297.5    |        159
        10     |   217     |    208      |        143
        11     |   175     |    175      |
        12     |   147     |    148      |        131
        13     |           |    129      |
        14     |           |    116      |        121
        15     |   100     |    100      |        100
        16     |    85.5   |             |
        17     |    70.5   |             |
        18     |    68     |     68      |         87
        19     |           |     65      |         78
        20     |    56     |     56      |         75
        21     |           |     53.3    |         78
        22     |    47     |     46      |         75
        23     |           |     45.5    |  Upper temperature
        24     |           |     42      |       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{10} with a sufficient degree of accuracy (see next
table). It seemed of importance to attempt to decide what the chemical
reaction underlying these reaction velocities is (if it is a chemical
reaction). Loeb and Wasteneys[255] investigated the temperature
coefficient for the rate of oxidations in the newly fertilized egg of
_Arbacia_ and found that the temperature coefficient Q{10} for that
process does not vary in the same way as the temperature coefficient
for cell division.

[255] _Loc. cit._

TABLE XI

TEMPERATURE COEFFICIENTS Q{10} FOR THE RATE OF SEGMENTATION AND
OXIDATIONS IN THE EGGS OF _Strongylocentrotus_ AND _Arbacia_

  -----------+--------------------+------------+-----------------
             |Q{10} for Rate of Segmentation in|Q{10} for Rate of
  Temperature+--------------------+------------+  Oxidations in
             |_Strongylocentrotus_|  _Arbacia_ |    _Arbacia_
  -----------+--------------------+------------+-----------------
    deg. 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
     11-21   |                    |     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 deg., for various
temperatures and does not show the variation from 7 or more to 2.2 for
Q{10} for the rate of segmentation.

Kanitz[256] has shown that in a graph in which the logarithms of the
segmentation velocities are drawn as ordinates and the temperatures as
abscissae 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 deg. and the second from 13-26 deg.; in
_Strongylocentrotus_ between 3-9 deg. and 9-20 deg.

[256] Kanitz, A., _loc. cit._, p. 123.

It was found in these experiments that if measurements of the Q{10}
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 development has been used to
find the conditions determining fluctuating variation. The reader
knows that by this expression are understood the differences between
individuals of a pure strain or breed. These variations are not
inherited, a fact contrary to the idea of Darwin, who assumed that by
the selection of extreme cases of fluctuating variation new varieties
could develop. 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
temperature coefficient might be used to test the idea. We have just
seen that the time required from insemination 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
observation 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 temperature, and that its temperature coefficient is
for each interval of 10 deg. practically identical with the temperature
coefficient of the segmentation for the same interval.[257] The slight
deviations are practically all in the same sense and accounted for by a
slight deficiency in the nature of the experiments. The 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. These two latter
coefficients are practically identical.

[257] Loeb, J., and Chamberlain, M. M., _Jour. Exper. Zool._, 1915,
xix., 559.

TABLE XII

    ---------------+-------------
                   | _Latitude
     _Temperature_ |     of
                   |  Variation_
    ---------------+-------------
        deg. C.    |  _Minutes_
            9      |    52.5
           10      |    39.5
           11      |    26.0
           12      |    22.5
           13      |    19.2
           14      |    17.5
           15      |    13.0
           18      |    12.0
           19      |    12.5
           20      |     9.6
           21      |     8.0
           22      |     7.8
           23      |     8.0
           24      |     8.0
           25      |     5.0
    ---------------+-------------

TABLE XIII

    --------------+------------------------------
                  |  TEMPERATURE COEFFICIENT OF
                  +-------------+----------------
     _Temperature | _Latitude   |
       Interval_  |     of      | _Segmentation_
                  |  Variation_ |
    --------------+-------------+----------------
        deg. C.   |             |
          9-19    |     4.2     |      4.7
         10-20    |     3.9     |      3.8
         11-21    |     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
    --------------+-------------+----------------

If we assume that the temperature coefficient for the segmentation
of the egg is that of a chemical reaction (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 influence of temperature
upon the organism is so constant that if disturbing factors are removed
it would be possible 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
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 disturbing
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 conditions
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. Koeroesy found among soldiers under
the most favourable 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
physicochemical laws might find it preposterous to imagine that the
rate of heart-beat could be used as a thermometer. Yet if we observe
the influence of temperature on the rate 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 demand.[258] 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 temperature 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.

[258] Loeb, J., and Ewald, W. F., _Biochem. Ztschr._, 1913, lviii., 179.

TABLE XIV

    ---------------+--------------------------------------------
     _Temperature_ | _Time Required for Nineteen Heart-beats in
                   |           the Embryo of Fundulus_
    ---------------+--------------------------------------------
        deg. C.    |                 _Seconds_
                   |
          30       |                   6.25
          25       |                   8.5
          20       |                  11.5
          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 _Fundulus_ the
heart-beat is a function almost if not exclusively 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 fluctuating 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 influences 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 temperature 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, _e. g._, the production of
CO{2} in seeds[259] and the assimilation of CO{2} by green plants.[260]
The writer would not be surprised if even the aberrations in the
colour of butterflies under the influence of temperature turned out
to be connected with the temperature coefficient. The experiments of
Dorfmeister, Weismann, Merrifield, Standfuss, and Fischer, on seasonal
dimorphism 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 pupae of _Vanessa
prorsa_ several weeks at a temperature of from 0 deg. to 1 deg.
Weismann succeeded in obtaining from the summer chrysalids specimens
which resembled the winter variety, _Vanessa levana_.

[259] Clausen, H., _Landwirtschaftl. Jahrb._, 1890, xix., 893.

[260] Matthaei, G. L. C., _Trans. Philosoph. Soc._, 1904, cxcvii., 47;
Blackman, F. F., _Ann. of Bot._, 1905, xix., 281.

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 extremely 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 submitted the pupae, and in the
vertical column below are found the varieties that were produced. In
the vertical column A are given the normal forms:

TABLE XV

 ------------+-------------+-------------+--------------+-------------+------------
  0 deg. to  |  0 deg. to  |      A      | +35 deg. to  | +36 deg. to | +42 deg. to
 -20 deg. C. | +10 deg. C. |   (Normal   | +37 deg. C.  | +41 deg. C. | +46 deg. C.
             |             |    Forms)   |              |             |
 ------------+-------------+-------------+--------------+-------------+------------
 _ichnusoides|_polaris_    |_urticae_    |_ichnusa_     |_polaris_    |_ichnusoides
   (nigrita)_|             |             |              |             |  (nigrita)_
 _antigone   |_fischeri_   |_io_         |   ----       |_fischeri_   |_antigone
   (iokaste)_|             |             |              |             |  (iokaste)_
 _testudo_   |_dixeyi_     |_polychloros_|_erythromelas_|_dixeyi_     |_testudo_
 _hygiaea_   |_artemis_    |_antiopa_    |_epione_      |_artemis_    |_hygiaea_
 _elymi_     |_wiskotti_   |_cardui_     |   ----       |_wiskotti_   |_elymi_
 _klymene_   |_merrifieldi_|_atalanta_   |   ----       |_merrifieldi_|_klymene_
 _weismanni_ |_porima_     |_prorsa_     |   ----       |_porima_     |_weismanni_
 ------------+-------------+-------------+--------------+-------------+------------

The reader will notice that the aberrations produced at a very
low temperature (from 0 deg. to -20 deg.C.) are absolutely identical
with the aberrations produced by exposing the pupae to extremely high
temperatures (42 deg. to 46 deg.C.). Moreover, the aberrations produced
by a moderately low temperature (from 0 deg. to 10 deg.C.) are identical
with the aberrations produced by a moderately high temperature (36 deg.
to 41 deg.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 physicochemical
point of view, we must realize that what we call life consists of
a series of chemical reactions, which are connected in a catenary
way; inasmuch as one reaction or group of reactions (_a_) (_e. g._,
hydrolyses) causes or furnishes the material for a second reaction
or group of reactions (_b_) (_e. g._, oxidations). 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 deg. and
lower near 30 deg. But we know also that the temperature coefficients
do not vary equally for the various physiological processes. It is,
therefore, to be expected that the temperature 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 (_b_). If therefore a certain substance is formed at the
normal temperature of the animal in such quantities as are needed for
the catenary reaction (_b_), 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 aberrant chemical effects, which
may underlie the colour aberrations observed by Fischer and other
experimenters.

It is important to notice that Fischer was also able to produce
aberrations through the application of narcotics. Wolfgang Ostwald has
produced experimentally, through variation of temperature, dimorphism
of form in _Daphnia_.

5. Next or equal in importance with the temperature 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 being
salt solutions differing in concentration but containing the three
salts NaCl, KCl, and CaCl{2} in about the same relative concentration,
namely 100 molecules NaCl: 2.2 molecules of KCl: 1.5 molecules of
CaCl{2}. This has suggested to some authors the poetical dream that
our home was once the ocean, but we cannot test the idea since
unfortunately we cannot experiment with the past. Plants, unicellular
fresh-water algae, and bacteria do not demand such a medium for their
existence.

Herbst had shown that when sea-urchin larvae were raised in a medium
in which only one of the constituents of the sea water was lacking
(not only NaCl, KCl, or CaCl{2}, but also Na{2}SO{4}, NaHCO{3}, or
Na{2}HPO{4}), 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 minutiae of the
external medium.

Experiments on a much more favourable animal 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{2} 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.[261] When we raise the eggs in a pure NaCl solution of a
concentration less than or equal to 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.

[261] Loeb, J., "The Poisonous Character of a Pure NaCl Solution."
_Am. Jour. Physiol._, 1900, iii., 329; _Arch. f. d. ges. Physiol._,
1901, lxxxviii., 68; _Am. Jour. Physiol._, 1902, vi., 411; _Biochem.
Zischr._, 1906, ii., 81.

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{2} which must be added
in order to allow fifty per cent. of the eggs to form embryos. (The
eggs were put into the solution an hour or two after fertilization.)

TABLE XVI

    ----------------+----------------------
     _Concentration | _Cc. m/16 CaCl{2}
           of       | Required for 50 c.c.
         _NaCl_     |     NaCl Solution_
    ----------------+----------------------
            m.      |
           3/8      |          0.1
           4/8      |          0.3
           5/8      |          0.5
           6/8      |          0.6
           7/8      |          0.9
           8/8      |        1.2-1.4
           9/8      |        1.8-2.0
          10/8      |        2.0-2.5
          11/8      |          2.0?
          12/8      |        3.0-3.5
          13/8      |          6.0
    ----------------+----------------------

This indicates that the quantity of CaCl{2} required to counteract the
injurious effects of a pure solution of NaCl increases approximately
in proportion to the square of the concentration of the NaCl
solution.[262] 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.

[262] Loeb, J., _Jour. Biol. Chem._, 1915, xxiii., 423.

It was found also that not only Ca but a large number 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;[263] 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 highest
concentration of NaCl solution in which the newly fertilized eggs of
_Fundulus_ can still form an embryo.[264]

    50 c.c. 10/8 m NaCl+4 c.c. m/1 MgCl{2}
    50 c.c. 14/8 m NaCl+1 c.c. m/1 CaCl{2}
    50 c.c. 11/8 m NaCl+1 c.c. m/1 SrCl{2}
    50 c.c.  7/8 m NaCl+1 c.c. m/1 BaCl{2}

[263] Loeb, J., "On the Physiological Effects of the Valency and
Possibly the Electrical Charges of Ions," _Am. Jour. Physiol._, 1902,
vi., 411.

[264] Loeb, J., _Jour. Biol. Chem._, 1914, xix., 431.

On the other hand it was seen that in all the chlorides with a
univalent cation, LiCl, KCl, RbCl, CsCl, NH{4}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.

TABLE XVII

CONCENTRATIONS AT WHICH THE EGGS NO LONGER ARE ABLE TO FORM EMBRYOS

    -------------------+-----------------------------
         _In the       | _In the Same Salts with the
        Pure Salts_    | Addition of 1 c.c. m CaCl{2}
                       |     to 50 c.c. Solution_
    -------------------+-----------------------------
    LiCl  about 6/32m  |           >5/8 m
    NaCl        m/2    |          >14/8 m
    KCl       >11/16 m |           >8/8 m
               <6/8 m  |
    RbCl       >8/8 m  |           >9/8 m
               <7/8 m  |
    CsCl       >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 eggs of the sea
urchin? Before we answer this question, 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,[265] possibly, as T. B. Robertson[266] 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, 1 c.c. 10/8 m CaCl{2} to 50 c.c. 3 m NaCl the eggs will float,
the heart will continue to beat normally and the embryo will continue
to develop for three days or more, because the calcium prevents the
NaCl from entering into the egg.[267] For if we put a newly hatched
embryo into 50 c.c. 3 m NaCl+1 c.c. 10/8 m CaCl{2} it will die almost
instantly; hence the membrane must have acted for three or more days as
a shield which prevented the NaCl from diffusing into the egg in the
presence of CaCl{2}.

[265] Loeb, J., _Arch. f. d. ges. Physiol._, 1905, cvii., 252.

[266] Robertson, T. B., _Ergeb. d. Physiol._, 1910, x., 216.

[267] Loeb, J., _Biochem. Ztschr._, 1912, xlvii., 127.

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 conditions 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[268] 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 concentration of NaCl
if some CaCl{2} is added. Wo. Ostwald[269] has shown the same for
a species of _Daphnia_. We, therefore, come to the conclusion that
the injurious 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 cation Ca and
Mg C{Na+K salts}/C{Ca+Mg salts} is of the greatest importance.

[268] Osterhout, W. J. V., _Bot. Gazette_, 1906, xlii., 127; 1907,
xliv., 257; _Jour. Biol., Chem._, 1906, i., 363.

[269] Ostwald, Wo., _Arch. f. d. ges. Physiol._, 1905, cvi., 568.

6. The importance of this quotient appears in the so-called "behaviour"
of marine animals. We have mentioned the newly hatched larvae of the
barnacle in connection with heliotropism. These larvae swim in a trough
of normal sea water at the surface, being either strongly positively or
negatively heliotropic. 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+KCl (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 bivalent 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{2} and CaCl{2}, 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 whether the quotient
C{NaCl+KCl}/C{MgCl{2}+CaCl{2}}, which just allowed all the animals to
swim at the surface, had a constant value. The MgCl{2}+CaCl{2} solution
was 3/8 m and contained the two metals in the proportion in which they
exist in the sea water; namely, 11.8 molecules MgCl{2} to 1.5 molecules
CaCl{2}. The next table gives the result.[270] 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.

[270] Loeb, J., _Jour. Biol. Chem._, 1915, xxiii., 423.

TABLE XVIII

    -----------+--------------+---------------+-----------
     _Number   |_Concentration|  _C.c. 3/8 m  |_Value of
        of     |      of      |CaCl{2}+MgCl{2}| C{Na+K}/
    Experiment_|   NaCl+KCl_  |   Required_   | C{Mg+Ca}_
    -----------+--------------+---------------+-----------
         1     |    {m/16     |      0.3      |   27.8
               |    {m/8      |    0.4-0.5    |   37.0
               |              |               |
         2     |    {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
               |              |               |
         4     |    {m/8      |      0.5      |   36.0
               |    {m/2      |    1.8-1.9    |   39.2
               |              |               |
         5     |    {m/4      |    0.8-0.9    |   39.2
               |    {m/2      |    1.6-1.7    |   40.3
               |              |               |
         6     |    {5/16 m   |      0.9      |   46.3
               |    {5/8 m    |      1.7      |   49.0
               |              |               |
         7     |    {3/16 m   |      0.6      |   41.7
               |    {6/8 m    |      2.4      |   41.7
    -----------+--------------+---------------+-----------

These experiments indicate that the ratio of C{Na+K}/C{Ca+Mg} remains
very nearly constant with varying concentrations of C{Na+K}.

In former experiments on jellyfish the writer had shown that there
exists an antagonism between Mg and Ca[271], and this observation
was subsequently confirmed by Meltzer and Auer[272] for mammals. It
was observed that in a solution of NaCl+KCl+MgCl{2} the larvae of
the barnacle were also not able to remain at the surface for more
than a few minutes, while an addition of some CaCl{2} made them swim
permanently at the surface. Various quantities of MgCl{2} were added to
a mixture of m/4 or m/2 NaCl+KCl, to find out how much CaCl{2}, was
required to allow them to swim permanently at the surface.

[271] Loeb, J., _Jour. Biol. Chem._, 1905-06, i., 427.

[272] Meltzer, S. J., and Auer, J., _Am. Jour. Physiol._, 1908, xxi.,
400.

TABLE XIX

  ----------------------------------------+---------------------------
                                          |   _C.c. of m/16 CaCl{2}
                                          |    Necessary to Induce
                                          |    the Majority of the
                                          |     Larvae to Swim in_
                                          +--------------+------------
                                          |  m/2 (Na+K)  | m/4 (Na+K)
  ----------------------------------------+--------------+------------
  50 c.c. NaCl+KCl+0.75 c.c. 3/8 m MgCl{2}|              |     0.2
  50 c.c. NaCl+KCl+ 1.5 c.c. 3/8 m MgCl{2}|      0.4     |     0.3
  50 c.c. NaCl+KCl+ 2.5 c.c. 3/8 m MgCl{2}|      0.4     |     0.4
  50 c.c. NaCl+KCl+ 5.0 c.c. 3/8 m MgCl{2}|    0.7-0.8   |   0.7-0.8
  50 c.c. NaCl+KCl+10.0 c.c. 3/8 m MgCl{2}|      1.6     |     1.6
  50 c.c. NaCl+KCl+15.0 c.c. 3/8 m MgCl{2}|      1.8     |
  50 c.c. NaCl+KCl+20.0 c.c. 3/8 m MgCl{2}|      1.8     |
  ----------------------------------------+--------------+------------

In order to interpret these figures correctly we must remember that
we are dealing with two different antagonisms, 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 concentrations of Mg between 2.5 and 10.0 c.c.
3/8 m MgCl{2}--which are those closest to the normal concentration of
Mg in the sea water--we notice that C{Ca} must vary in proportion to
C{Mg}. If we now combine the results of this and the previous paragraph
we may 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._[273] This law of physiologically
balanced solutions seems to be the general expression of the effect of
changes in the constitution of the salt solutions for marine or all
aquatic organisms.

[273] This theory was first expressed by the writer in _Am. Jour.
Physiol._, 1900, iii., 434.

This chapter would not be complete without an intimation of the role of
buffers in the sea water and the blood, by which the reaction of these
media is prevented from changing in a way injurious to the organism.
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.[274] The ratio in which the salts of
the different metals exist in the sea water is another. It is obvious
that the quantitative laws prevailing in the effect of environment
upon organisms leave no more room for the interference of a "directing
force" of the vitalist than do the laws of the motion of the solar
system.

[274] Henderson, L., _The Fitness of the Environment_. See also
Michaelis, L., _Die Wasserstoffionenconzentration_. Berlin. 1914.




CHAPTER XII

ADAPTATION TO ENVIRONMENT


1. It is assumed by certain biologists that the environment 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 mechanism 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,[275] _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 that
the question of use or disuse has nothing to do with the production of
certain harmless chemical compounds in the body. A much more striking
example is offered in the case of galvanotropism. Many organisms
show the phenomenon of galvanotropism, yet, as the writer pointed
out years ago, galvanotropism 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 laboratory 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 responsible for the solar systems. From this viewpoint it
is comprehensible why utterly useless tropisms or structures should
occur in organisms.

[275] Loeb, J., _Der Heliotropismus der Tiere and seine Uebereinstimmung
mit dem Heliotropismus der Pflanzen_. Wuerzburg, 1890 (appeared in 1889).

2. A famous case for the apparent adaptation of animals to environment
has been the blind cave animals. 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 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 _Spelerpes maculicauda_ and _Spelerpes
stejnegeri_. Two others living in caves have quite degenerate eyes,
_Typhlotriton spelaeus_ 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 Amblyopsidae. 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 caves also might have abnormal eyes even if
they had never lived in caves.

Through these facts the old idea becomes questionable, 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.[276] 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.

[276] Loeb, J., _Biol. Bull._, 1915, xxix., 50.

Another method of producing blind fish embryos consists in exposing the
egg immediately, or soon after fertilization, to a temperature between
0 deg. and 2 deg. 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_, _i. e._, a number of them have quite
degenerated eyes. If the eggs have once formed an embryo they can be
kept at the temperature of 0 deg. 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 reaction 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 degeneration which was followed by a complete regeneration.
He showed that this regeneration took place in complete darkness and
that the transplanted eyes remained normal in salamanders kept in
the dark for fifteen 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
consequently 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 absence of
light does not usually interfere with the circulation 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 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 hereditary. 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, however, one point unexplained; namely, the
greater frequency 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.[277] These
animals are all negatively 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 Amblyopsidae 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.

[277] Cuenot has proposed the term preadaptation for such cases and
this term expresses the situation correctly. Cuenot, L., _La Genese des
Especes animales_. Paris, 1911.

Only those animals can thrive in caves which for their feeding and
mating do not depend upon visual mechanisms; 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. This seems 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 competition of seeing forms,
than in the open. In man 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[278] 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.
According 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 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 alteration 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.

[278] Kammerer, P., _Arch. f. Entwcklngsmech._, 1912, xxxiii., 349.

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;[279] and the time of exposure
may be rather short according to Goldfarb.[280] 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 evidence 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.

[279] Loeb, J., _Arch. d. f. ges. Physiol._, 1896, lxiii., 273.

[280] Goldfarb, A. J., _Jour. Exper. Zool._, 1906, iii., 129; 1910,
viii., 133.

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 eyes can survive in a cave while they can only
exceptionally survive in the open. The cause of the degeneration 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 a
lens. Wherever the optic cup of the embryo is transplanted under the
epithelium the latter will be transformed into a transparent lens.
When the upper edge of the iris is injured 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 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 conditions 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, however, 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 investigator examining the salinity of the water
and not knowing 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 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 concentration of the natural sea water
is a little over that of a m/2 solution of NaCl+KCl+CaCl{2} in the
proportion 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{2}
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[281] 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{2} for a month (or possibly indefinitely; the experiment
was discontinued after that period). When a 10/8 m solution 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 fish is naturally
immune to a 9/8 m NaCl+KCl+CaCl{2} solution, by the method of slowly
raising the concentration 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{2} 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{2} solution is weakened.

[281] Loeb, J., _Biochem. Ztschr._, 1913, liii., 391.

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 comparatively
impermeable skin whose permeability is not seriously altered by
sudden changes within these limits of concentration; while if these
limits are exceeded 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
seen that the fish is naturally immune to a 9/8 m solution. 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{2} is added. The explanation of this fact is that the pure NaCl
solution is able to diffuse into the tissues of the animal while
the addition of a trace of CaCl{2} renders the membrane 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 concentration 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 concentrations 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 previously in a weaker NaCl solution they
die if anything 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
(or with a mixture of NaCl+KCl+CaCl{2}) of a higher concentration so
that they become adapted to a 10/8 m solution of NaCl+KCl+CaCl{2} 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.[282]

[282] Loeb, J., _Biochem. Ztschr._, 1913, liii., 391.

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{2} 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 organisms to certain poisons. If
the writer understands these experiments correctly they consisted
in infecting 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. According 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 infected mice the weaker populations of trypanosomes are
killed and only the more resistant survive. These of course continue to
retain their resistance if transplanted to hosts of the same species.
According to this 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 tsetse 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 deg. 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 transferred from a temperature of
22 deg. to that of 38 deg. no longer formed pigment and trimethylamine.
After the cocci had been cultivated for ten or fifteen generations at 38
deg. they failed to form pigment even when transferred back to 22 deg. C.
Dieudonne[283] used _Bacillus fluorescens_ for similar purposes. At
22 deg. it forms a fluorescing pigment and trimethylamine, but not at 35
deg. By constantly cultivating this bacillus at 35 deg. Dieudonne found
that after the fifteenth generation had been cultivated at 35 deg. the
bacillus produced pigment and trimethylamine at 35 deg. Davenport and
Castle[284] found that tadpoles of a frog kept at 15 deg. went into heat
rigour at 40.3 deg. C., while those kept for twenty-eight days at 25 deg.
were not affected by this temperature but went into heat rigour at 43.5
deg. When the latter tadpoles were put back for seventeen days to a
temperature of 15 deg. they had lost their resistance to high temperature
partially, but not completely, since they went into heat rigour at 41.6
deg. 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[285], who found that the coagulation temperature of
their muscle plasm is not altered by keeping cold-blooded animals at
different temperatures.

[283] Dieudonne, A., _Arb. a. d. kais. Gesndhtsmt._, 1894, ix., 492.

[284] Davenport, C. B., and Castle, W. E., _Arch. f. Entwcklngsmech._,
1896, ii., 227.

[285] Kryz, F., _Arch. f. Entwcklngsmech._, 1907, xxiii., 560.

Loeb and Wasteneys[286] found that _Fundulus_ taken from a low
temperature of 10 deg. C. die in less than two hours when suddenly
transferred to sea water of 29 deg. C.; and in a few minutes if suddenly
transferred to a temperature of 35 deg. C. If, however, the fish were
transferred to a temperature of 27 deg. C. for forty hours they could
live indefinitely in sea water of 35 deg. By exposing the fish each day
two hours to a gradually rising temperature they could render them
resistant to a temperature of 39 deg.. The remarkable fact was that fish
if once made resistant to a high temperature (35 deg.) did not lose this
resistance when kept for four weeks at from 10 deg. to 14 deg. C. Control
fish taken from the same temperature died in from two to four minutes;
immunized fish taken from 10 deg. and put directly to 35 deg. C. lived
for many hours or indefinitely. They will even retain this immunity when
kept for two weeks at a temperature of 0.4 deg. C.

[286] Loeb, J., and Wasteneys, H., _Jour. Exper. Zool._, 1912, xii.,
543.

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 sudden increase in temperature brings about
certain changes in the cells (_e. g._, an increase in permeability or
destruction 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 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 Dieudonne and many others.

6. The theory of an adapting effect of the environment 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-Sequard'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
secondary 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 secondary 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[287] that the male offspring of such
couples will in the third generation produce the swelling 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 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.[288]

[287] Kammerer, P., _Arch. f. Entwcklngsmech._, 1909, xxviii., 448.

[288] Bateson, W., _Problems of Genetics_, pp. 201-202. Yale University
Press, 1913.

More recently the same author has reported another hereditary
morphological change brought about by outside conditions.[289] A
certain salamander (_Salamandra 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
induced changes are hereditary. Here again we are dealing with the
inheritance of an acquired morphological character.

[289] Kammerer, P., _Arch. f. Entwcklngsmech._, 1913, xxxvi., 4.

Megusar[290] 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
telephotography, for which no physical explanation has yet been
found.[291] This phenomenon, however, does not lead to any hereditary
change of colour.

[290] Werner, F., _Biol. Centralbl._, 1915, xxxv., 176.

[291] Loeb, J., _The Mechanistic Conception of Life_. Chicago, 1912.

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 produces 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 experiments--or
by exposing butterflies to extreme temperatures, 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 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 offspring 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 trypanosomes or Kammerer's statements
in regard to the inheritance 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 perpetuate 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 which animals
get their food and the suitable conditions of preservation. Can we
understand all these adaptations, without a belief in the heredity
of acquired characters? 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 assumption 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 adaptation 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 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 spermatozoon, and yet we can imitate
the activating effect of a spermatozoon on the egg by definite chemical
compounds, which leads to the suggestion that the activating effect of
the spermatozoon 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 response
is due (Chapter X) to the presence of a photosensitive substance
present in almost all green plants.

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 existence 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 disharmonies 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 existence. We have mentioned the fact observed by
Moenkhouse, the writer, and Newman, that it is possible to fertilize
the eggs of each marine bony fish with the 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 hybridizations,
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 possibly 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 adequate to account for the Mendelian numbers.




CHAPTER XIII

EVOLUTION


Darwin's work has been compared to that of Copernicus 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 stagnation,
squalor, immorality, and misery of the Middle Ages. Copernicus and
Galileo were the first to deliver the intellect from the idea of
a universe 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
humanity.

Darwin assumed that the small fluctuating variations could accumulate
to larger variations and thus cause new forms to originate.

It was the merit of de Vries[292] to have pointed out that fluctuating
variations are not hereditary and hence could not have played the role
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 accidental 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 mutation. 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 or a
modification of a Mendelian factor. If we 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.

[292] de 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.

This way of looking at the origin of variation has had the effect of
putting an end to the vague speculations 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.[293] 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.

[293] For a critical discussion of the details, see Bateson, W.,
_Problems of Genetics_, New Haven, 1913, Chapter X.

It is, however, still uncertain whether the mere addition or loss of
Mendelian characters can lead to the origin of new species. Species
specificity is determined by specific proteins (Chapter III.), while
some Mendelian characters at least seem to be determined by hormones or
substances which need neither be proteins nor specific for the species.




CHAPTER XIV

DEATH AND DISSOLUTION OF THE ORGANISM


1. 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 expression 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 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.[294] This is in harmony with the fact that many parasites
live in the intestine without being digested as long as they are
alive. Fermi concluded 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
(perhaps because the enzyme cannot attach itself to it?), 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 cannot 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. Fredericq, 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 suggestion that a membrane
impermeable for trypsin protects the cells would explain why living
protozoa are not digested by trypsin, but it leaves another fact
unexplained, namely, the autodigestion of all the cells after death by
enzymes contained in the cells themselves.

[294] Fermi, C., _Centralbl. f. Bacteriologie_, Abt. 1, 1910, lvi., 55.

2. The disintegration of the body after death is not caused exclusively
or even chiefly by the digestive enzymes of the intestinal tract or
the micro-organisms entering the dead body from the outside, but by
the enzymes contained in the cells themselves. This phenomenon of
autolysis[295] was first characterized by Hoppe-Seyler.[296]

    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 and
    soaps. This maceration, identical with the pathological conception
    of softening, is accomplished without giving rise to ill odour and
    is a process similar to the one resulting from the action of water,
    acids, and digestive enzymes.

[295] Levene, P. A., _Autolysis_. The Harvey Lectures, 1905-1906, p.
73, gives a full account of the work on this subject up to 1905.

[296] Hoppe-Seyler, F., _Tuebinger med.-chem. Untersuchungen_, 1871, P.
499.

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, especially 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.[297]

[297] Levene, P. A., _Am. Jour. Physiol._, 1904, xii., 276.

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 consequence of the lack of oxidations. The presence
of antiferments must continue after death and cannot be the cause which
prevents the self-digestion during life, since nothing indicates the
destruction of the hypothetical antidigestive enzymes through lack of
oxygen. The recent work of Bradley and Morse[298] and of Bradley[299]
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{2}, 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 formation of
lactic acid continues, but the acid is not oxidized to CO{2} 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 contained in the cells themselves could not attack formerly,
becomes possible. Acid increases the digestibility of a protein,
probably by salt formation. Theoretically we should not be surprised
that while in the liver an increase in the C{H} favours autolysis in
other tissues the same result is produced by the reverse effect. We
might say that the preservation of a certain C{H} probably at or near
the point of neutrality during life prevents self-digestion, while
the gross alteration of the C{H} in either direction after death (or
after the cessation 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 C{H} in the tissues.

[298] Bradley, H. C., and Morse, M., _Jour. Biol. Chem._, 1915, xxi.,
209.

[299] Bradley, H. C., _ibid._, 1915, xxii., 113.

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" molecule.
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: _la vie, c'est la creation_. During life, when nutritive
material is abundant, through the reversible action of certain
enzymes, synthetic compounds 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 C{H} 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 irreversible 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_.[300]

[300] Loeb, J., _Arch. f. d. ges. Physiol._, 1895, lxii., 249.

[Illustration: FIG. 48]

[Illustration: FIG. 49]

[Illustration: FIG. 50]

[Illustration: FIG. 51]

    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 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
    irreversible 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) responsible for death under such
    conditions? In order to obtain an answer to this question, the
    writer investigated the effect of the lack of oxygen upon the
    heart-beat of the embryo of the same fish _Ctenolabrus_. 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, comparable 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 embryo can
    continue to beat for about twelve hours in a current 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 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[301]; as a rule the
    membrane of the infusorian bursts or breaks at one point, whereby
    the liquid contents flow out. Hardesty and the writer found
    that Paramoecium becomes more strongly vacuolized when deprived
    of oxygen, and at last bursts. Amoebae 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 observation. 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.[302]

[301] Budgett, S. P., _Am. Jour. Physiol._, 1898, i., 210.

[302] Loeb, J., _The Dynamics of Living Matter_, New York, 1906, pp.
19-21.

The phenomenon of rapid disintegration when deprived of oxygen (or
in the presence of KCN) seems to be general as Child[303] has shown
in extensive experiments. Child has used it to show that younger
animals disintegrate more rapidly than older or larger ones, and he
uses this fact for a theory of senescence. He connects the more rapid
disintegration of the young animal with a greater metabolism.[304]
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 liquefied. 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.

[303] Child, C. M., _Senescence and Rejuvenescence_, Chicago, 1915.

[304] It 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.

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 _Ctenolabrus_) 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 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 is 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 characteristic as any morphological peculiarity of a species.
No species can exist unless the natural life of its individuals
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 Psychidae, 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.

In the Zoological 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[305] 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 ephemeridae 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 contained 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 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 wisdom 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.

[305] Metchnikoff, E., _Ann. d. l'Inst. Pasteur_, 1915, xxix., 477.

5. Minot had tried to prove that the death of metazoa is due to the
greater differentiation and specialization 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 discussion 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.

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[306] observed that eggs which ripen
disintegrate very rapidly when not fertilized. This disintegration may
be due to a process of autolysis, which 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.

[306] Loeb, J., _Biol. Bull._, 1902, iii., 295.

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 aerobe the mature unfertilized egg is an
anaerobe. This latter statement, however, becomes doubtful since the
presence of oxygen may help the disintegration only indirectly 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 short and that the entrance of a spermatozoon 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 accompanies 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 coefficient for the duration of life or at least be able
to show that if all other conditions are the same the duration of life
is for a given organism a function of temperature. The writer[307]
investigated the duration of life of fertilized and unfertilized eggs
of _Strongylocentrotus purpuratus_ for the upper temperature limits.

[307] Loeb, J., _Arch. f. d. ges. Physiol._, 1908, cxxiv., 411.

TABLE XX

    -------------+-----------------------------------------------
                 |Duration of life of the eggs of _S. purpuratus_
    -------------+-----------------------+-----------------------
    _Temperature_|    _Unfertilized_     |     _Fertilized_
    -------------+-----------------------+-----------------------
       deg. C.   |       _Minutes_       |       _Minutes_
                 |                       |
         32      |         { > 1-1/6     |            1-1/2
                 |         { < 2         |
                 |                       |
         31      |                       |        { > 2-1/4
                 |                       |        { < 3
                 |                       |
         30      |         { > 3         |        { > 4
                 |         { < 5         |        { < 5
                 |                       |
         29      |                       |        { > 6
                 |                       |        { < 7
                 |                       |
         28      |        {  > 8         |       { > 11
                 |        { < 10         |       { < 13
                 |                       |
         27      |      about 18         |       { > 20
                 |                       |       { < 22
                 |                       |
         26      |        { > 35         |       { > 35
                 |        { < 40         |       { < 40
                 |                       |
         25      |                       |       { > 76
                 |                       |       { < 81
                 |                       |
         24      |       { > 168         |      { > 192
                 |       { < 200         |      { < 209
                 |                       |
                 |                       |        _Hours_
                 |                       |
         22      |                       |           10-1/5
                 |                       |
         21      |                       |           24
                 |                       |
         20      |                       |           72
    -------------+-----------------------+-----------------------

These observations show a very high temperature coefficient near the
upper temperature limit, and this may account at least partly for the
fact that in tropical seas the pelagic fauna is so much more limited
than in polar seas.[308] It is quite probable that the high temperature
coefficients at the utmost limits are only an expression of the
coagulation time of certain proteins.

[308] K. Brandt ("Ueber den Nitratgehalt des Ozeanwassers and 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.

P. and N. Rau state that in the cold certain butterflies 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 _Drosophila_. Newly hatched flies were
kept first without food except water and air at 34 deg., 28 deg., 24
deg., 19 deg., 14 deg., and 10 deg., and second with cane sugar. The
average duration of life was as follows:

  _With water_         days    _With cane sugar_   days
    34 deg.             2.1                         6.2
    28 deg.             2.4                         7.2
    24 deg.             2.4                         9.4
    19 deg.             4.1                        12.3
    14 deg.             8.3
    10 deg.            11.9

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
experiments 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. Metchnikoff[309] has furnished the scientific facts for our
understanding of senescence. He has demonstrated that the changes
in tissue which give rise to phenomena of senility are due to the
action of phagocytes. 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 tadpole 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 simultaneously
and that both must be caused by a constituent of the blood. Such a
constituent may be responsible for phagocytosis and autolysis in
the organs undergoing 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 suppose 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.

[309] Metchnikoff, E., _The Prolongation of Life_. New York, 1907.

Certain plants like the Californian _Sequoia gigantea_ may be
considered as practically immortal since they live several thousands
of years; other plants, the annuals, die after fructification.
Metchnikoff quotes from a letter by de Vries that this author prolonged
the life of _OEnotheras_ 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 addition 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. _See_ Balanced salt solutions.

  _Antennularia antennina_, 194, 196

  Apes, blood relationship to man, 54, 56 ff.

  Apolant, 45

  _Arbacia_, 75 ff., 96, 99, 101, 111, 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;
    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 determination of sex, 125

  Artificial production of life, 38-39

  Assimilation of CO{2} without chlorophyll, 17 ff.

  _Asterias_, 49, 81, 110, 363;
    _ochracea_, 73 ff.;
    _capitata_, 74

  _Asterina_, 75, 81, 110

  Astrospheres, 115 ff., 192

  Auer, J., 315

  Autolysis, 351 ff.

  _Avena_, 263


  _B. coli communis_, 36;
    _typhosus_, 36;
    _fluorescens_, 334

  Bacteria, growth of, 15 ff., 29, 71 ff.;
    specificity in, 41 ff.

  "_Bacterio-purpurin_," 41

  Balanced salt solutions, 307-317;
    theory of, 317;
    and adaptation, 331 ff.

  _Balanus_, 259

  Baltzer, F., 215 ff.

  Bancroft, F. W., 70, 125, 127, 264, 269 ff.

  Bang, 63

  Bardeen, C. R., 174 ff.

  Barnacle, larvae 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

  Beijerinck, M., 20

  Berkeley, Lord, 111

  Bernard, Claude, 2 ff., 26, 159, 350, 354, 355, 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 reaction, 61 ff.;
    hemoglobin crystals, 64 ff.

  Blood serum, precipitin reaction of, 54 ff.;
    effect of, on unfertilized eggs, 101 ff., 124

  Blowfly, heliotropism of larvae 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 ff., 177

  Buchner, 24

  Budgett, 358

  Buller, 93

  Bunsen-Roscoe, law of, 11, 256 ff., 261, 263, 264

  Burrows, 31


  _Campanularia_, 178, 181

  Cannon, W. B., 285

  _Carcinus maenas_, 217

  _Cardamine pratensis_, 90

  Carrel, 31

  _Cassia bicapsularis_, 37

  Castle, W. E., 89 ff., 335

  Caullery, M., 158, 180, 217

  Cave animals, 319 ff.

  Cell division, 15, 29, 129 ff.;
    suppression of, 113 ff.

  Cells, nutritive media of, 15 ff;
    immortality of, 30 ff.;
    migrating, 44;
    mesenchyme, 51 ff., 130 ff., 147, 155 ff.

  _Cerianthus membranaceus_, 171 ff., 188, 361

  _Chaetopterus_, 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, role 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

  Crossing over of chromosomes, 241 ff.

  Crystals, differences between living organisms and, 14 ff.

  _Ctenolabrus_, 355, 357, 359

  _Ctenophores_, 142

  Cuenot, L., 12, 324

  Cullen, G. E., 24, 291

  _Cuma rathkii_, 318

  _Cyanophyceae_, 287

  _Cytisus biflorus_, 37

  Cytoplasm 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, 110, 111, 123, 126, 186

  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

  Dieudonne, 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 ff., 147, 150, 169 ff.,
               180 ff., 184 ff.

  _Drosophila ampelophila_, 204 ff., 237, 243, 322, 347, 366

  Duclaux, E., 288, 289

  v. Dungern, 80

  Duration of life, 360 ff.

  Durham, 249

  Dutrochet, 154

  Dzierzon, 208


  Ectoderm formation, 130 ff.

  Egg, as the future embryo, 8, 9, 70, 126, 151;
    artificial parthenogenesis of, 95 ff.;
    organisms from, 128 ff.;
    determining unity of organism, 151-152;
    chromosomes 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., 341;
    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 permeability, 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 regeneration in _Bryophyllum_, 161 ff.

  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

  Fredericq, 351

  Free-martin, cause of sterility, 218-219

  Friedenthal, H., 53 ff., 60

  Frisch, K., 278, 279

  Froeschel, 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.


  Galileo, 346

  Galvanotropism, 11, 270 ff., 319

  Gay, F. P., 62 ff.

  Generation, spontaneous, 14 ff., 34

  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;
    influence of cell size, 187

  Gudernatsch, J. F., 155, 255, 342

  Guyer, 124

  Gynandromorphism, 209


  Haeckel, 346

  Half-embryos and whole embryos, 141, 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

  Hess, C., 278

  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.

  Huxley, 346

  Hybridization, heterogeneous, in sea urchins, 48 ff., 73 ff.;
    in fishes, 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 ff.

  Inhibition of regeneration in _Bryophyllum_, 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.


  Jacoby, 352

  Janda, V., 219 ff.

  Jansky, 67

  Janssens, 242

  Jennings, H. S., 264 ff.

  Jensen, 45

  Joest, 46

  Johannsen, W., 42, 333

  Jones, 352

  Jost, 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. Knaffl, E., 106

  Knowlton, E. P., 292

  Kofoid, C. A., 143

  v. Koeroesy, 300

  Korschelt, 361

  Krakatau, 21

  Kraus, 54 ff.

  Krogh, 292

  Kryz, F., 335

  Kupelwieser, H., 75


  Lack of oxygen, influence on disintegration of tissue, 355 ff.

  Ladoff, S., 224

  Lamarck, 6

  _Laminaria_, 165

  Landois, L., 53

  Landsteiner, 66

  _Lanice_, 143 ff.

  Lankester, E. R., 41

  Leathes, J. B., 63

  _Leucaena leucocephala_, 37

  Levene, 351, 352

  Lewis, 183, 344, 364

  Light, influence on organ formation, in cave animals, 319 ff.;
    in _Proteus_, 325;
    in _Eudendrium_, 326.
    _See also_ Heliotropism.

  Lillie, F. R., 80, 82 ff., 87 ff., 93, 134, 191, 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 characters 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

  _Lygaeus_, 201

  _Lymantria dispar_, 220

  _Lymnaeus_, 142

  Lymphocytes, role of, 45 ff.

  Lyon, E. P., 134 ff.


  Macfadyen, A., 36

  Maeterlinck, 255

  Magnus, W., 60

  Maltase, synthetic action of, 25

  Marchal, P., 222 ff., 254

  _Margelis_, 192

  Mass of chromatin and of cytoplasm, 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.;
    artificial, 98 ff.

  Mendel, G., 23, 229 ff.

  Mendelian characters, and evolution, 70, 348;
    and internal secretions, 243, 348;
    and enzymes, 247, 248, 249

  Mendelian, factors of heredity, 4 ff., 68, 151 ff.;
    mutation, 66;
    dominant, 90;
    segregation, 229 ff.
    _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 induced by thyroid, 155, 156

  Metchnikoff, 361 ff., 367 ff.

  Michaelis, L., 62, 317

  _Micrococcus prodigiosus_, 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

  Muller, H. J., 229, 231 ff.

  Murphy, J. B., 44 ff.

  Mutation, 6, 42, 243; and evolution, 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

  _Nostocaceae_, 21

  Nussbaum, M., 149

  Nuttall, G. H. F., 56 ff.


  _Ocneria dispar_, 225

  _OEnotherus_, 369

  Onslow, H., 249

  Organ-forming substances or hormones in regeneration, 154 ff.;
    causing metamorphosis in tadpoles, 155-157;
    decidua formation, 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


  _Palaemon_, 193

  _Palaemonetas_, 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 ff

  Pauli, W., 289

  Pavy, 350

  Payne, F., 322

  Pearl, R., 203, 244

  _Penicillium_, 289

  _Pennaria_, 192

  Pepsin, synthetic action of, 28, 62, 63

  Pfeffer, 92 ff.

  Phagocytosis, 367

  _Planaria_, 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, 230 ff.

  _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, role in transmission of spores through
                      interstellar space, 34 ff.

  _Rana, esculenta_, 46;
    _palustris_, 46;
    _virescens_, 46

  Rate of segmentation, a non-Mendelian hereditary character, 246

  Rau, 366

  Reaction, tropistic, 11 ff., 92 ff., 147, 178, 187, 255 ff.;
    precipitin, 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 Planarians, 173-176;
    in _Alpheus_, 176;
    and autolysis, 178-181;
    of lens, 182, 183;
    external influences on, 192 ff.;
    of gonads in hermaphrodites, 219

  Regulation, 139, 140, 141;
    in regeneration, _see_ Regeneration.

  Reichert, E. T., 64 ff.

  _Reseda_, 90

  Resistance of spores, 36;
    seeds, 36 ff.

  Reversibility of development, in _Campanularia_, 178 ff.;
    in Ascidians, 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;
    _cerevisiae_, 60

  _Sacculina_, 216 ff.

  Sachs, 88

  v. Sachs, J., 145, 154 ff., 159, 161, 184

  _Salamandra maculosa_, 339

  Salkowski, 352

  Salts required for life, 306 ff.

  Sansum, W. D., 64

  _Schizophyceae_, 21

  Schleip, W., 213

  Schoenbein, 358

  Schottelius, 334, 337

  Schroeder, 14, 33

  Schultze, O., 141

  Schuetze, 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, 125;
    of twins, 211

  Sex chromosome, 199 ff.

  Sex determination, cytological basis of, 198 ff.;
    physiological basis of, 214 ff.

  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, 40 ff.;
    specificity of, 41 ff.;
    incompatibility 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.;
    cultivating 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 differentiation, 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;
    larvae of, 49 ff.

  Sturtevant, A. H., 229 ff.

  Styela, 146

  Sulphur bacteria, 19 ff.

  Supergenes, 5, 9, 136, 319

  Sutton, W. S., 68, 233 ff.

  Synthesis of living matter, by micro-organisms, 15 ff.;
    by enzymes, 24 ff.

  Synthetic action of enzymes, 23 ff., 38


  _Taenia_, 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 heliotropism, 280;
    upper limit for organisms, 287 ff.;
    effect on life, 288 ff.;
    on butterflies, 303 ff.;
    adaptation to, 334 ff.

  Temperature coefficient, 290 ff., 305;
    for enzyme, 291;
    for development, 292 ff.;
    for oxidations, 295;
    and fluctuating variation, 296 ff.;
    for heart-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.;
    specificity 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, 148;
    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 development, 147;
    of cave animals, 324

  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. Uexkuell, J., 4 ff., 128, 139

  Uhlenhuth, E., 157, 183, 187

  Uhlenhuth, P., 55, 58, 66, 322

  Underhill, F. P., 23


  _Vanessa, prorsa_, 303;
    _levana_, 303

  Vaney, 217

  Van Slyke, D. D., 22, 24, 291

  van't Hoff, 24 ff., 290, 292, 296

  Variation, 6, 297 ff., 346-348

  Vitzou, 159

  _Volvox_, 280


  Walcott, 42, 61

  Warburg, O., 117 ff.

  Warming, 41

  Wasps, polymorphism in, 222-224;
    sex determination, 255 ff.

  Wassermann, 55

  Wasteneys, H., 29, 82, 87, 112, 113, 117, 191, 277, 293, 295, 335, 364

  Weiggert, 188

  Weismann, 7, 30, 303

  Wells, H. G., 62, 69

  Welsh, D. A., 60

  Werner, F., 340

  Wheeler, W. M., 43

  White, J., 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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American Race," etc. Edited by LIVINGSTON FARRAND, Columbia University.
8^o.

    "Professor Brinton his shown in this volume an intimate and
    appreciative knowledge of all the important anthropological
    theories. No one seems to have been better acquainted with the very
    great body of facts represented by these sciences."--_Am. Journal
    of Sociology._

=11.=--=Experiments on Animals.= By STEPHEN PAGET. With an Introduction
by Lord Lister. Illustrated. 8^o.

    "To a large class of readers this presentation will be attractive,
    since it gives to them in a nut-shell the meat of a hundred
    scientific dissertations in current periodical literature. The
    volume has the authoritative sanction of Lord Lister."--_Boston
    Transcript._

=12.=--=Infection and Immunity.= With Special Reference to the
Prevention of Infectious Diseases. By GEORGE M. STERNBERG, M.D., LL.D.,
Surgeon-General U. S. Army (Retired). Illustrated. 8^o.

    "A distinct public service by an eminent authority. This admirable
    little work should be a part of the prescribed reading of the head
    of every institution in which children of youths are gathered.
    Conspicuously useful."--_N. Y. Times._

=13.=--=Fatigue.= By A. MOSSO, Professor of Physiology in the
University of Turin. Translated by MARGARET DRUMMOND, M.A., and W.
B. DRUMMOND, M.B., C.M., F.R.C.P.E.; extra Physician, Royal Hospital
for Sick Children, Edinburgh; Author of "The Child, His Nature and
Nurture." Illustrated. 8^o.

    "A book for the student and for the instructor, full of interest,
    also for the intelligent general reader. The subject constitutes
    one of the most fascinating chapters in the history of medical
    science and of philosophical research."--_Yorkshire Post._ #/

=14.=--=Earthquakes.= In the Light of the New Seismology. By CLARENCE
E. DUTTON, Major, U. S. A. Illustrated. 8^o.

    "The book summarizes the results of the men who have accomplished
    the great things in their pursuit of seismological knowledge.
    It is abundantly illustrated and it fills a place unique in the
    literature of modern science."--_Chicago Tribune._

=15.=--=The Nature of Man.= Studies in Optimistic Philosophy. By ELIE
METCHNIKOFF, Professor at the Pasteur Institute. Translation and
introduction by P. CHAMBERS MITCHELL, M.A., D.Sc. Oxon. Illustrated.
8^o.

    "A book to be set side by side with Huxley's Essays, whose
    spirit it carries a step further on the long road towards its
    goal."--_Mail and Express._

=16.=--=The Hygiene of Nerves and Mind in Health and Disease.= By
AUGUST FOREL, M.D., formerly Professor of Psychiatry in the University
of Zurich. Authorized Translation. 8^o.

    A comprehensive and concise summary of the results of science in
    its chosen field. Its authorship is a guarantee that the statements
    made are authoritative as far as the statement of an individual can
    be so regarded.

=17.=--=The Prolongation of Life.= Optimistic Essays. By ELIE
METCHNIKOFF, Sub-Director of the Pasteur Institute. Author of "The
Nature of Man," etc. 8^o. Illustrated. Net, $2.50. Popular Edition.
With an introduction by Prof. CHARLES S. MINOT.

    In his new work Professor Metchnikoff expounds at greater length,
    in the light of additional knowledge gained in the last few years,
    his main thesis that human life is not only unnaturally short but
    unnaturally burdened with physical and mental disabilities. He
    analyzes the causes of these disharmonies and explains his reasons
    for hoping that they may be counteracted by a rational hygiene.

=18.=--=The Solar System.= A Study of Recent Observations. By Prof.
CHARLES LANE POOR, Professor of Astronomy in Columbia University. 8^o.
Illustrated.

    The subject is presented in untechnical language and without the
    use of mathematics. Professor Poor shows by what steps the precise
    knowledge of to-day has been reached and explains the marvellous
    results of modern methods and modern observations.

=19.=--=Heredity.= By J. ARTHUR THOMSON, M.A., Professor of Natural
History in the University of Aberdeen; Author of "The Science of Life,"
etc. 8^o. Illustrated.

    The aim of this work is to expound, in a simple manner, the facts
    of heredity and inheritance as at present known, the general
    conclusions which have been securely established, and the more
    important theories which have been formulated.

=20.=--=Climate--Considered Especially in Relation to Man.= By
ROBERT DECOURCY WARD, Assistant Professor of Climatology in Harvard
University. 8^o. Illustrated.

    This volume is intended for persons who have not had special
    training in the technicalities of climatology. Climate covers a
    wholly different field from that included in the meteorological
    text-books. It handles broad questions of climate in a way which
    has not been attempted in a single volume. The needs of the teacher
    and student have been kept constantly in mind.

=21.=--=Age, Growth, and Death.= By CHARLES S. MINOT, James Stillman
Professor of Comparative Anatomy in Harvard University, President
of the Boston Society of Natural History, and Author of "Human
Embryology," "A Laboratory Text-book of Embryology," etc. 8^o.
Illustrated.

    This volume deals with some of the fundamental problems of biology,
    and presents a series of views (the results of nearly thirty years
    of study), which the author has correlated for the first time in
    systematic form. #/

=22.=--=The Interpretation of Nature.= By C. LLOYD MORGAN, LL.D.,
F.R.S. Crown 8vo.

    Dr. Morgan seeks to prove that a belief in purpose as the causal
    reality of which nature is an expression is not inconsistent with a
    full and whole-hearted acceptance of the explanations of naturalism.

=23.=--=Mosquito Life.= The Habits and Life Cycles of the Known
Mosquitoes of the United States; Methods for their Control; and Keys
for Easy Identification of the Species in their Various Stages. An
account based on the investigation of the late James William Dupree,
Surgeon-General of Louisiana, and upon the original observations
by the Writer. By EVELYN GROESBEECK MITCHELL, A.B., M.S. With 64
Illustrations. 8^o.

    This volume has been designed to meet the demand of the constantly
    increasing number of students for a work presenting in compact form
    the essential facts so far made known by scientific investigation
    in regard to the different phases of this, as is now conceded,
    important and highly interesting subject. While aiming to keep
    within reasonable bounds, that it may be used for work in the field
    and in the laboratory, no portion of the work has been slighted, or
    fundamental information omitted, in the endeavor to carry this plan
    into effect.

=24.=--=Thinking, Feeling, Doing.= An Introduction to Mental Science.
By E. W. SCRIPTURE, Ph.D., M.D., Assistant Neurologist Columbia
University, formerly Director of the Psychological Laboratory at Yale
University. 189 Illustrations. 2d Edition, Revised and Enlarged. 8^o.

    "The chapters on Time and Action, Reaction Time, Thinking Time,
    Rhythmic Action, and Power and Will are most interesting. This book
    should be carefully read by every one who desires to be familiar
    with the advances made in the study of the mind, which advances,
    in the last twenty-five years, have been quite as striking and
    epoch-making as the strides made in the more material lines of
    knowledge."--_Jour. Amer. Med. Ass'n._, Feb. 22, 1908.

=25.=--=The World's Gold.= By L. DE LAUNAY, Professor at the Ecole
Superieure des Mines. Translated by Orlando Cyprian Williams. With an
Introduction by Charles A. Conant, author of "History of Modern Banks
of Issue," etc. 8^o.

    M. de Launay is a professor of considerable repute not only in
    France, but among scientists throughout the world. In this work
    he traces the various uses and phases of gold; first its geology;
    secondly, its extraction; thirdly, its economic value.

=26.=--=The Interpretation of Radium.= By FREDERICK SODDY, Lecturer
in Physical Chemistry in the University of Glasgow. Third Edition,
rewritten, with data brought down to 1912. 8^o With 33 Diagrams and
Illustrations.

    As the application of the present-day interpretation of Radium
    (that it is an element undergoing spontaneous disintegration) is
    not confined to the physical sciences, but has a wide and general
    bearing upon our whole outlook on Nature, Mr. Soddy has presented
    the subject in non-technical language, so that the ideas involved
    are within reach of the lay reader. No effort has been spared to
    get to the root of the matter and to secure accuracy, so that
    the book should prove serviceable to other fields of science and
    investigation, as well as to the general public.

=27.=--=The Social Evil.= With Special Reference to Conditions Existing
in the City of New York. A Report Prepared in 1902 under the Direction
of the Committee of Fifteen. Second Edition, Revised, with New Material
Covering the Years 1902-1911. Edited by EDWIN R. A. SELIGMAN, LL.D.,
McVickar Professor of Political Economy in Columbia University. 8vo.

    A study that is far from being of merely local interest and
    application. The problem is considered in all its aspects and, for
    this purpose, reference has been made to conditions prevailing in
    other communities and to the different attempts foreign cities have
    made to regulate vice.

=28.=--=Microbes and Toxins.= By ETIENNE BURNET, of the Pasteur
Institute, Paris. With an Introduction by Elie Metchnikoff,
Sub-Director of the Pasteur Institute, Paris. With about 71
Illustrations.

    A well-known English authority said in recommending the volume:
    "Incomparably the best book there is on this tremendously important
    subject. In fact, I am assured that nothing exists which gives
    anything like so full a study of microbiology." In the volume are
    considered the general functions of microbes, the microbes of the
    human system, the form and structure of microbes, the physiology of
    microbes, the pathogenic protozoa, toxins, tuberculin and mallein,
    immunity, applications of bacteriology, vaccines and serums,
    chemical remedies, etc. #/

=29.=--=Problems of Life and Reproduction.= By MARCUS HARTOG, D. Sc.,
Professor of Zoology in University College, Cork. 8vo.

    The author uses all the legitimate arms of scientific controversy
    in assailing certain views that have been widely pressed on
    the general public with an assurance that must have given
    many the impression that they were protected by the universal
    consensus of biologists. Among the subjects considered are: "The
    Cellular Pedigree and the Problem of Heredity"; "The Relation
    of Brood-Formation to Ordinary Cell-Division"; "The New Force,
    Mitokinetism"; "Nuclear Reduction and the Function of Chroism";
    "Fertilization"; "The Transmission of Acquired Characters";
    "Mechanism and Life"; "The Biological Writings of Samuel Butler";
    "Interpolation in Memory"; "The Teaching of Nature Study."

=30.=--=Problems of the Sexes.= By JEAN FINOT, Author of "The Science
of Happiness," etc. Translated under authority by Mary J. Safford. 8vo.

    A masterly presentation of the attitude of the ages toward women
    and an eloquent plea for her further enfranchisement from imposed
    and unnatural limitations. The range of scholarship that has been
    enlisted in the writing may well excite one's wonder, but the tone
    of the book is popular and its appeal is not to any small section
    of the reading public but to all the classes and degrees of an age
    that, from present indications, will go down in history as the
    century of Woman.

=31.=--=The Positive Evolution of Religion.= Its Moral and Social
Reaction. By FREDERIC HARRISON. 8vo.

    The author has undertaken to estimate the moral and social reaction
    of various forms of Religion--beginning with Nature Worship,
    Polytheism, Catholicism, Protestantism, and Deism. The volume may
    be looked upon as the final word, the summary of the celebrated
    author's philosophy--a systematic study of the entire religious
    problem.

=32.=--=The Science of Happiness.= By JEAN FINOT, Author of "Problems
of the Sexes," etc. Translated from the French by Mary J. Safford. 8^o.

    In this work, which was crowned by the Academy, the author
    considers a subject, the solution of which offers more enticement
    to the well-wisher of the race than the gold of the Incas did to
    the treasure-seekers of Spain, who themselves doubtless looked
    upon the coveted yellow metal, however mistakenly, as a key to the
    happiness which all are trying to find. "Amid the noisy tumult of
    life, amid the dissonance that divides man from man," remarks M.
    Finot, "the Science of Happiness tries to discover the divine link
    which binds humanity to happiness through the soul and through the
    union of souls." The author considers the nature of happiness and
    the means of its attainment, as well as many allied questions.

=33.=--=Genetic Theory of Reality.= Being the Outcome of Genetic Logic
as Issuing in the Aesthetic Theory of Reality Called Pancalism. By
JAMES MARK BALDWIN, Ph.D., D.Sc., LL.D., Foreign Correspondent of the
Institute of France, Author of "History of Psychology," etc.

    The author here states the general results of the extended studies
    in genetic and social science and anthropology made by him and
    others, and gives a critical account of the history of the
    interpretation of nature and man, both racial and philosophical.

    The book offers an _Introduction to Philosophy_ from a new point of
    view. It contains, also, a valuable glossary of the terms employed
    in these and similar discussions.

=34.=--=Mosquito Control in Panama.= The Eradication of Malaria and
Yellow Fever in Cuba and Panama. By J. A. LE PRINCE, C.E., A. M.,
Chief Sanitary Inspector, Isthmian Canal Commission, 1904-1914, and
A. J. ORENSTEIN, M.D., Assistant Chief Sanitary Inspector, Isthmian
Canal Commission. With an introduction by L. O. HOWARD, Ph.D.,
Entomologist and Chief, Bureau of Entomology, United States Department
of Agriculture. 8^o. 95 illustrations.

    Mr. Le Prince's books will be not only of great practical
    importance as a guide to future work of the same character,
    especially in the Tropics, but also of permanent historic value.

=35.=--=The Organism as a Whole.= From a Physico-Chemical Viewpoint. By
JACQUES LOEB, Author of "Comparative Physiology of the Brain." 8^o.

    The author accounts for the harmonious character of the organism on
    a purely physico-chemical basis, without the assumption of design
    on the one hand, and without the formulation of too definite a
    theory of evolution on the other. The book contains, in addition to
    the text, all the necessary illustrations.




      *      *      *      *      *      *




Transcriber's note:

The oe-ligature is replaced by the two two letters oe.

Footnotes have been repositioned to below the paragraph of reference or
below the relevant block quote.

A small number of spelling anomalies were noted and these have mostly
been corrected, but a few that possibly represent authentic contemporary
alternatives have been left unchanged - see below.

Corrections:

  spermatozoon-->spermatozoon
  i.e.-->i. e.
  e.g.-->e. g.
  nermaphrodite-->hermaphrodite
  suceeded-->succeeded
  of-->of
  tryosinase-->tyrosinase
  in-as-much-->inasmuch
  ultra-violet-->ultraviolet
  view-point-->viewpoint
  Fredericq-->Fredericq
  Koroesy-->Koeroesy
  Sitzngsber-->Sitzungsber
  negaceros-->megaceros

Variants:

  clew/clue
  Entswcklngsmech/Entwcklngsmech/Entwicklngsmech
  peroxidase/peroxydase (latter spelling in quoted text.)
  20 deg.C/20 deg. C (spaced and unspaced temperature specifications)
  8vo/8^o



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