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., _Leçons sur les Phénomènes 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. Uexküll,[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. Uexküll suggests

a kind of Platonic “idea” as a peculiar characteristic of life which

accounts for the purposeful character of the organism.

v. Uexküll 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. Uexküll’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. Uexküll 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. Uexküll, J., _Bausteine zu einer biologischen Weltanschauung_.

München, 1913.

[4] v. Uexküll, J., _Bausteine zu einer biologischen Weltanschauung_.

München, 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. Uexküll 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. Uexküll 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 rôle 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 spermatozoön 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 spermatozoön 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

rôle 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.

Cuénot 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 sér., 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 rôle in the alcoholic fermentation of sugar.

Chlorophyll, under the influence of the red rays of light, manufactures

the sugars from the CO₂ 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 CO₂ 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

CO₂.”[9] “Since the oxidation of NH₃ 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 CaSO₄ 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) 2H₂S + O₂ = 2H₂O + S₂

(2) S₂ + 3O₂ + 2H₂O = 2H₂SO₄

The sulphuric acid is at once neutralized by carbonates.

Winogradsky assumes that the oxidation of H₂S by the sulphur bacteria

is the source of energy which plays the same rôle as the oxidation

of NH₃ 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 CO₂ 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

Na₂S₂O₃, is added to sea water. These bacteria can only develop if CO₂

from the air is admitted or when carbonates are present. For these

organisms the CO₂ cannot be replaced by glucose, urea, or other organic

substances. Such bacteria must therefore possess the power of producing

sugar and starch from CO₂ 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, SO₄, CO₂,

and O₂ besides NH₃, or SH₂, 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 algæ 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 algæ referred to were according to Euler of the nostoc

type. Nostoc does not require sugar, since it can produce that compound

from the CO₂ 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 _Nostocaceæ_ generally

appear as the first settlers on sand the conclusion has been drawn that

they or the group of _Schizophyceæ_ 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., _Über die zeitlichen Eigenschaften der

Entwicklungsvorgänge_, 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, _Über die Entstehung von Bindegewebe, Leucocyten und

rothen Blutkörperchen aus Epithel und über eine Methode isolierte

Gewebsteile zu züchten_. 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° 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° 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_, _Leucæna 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 générale 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 CO₂, 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., _Beiträge 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 algæ 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 rôle 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., _Einführung 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 spermatozoön enters. The entrance

of a spermatozoön 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 larvæ from a sea urchin

(Strongylocentrotus purpuratus) ♀ and a starfish (Asterias) ♂. (Front

view.)]

[Illustration: FIG. 2. Five-days-old larvæ of Strongylocentrotus

purpuratus produced by artificial parthenogenesis. (Side view.) The

larvæ 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 spermatozoön is seen very distinctly in

the primitive skeleton formed by the larva.[36] (Fig. 3.) In the case

of the heterogeneous hybridization the spermatozoön 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 larvæ of two closely related forms

of sea urchins (S. purpuratus ♀ and S. franciscanus ♂). 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 larvæ.

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_ ♀ and _Menidia_ ♂ have usually no

circulation of blood, although the heart is formed and beats and

blood-vessels and blood cells are formed; the eyes are often incomplete

or abnormal though they may be normal at first; the growth of the

embryo is mostly retarded. 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_

♀ and _S. franciscanus_ ♂ 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_

♀ and _S. purpuratus_ ♂, the development is at first normal, but

beginning with the time of mesenchyme formation the majority of larvæ

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 rôle 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°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 Schütze 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 cerevisiæ_) 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 algæ

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°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 spermatozoön,

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 spermatozoön to develop into an embryo,[59] while we can

induce the egg to develop into an embryo without a spermatozoön. 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. Zoöl._, 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 spermatozoön 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

spermatozoön--enters the female gamete--the egg--and this starts the

development. In the case of most animals the egg cannot develop unless

the spermatozoön enters. The question arises: How does the spermatozoön

activate the egg? And also how does it happen that the spermatozoön

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 spermatozoön 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 spermatozoön enters the egg through the so-called fertilization

cone, _i. e._, a protoplasmic process comparable to the pseudopodium of

an amœboid cell. The analogy of the process of phagocytosis--_i. e._,

the taking up of particles by an amœboid cell--and that of the

engulfing of the spermatozoön 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 spermatozoön

which in the case of _S. purpuratus_ ♀ and _Asterias_ ♂ 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 spermatozoön 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 spermatozoön 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 MgCl₂

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 spermatozoön

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+MgCl₂,

containing in addition one drop of a N/100 solution of NaOH (or NH₃ or

benzylamine or butylamine) or eight drops of m/100 NaHCO₃, 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+MgCl₂+CaCl₂ (in the

proportion in which these salts exist in the sea water) or into a

neutral mixture of NaCl+MgCl₂+KCl+CaCl₂. 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 CaCl₂ we

establish the same block to the entrance of the spermatozoön 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 spermatozoön into an egg of its own species

may consist in an agglutination between spermatozoön 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 spermatozoön into the egg which takes place if two different

types of sperm are mixed. He had found that the sperm of the annelid

_Chætopterus_ 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 _Chætopterus_ and the sperm of

sea urchins are mixed the mixture is not able to induce development or

membrane formation, since now neither spermatozoön 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 (_Chætopterus_) 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 spermatozoön. The

block can be removed mechanically by washing.

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

[68] Loeb, J., _Jour. Exper. Zoöl._, 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 spermatozoön 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 spermatozoön 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 spermatozoön to make the latter stick

to the surface of the egg and agglutinate and that later forces of a

different character bring the spermatozoön into the egg. The fact that

under normal conditions a very slight degree of motility on the part of

the spermatozoön 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. Zoöl._, 1914, xvi., 523.

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

TABLE V

SPECIFICITY OF ACTIVATION OF SPERM BY EGGS

--------------+------------+-------------+---------------+-------------

|_Asterias_♂ | _Asterina_♂ |_Franciscanus_♂|_Purpuratus_♂

| | | |

--------------+------------+-------------+---------------+-------------

_Asterias_ |_Immediately|No |Moderately |Slight effect

♀ (immature) | very | activation. | active. | in immediate

| motile._ | | | contact with

| | | | egg.

| | | |

_Asterina_ | Not motile.|_Violent |_Violent |Slight effect

♀ (immature) | | activity._ | activity._ | only near

| | | | the egg.

| | | |

_Franciscanus_|Slightly |No motility. |_Immediately |_Immediately

♀ (mature) | motile. | | active._ | active._

| | | |

| | | |

_Purpuratus_ |Slightly |Slight effect|_Immediately |_Immediately

♀ (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.

Zoöl._, 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 spermatozoön 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. Zoöl_., 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 larvæ; and if the spermatozoön 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 spermatozoön 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 spermatozoön 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

spermatozoön 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.