The Mechanistic Conception of Life

These observations on the sea-urchin egg, therefore, suggest the possibility that the combination of the three salts in their definite proportion and concentration has the function of forming a surface film of a definite structure or texture, around the protoplasm of each cell, by which the protoplasm is kept together, protected against and separated from the surrounding media. The previously mentioned observation of Herbst again shows the important role of calcium in this process.

The objection might be raised that the beneficial action of the three salts could only be proved on marine animals or on tissues of higher animals, which are said to be '^ adapted" to a mixture of NaCl, KCl, and CaCl., in definite proportions. Experiments on fresh-water organisms, for which ^'adaptation" to a mixture of NaCl, KCl, and CaCl, in these definite proportions cannot be claimed, show that this objection is not valid. Ostwald worked with fresh-water crustaceans which he put into mixtures of various salts. It was found that these animals live longer in a mixture of NaCl + KCl +CaClo than in a solution of NaCl, or NaCl+KCl, or NaCl+CaCl, of the same osmotic pressure.

Osterhout was able to show that the spores of a certain variety of Vaucheria die in a pure 3/32 m solution of NaCl in 10 to 20 minutes, while they live in 100 c.c. 3/32 m NaCl + 1 c.c. 3/32 CaClg 2 to 4 weeks, and in 100 c.c. 3/32 m NaCl + 1 c.c. 3/32 m CaCl,+2.2 c.c. 3/32 m KCl 6 to 8 weeks. The reaction of the solution was strictly neutral and the NaCl the purest obtainable. The results remained the same after the NaCl had been recrystallized six times. Experiments with Spirogyra gave a similar result. The solutions were all 3/32 m. In NaCl the Spirogyra died in 18 hours; in NaCl+KCl in two days; in NaCl+KCl+CaCl, they lived 65 days. Osterhout caused wheat grains to develop in such solutions and measured the total length of the roots formed.

These cases, to which many other similar observations might be added, prove that the life-preserving effect of the combination of NaCl + KCl +CaCL in definite proportions is not due to the fact that organisms are ''adapted" to this mixture but to a specific protective effect of the combination of the three It seems, therefore, to be a general fact that wherever tissues or animals require a medium of a comparatively high osmotic pressure — like our tissues — their life lasts much longer in a mixture of NaCl+KCl+CaCl, in the proportion in which these salts exist in the blood and in the ocean, than in any other osmotic solution, even a pure solution of NaCl. But the reader has noticed that there are considerable differences in the resistance of various organisms to abnormal solutions . While a marine Gammarus dies in half an hour in an isotonic solution of NaCl or cane sugar, red blood corpuscles or even the muscle of a frog can be kept for a day or longer in such a solution (of course even the muscle of a frog lives longer if the NaCl solution contains in addition KCl or CaClg). What causes this difference?

Six years ago I found that the unfertilized eggs of the seaurchin (Strongylocentrotus purpuratus) can keep alive and remain apparently intact in a pure neutral solution of CaCl, or of NaCl for several days at a temperature of 15°, while the fertilized eggs of the same female are killed in a pure neutral solution of CaClo in a few hours. The same difference is found for other salts also. What causes this difference? Several authors have suggested that it is due to the fact that the fertilized egg is more permeable to salts than the unfertilized egg. But recent experiments by Warburg, which were confirmed and amplified by Harvey, make it doubtful whether the salts which are not soluble in fats can enter the fertilized egg at all. I believe that the explanation of the difference is much more simple. The unfertilized egg is surrounded by a cortical layer and this layer is destroyed or modified in the process of fertilization. One result of this modification is the formation of the fertilization membrane, for which I have been able to

show that it is readily permeable for salts. As long as the cortical layer of the unfertilized egg is intact, it prevents the surrounding salt solution from coming in contact with the protoplasm or at least it retards this process. If, however, the cortical layer is destroyed by fertilization the surrounding salt solution comes directly in contact with the protoplasm and if the solution is abnormal it can cause the disintegration of the surface layer of the protoplasm.

I am inclined to believe that differences in the resisting power of various cells or organisms to abnormal salt solutions are primarily due to differences in the constitution of the protective envelopes of the animals or the cells. Microorganisms which can live in strong organic acids or salt solutions of a high concentration probably possess a surface layer which shuts off their protoplasm from contact with the solution. For the protoplasm of muscle the rather tough sarcolemma forms not an absolute but nevertheless an effective wall against the surrounding solution.

But aside from differences of this kind there are other conditions which influence the degree of resistance of cells to various solutions. I have foimd that the fertilized eggs of the seaurchin will live longer in abnormal salt solutions if the oxidations in the egg are stopped, either by the withdrawal of oxygen or the addition of KCN or NaCN. Warburg and Meyerhof have dra^\Tl the conclusion that in a pure NaCl solution the rate of oxidations of the egg of Strongylocentfotus is increased and that it is this increase in the rate of oxidations which kills the eggs. But this increase of oxidations cannot be observed in the eggs of Arbacia when they are put into a pure NaCl solution and, moreover, lack of oxygen prolongs the life of the fertilized egg just as well in solutions of NaCl-f-CaClg or of NaCl+BaCl,, for which salts these authors do not claim that they can raise the rate of oxidations of the egg. I am inclined to believe that during or previously to cell-division, besides

phenomena of streaming inside the cell, changes in the surface film of the protoplasm occur, whereby this film is more easily injured by the salts. If we suppress the oxidations we suppress also the processes leading to cell-division and thereby retard the deleterious action of the abnormal salt solution upon the surface layer of the protoplasm of the egg. If we now raise the question as to why salts are necessary for the preservation of the life of the cell we can point to a number of cases in which this answer seems clear. Each cell may be considered a chemical factory, in which the work can only go on in the proper way, if the diffusion of substances through the cell-wall is restricted. This diffusion depends on the nature of the surface layer of the cell. Overton and others assume that this layer consists of a continuous membrane of fat or lipoids. This assumption is not compatible with two facts, namely, that water diffuses very rapidly into the cell, and second, that life depends upon an exchange of watersoluble and not of fat-soluble substances between the cells and the surrounding liquid. The above-mentioned facts of the antagonism between acids and salts suggest the idea that the surface film of cells consists exclusively or essentially of certain proteins.

The experiments mentioned in this paper indicate that the role of salts in the preservation of life consists in the ^'tanning" effect which they have upon the surface films of the cells, whereby these films acquire those physical qualities of durability and comparative impermeability, without which the cell cannot exist. On this assumption we can understand that neutral salts should be necessary for the preservation of life although they do not furnish energy.

As far as the dynamical effects of salts are concerned it is not impossible that some of them belong also to the type of those mentioned in this paper. The fact that the addition of calcium to an NaCl solution prevents the twitchings of the muscle, which occur in the pure NaCl solution, suggests the possibility that the CaCl, merely prevents or retards the diffusion of NaCl through the sarcolemma. But other effects of salts, e.g., the apparent dependence of contractility of the muscle upon the presence of NaCl, or the role of PO^, do not find their explanation in the facts discussed here.

What the biologist calls the natural environment of an animal is from a physical point of view a rather rigid combination of definite forces. It is obvious that by a purposeful and systematic variation of these and by the application of other forces in the laboratory, results must be obtainable which do not appear in the natural environment. This is the reasoning underlying the modern development of the study of the effect of environment upon animal life. It was perhaps not the least important of Darwin's services to science that the boldness of his conceptions gave to the experimental biologist courage to enter upon the attempt of controlling at will the life phenomena of animals, and of bringing about effects which cannot be expected in nature.

The systematic physico-chemical analysis of the effect of outside forces upon the form and reactions of animals is also our only means of unraveling the mechanism of heredity beyond the results which can be obtained by a mere cytological investigation. The manner in which a germ cell can force upon the adult certain characters will not be understood until we succeed in varying and controlling hereditary characteristics; and this can only be accomplished on the basis of a systematic study of the effects of chemical and physical forces upon living matter.

Owing to limitation of space this sketch is necessarily very incomplete, and it must not be inferred that studies which are 1 Reprinted from Darwin and Modern Science (1909), by courtesy of Professor A. C. Seward, of the University of Cambridge, England. not mentioned here were considered to be of minor importance. All the writer could hope to do was to bring together a few instances of the experimental analysis of the effect of environment, which indicate the nature and extent of our control over life phenomena and which also have some relation to the work of Darwin. In the selection of these instances preference is given to those problems which are not too technical for the general reader.

The forces, the influence of which we shall discuss, are in succession chemical agencies, temperature, light, and gravitation. We shall also treat separately the effect of these forces upon form and instinctive reactions. a) Heterogeneous hybridization. — It was held until recently that hybridization is not possible except between closely related species and that even among these a successful hybridization cannot always be counted upon. This view was well supported by experience. It is, for instance, well known that the majority of marine animals lay their unfertilized eggs in the ocean and that the males shed their sperm also into the sea-water. The numerical excess of the spermatozoa over the ova in the seawater is the only guaranty that the eggs are fertilized, for the spermatozoa are carried to the eggs by chance and are not attracted by the latter. This statement is the result of numerous experiments by various authors, and is contrary to common beUef. As a rule all or the majority of individuals of a species in a given region spawn on the same day, and when this occurs the sea-water constitutes a veritable suspension of sperm. It has recently been showni by experiment that in fresh sea-water the sperm may live and retain its fertilizing power for several days. It is thus unavoidable that at certain periods more than one kind of spermatozoa is suspended in the sea-water and it is a matter of surprise that the most heterogeneous hybridizations do not

constantly occur. The reason for this becomes obvious when we bring together mature eggs and equally mature and active sperm of different families. When this is done no egg is, as a rule, fertilized. The eggs of a sea-urchin can be fertilized by sperm of their o^\^l species, or, though in smaller numbers, by the sperm of other species of sea-urchins, but not by the sperm of other groups of echinoderms, e.g., star-fish, brittle-stars, holothurians, or crinoids, and still less by the sperm of more distant groups of animals. The consensus of opinion seemed to be that the spermatozoon must enter the egg through a narrow opening or canal, the so-called micropyle, and that the micropyle allowed only the spermatozoa of the same or of a closely related species to enter the egg.

It seemed to the writer that the cause of this limitation of hybridization might be of another kind and that by a change in the constitution of the sea-water it might be possible to bring about heterogeneous hybridizations, which in normal seawater are impossible. This assumption proved correct. Sea-water has a faintly alkaline reaction (in terms of the physical chemist its concentration of hydroxy 1 ions is about 10~^ n at Pacific Grove, California, and about 10~^ n at Woods Hole, Massachusetts). If we slightly raise the alkalinity of the seawater by adding to it a small but definite quantity of sodium hydroxide or some other alkali, the eggs of the sea-urchin can be fertilized with the sperm of widely different groups of animals. In 1903 it was sho\\Ti that if we add from about 0.5 to 0.8 c.c. n/10 sodium hydroxide to 50 c.c. of sea-water, the eggs of Strongylocentrotus purpuratus (a sea-urchin which is found on the coast of California) can be fertilized in large quantities by the sperm of various kinds of star-fish, brittlestars, and holothurians; while in normal sea-water or with less sodium hydroxide not a single egg of the same female could be fertilized with the star-fish sperm which proved effective in the hyperalkaline sea-water. The sperm of the various forms

of star-fish was not equally effective for these hybridizations; the sperm of Asterias ochracea and A. capitata gave the best results, since it was possible to fertilize from 50 per cent to 100 per cent of the sea-urchin eggs, while the sperm of Pycnopodia and Asterina fertilized only 10 or 2 per cent respectively of the same eggs. Godlewski used the same method for the hybridization of the sea-urchin eggs with the sperm of a crinoid {Antedon rosacea) . Kupelwieser afterward obtained results which seemed to indicate the possibility of fertilizing the eggs of Strongylocentrotus with the sperm of a mollusk (Mytilus) . Recently, the writer succeeded in fertilizing the eggs of Strongylocentrotus franciscanus with the sperm of a mollusk — Chlorostoma. This result could only be obtained in sea-water the alkalinity of which had been increased (through the addition of 0.8 c.c. n/10 sodium hydroxide to 50 c.c. of seawater). We thus see that by increasing the alkalinity of the sea-water it is possible to effect heterogeneous hybridizations which are at present impossible in the natural environment of these animals.

It is, however, conceivable that in former periods of the earth's history such heterogeneous hybridizations were possible. It is known that in solutions like seawater the degree of alkalinity must increase when the amount of carbon dioxide in the atmosphere is diminished. If it be true, as Arrhenius assumes, that the Ice age was caused or preceded by a diminution in the amount of carbon dioxide in the air, such a diminution must also have resulted in an increase of the alkalinity of the sea-water, and one result of such an increase must have been to render possible heterogeneous hybridizations in the ocean which in the present state of alkalinity are practically excluded.

But granted that such hybridizations were possible, would they have influenced the character of the fauna ? In other words, are the hybrids between sea-urchin and star-fish, or better still, between sea-urchin and mollusks, capable of development. and if so, what is their character ? In all cases of heterogeneous hybridization the vitality of the egg or the embryo seems weakened and it is still doubtful whether any heterogeneous hybrid can reach maturity. The number of experiments is still limited and this statement is therefore not yet final.

So far as the question of heredity is concerned, all the experiments on heterogeneous hybridization of the egg of the sea-urchin with the sperm of star-fish, brittle-stars, crinoids, and moUusks have led to the same result, namely, that the larvae have purely maternal characteristics and differ in no way from the pure breed of the form from which the egg is taken. By way of illustration it may be said that the larvae of the sea-urchin reach on the third day or earlier (according to species and temperature) the so-called pluteus stage, in which they possess a typical skeleton (Fig. 10, p. 11); while neither the larvae of the star-fish nor those of the molluskform a skeleton at the corresponding stage. It was, therefore, a matter of some interest to find out whether or not the larvae produced by the fertilization of the sea-urchin egg with the sperm of star-fish or mollusk would form the normal and typical pluteus skeleton. This was invariably the case in the experiments of Godlewski, Kupelwieser, Hagedoorn, and the writer. These hybrid larvae were exclusively maternal in character.

It might be argued that in the case of heterogeneous hybridization the sperm nucleus does not fuse with the egg nucleus, and that, therefore, the spermatozoon cannot transmit its hereditary substances to the larvae. But these objections are refuted by Godlewski's experiments, in which he showed definitely that if the egg of the sea-urchin is fertilized with the sperm of a crinoid the fusion of the egg nucleus and sperm nucleus takes place in the normal way.

h) Artificial parthenogenesis. — Possibly in no other field of biology has our ability to control life phenomena by outside conditions been proved to such an extent as in the domain of fertilization. The reader knows that the eggs of the overwhelming majority of animals cannot develop miless a spermatozoon enters them. In this case a living agency is the cause of development and the problem arises whether it is possible to accomplish the same result through the application of wellknowTi physico-chemical agencies. This is, indeed, true, and during the last ten years living larvae have been produced by chemical agencies from the unfertilized eggs of sea-urchins, star-fish, holothurians, and a number of annelids and mollusks; in fact this holds true in regard to the eggs of practically all forms of animals with which such experiments have been tried long enough. In each form the method of procedure is somewhat different and a long series of experiments is often required before the successful method is found.

The facts of artificial parthenogenesis, as the chemical fertilization or activation of the egg is called, have, perhaps, some bearing on the problem of evolution. If we wish to form a mental image of the process of evolution we have to reckon with the possibility that parthenogenetic propagation may have preceded sexual reproduction. This suggests also the possibility that at that period outside forces may have supplied the conditions for the development of the egg which at present the spermatozoon has to supply. For this, if for no other reason, a brief consideration of the means of artificial parthenogenesis may be of interest to the student of evolution.

It seemed necessary in these experiments to imitate as completely as possible by chemical agencies the effects of the spermatozoon upon the egg. When a spermatozoon enters the egg of a sea-urchin or certain star-fish or annelids, the immediate effect is a characteristic change of the surface of the egg, namely, the formation of the so-called membrane of fertilization (Figs. 1 and 2). The writer found that we can produce this membrane in the unfertilized egg by certain acids, especially the monobasic acids of the fatty series, e.g., formic, acetic, propionic,

butyric, etc. Carbon dioxide is also very efficient in this direction. It was also found that the higher acids are more efficient than the lower ones, and it is possible that the spermatozoon induces membrane formation by carrying into the egg a higher fatty acid, namely oleic acid or one of its salts or esters. The physico-chemical process which underlies the formation of the membrane seems to be the cause of the development of the egg. In all cases in which the unfertilized egg has been treated in such a way as to cause it to form a membrane it begins to develop. For the eggs of certain animals membrane formation is all that is required to induce a complete development of the unfertilized egg, e.g., in the star-fish and certain annelids. For the eggs of other animals a second treatment is necessary. Thus the unfertilized eggs of the sea-urchin Strongylocentrotus purpuratus of the Californian coast begin to develop when membrane formation has been induced by treatment ^vith a fatty acid, e.g., butyric acid; but the development soon ceases and the eggs perish in the early stages of segmentation, or after the first nuclear division. But if we treat the same eggs after membrane formation, for from thirty-five to fifty-five minutes (at 15° C.) with sea-water the concentration (osmotic pressure) of which has been raised through the addition of a definite amount of some salt or sugar, the eggs will segment and develop normally, when transferred back to normal sea-water. If care is taken, practically all the eggs can be caused to develop into plutei, the majority of which may be perfectly normal and may live as long as larvae produced from eggs fertilized with sperm.

It is possible that the sea-urchin egg is injured in the process of membrane formation. The nature of this injury became clear when it was discovered that all the agencies which cause hemolysis, i.e., the destruction of the red blood corpuscles, also cause membrane formation in unfertilized eggs, e.g., fatty acids or ether, alcohols or chloroform, etc., or saponin, solanin, digitalin, bile salts, and alkali. It thus happens that the phenomena of artificial parthenogenesis are linked together with the phenomena of hemolysis which at present play so important a role in the study of immunity. The difference between cytolysis (or hemolysis) and fertilization seems to be this, that the latter is caused by a superficial cytolysis of the egg, while if the cytol>i:ic agencies have time to act on the whole egg the latter is completely destroyed. If we put unfertilized eggs of a sea-urchin into sea-water which contains a trace of saponin we notice that, after a few minutes, all the eggs form the typical membrane of fertilization. If the eggs are then taken out of the saponin solution, freed from all traces of saponin by repeated washing in normal sea-water, and transferred to the hypertonic sea-Avater for from thirty-five to fifty-five minutes, they develop into larvae. If, however, they are left in the sea-water containing the saponin they undergo, a few minutes after membrane formation, the disintegration knoTvn in pathology as cytolysis. Membrane formation is, therefore, caused by a superficial or incomplete cytolysis. It is possible that the subsequent treatment of the egg with hypertonic sea-water is partly needed to overcome the destructive effects of this cytolysis of the cortical layer.

Many pathologists assume that hemolysis or cytolysis is due to a liquefaction of certain fatty or fat-hke compounds, the so-called lipoids, in the cell. If this view is correct, it would be necessary to ascribe the fertilization of the egg to the same process. The analogy between hemolysis and fertilization throws, possibly, some light on a curious observation. It is well knoAATi that the blood corpuscles, as a rule, undergo cytolysis if injected into the blood of an animal which belongs to a different family. The writer found last year that the blood of mammals, e.g., the rabbit, pig, and cattle, causes the egg of Strongylocentrotus to form a typical fertilization membrane. If such eggs are afterward treated for a short period with hj^Dertonic sea-water they

develop into normal larvae (plutei). Some substance contained in the blood causes, presumably, a superficial cytolysis of the egg and thus starts its development. We can also cause the development of the sea-urchin egg without membrane formation. The early experiments of the writer were done in this way and many experimenters still use such methods. It is probable that in this case the mechanism of fertilization is essentially the same as in the case where the membrane formation is brought about, with this difference only, that the cytolytic effect is less when no fertilization membrane is formed. This inference is corroborated by observations on the fertilization of the sea-urchin egg with ox blood. It very frequently happens that not all of the eggs form membranes in this process. Those eggs which form membranes begin to develop, but perish if they are not treated ^vith h^'pertonic sea-water. Some of the other eggs, however, which do not form membranes, develop directly into normal larvae without any treatment with hypertonic sea-water, provided they are exposed to the blood for only a few minutes. Presumably some blood enters the eggs and causes the cj'tolytic effects in a less degree than is necessary for membrane formation, but in a sufficient degree to cause their development. The slightness of the cytolytic effect allows the egg to develop without treatment with hypertonic sea-water.

Since the entrance of the spermatozoon causes that degree of cytolysis which leads to membrane formation, it is probable that, in addition to the cytolytic or membrane-forming substance (presumably a higher fatty acid), it carries another substance into the egg which counteracts the deleterious effects underh'ing or following membrane formation. The question may be raised whether the larvae produced by artificial parthenogenesis can reach the mature stage. This question may be answered in the affirmative, since Delage has succeeded in raising several parthenogenetic sea-urchin larvae