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Darwin and Modern Science (1909)

Edited by A.C. Seward


XIV. EXPERIMENTAL STUDY OF THE INFLUENCE OF ENVIRONMENT ON ANIMALS.

By JACQUES LOEB, M.D.
Professor of Physiology in the University of California.

I. INTRODUCTORY REMARKS.

B
hat 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 effects 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 unravelling the mechanism of heredity beyond the scope of the Mendelian law. 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 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.

II. THE EFFECTS OF CHEMICAL AGENCIES.

(a) HETEROGENEOUS HYBRIDISATION.

It was held until recently that hybridisation is not possible except between closely related species and that even among these a successful hybridisation 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 unfertilised 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 sea-water is the only guarantee that the eggs are fertilised, 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 belief. 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 been shown by experiment that in fresh sea-water the sperm may live and retain its fertilising power for several days. It is thus unavoidable that at certain periods more than one kind of spermatozoon is suspended in the sea-water and it is a matter of surprise that the most heterogeneous hybridisations do not constantly occur. The reason for this becomes obvious if we bring together mature eggs and equally mature and active sperm of a different family. When this is done no egg is, as a rule, fertilised. The eggs of a sea-urchin can be fertilised by sperm of their own 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. starfish, 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 hybridisation might be of another kind and that by a change in the constitution of the sea-water it might be possible to bring about heterogenous hybridisations, which in normal sea-water are impossible. This assumption proved correct. Sea-water has a faintly alkaline reaction (in terms of the physical chemist its concentration of hydroxyl ions is about (10 to the power minus six)N at Pacific Grove, California, and about (10 to the power minus 5)N at Woods Hole, Massachusetts). If we slightly raise the alkalinity of the sea-water by adding to it a small but definite quantity of sodium hydroxide or some other alkali, the eggs of the sea-urchin can be fertilised with the sperm of widely different groups of animals, possibly with the sperm of any marine animal which sheds it into the ocean. In 1903 it was shown that if we add from about 0.5 to 0.8 cubic centimetre N/10 sodium hydroxide to 50 cubic centimetres of sea-water, the eggs of Strongylocentrotus purpuratus (a sea-urchin which is found on the coast of California) can be fertilised in large quantities by the sperm of various kinds of starfish, brittle- stars and holothurians; while in normal sea-water or with less sodium hydroxide not a single egg of the same female could be fertilised with the starfish sperm which proved effective in the hyper-alkaline sea-water. The sperm of the various forms of starfish was not equally effective for these hybridisations; the sperm of Asterias ochracea and A. capitata gave the best results, since it was possible to fertilise 50 per cent or more of the sea-urchin eggs, while the sperm of Pycnopodia and Asterina fertilised only 2 per cent of the same eggs.

Godlewski used the same method for the hybridisation of the sea-urchin eggs with the sperm of a crinoid (Antedon rosacea). Kupelwieser afterwards obtained results which seemed to indicate the possibility of fertilising the eggs of Strongylocentrotus with the sperm of a mollusc (Mytilus.) Recently, the writer succeeded in fertilising the eggs of Strongylocentrotus franciscanus with the sperm of a mollusc--Chlorostoma. This result could only be obtained in sea-water the alkalinity of which had been increased (through the addition of 0.8 cubic centimetre N/10 sodium hydroxide to 50 cubic centimetres of sea-water). We thus see that by increasing the alkalinity of the sea-water it is possible to effect heterogeneous hybridisations 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 hybridisations were possible. It is known that in solutions like sea-water 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 hybridisations in the ocean which in the present state of alkalinity are practically excluded.

But granted that such hybridisations were possible, would they have influenced the character of the fauna? In other words, are the hybrids between sea-urchin and starfish, or better still, between sea-urchin and mollusc, capable of development, and if so, what is their character? The first experiment made it appear doubtful whether these heterogeneous hybrids could live. The sea-urchin eggs which were fertilised in the laboratory by the spermatozoa of the starfish, as a rule, died earlier than those of the pure breeds. But more recent results indicate that this was due merely to deficiencies in the technique of the earlier experiments. The writer has recently obtained hybrid larvae between the sea-urchin egg and the sperm of a mollusc (Chlorostoma) which, in the laboratory, developed as well and lived as long as the pure breeds of the sea-urchin, and there was nothing to indicate any difference in the vitality of the two breeds.

So far as the question of heredity is concerned, all the experiments on heterogeneous hybridisation of the egg of the sea-urchin with the sperm of starfish, brittle-stars, crinoids and molluscs, 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; while neither the larvae of the starfish nor those of the mollusc form 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 fertilisation of the sea-urchin egg with the sperm of starfish or mollusc 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 hybridisation 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 fertilised with the sperm of a crinoid the fusion of the egg-nucleus and sperm-nucleus takes place in the normal way. It remains for further experiments to decide what the character of the adult hybrids would be.

(b). 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 fertilisation. The reader knows that the eggs of the overwhelming majority of animals cannot develop unless 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 well-known physico-chemical agencies. This is, indeed, true, and during the last ten years living larvae have been produced by chemical agencies from the unfertilised eggs of sea-urchins, starfish, holothurians and a number of annelids and molluscs; 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 fertilisation 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 starfish or annelids, the immediate effect is a characteristic change of the surface of the egg, namely the formation of the so-called membrane of fertilisation. The writer found that we can produce this membrane in the unfertilised 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 unfertilised 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 unfertilised egg, e.g. in the starfish and certain annelids. For the eggs of other animals a second treatment is necessary, presumably to overcome some of the injurious effects of acid treatment. Thus the unfertilised eggs of the sea-urchin Strongylocentrotus purpuratus of the Californian coast begin to develop when membrane-formation has been induced by treatment with 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 35 to 55 minutes (at 15 deg 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 fertilised with sperm.

It is obvious that the sea-urchin egg is injured in the process of membrane-formation and that the subsequent treatment with a hypertonic solution only acts as a remedy. The nature of this injury became clear when it was discovered that all the agencies which cause haemolysis, i.e. the destruction of the red blood corpuscles, also cause membrane-formation in unfertilised 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 haemolysis which at present play so important a role in the study of immunity. The difference between cytolysis (or haemolysis) and fertilisation seems to be this, that the latter is caused by a superficial or slight cytolysis of the egg, while if the cytolytic agencies have time to act on the whole egg the latter is completely destroyed. If we put unfertilised 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 fertilisation. 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-water for from 35 to 55 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 known in pathology as CYTOLYSIS. Membrane-formation is, therefore, caused by a superficial or incomplete cytolysis. The writer believes that the subsequent treatment of the egg with hypertonic sea-water is needed only to overcome the destructive effects of this partial cytolysis. The full reasons for this belief cannot be given in a short essay.

Many pathologists assume that haemolysis or cytolysis is due to a liquefaction of certain fatty or fat-like compounds, the so-called lipoids, in the cell. If this view is correct, it would be necessary to ascribe the fertilisation of the egg to the same process.

The analogy between haemolysis and fertilisation throws, possibly, some light on a curious observation. It is well known 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 fertilisation-membrane. If such eggs are afterwards treated for a short period with hypertonic 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 fertilisation 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 fertilisation-membrane is formed. This inference is corroborated by observations on the fertilisation 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 with hypertonic 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 cytolytic 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 cytolytic effects underlying 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 beyond the metamorphosis into the adult stage and since in all the experiments made by the writer the parthenogenetic plutei lived as long as the plutei produced from fertilised eggs.

(c). ON THE PRODUCTION OF TWINS FROM ONE EGG THROUGH A CHANGE IN THE CHEMICAL CONSTITUTION OF THE SEA-WATER.

The reader is probably familiar with the fact that there exist two different types of human twins. In the one type the twins differ as much as two children of the same parents born at different periods; they may or may not have the same sex. In the second type the twins have invariably the same sex and resemble each other most closely. Twins of the latter type are produced from the same egg, while twins of the former type are produced from two different eggs.

The experiments of Driesch and others have taught us that twins originate from one egg in this manner, namely, that the first two cells into which the egg divides after fertilisation become separated from each other. This separation can be brought about by a change in the chemical constitution of the sea-water. Herbst observed that if the fertilised eggs of the sea- urchin are put into sea-water which is freed from calcium, the cells into which the egg divides have a tendency to fall apart. Driesch afterwards noticed that eggs of the sea-urchin treated with sea-water which is free from lime have a tendency to give rise to twins. The writer has recently found that twins can be produced not only by the absence of lime, but also through the absence of sodium or of potassium; in other words, through the absence of one or two of the three important metals in the sea-water. There is, however, a second condition, namely, that the solution used for the production of twins must have a neutral or at least not an alkaline reaction.

The procedure for the production of twins in the sea-urchin egg consists simply in this:--the eggs are fertilised as usual in normal sea-water and then, after repeated washing in a neutral solution of sodium chloride (of the concentration of the sea-water), are placed in a neutral mixture of potassium chloride and calcium chloride, or of sodium chloride and potassium chloride, or of sodium chloride and calcium chloride, or of sodium chloride and magnesium chloride. The eggs must remain in this solution until half an hour or an hour after they have reached the two-cell stage. They are then transferred into normal sea-water and allowed to develop. From 50 to 90 per cent of the eggs of Strongylocentrotus purpuratus treated in this manner may develop into twins. These twins may remain separate or grow partially together and form double monsters, or heal together so completely that only slight or even no imperfections indicate that the individual started its career as a pair of twins. It is also possible to control the tendency of such twins to grow together by a change in the constitution of the sea-water. If we use as a twin-producing solution a mixture of sodium, magnesium and potassium chlorides (in the proportion in which these salts exist in the sea-water) the tendency of the twins to grow together is much more pronounced than if we use simply a mixture of sodium chloride and magnesium chloride.

The mechanism of the origin of twins, as the result of altering the composition of the sea-water, is revealed by observation of the first segmentation of the egg in these solutions. This cell-division is modified in a way which leads to a separation of the first two cells. If the egg is afterwards transferred back into normal sea-water, each of these two cells develops into an independent embryo. Since normal sea-water contains all three metals, sodium, calcium, and potassium, and since it has besides an alkaline reaction, we perceive the reason why twins are not normally produced from one egg. These experiments suggest the possibility of a chemical cause for the origin of twins from one egg or of double monstrosities in mammals. If, for some reason, the liquids which surround the human egg a short time before and after the first cell-division are slightly acid, and at the same time lacking in one of the three important metals, the conditions for the separation of the first two cells and the formation of identical twins are provided.

In conclusion it may be pointed out that the reverse result, namely, the fusion of normally double organs, can also be brought about experimentally through a change in the chemical constitution of the sea-water. Stockard succeeded in causing the eyes of fish embryos (Fundulus heteroclitus) to fuse into a single cyclopean eye through the addition of magnesium chloride to the sea-water. When he added about 6 grams of magnesium chloride to 100 cubic centimetres of sea-water and placed the fertilised eggs in the mixture, about 50 per cent of the eggs gave rise to one-eyed embryos. "When the embryos were studied the one-eyed condition was found to result from the union or fusion of the 'anlagen' of the two eyes. Cases were observed which showed various degrees in this fusion; it appeared as though the optic vessels were formed too far forward and ventral, so that their antero-ventro-median surfaces fused. This produces one large optic cup, which in all cases gives more or less evidence of its double nature." (Stockard, "Archiv f. Entwickelungsmechanik", Vol. 23, page 249, 1907.)

We have confined ourselves to a discussion of rather simple effects of the change in the constitution of the sea-water upon development. It is a priori obvious, however, that an unlimited number of pathological variations might be produced by a variation in the concentration and constitution of the sea-water, and experience confirms this statement. As an example we may mention the abnormalities observed by Herbst in the development of sea-urchins through the addition of lithium to sea-water. It is, however, as yet impossible to connect in a rational way the effects produced in this and similar cases with the cause which produced them; and it is also impossible to define in a simple way the character of the change produced.

III. THE INFLUENCE OF TEMPERATURE.

(a) THE INFLUENCE OF TEMPERATURE UPON THE DENSITY OF PELAGIC ORGANISMS AND THE DURATION OF LIFE.

It has often been noticed by explorers who have had a chance to compare the faunas in different climates that in polar seas such species as thrive at all in those regions occur, as a rule, in much greater density than they do in the moderate or warmer regions of the ocean. This refers to those members of the fauna which live at or near the surface, since they alone lend themselves to a statistical comparison. In his account of the Valdivia expedition, Chun (Chun, "Aus den Tiefen des Weltmeeres", page 225, Jena, 1903.) calls especial attention to this quantitative difference in the surface fauna and flora of different regions. "In the icy water of the Antarctic, the temperature of which is below 0 deg C., we find an astonishingly rich animal and plant life. The same condition with which we are familiar in the Arctic seas is repeated here, namely, that the quantity of plankton material exceeds that of the temperate and warm seas." And again, in regard to the pelagic fauna in the region of the Kerguelen Islands, he states: "The ocean is alive with transparent jelly fish, Ctenophores (Bolina and Callianira) and of Siphonophore colonies of the genus Agalma."

The paradoxical character of this general observation lies in the fact that a low temperature retards development, and hence should be expected to have the opposite effect from that mentioned by Chun. Recent investigations have led to the result that life-phenomena are affected by temperature in the same sense as the velocity of chemical reactions. In the case of the latter van't Hoff had shown that a decrease in temperature by 10 degrees reduces their velocity to one half or less, and the same has been found for the influence of temperature on the velocity of physiological processes. Thus Snyder and T.B. Robertson found that the rate of heartbeat in the tortoise and in Daphnia is reduced to about one-half if the temperature is lowered 10 deg C., and Maxwell, Keith Lucas, and Snyder found the same influence of temperature for the rate with which an impulse travels in the nerve. Peter observed that the rate of development in a sea-urchin's egg is reduced to less than one-half if the temperature (within certain limits) is reduced by 10 degrees. The same effect of temperature upon the rate of development holds for the egg of the frog, as Cohen and Peter calculated from the experiments of O. Hertwig. The writer found the same temperature- coefficient for the rate of maturation of the egg of a mollusc (Lottia).

All these facts prove that the velocity of development of animal life in Arctic regions, where the temperature is near the freezing point of water, must be from two to three times smaller than in regions where the temperature of the ocean is about 10 deg C. and from four to nine times smaller than in seas the temperature of which is about 20 deg C. It is, therefore, exactly the reverse of what we should expect when authors state that the density of organisms at or near the surface of the ocean in polar regions is greater than in more temperate regions.

The writer believes that this paradox finds its explanation in experiments which he has recently made on the influence of temperature on the duration of life of cold-blooded marine animals. The experiments were made on the fertilised and unfertilised eggs of the sea-urchin, and yielded the result that for the lowering of temperature by 1 deg C. the duration of life was about doubled. Lowering the temperature by 10 degrees therefore prolongs the life of the organism 2 to the power 10, i.e. over a thousand times, and a lowering by 20 degrees prolongs it about one million times. Since this prolongation of life is far in excess of the retardation of development through a lowering of temperature, it is obvious that, in spite of the retardation of development in Arctic seas, animal life must be denser there than in temperate or tropical seas. The excessive increase of the duration of life at the poles will necessitate the simultaneous existence of more successive generations of the same species in these regions than in the temperate or tropical regions.

The writer is inclined to believe that these results have some bearing upon a problem which plays an important role in theories of evolution, namely, the cause of natural death. It has been stated that the processes of differentiation and development lead also to the natural death of the individual. If we express this in chemical terms it means that the chemical processes which underlie development also determine natural death. Physical chemistry has taught us to identify two chemical processes even if only certain of their features are known. One of these means of identification is the temperature coefficient. When two chemical processes are identical, their velocity must be reduced by the same amount if the temperature is lowered to the same extent. The temperature coefficient for the duration of life of cold-blooded organisms seems, however, to differ enormously from the temperature coefficient for their rate of development. For a difference in temperature of 10 deg C. the duration of life is altered five hundred times as much as the rate of development; and, for a change of 20 deg C., it is altered more than a hundred thousand times as much. From this we may conclude that, at least for the sea-urchin eggs and embryo, the chemical processes which determine natural death are certainly not identical with the processes which underlie their development. T.B. Robertson has also arrived at the conclusion, for quite different reasons, that the process of senile decay is essentially different from that of growth and development.

(b) CHANGES IN THE COLOUR OF BUTTERFLIES PRODUCED THROUGH THE INFLUENCE OF TEMPERATURE.

The experiments of Dorfmeister, Weismann, Merrifield, Standfuss, and Fischer, on seasonal dimorphism and the aberration of colour in butterflies have so often been discussed in biological literature that a short reference to them will suffice. By seasonal dimorphism is meant the fact that species may appear at different seasons of the year in a somewhat different form or colour. Vanessa prorsa is the summer form, Vanessa levana the winter form of the same species. By keeping the pupae of Vanessa prorsa several weeks at a temperature of from 0 deg to 1 deg Weismann succeeded in obtaining from the summer chrysalids specimens which resembled the winter variety, Vanessa levana.

If we wish to get a clear understanding of the causes of variation in the colour and pattern of butterflies, we must direct our attention to the experiments of Fischer, who worked with more extreme temperatures than his predecessors, and found that almost identical aberrations of colour could be produced by both extremely high and extremely low temperatures. This can be clearly seen from the following tabulated results of his observations. At the head of each column the reader finds the temperature to which Fischer submitted the pupae, and in the vertical column below are found the varieties that were produced. In the vertical column A are given the normal forms:

(Temperatures in deg C.)
0 to -20     0 to +10    A.           +35 to +37    +36 to +41  +42 to +46
                        (Normal forms)

ichnusoides  polaris     urticae      ichnusa       polaris     ichnusoides
  (nigrita)                                                       (nigrita)

antigone     fischeri    io             -           fischeri    antigone
  (iokaste)                                                       (iokaste)

testudo      dixeyi      polychloros  erythromelas  dixeyi      testudo

hygiaea      artemis     antiopa      epione        artemis     hygiaea

elymi        wiskotti    cardui         -           wiskotti    elymi

klymene      merrifieldi atalanta       -           merrifieldi klymene

weismanni    porima      prorsa         -           porima      weismanni

The reader will notice that the aberrations produced at a very low temperature (from 0 to -20 deg C.) are absolutely identical with the aberrations produced by exposing the pupae to extremely high temperatures (42 to 46 deg C.). Moreover the aberrations produced by a moderately low temperature (from 0 to 10 deg C.) are identical with the aberrations produced by a moderately high temperature (36 to 41 deg C.)

From these observations Fischer concludes that it is erroneous to speak of a specific effect of high and of low temperatures, but that there must be a common cause for the aberration found at the high as well as at the low temperature limits. This cause he seems to find in the inhibiting effects of extreme temperatures upon development.

If we try to analyse such results as Fischer's from a physico-chemical point of view, we must realise that what we call life consists of a series of chemical reactions, which are connected in a catenary way; inasmuch as one reaction or group of reactions (a) (e.g. hydrolyses) causes or furnishes the material for a second reaction or group of reactions (b) (e.g. oxydations). We know that the temperature coefficient for physiological processes varies slightly at various parts of the scale; as a rule it is higher near 0 and lower near 30 deg. But we know also that the temperature coefficients do not vary equally from the various physiological processes. It is, therefore, to be expected that the temperature coefficients for the group of reactions of the type (a) will not be identical through the whole scale with the temperature coefficients for the reactions of the type (b). If therefore a certain substance is formed at the normal temperature of the animal in such quantities as are needed for the catenary reaction (b), it is not to be expected that this same perfect balance will be maintained for extremely high or extremely low temperatures; it is more probable that one group of reactions will exceed the other and thus produce aberrant chemical effects, which may underlie the colour aberrations observed by Fischer and other experimenters.

It is important to notice that Fischer was also able to produce aberrations through the application of narcotics. Wolfgang Ostwald has produced experimentally, through variation of temperature, dimorphism of form in Daphnia. Lack of space precludes an account of these important experiments, as of so many others.

IV. THE EFFECTS OF LIGHT.

At the present day nobody seriously questions the statement that the action of light upon organisms is primarily one of a chemical character. While this chemical action is of the utmost importance for organisms, the nutrition of which depends upon the action of chlorophyll, it becomes of less importance for organisms devoid of chlorophyll. Nevertheless, we find animals in which the formation of organs by regeneration is not possible unless they are exposed to light. An observation made by the writer on the regeneration of polyps in a hydroid, Eudendrium racemosum, at Woods Hole, may be mentioned as an instance of this. If the stem of this hydroid, which is usually covered with polyps, is put into an aquarium the polyps soon fall off. If the stems are kept in an aquarium where light strikes them during the day, a regeneration of numerous polyps takes place in a few days. If, however, the stems of Eudendrium are kept permanently in the dark, no polyps are formed even after an interval of some weeks; but they are formed in a few days after the same stems have been transferred from the dark to the light. Diffused daylight suffices for this effect. Goldfarb, who repeated these experiments, states that an exposure of comparatively short duration is sufficient for this effect, it is possible that the light favours the formation of substances which are a prerequisite for the origin of polyps and their growth.

Of much greater significance than this observation are the facts which show that a large number of animals assume, to some extent, the colour of the ground on which they are placed. Pouchet found through experiments upon crustaceans and fish that this influence of the ground on the colour of animals is produced through the medium of the eyes. If the eyes are removed or the animals made blind in another way these phenomena cease. The second general fact found by Pouchet was that the variation in the colour of the animal is brought about through an action of the nerves on the pigment-cells of the skin; the nerve-action being induced through the agency of the eye.

The mechanism and the conditions for the change in colouration were made clear through the beautiful investigations of Keeble and Gamble, on the colour-change in crustaceans. According to these authors the pigment-cells can, as a rule, be considered as consisting of a central body from which a system of more or less complicated ramifications or processes spreads out in all directions. As a rule, the centre of the cell contains one or more different pigments which under the influence of nerves can spread out separately or together into the ramifications. These phenomena of spreading and retraction of the pigments into or from the ramifications of the pigment-cells form on the whole the basis for the colour changes under the influence of environment. Thus Keeble and Gamble observed that Macromysis flexuosa appears transparent and colourless or grey on sandy ground. On a dark ground their colour becomes darker. These animals have two pigments in their chromatophores, a brown pigment and a whitish or yellow pigment; the former is much more plentiful than the latter. When the animal appears transparent all the pigment is contained in the centre of the cells, while the ramifications are free from pigment. When the animal appears brown both pigments are spread out into the ramifications. In the condition of maximal spreading the animals appear black.

This is a comparatively simple case. Much more complicated conditions were found by Keeble and Gamble in other crustaceans, e.g. in Hippolyte cranchii, but the influence of the surroundings upon the colouration of this form was also satisfactorily analysed by these authors.

While many animals show transitory changes in colour under the influence of their surroundings, in a few cases permanent changes can be produced. The best examples of this are those which were observed by Poulton in the chrysalids of various butterflies, especially the small tortoise-shell. These experiments are so well known that a short reference to them will suffice. Poulton (Poulton, E.B., "Colours of Animals" (The International Scientific Series), London, 1890, page 121.) found that in gilt or white surroundings the pupae became light coloured and there was often an immense development of the golden spots, "so that in many cases the whole surface of the pupae glittered with an apparent metallic lustre. So remarkable was the appearance that a physicist to whom I showed the chrysalids, suggested that I had played a trick and had covered them with goldleaf." When black surroundings were used "the pupae were as a rule extremely dark, with only the smallest trace, and often no trace at all, of the golden spots which are so conspicuous in the lighter form." The susceptibility of the animal to this influence of its surroundings was found to be greatest during a definite period when the caterpillar undergoes the metamorphosis into the chrysalis stage. As far as the writer is aware, no physico-chemical explanation, except possibly Wiener's suggestion of colour-photography by mechanical colour adaptation, has ever been offered for the results of the type of those observed by Poulton.

V. EFFECTS OF GRAVITATION.

(a) EXPERIMENTS ON THE EGG OF THE FROG.

Gravitation can only indirectly affect life-phenomena; namely, when we have in a cell two different non-miscible liquids (or a liquid and a solid) of different specific gravity, so that a change in the position of the cell or the organ may give results which can be traced to a change in the position of the two substances. This is very nicely illustrated by the frog's egg, which has two layers of very viscous protoplasm one of which is black and one white. The dark one occupies normally the upper position in the egg and may therefore be assumed to possess a smaller specific gravity than the white substance. When the egg is turned with the white pole upwards a tendency of the white protoplasm to flow down again manifests itself. It is, however, possible to prevent or retard this rotation of the highly viscous protoplasm, by compressing the eggs between horizontal glass plates. Such compression experiments may lead to rather interesting results, as O. Schultze first pointed out. Pflueger had already shown that the first plane of division in a fertilised frog's egg is vertical and Roux established the fact that the first plane of division is identical with the plane of symmetry of the later embryo. Schultze found that if the frog's egg is turned upside down at the time of its first division and kept in this abnormal position, through compression between two glass plates for about 20 hours, a small number of eggs may give rise to twins. It is possible, in this case, that the tendency of the black part of the egg to rotate upwards along the surface of the egg leads to a separation of its first cells, such a separation leading to the formation of twins.

T.H. Morgan made an interesting additional observation. He destroyed one half of the egg after the first segmentation and found that the half which remained alive gave rise to only one half of an embryo, thus confirming an older observation of Roux. When, however, Morgan put the egg upside down after the destruction of one of the first two cells, and compressed the eggs between two glass plates, the surviving half of the egg gave rise to a perfect embryo of half size (and not to a half embryo of normal size as before.) Obviously in this case the tendency of the protoplasm to flow back to its normal position was partially successful and led to a partial or complete separation of the living from the dead half; whereby the former was enabled to form a whole embryo, which, of course, possessed only half the size of an embryo originating from a whole egg.

(b) EXPERIMENTS ON HYDROIDS.

A striking influence of gravitation can be observed in a hydroid, Antennularia antennina, from the bay of Naples. This hydroid consists of a long straight main stem which grows vertically upwards and which has at regular intervals very fine and short bristle-like lateral branches, on the upper side of which the polyps grow. The main stem is negatively geotropic, i.e. its apex continues to grow vertically upwards when we put it obliquely into the aquarium, while the roots grow vertically downwards. The writer observed that when the stem is put horizontally into the water the short lateral branches on the lower side give rise to an altogether different kind of organ, namely, to roots, and these roots grow indefinitely in length and attach themselves to solid bodies; while if the stem had remained in its normal position no further growth would have occurred in the lateral branches. From the upper side of the horizontal stem new stems grow out, mostly directly from the original stem, occasionally also from the short lateral branches. It is thus possible to force upon this hydroid an arrangement of organs which is altogether different from the hereditary arrangement. The writer had called the change in the hereditary arrangement of organs or the transformation of organs by external forces HETEROMORPHOSIS. We cannot now go any further into this subject, which should, however, prove of interest in relation to the problem of heredity.

If it is correct to apply inferences drawn from the observation on the frog's egg to the behaviour of Antennularia, one might conclude that the cells of Antennularia also contain non-miscible substances of different specific gravity, and that wherever the specifically lighter substance comes in contact with the sea-water (or gets near the surface of the cell) the growth of a stem is favoured; while contact with the sea-water of the specifically heavier of the substances, will favour the formation of roots.

VI. THE EXPERIMENTAL CONTROL OF ANIMAL INSTINCTS.

(a) EXPERIMENTS ON THE MECHANISM OF HELIOTROPIC REACTIONS IN ANIMALS.

Since the instinctive reactions of animals are as hereditary as their morphological character, a discussion of experiments on the physico- chemical character of the instinctive reactions of animals should not be entirely omitted from this sketch. It is obvious that such experiments must begin with the simplest type of instincts, if they are expected to lead to any results; and it is also obvious that only such animals must be selected for this purpose, the reactions of which are not complicated by associative memory, or, as it may preferably be termed, associative hysteresis.

The simplest type of instincts is represented by the purposeful motions of animals to or from a source of energy, e.g. light; and it is with some of these that we intend to deal here. When we expose winged aphides (after they have flown away from the plant), or young caterpillars of Porthesia chrysorrhoea (when they are aroused from their winter sleep) or marine or freshwater copepods and many other animals, to diffused daylight falling in from a window, we notice a tendency among these animals to move towards the source of light. If the animals are naturally sensitive, or if they are rendered sensitive through the agencies which we shall mention later, and if the light is strong enough, they move towards the source of light in as straight a line as the imperfections and peculiarities of their locomotor apparatus will permit. It is also obvious that we are here dealing with a forced reaction in which the animals have no more choice in the direction of their motion than have the iron filings in their arrangement in a magnetic field. This can be proved very nicely in the case of starving caterpillars of Porthesia. The writer put such caterpillars into a glass tube the axis of which was at right angles to the plane of the window: the caterpillars went to the window side of the tube and remained there, even if leaves of their food-plant were put into the tube directly behind them. Under such conditions the animals actually died from starvation, the light preventing them from turning to the food, which they eagerly ate when the light allowed them to do so. One cannot say that these animals, which we call positively helioptropic, are attracted by the light, since it can be shown that they go towards the source of the light even if in so doing they move from places of a higher to places of a lower degree of illumination.

The writer has advanced the following theory of these instinctive reactions. Animals of the type of those mentioned are automatically orientated by the light in such a way that symmetrical elements of their retina (or skin) are struck by the rays of light at the same angle. In this case the intensity of light is the same for both retinae or symmetrical parts of the skin.

This automatic orientation is determined by two factors, first a peculiar photo-sensitiveness of the retina (or skin), and second a peculiar nervous connection between the retina and the muscular apparatus. In symmetrically built heliotropic animals in which the symmetrical muscles participate equally in locomotion, the symmetrical muscles work with equal energy as long as the photo-chemical processes in both eyes are identical. If, however, one eye is struck by stronger light than the other, the symmetrical muscles will work unequally and in positively heliotropic animals those muscles will work with greater energy which bring the plane of symmetry back into the direction of the rays of light and the head towards the source of light. As soon as both eyes are struck by the rays of light at the same angle, there is no more reason for the animal to deviate from this direction and it will move in a straight line. All this holds good on the supposition that the animals are exposed to only one source of light and are very sensitive to light.

Additional proof for the correctness of this theory was furnished through the experiments of G.H. Parker and S.J. Holmes. The former worked on a butterfly, Vanessa antiope, the latter on other arthropods. All the animals were in a marked degree positively heliotropic. These authors found that if one cornea is blackened in such an animal, it moves continually in a circle when it is exposed to a source of light, and in these motions the eye which is not covered with paint is directed towards the centre of the circle. The animal behaves, therefore, as if the darkened eye were in the shade.

(b) THE PRODUCTION OF POSITIVE HELIOTROPISM BY ACIDS AND OTHER MEANS AND THE PERIODIC DEPTH-MIGRATIONS OF PELAGIC ANIMALS.

When we observe a dense mass of copepods collected from a freshwater pond, we notice that some have a tendency to go to the light while others go in the opposite direction and many, if not the majority, are indifferent to light. It is an easy matter to make the negatively heliotropic or the indifferent copepods almost instantly positively heliotropic by adding a small but definite amount of carbon-dioxide in the form of carbonated water to the water in which the animals are contained. If the animals are contained in 50 cubic centimetres of water it suffices to add from three to six cubic centimetres of carbonated water to make all the copepods energetically positively heliotropic. This heliotropism lasts about half an hour (probably until all the carbon-dioxide has again diffused into the air.) Similar results may be obtained with any other acid.

The same experiments may be made with another freshwater crustacean, namely Daphnia, with this difference, however, that it is as a rule necessary to lower the temperature of the water also. If the water containing the Daphniae is cooled and at the same time carbon-dioxide added, the animals which were before indifferent to light now become most strikingly positively heliotropic. Marine copepods can be made positively heliotropic by the lowering of the temperature alone, or by a sudden increase in the concentration of the sea-water.

These data have a bearing upon the depth-migrations of pelagic animals, as was pointed out years ago by Theo. T. Groom and the writer. It is well known that many animals living near the surface of the ocean or freshwater lakes, have a tendency to migrate upwards towards evening and downwards in the morning and during the day. These periodic motions are determined to a large extent, if not exclusively, by the heliotropism of these animals. Since the consumption of carbon-dioxide by the green plants ceases towards evening, the tension of this gas in the water must rise and this must have the effect of inducing positive heliotropism or increasing its intensity. At the same time the temperature of the water near the surface is lowered and this also increases the positive heliotropism in the organisms.

The faint light from the sky is sufficient to cause animals which are in a high degree positively heliotropic to move vertically upwards towards the light, as experiments with such pelagic animals, e.g. copepods, have shown. When, in the morning, the absorption of carbon-dioxide by the green algae begins again and the temperature of the water rises, the animals lose their positive heliotropism, and slowly sink down or become negatively heliotropic and migrate actively downwards.

These experiments have also a bearing upon the problem of the inheritance of instincts. The character which is transmitted in this case is not the tendency to migrate periodically upwards and downwards, but the positive heliotropism. The tendency to migrate is the outcome of the fact that periodically varying external conditions induce a periodic change in the sense and intensity of the heliotropism of these animals. It is of course immaterial for the result, whether the carbon-dioxide or any other acid diffuse into the animal from the outside or whether they are produced inside in the tissue cells of the animals. Davenport and Cannon found that Daphniae, which at the beginning of the experiment, react sluggishly to light react much more quickly after they have been made to go to the light a few times. The writer is inclined to attribute this result to the effect of acids, e.g. carbon-dioxide, produced in the animals themselves in consequence of their motion. A similar effect of the acids was shown by A.D. Waller in the case of the response of nerve to stimuli.

The writer observed many years ago that winged male and female ants are positively helioptropic and that their heliotropic sensitiveness increases and reaches its maximum towards the period of nuptial flight. Since the workers show no heliotropism it looks as if an internal secretion from the sexual glands were the cause of their heliotropic sensitiveness. V. Kellogg has observed that bees also become intensely positively heliotropic at the period of their wedding flight, in fact so much so that by letting light fall into the observation hive from above, the bees are prevented from leaving the hive through the exit at the lower end.

We notice also the reverse phenomenon, namely, that chemical changes produced in the animal destroy its heliotropism. The caterpillars of Porthesia chrysorrhoea are very strongly positively heliotropic when they are first aroused from their winter sleep. This heliotropic sensitiveness lasts only as long as they are not fed. If they are kept permanently without food they remain permanently positively heliotropic until they die from starvation. It is to be inferred that as soon as these animals take up food, a substance or substances are formed in their bodies which diminish or annihilate their heliotropic sensitiveness.

The heliotropism of animals is identical with the heliotropism of plants. The writer has shown that the experiments on the effect of acids on the heliotropism of copepods can be repeated with the same result in Volvox. It is therefore erroneous to try to explain these heliotropic reactions of animals on the basis of peculiarities (e.g. vision) which are not found in plants.

We may briefly discuss the question of the transmission through the sex cells of such instincts as are based upon heliotropism. This problem reduces itself simply to that of the method whereby the gametes transmit heliotropism to the larvae or to the adult. The writer has expressed the idea that all that is necessary for this transmission is the presence in the eyes (or in the skin) of the animal of a photo-sensitive substance. For the transmission of this the gametes need not contain anything more than a catalyser or ferment for the synthesis of the photo-sensitive substance in the body of the animal. What has been said in regard to animal heliotropism might, if space permitted, be extended, mutatis mutandis, to geotropism and stereotropism.

(c) THE TROPIC REACTIONS OF CERTAIN TISSUE-CELLS AND THE MORPHOGENETIC EFFECTS OF THESE REACTIONS.

Since plant-cells show heliotropic reactions identical with those of animals, it is not surprising that certain tissue-cells also show reactions which belong to the class of tropisms. These reactions of tissue-cells are of special interest by reason of their bearing upon the inheritance of morphological characters. An example of this is found in the tiger-like marking of the yolk-sac of the embryo of Fundulus and in the marking of the young fish itself. The writer found that the former is entirely, and the latter at least in part, due to the creeping of the chromatophores upon the blood-vessels. The chromatophores are at first scattered irregularly over the yolk-sac and show their characteristic ramifications. There is at that time no definite relation between blood-vessels and chromatophores. As soon as a ramification of a chromatophore comes in contact with a blood- vessel the whole mass of the chromatophore creeps gradually on the blood- vessel and forms a complete sheath around the vessel, until finally all the chromatophores form a sheath around the vessels and no more pigment cells are found in the meshes between the vessels. Nobody who has not actually watched the process of the creeping of the chromatophores upon the blood- vessels would anticipate that the tiger-like colouration of the yolk-sac in the later stages of the development was brought about in this way. Similar facts can be observed in regard to the first marking of the embryo itself. The writer is inclined to believe that we are here dealing with a case of chemotropism, and that the oxygen of the blood may be the cause of the spreading of the chromatophores around the blood-vessels. Certain observations seem to indicate the possibility that in the adult the chromatophores have, in some forms at least, a more rigid structure and are prevented from acting in the way indicated. It seems to the writer that such observations as those made on Fundulus might simplify the problem of the hereditary transmission of certain markings.

Driesch has found that a tropism underlies the arrangement of the skeleton in the pluteus larvae of the sea-urchin. The position of this skeleton is predetermined by the arrangement of the mesenchyme cells, and Driesch has shown that these cells migrate actively to the place of their destination, possibly led there under the influence of certain chemical substances. When Driesch scattered these cells mechanically before their migration, they nevertheless reached their destination.

In the developing eggs of insects the nuclei, together with some cytoplasm, migrate to the periphery of the egg. Herbst pointed out that this might be a case of chemotropism, caused by the oxygen surrounding the egg. The writer has expressed the opinion that the formation of the blastula may be caused generally by a tropic reaction of the blastomeres, the latter being forced by an outside influence to creep to the surface of the egg.

These examples may suffice to indicate that the arrangement of definite groups of cells and the morphological effects resulting therefrom may be determined by forces lying outside the cells. Since these forces are ubiquitous and constant it appears as if we were dealing exclusively with the influence of a gamete; while in reality all that it is necessary for the gamete to transmit is a certain form of irritability.

(d) FACTORS WHICH DETERMINE PLACE AND TIME FOR THE DEPOSITION OF EGGS.

For the preservation of species the instinct of animals to lay their eggs in places in which the young larvae find their food and can develop is of paramount importance. A simple example of this instinct is the fact that the common fly lays its eggs on putrid material which serves as food for the young larvae. When a piece of meat and of fat of the same animal are placed side by side, the fly will deposit its eggs upon the meat on which the larvae can grow, and not upon the fat, on which they would starve. Here we are dealing with the effect of a volatile nitrogenous substance which reflexly causes the peristaltic motions for the laying of the egg in the female fly.

Kammerer has investigated the conditions for the laying of eggs in two forms of salamanders, e.g. Salamandra atra and S. maculosa. In both forms the eggs are fertilised in the body and begin to develop in the uterus. Since there is room only for a few larvae in the uterus, a large number of eggs perish and this number is the greater the longer the period of gestation. It thus happens that when the animals retain their eggs a long time, very few young ones are born; and these are in a rather advanced stage of development, owing to the long time which elapsed since they were fertilised. When the animal lays its eggs comparatively soon after copulation, many eggs (from 12 to 72) are produced and the larvae are of course in an early stage of development. In the early stage the larvae possess gills and can therefore live in water, while in later stages they have no gills and breathe through their lungs. Kammerer showed that both forms of Salamandra can be induced to lay their eggs early or late, according to the physical conditions surrounding them. If they are kept in water or in proximity to water and in a moist atmosphere they have a tendency to lay their eggs earlier and a comparatively high temperature enhances the tendency to shorten the period of gestation. If the salamanders are kept in comparative dryness they show a tendency to lay their eggs rather late and a low temperature enhances this tendency.

Since Salamandra atra is found in rather dry alpine regions with a relatively low temperature and Salamandra maculosa in lower regions with plenty of water and a higher temperature, the fact that S. atra bears young which are already developed and beyond the stage of aquatic life, while S. maculosa bears young ones in an earlier stage, has been termed adaptation. Kammerer's experiments, however, show that we are dealing with the direct effects of definite outside forces. While we may speak of adaptation when all or some of the variables which determine a reaction are unknown, it is obviously in the interest of further scientific progress to connect cause and effect directly whenever our knowledge allows us to do so.

VII. CONCLUDING REMARKS.

The discovery of De Vries, that new species may arise by mutation and the wide if not universal applicability of Mendel's Law to phenomena of heredity, as shown especially by Bateson and his pupils, must, for the time being, if not permanently, serve as a basis for theories of evolution. These discoveries place before the experimental biologist the definite task of producing mutations by physico-chemical means. It is true that certain authors claim to have succeeded in this, but the writer wishes to apologise to these authors for his inability to convince himself of the validity of their claims at the present moment. He thinks that only continued breeding of these apparent mutants through several generations can afford convincing evidence that we are here dealing with mutants rather than with merely pathological variations.

What was said in regard to the production of new species by physico- chemical means may be repeated with still more justification in regard to the second problem of transformation, namely the making of living from inanimate matter. The purely morphological imitations of bacteria or cells which physicists have now and then proclaimed as artificially produced living beings; or the plays on words by which, e.g. the regeneration of broken crystals and the regeneration of lost limbs by a crustacean were declared identical, will not appeal to the biologist. We know that growth and development in animals and plants are determined by definite although complicated series of catenary chemical reactions, which result in the synthesis of a DEFINITE compound or group of compounds, namely, NUCLEINS.

The nucleins have the peculiarity of acting as ferments or enzymes for their own synthesis. Thus a given type of nucleus will continue to synthesise other nuclein of its own kind. This determines the continuity of a species; since each species has, probably, its own specific nuclein or nuclear material. But it also shows us that whoever claims to have succeeded in making living matter from inanimate will have to prove that he has succeeded in producing nuclein material which acts as a ferment for its own synthesis and thus reproduces itself. Nobody has thus far succeeded in this, although nothing warrants us in taking it for granted that this task is beyond the power of science.



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