Chapter X: "On the Imperfection of the Geological Record," and Chapter (3)

The study of the physiology of form-development in a pure species has already yielded results and makes slow but sure progress. The physiology of the possibility of the transformation of one species into another is based, as yet, rather on pious hope than on accomplished fact. From the first it appeared to be hopeless to investigate physiologically the origin of Linnean species and at the same time that of the natural system, an aim which Darwin had before him in his enduring work. The historical sequence of events, of which an organism is the expression, can only be treated hypothetically with the help of facts supplied by comparative morphology, the history of development, geographical distribution, and palaeontology. (See Lotsy, "Vorlesungen" (Jena, I. 1906, II. 1908), for summary of the facts.) A glance at the controversy which is going on today in regard to different hypotheses shows that the same material may lead different investigators to form entirely different opinions. Our ultimate aim is to find a solution of the problem as to the cause of the origin of species. Indeed such attempts are now being made: they are justified by the fact that under cultivation new and permanent strains are produced; the fundamental importance of this was first grasped by Darwin. New points of view in regard to these lines of inquiry have been adopted by H. de Vries who has succeeded in obtaining from Oenothera Lamarckiana a number of constant "elementary" species. Even if it is demonstrated that he was simply dealing with the complex splitting up of a hybrid (Bateson, "Reports to the Evolution Committee of the Royal Society", London, 1902; cf. also Lotsy, "Vorlesungen", Vol. I. page 234.), the facts adduced in no sense lose their very great value.

We must look at the problem in its simplest form; we find it in every case where a new race differs essentially from the original type in a single character only; for example, in the colour of the flowers or in the petalody of the stamens (doubling of flowers). In this connection we must keep in view the fact that every visible character in a plant is the resultant of the cooperation of specific structure, with its various potentialities, and the influence of the environment. We know, that in a pure species all characters vary, that a blue-flowering Campanula or a red Sempervivum can be converted by experiment into white-flowering forms, that a transformation of stamens into petals may be caused by fungi or by the influence of changed conditions of nutrition, or that plants in dry and poor soil become dwarfed. But so far as the experiments justify a conclusion, it would appear that such alterations are not inherited by the offspring. Like all other variations they appear only so long as special conditions prevail in the surroundings.

It has been shown that the case is quite different as regards the white-flowering, double or dwarf races, because these retain their characters when cultivated under practically identical conditions, and side by side with the blue, single-flowering or tall races. The problem may therefore be stated thus: how can a character, which appears in the one case only under the strictly limited conditions of the experiment, in other cases become apparent under the very much wider conditions of ordinary cultivation? If a character appears, in these circumstances, in the case of all individuals, we then speak of constant races. In such simple cases the essential point is not the creation of a new character but rather an ALTERATION OF THIS CHARACTER IN ACCORDANCE WITH THE ENVIRONMENT. In the examples mentioned the modified character in the simple varieties (or a number of characters in elementary species) appears more or less suddenly and is constant in the above sense. The result is what de Vries has termed a Mutation. In this connection we must bear in mind the fact that no difference, recognisable externally, need exist between individual variation and mutation. Even the most minute quantitative difference between two plants may be of specific value if it is preserved under similar external conditions during many successive generations. We do not know how this happens. We may state the problem in other terms; by saying that the specific structure must be altered. It is possible, to some extent, to explain this sudden alteration, if we regard it as a chemical alteration of structure either in the specific qualities of the proteids or of the unknown carriers of life. In the case of many organic compounds their morphological characters (the physical condition, crystalline form, etc.) are at once changed by alteration of atomic relations or by incorporation of new radicals. (For instance ethylchloride (C2H5Cl) is a gas at 21 deg C., ethylenechloride (C2H4Cl2) a fluid boiling at 84 deg C., beta trichlorethane (C2H3Cl3) a fluid boiling at 113 deg C., perchlorethane (C2Cl6) a crystalline substance. Klebs, ("Willkurliche Entwickelungsanderungen" page 158.) Much more important, however, would be an answer to the question, whether an individual variation can be converted experimentally into an inherited character--a mutation in de Vries's sense.

In all circumstances we may recognise as a guiding principle the assumption adopted by Lamarck, Darwin, and many others, that the inheritance of any one character, or in more general terms, the transformation of one species into another, is, in the last instance, to be referred to a change in the environment. From a causal-mechanical point of view it is not a priori conceivable that one species can ever become changed into another so long as external conditions remain constant. The inner structure of a species must be essentially altered by external influences. Two methods of experimental research may be adopted, the effect of crossing distinct species and, secondly, the effect of definite factors of the environment.

The subject of hybridisation is dealt with in another part of this essay. It is enough to refer here to the most important fact, that as the result of combinations of characters of different species new and constant forms are produced. Further, Tschermack, Bateson and others have demonstrated the possibility that hitherto unknown inheritable characters may be produced by hybridisation.

The other method of producing constant races by the influence of special external conditions has often been employed. The sporeless races of Bacteria and Yeasts (Cf. Detto, "Die Theorie der direkten Anpassung... ", pages 98 et seq., Jena, 1904; see also Lotsy, "Vorlesungen", II. pages 636 et seq., where other similar cases are described.) are well known, in which an internal alteration of the cells is induced by the influence of poison or higher temperature, so that the power of producing spores even under normal conditions appears to be lost. A similar state of things is found in some races which under certain definite conditions lose their colour or their virulence. Among the phanerogams the investigations of Schubler on cereals afford parallel cases, in which the influence of a northern climate produces individuals which ripen their seeds early; these seeds produce plants which seed early in southern countries. Analogous results were obtained by Cieslar in his experiments; seeds of conifers from the Alps when planted in the plains produced plants of slow growth and small diameter.

All these observations are of considerable interest theoretically; they show that the action of environment certainly induces such internal changes, and that these are transmitted to the next generation. But as regards the main question, whether constant races may be obtained by this means, the experiments cannot as yet supply a definite answer. In phanerogams, the influence very soon dies out in succeeding generations; in the case of bacteria, in which it is only a question of the loss of a character it is relatively easy for this to reappear. It is not impossible, that in all such cases there is a material hanging-on of certain internal conditions, in consequence of which the modification of the character persists for a time in the descendants, although the original external conditions are no longer present.

Thus a slow dying-out of the effect of a stimulus was seen in my experiments on Veronica chamaedrys. (Klebs, "Kunstliche Metamorphosen", Stuttgart, 1906, page 132.) During the cultivation of an artificially modified inflorescence I obtained a race showing modifications in different directions, among which twisting was especially conspicuous. This plant, however, does not behave as the twisted race of Dipsacus isolated by de Vries (de Vries, "Mutationstheorie", Vol. II. Leipzig, 1903, page 573.), which produced each year a definite percentage of twisted individuals. In the vegetative reproduction of this Veronica the torsion appeared in the first, also in the second and third year, but with diminishing intensity. In spite of good cultivation this character has apparently now disappeared; it disappeared still more quickly in seedlings. In another character of the same Veronica chamaedrys the influence of the environment was stronger. The transformation of the inflorescences to foliage-shoots formed the starting-point; it occurred only under narrowly defined conditions, namely on cultivation as a cutting in moist air and on removal of all other leaf-buds. In the majority (7/10) of the plants obtained from the transformed shoots, the modification appeared in the following year without any interference. Of the three plants which were under observation several years the first lost the character in a short time, while the two others still retain it, after vegetative propagation, in varying degrees. The same character occurs also in some of the seedlings; but anything approaching a constant race has not been produced.

Another means of producing new races has been attempted by Blaringhem. (Blaringhem, "Mutation et Traumatisme", Paris, 1907.) On removing at an early stage the main shoots of different plants he observed various abnormalities in the newly formed basal shoots. From the seeds of such plants he obtained races, a large percentage of which exhibited these abnormalities. Starting from a male Maize plant with a fasciated inflorescence, on which a proportion of the flowers had become male, a new race was bred in which hermaphrodite flowers were frequently produced. In the same way Blaringhem obtained, among other similar results, a race of barley with branched ears. These races, however, behaved in essentials like those which have been demonstrated by de Vries to be inconstant, e.g. Trifolium pratense quinquefolium and others. The abnormality appears in a proportion of the individuals and only under very special conditions. It must be remembered too that Blaringhem worked with old cultivated plants, which from the first had been disposed to split into a great variety of races. It is possible, but difficult to prove, that injury contributed to this result.

A third method has been adopted by MacDougal (MacDougal, "Heredity and Origin of species", "Monist", 1906; "Report of department of botanical research", "Fifth Year-book of the Carnegie Institution of Washington", page 119, 1907.) who injected strong (10 percent) sugar solution or weak solutions of calcium nitrate and zinc sulphate into young carpels of different plants. From the seeds of a plant of Raimannia odorata the carpels of which had been thus treated he obtained several plants distinguished from the parent-forms by the absence of hairs and by distinct forms of leaves. Further examination showed that he had here to do with a new elementary species. MacDougal also obtained a more or less distinct mutant of Oenothera biennis. We cannot as yet form an opinion as to how far the effect is due to the wound or to the injection of fluid as such, or to its chemical properties. This, however, is not so essential as to decide whether the mutant stands in any relation to the influence of external factors. It is at any rate very important that this kind of investigation should be carried further.

If it could be shown that new and inherited races were obtained by MacDougal's method, it would be safe to conclude that the same end might be gained by altering the conditions of the food-stuff conducted to the sexual cells. New races or elementary species, however, arise without wounding or injection. This at once raises the much discussed question, how far garden-cultivation has led to the creation of new races? Contrary to the opinion expressed by Darwin and others, de Vries ("Mutationstheorie", Vol. I. pages 412 et seq.) tried to show that garden-races have been produced only from spontaneous types which occur in a wild state or from sub-races, which the breeder has accidentally discovered but not originated. In a small number of cases only has de Vries adduced definite proof. On the other side we have the work of Korschinsky (Korschinsky, "Heterogenesis und Evolution", "Flora", 1901.) which shows that whole series of garden-races have made their appearance only after years of cultivation. In the majority of races we are entirely ignorant of their origin.

It is, however, a fact that if a plant is removed from natural conditions into cultivation, a well-marked variation occurs. The well-known plant-breeder L. de Vilmorin (L. de Vilmorin, "Notices sur l'amelioration des plantes", Paris, 1886, page 36.), speaking from his own experience, states that a plant is induced to "affoler," that is to exhibit all possible variations from which the breeder may make a further selection only after cultivation for several generations. The effect of cultivation was particularly striking in Veronica chamaedrys (Klebs, "Kunstliche Metamorphosen", Stuttgart, 1906, page 152.) which, in spite of its wide distribution in nature, varies very little. After a few years of cultivation this "good" and constant species becomes highly variable. The specimens on which the experiments were made were three modified inflorescence cuttings, the parent-plants of which certainly exhibited no striking abnormalities. In a short time many hitherto latent potentialities became apparent, so that characters, never previously observed, or at least very rarely, were exhibited, such as scattered leaf-arrangement, torsion, terminal or branched inflorescences, the conversion of the inflorescence into foliage-shoots, every conceivable alteration in the colour of flowers, the assumption of a green colour by parts of the flowers, the proliferation of flowers.

All this points to some disturbance in the species resulting from methods of cultivation. It has, however, not yet been possible to produce constant races with any one of these modified characters. But variations appeared among the seedlings, some of which, e.g. yellow variegation, were not inheritable, while others have proved constant. This holds good, so far as we know at present, for a small rose-coloured form which is to be reckoned as a mutation. Thus the prospect of producing new races by cultivation appears to be full of promise.

So long as the view is held that good nourishment, i.e. a plentiful supply of water and salts, constitutes the essential characteristic of garden-cultivation, we can hardly conceive that new mutations can be thus produced. But perhaps the view here put forward in regard to the production of form throws new light on this puzzling problem.

Good manuring is in the highest degree favourable to vegetative growth, but is in no way equally favourable to the formation of flowers. The constantly repeated expression, good or favourable nourishment, is not only vague but misleading, because circumstances favourable to growth differ from those which promote reproduction; for the production of every form there are certain favourable conditions of nourishment, which may be defined for each species. Experience shows that, within definite and often very wide limits, it does not depend upon the ABSOLUTE AMOUNT of the various food substances, but upon their respective degrees of concentration. As we have already stated, the production of flowers follows a relative increase in the amount of carbohydrates formed in the presence of light, as compared with the inorganic salts on which the formation of albuminous substances depends. (Klebs, "Kunstliche Metamorphosen", page 117.) The various modifications of flowers are due to the fact that a relatively too strong solution of salts is supplied to the rudiments of these organs. As a general rule every plant form depends upon a certain relation between the different chemical substances in the cells and is modified by an alteration of that relation.

During long cultivation under conditions which vary in very different degrees, such as moisture, the amount of salts, light intensity, temperature, oxygen, it is possible that sudden and special disturbances in the relations of the cell substances have a directive influence on the inner organisation of the sexual cells, so that not only inconstant but also constant varieties will be formed.

Definite proof in support of this view has not yet been furnished, and we must admit that the question as to the cause of heredity remains, fundamentally, as far from solution as it was in Darwin's time. As the result of the work of many investigators, particularly de Vries, the problem is constantly becoming clearer and more definite. The penetration into this most difficult and therefore most interesting problem of life and the creation by experiment of new races or elementary species are no longer beyond the region of possibility.

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.

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 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.