Individuality in Organisms

If metabolic gradients are of such fundamental importance in the organic individual it should be possible to discover various proofs or indications of their existence. This chapter is a survey of some of the experimental and observational evidence for the existence of metabolic gradients. The resistance or susceptibility of living protoplasm to various poisons can be used, with certain precautions and within certain limits, as an index of its metabolic condition. This method, which may be called the susceptibility method, makes it possible, particularly in early stages of development and in small, simple animals, to compare the susceptibilities and so to obtain a general idea of the differences in metabolic activity of different regions of the body of a single organism. Many different substances may be used as reagents for determining susceptibility, such, for example, as the alcohols, ethers, and other narcotics, and acids and alkalies. Various products of metabolism, among them carbon dioxide, and certain conditions, such as lack of oxygen, serve the same purpose. But the cyanides, which are powerful poisons, are in many respects the most satisfactory reagents, and they have been used in most of my experiments.

The relation between metabolic activity and susceptibility to these substances is primarily quantitative, the degree of susceptibihty depending upon the rate or intensity of metaboKsm or of certain fundamental metabolic reactions. In aqueous concentrations of a given reagent which kill within a few hours, the susceptibihty varies directly with the general metabohc rate; the higher the rate of metabohc activity, the sooner does death occur. In very low concentrations, however, to which the organism is able to acclimate or accustom itself to some extent, we find the relation reversed. The higher the metabolic rate, the greater the degree of acclimation and therefore the less the susceptibility and the later the occurrence of death. These two methods of comparing susceptibilities I have called the direct and the acclimation method.

The question how these various substances act upon the living organism, whether they enter directly into the chemical reactions or whether they change the physical condition of the protoplasm or certain of its constituents in such a way that the reactions cannot continue, has long been and is still the subject of discussion, but cannot be considered here. Whatever the nature of their action, there can be no doubt concerning the general relation between susceptibility to them and metabolic condition, although under certain conditions the relation may be masked or altered by certain incidental factors.

For the direct form of the method, which is the simplest and most widely applicable, the procedure consists in the immersion ofthe animals to be examined, either singly or in lots, in a concentration of cyanide or other reagent used, which has been previously determined as a concentration which will kill the animals in the course of a few hours under the given conditions of temperature, etc. In many of the lower animals death is followed at once or in a few moments by a visible disintegration and complete loss of structure and form of the part concerned, and in such cases the progress of death can be directly observed. In other cases other means of determining the death-point may be employed or the animals may be removed from the reagent at definite intervals and the progress of death, and so the susceptibility, determined by observing whether and to what extent recovery occurs in each case. When the method is used in this way regions of high metabolic rate die earlier than those of low rate.

In the indirect or acclimation form of the method we find that the degree of acclimation varies with metabolic rate. With this form of the method regions of high metabolic rate are least susceptible in the long run because they become acclimated more readily, while regions of lower metabolic rate undergo less acclimation and so are inhibited to a greater degree and may even die. The susceptibility gradients observed with these two modifications of the method are themselves opposite in direction, but are different expressions of the same metabolic gradient.^

Several species of the fiatworm Planaria constituted the material for my first observations on susceptibility gradients. The results obtained were so definite and ^ For more extended discussions of this method see Child, Senescence and Rejuvenescence, 1915, chap, iii; also "Studies on the Dynamics of Morphogenesis and Inheritance in Experimental Reproduction, V," Jour, of Exper. ZooL, XIV, 1913. striking in character that the desirability of comparative study of different forms at once became evident. Up to the present time some fifty species of animals from various groups have been examined by means of the susceptibility method, either in the adult or embryonic stages or in both, in the attempt to determine to what extent regional differences or gradients in metabolic condition with respect to the axial or any other directions in the body are characteristic features of the animal organism.'

In each form examined a more or less distinct and regular gradient in susceptibility has been observed in the direction of the major axis of the body and in many cases gradients in the direction of the minor axes and of the axes of various organs and parts as well.^ ^ The forma examined include twelve species of ciliate infusoria among the protozoa, the post-embryonic or adult stages of the freshwater hydra, and three species of hydroids among coelentrates; one ctenophore, eleven species of turbellaria, and certain larval stages of one trematode among the flatworms. Dr. L. H. Hyman, workmg under my direction, has examined in the same way nine species of oligochete annelids and one polychete. Susceptibility studies have been made upon the eggs and embryonic or larval stages of the following forms: starfish, sea-urchin, the polychete annelids Nereis, Chaetopterus, Arenicola, Hydroides among the invertebrates, and two species of fishes and the salamander and frog among the vertebrates.

2 The data concerning susceptibility gradients, so far as they have been pubhshed, will be found in the following papers: Child, "Studies on the Dynamics of Morphogenesis and Inheritance in Experimental Reproduction, I-V, VII, VHI," Jour, of Exper. ZooL, X, XI, XIII, XIV, XVI, XVII, 1911-14; "Studies, etc., VI," Archh fur Entwickelirngsmechanik, XXXVII, 1913; "Certain Dynamic Factors in Experimental Reproduction and Their Significance for the Problems of Reproduction and Development," Archiv fiir Entwickelungsmechanik, XXXV, 1913; "Susceptibility Gradients in Animals," Science, XXXIX, No. 993, 1914; "The Axial Gradient in Ciliate Infusoria," Biol. Bull., XXVI, 1914; "Axial Gradients in the Early Development of the Starfish," Amer. Jour, of Physiol., XXXVII, 1915.

In organisms or parts with a radial structure gradients in susceptibility may commonly appear in the direction of the radial axis, and in those animals and developmental stages where the outer body surface consists of active living cells and is not covered by a heavy cuticle or exoskeleton a susceptibility gradient from the surface inward has been frequently observed. In the simpler multicellular animals and in those unicellular organisms which possess definite permanent axes, the susceptibihty gradients along the main body axes often persist from the beginning of development throughout Ufe without essential change, but in many cases they undergo various changes during the course of development: they may disappear and new gradients arise with advancing differentiation and the appearance of new organs, or they may undergo reversal in direction in some or most of the tissues of the body. In all cases, however, so far as observed, such changes occur in a definite and orderly way, so that the relation between the original and the final condition is essentially constant and characteristic for a given species. In spite of the developmental alterations, it is true, as far as observations go at present, that for each of the main axes of the body a defuiite susceptibility gradient exists, at least during the earlier stages following the appearance of the axis, and a definite relation exists between the direction of the gradient from high to low susceptibility along a given axis and the course of development and differentiation and the functional correlation of different parts with reference to the same axis.

The following figures will serve to show something of the definiteness of the gradient along the apico-basal axis in single cells. Figs. 3-7 show the course of death and disintegration along the axis in Stentor coendeiis, one of the common infusoria. Fig. 3 represents the normal animal in extended condition, showing the Figs. 3-7. — Axial susceptibility gradient of Stentor in cyanide: Fig. 3, intact animal; Fig. 4, beginning of disintegration; Figs. 5-7, successive stages of disintegration.

flattened peristome at the free apical end with its spiral of large cilia, the shorter cilia over other parts, the longitudinal striations or fibrillae, and the elongated basal region with organ of attachment . In cyanide the body undergoes some contraction, death begins at the apical end (Fig. 4) and is accompanied by the instantaneous loss of all movement and disintegration of structure in the part concerned, and the protoplasm swells and spreads out in the water, as indicated by the dotted outline in Fig. 4. Other parts remain intact and the cilia continue to vibrate. From the apical region death and disintegration proceed along the body as shown in Figs. 5-7, the line of demarcation between the dead and disintegrated and the living portions remaining distinct at all times until the progress of death ends at the basal end of the body. The rate of progress of death over the whole body may vary from a few seconds to five or ten minutes, according to concentration of cyanide used, temperature, and other conditions. Deviations from this course are very rare and are probably the result of local stimulations of one part or another of the body.

In Fig. 8 the beginning of death and disintegration in the unfertilized starfish egg is shown. The region of the egg where disintegration begins is that region where the nucleus lies nearest the surface. When the egg develops this region gives rise to the apical end of the embryo and larva. From this region disintegration proceeds through the egg along the axis determined by the eccentric position of the nucleus (Fig. 9), and this axis corresponds with the major axis of the embryo and larva. The same susceptibility gradient also appears in embryonic and early larval stages. In these cases the death gradient does not indicate the presence of more than one axis. In many forms other axes are also indicated by the course of death. In the embryo of the

frog, for example, which is bilaterally symmetrical and in which three axes, the major or longitudinal axis and the minor transverse and dorso-ventral axes, are distinguishable in the arrangement of parts, disintegration begins first of all at the anterior end and proceeds posteriorly, and at any level of the body it begins in the median dorsal region and proceeds laterally and ventrally. The susceptibiHty gradients in particular organs or parts of the body also show a relation to the axes of these

Figs. 8, 9. — Axial susceptibility gradient of starfish egg in cyanide parts. In the elongated tentacles of hydra and various sea-anemones, for example, death begins at the tip and proceeds toward the base, and in nerves, so far as examined, a susceptibility gradient exists and death proceeds in the direction of conduction. Many other examples might be cited to show the relation between the progress of death over the body and the axes with reference to which an order in the course of development, the arrangement of parts, or the behavior of the organism can be distinguished. For the present, however, it must suffice to say that the results

of experimentation along this line have demonstrated beyond a doubt the existence of such gradients as a general feature of the constitution of the animal body. Such susceptibility gradients may be demonstrated, not only by the course of death over the body, but by the different degrees of retardation or inhibition of growth and development at different levels under the same experimental conditions. I have described such retardation or inhibition gradients as observed in the flatworm Planaria,^ and in the development of the seaurchin I have found it possible to alter and control to a high degree the form and proportions of the larva through the differences in susceptibility along the axes to various reagents. Such gradients are also very clearly evident in many cases described by various authors of the effect of external conditions of various kinds of development. The abnormal forms produced in such experiments almost invariably indicate the existence of axial differences in susceptibility. The gradient which appears in such cases is usually the acclimation gradient, the regions of highest metabolic rate being least susceptible and so least affected, but if the external factor acts with sufficient intensity or if acclimation does not occur, the differences in susceptibility are parallel with the metabolic gradient itself. In the embryo of the frog, which has been much used for experiments of this sort, various experimental conditions may retard or inhibit developmental processes in the

^ Child, "Studies on the Dynamics of Morphogenesis and Inheritance in Experimental Reproduction, IV, Certain Dynamic Factors in the Regulatory Morphogenesis of Planaria dorotocephala in Relation to the Axial Gradient," Jour, of Ex per. ZooL, XIII, 1912. posterior region of the body while in the anterior region development proceeds more or less normally. In such cases the posterior regions, which possess a lower metabolic rate than anterior regions, do not acclimate to the conditions as readily as the latter and are therefore retarded or inhibited to a greater extent in their development. Such embryos produce certain characteristic forms of monsters, more or less completely normal anteriorly and increasingly abnormal in the posterior direction. Where acclimation does not play a part the anterior regions of the embryo may be most, the posterior least, affected and another type of monsters results. In many of these monstrous forms the symmetry gradients as well as the major gradient appear more or less clearly

In fact the field of teratogeny, the experimental production of monstrous or abnormal forms, contains a large amount of evidence for the existence of susceptibility gradients, but neither the relation between susceptibility and metabolic rate nor the existence of the metabolic gradients has been recognized by the investigators in this field. There is no doubt that further experiments directly concerned with the problem of susceptibility and metabolic gradients will afford even more definite and positive results.

These gradients in susceptibility indicate the existence in the animal organism of more or less definite metabolic gradients essentially quantitative in nature. In other words, we find a definite order in the gradation of rate or intensity of general metabolic activity in directions coinciding with those in which an orderly sequence of events and arrangement of parts or an orderly behavior of the organism in other respects are distinguishable. Alteration or even reversal of certain gradients during development in some cases makes it necessary to distinguish between the primary gradients, existing at the beginning or in the early stages of development, and the secondary gradients, which arise by alteration of the primary.

The primary relations between the most conspicuous metabolic gradients and the chief axes of the individual is briefly as follows. The major axis is represented by a gradient in which the apical region is always primarily the region of highest, and the basal, that of the lowest, rate of reaction. Stated in different terms, the region of highest metabolic rate in this gradient always gives rise in development to the apical region or head of the animal, the region of lowest rate to the basal or posterior end. In radial gradients the region of highest rate may be either peripheral or central according to the character of the radius. In bilaterally symmetrical animals the relations differ in different cases. In at least most bilaterally symmetrical invertebrates the median ventral region is primarily the apical region of the minor body axes, and from this region gradients of decreasing rate extend laterally and dorsally. In the vertebrates, on the other hand, the median dorsal region is primarily the apical region, and gradients of decreasing rate extend laterally and centrally. The fact must be emphasized that these are the general and primary relations and that they may be altered in various, but always orderly and definite, ways during the development of the individual.

These facts indicate very clearly that the chief axes of the animal body are represented dynamically by metabohc gradients and that each organ or part arises in a relation to one or more of these gradients which is definite and characteristic for each kind of organism. The relation of the central nervous system to these gradients is highly significant. The apical portion of the central nervous system, the cephalic ganglion or brain, always arises in the region of highest metabolic rate in the whole body, the apical region of the major axis, and such portions of the central nervous system as appear in other parts of the body, e.g., the longitudinal ganglionic nerve cords of various invertebrates and the spinal cord of vertebrates, always arise in the regions of highest rate in the minor axial gradients. In the bilateral invertebrates this is the median ventral, in the vertebrates the median dorsal, region. In short, it may be said that where a central nervous system is present it is the organ characteristic of the apical, i.e., the dominant, region in each of the chief axial metabolic gradients. The functional dominance of the central nervous system in the later Kfe of the animal is then simply a more highly specialized expression of the primary relation of dominance and subordination existing at the beginning of individuation between regions of high and those of lower metabohc rate.

As regards plants, I have as yet examined only some fifteen species of marine algae, but in all of these the apical region of each axis shows the highest susceptibility to the higher concentrations of cyanides and the susceptibility decreases very markedly in the basal direction. In these plants there is no such disintegration at death as in the lower animals, although in the more transparent forms the breaking up and coagulation of the protoplasm can be observed inside the cell. By first

staining the plants with neutral red and then killing with cyanide or some other reagent the susceptibility gradient can be made visible, for as the cells die the red of the stain at first becomes deeper because of increasing acidity, then changes to yellow as the alkali of the solution enters, and finally all color disappears. The susceptibility gradients do not constitute the only experimental evidence for the existence of metabolic gradients in the organism. Estimations of carbondioxide production by means of the Tashiro biometer,^ which were made by Dr. Tashiro at my request, have confirmed the results obtained by the susceptibility method in all cases subjected to this test. The gradient in carbon-dioxide production is similar to the gradient in metabolic rate indicated by the differences in susceptibility. On the other hand, in the case of certain nerves I have been able to confirm Tashiro 's recent discovery of a gradient in carbon-dioxide production in the direction of conduction of the impulse along the fiber by the demonstration of a gradient in susceptibility in the same direction, and have found a similar susceptibility gradient in certain other nerves for which carbon-dioxide production has not been determined The gradient in the production of carbon dioxide indicates the existence of a gradient in the rate or intensity of the respiratory processes, the oxidations, the region

'Tashiro, "A New Method and Apparatus for the Estimation of Exceedingly Minute Quantities of Carbon Dioxide," Amer. Jour, of Physiol., XXXII, 1913. of highest carbon-dioxide production being the region of highest respiratory rate. Since the oxidations are unquestionably reactions of fundamental importance in the metabolic reaction system, the estimations of carbon-dioxide production lead to the same conclusions concerning the existence of metabolic gradients as do the results obtained by the susceptibility method.

So far as technical and other sources of error can be eliminated, the rate of oxygen consumption of different parts of the body may be used like the rate of carbondioxide production as a measure of respiratory activity. The use of this method in animal physiology has been such that the data, while of great value for various other purposes, have in most cases no bearing upon the problem of metabolic gradients. In the plants, however, the rate oi both oxygen consumption and carbon-dioxide production have been found to differ in different parts in such a way as to indicate very clearly the existence in the plant-body of metabolic gradients. The growing bud, for example, respires at a higher rate than the full-grown stem or leaf.

Differences in electrical potential indicating differences of some kind in chemical or physical activity are known to occur very generally in different parts of both animal and plant organisms and even in different parts of the same organ or cell. The presence of these electrical differences gives no clue to the exact nature of the physical or chemical differences which produce them, but it is becoming more and more evident that in both animals and plants they are to a large extent associated with differences in metabolic activity. So far as this is the case, we should expect in general that

parts with a higher respiratory rate would appear by the usual methods as electro-negative to regions of lower rate. Some twelve years ago Mathews* observed a difference in electrical potential along the main axis of certain simple animals, the hydroids, the parts nearer the apical end being electro-negative to those nearer the basal end. In these forms the susceptibility method indicates that the metabolic rate decreases from the apical toward the basal end; that is, in the same direction as the decrease in electro-negativity. Probably a similar electrical gradient exists in nerves, although in the nerves of the higher animals the change is undoubtedly very slight within the physiological limits of length. As regards the plants also various data on the differences of electric potential suggest the existence of metabohc gradients, although the fact that the observations were made with other objects in view often leaves the evidence inconclusive as regards the matter of gradients.

In the early stages of development of the starfish I have been able to make the axial metabolic gradient directly visible to the eye by differential staining in the living animal,'' the stain in this case consisting of a colored precipitate formed within the cells by the oxidation of certain substances added to the water. The rate of formation of this precipitate in different cells differs with the amount or activity of enzymes or other conditions which influence the rate of oxidation. In those

=* Child, "Axial Gradients in the Early Development of the Starfish," ibid.,XXXYll, 191 5. cells where the rate of oxidation is highest the precipitate is formed most rapidly and vice versa. In the starfish embryos and early larvae the precipitate appears first in the cells of the apical region, and a very definite color gradient along the main axis arises in living animals, while in animals which have been killed before staining no gradient appears. This method is undoubtedly capable of wide application.

These various methods and results indicate the possibilities of demonstrating the existence of the metaboHc gradients in organisms by biochemical and physiological methods. Unquestionably future investigation will give us much more accurate and extensive data than we possess at present. Gradients in rate of cell division, size of cells, condition or amount of protoplasm in the cells, rate of growth, and rate and sequence of differentiation are very characteristic features of both animal and plant development. Such gradients are definitely related to the axes of the individual or its parts, and are evidently expressions of axial metabolic gradients. While the existence of such gradients indicates the existence of gradients in activity of some sort, the various kinds of gradients are not all necessarily present where metabolic gradients exist. In some cases the visible gradient may be a gradient in rate of growth or in protoplasmic constitution; in still others a gradient in sequence of differentiation, etc., and sometimes metabolic gradients exist without any structural indications of their presence.

At best these various kinds of gradients are merely general indications of differences in metabolic rate, and undoubtedly in many cases the visible differences along an axis represent something more than differences in metabolic rate. The important point is that visible indications of graded differences in metabolic rate occur so generally in definite relations to the chief axes of the body. In the animal egg a gradient in the distribution of the yolk is often visible before development begins, and in such cases that part of the egg which gives rise to the apical region of the embryo contains less yolk than the basal region.' Associated with this gradient in most cases we find differences in the size of cells appearing in very early embryonic stages. In the egg of the frog, which is an excellent example of this sort of egg, the yolk gradient is very distinct, and the early developmental stages show a gradient in the same direction in the rate of cell division and the size of the cells formed (Figs. lo, ii). The yolk gradient and the associated gradient in cell division differ widely in different kinds of eggs: in some cases only the apical region of the egg divides at all, other parts serving as a source of nutrition which is gradually used up during development. At the other extreme are cases in which no yolk gradient is distinguishable and differences in division rate and size of cells do not become evident until later stages.