The Organism as a Whole, from a Physicochemical Viewpoint

The process of heat coagulation of colloids is also a function of time. If the exposure to high temperature is not sufficiently long, only part of the colloid coagulates ; in this case an organism may again recover. Inside of these upper and lower temperature limits we find that life phenomena are influenced by temperature in such a way that their rate is about doubled for an increase of the temperature of 10° C., and that this temperature coefficient for 10°, QIO, very often steadily diminishes from the lower to the higher temperature; so that near the lower temperature limit it becomes often considerably greater than 2 and near the higher temperature limit it becomes very often less than 2.1 This influence of temperature is so general that we are bound to associate it with an equally general feature of life phenomena; and such a feature would be most likely the chemical reactions. It is known through the work of Berthelot, van't Hoff, and Arrhenius that the temperature coefficient for the velocity of chemical reactions is also generally of about the same order of magnitude; namely ^ 2 for a difference of 10°. In chemical reactions there is also a tendency for QIO to become larger for lower temperature, and coefficients of QIO about 5 or 6 have repeatedly been found for purely chemical reactions between o° and 10°, e. g., for the inversion of cane sugar by the hydrogen ion. The temperature coefficient for the reaction velocity of ferments shows the same diminution of QIO with

1 A full discussion of the literature on temperature coefficients is given in A. Kanitz's book on Temperatur und Lebensvorgdnge, Berlin, 1915, rising temperature which is also noticed in most life phenomena. Thus Van Slyke and Cullen1 found that the reaction rate of the enzyme urease "is nearly doubled by every 10° rise in temperature between 10° and 50°. Within this range the temperature coefficient is nearly constant and averages 1.91. From o° to 10° it is 2.80, from 50° to 60° it is only 1.09. The optimum is at about 55°." The rapid fall of the temperature coefficient for enzyme action at the upper temperature limit has been ascribed by Tammann to a progressive destruction of the active mass of enzyme by the higher temperature (by hydrolysis). This will, however, not account for the high value of the coefficient near the lower limit. But is it not imaginable that at low temperature an aggregation of the enzyme particles exists which is also equivalent to a diminution of the active mass of the enzyme and that this aggregation is gradually dispersed by the rising temperature? This would account for the fact that at a temperature near o°C life phenomena stop because the enzymes are all in a state of aggregation or gelation; that then more and more are dissolved and the rate of chemical reaction increases since the mass of enzyme particles increases until all the enzyme molecules are dissolved or rendered active. Under this assumption three processes are superposed in the variation of the value

of QIO with temperature: (i) the supposed increase in the number of available ferment molecules with increasing temperature near the lower temperature limit; (2) the temperature coefficient of the reaction velocity which is nearly = 2 for io°C.; (3) the diminution of the number of available ferment molecules by hydrolysis or some other action of the increasing temperature. This latter is noticeable near the upper temperature limit. The reason that I and 3 interfere more strongly in life phenomena than in the chemical reactions of crystalloid substances may possibly be accounted for by the fact that the enzymes and most of the constituents of living matter are colloidal, i. e., consist of particles of a considerably greater order of magnitude than the molecules of crystalloids. x

We will now show the role of the temperature coefficient upon phenomena of development. F. R. Lillie and Knowlton2 first determined the influence of temperature upon the development of the egg of the frog and showed that it was of the same nature as that of a chemical reaction. These experiments were repeated a year later by 0. Hertwig.3 1 These considerations may meet the objections of Krogh to the application of the van't Hoff rule of temperature effect on reaction velocity to life phenomena. See also the discussion of this subject in Kanitz's book.

The time required for the eggs to reach definite stages was measured for different temperatures and it was found that the temperature coefficient QJO between 2.5° and 6° was equal to 10 or more; between 6° and 15° it was between 2.6 and 4.5; between 10° and 20° it was 2.9 to 3.3, and between 20° and 24° it was between 1.4 and 2.0. To anybody who has worked on this problem it is obvious that no exact figures can be obtained in this way, since the point when a certain stage of development is reached is not so sharply defined as to exclude a certain latitude of arbitrariness. The writer found that very exact figures can be obtained on the influence of temperature upon development of the sea-urchin egg by measuring the time from insemination to the first cell division. Such experiments were carried out in a cold-water form Strongylocentrotus purpuratus and a form living in warmer water, Arbacia.1 The figures on Arbacia have been verified by different observers in different years.

INFLUENCE OF TEMPERATURE UPON THE TIME (IN MINUTES) REQUIRED These figures permitted the determination of the temperature coefficients QIO with a sufficient degree of accuracy (see next table). It seemed of importance to attempt to decide what the chemical reaction underlying these reaction velocities is (if it is a chemical reaction). Loeb and Wasteneys1 investigated the temperature coefficient for the rate of oxidations in the newly fertilized egg of Arbacia and found that the temperature coefficient QIO for that process does not vary in the same way as the temperature coefficient for cell division.

It is obvious that the temperature coefficient of the rate of oxidations is remarkably constant, about 2 for 10°, for various temperatures and does not show the variation from 7 or more to 2.2 for QIO for the rate of segmentation. Kanitz1 has shown that in a graph in which the logarithms of the segmentation velocities are drawn as ordinates and the temperatures as abscissas the logarithms form two straight lines which are joined at an angle. According to the law of van't Hoff and Arrhenius concerning the influence of temperature upon velocities of chemical reactions the logarithms should lie in a straight line. We are dealing therefore in these cases with two exponential curves, one representing in Arbacia the interval 7-13° and the second from 13-26°; in Strongylocentrotus between 3-9° and 9-20°.

It was found in these experiments that if measurements of the QIO of later stages of development are attempted the variations due to unavoidable difficulties become too great to permit an equal degree of reliability in the determinations. The vast importance of this influence of temperature upon the rate of development is seen in the fact that in addition to the food supply the rate of the maturing of plants and animals depends on this factor. 2. This influence of temperature upon development has been used to find the conditions determining fluctuating variation. The reader knows that by this expression are understood the differences between individuals of a pure strain or breed. These variations

are not inherited, a fact contrary to the idea of Darwin, who assumed that by the selection of extreme cases of fluctuating variation new varieties could develop. What is the basis of this fluctuating variation? The writer concluded that if fluctuating variations were due to a slight variation in the quantity of a specific substance — in some cases an enzyme — required for the formation of a hereditary character, the temperature coefficient might be used to test the idea. We have just seen that the time required from insemination until the cell division of the first egg occurs is very sharply defined for each temperature. If a large number e.g. one hundred or more eggs are under observation simultaneously in a microscopic field it can be seen that they do not all segment at the same time but in succession; this is the expression of fluctuating variation. Miss Chamberlain and the writer have measured the time which elapses between the moment the first egg of such a group segments and the moment the last egg begins its segmentation, and found that this latitude of variation is also very definite for each temperature, and that its temperature coefficient is for each interval of 10° practically identical with the temperature coefficient of the segmentation for the same interval.1 The slight deviations are practically all in the same sense and accounted for by a slight deficiency in the nature of the experiments. The 1 Loeb, J., and Chamberlain, M. M., Jour. Exper. Zool.t 1915, xix., 559.

two following tables give the latitude of variations for different temperatures for the first segmentation in Arbacia and the temperature coefficient for this latitude and the rate of segmentation. These two latter coefficients are practically identical. If we assume that the temperature coefficient for the segmentation of the egg is that of a chemical reaction (other than oxidation) underlying the process of segmentation, the fluctuating variation in the time of the segmentations of the various eggs fertilized at the same time is due to the fact that the mass of the enzyme controlling that reaction varies within definite limits in different eggs. The first egg segmenting at a given temperature has the maximal, the last egg segmenting has the minimal mass of enzyme. It should be added that the time of the first segmentation is determined by the cytoplasm and is not a Mendelian character, as was stated in a previous chapter.

3. The point of importance to us is that the influence of temperature upon the organism is so constant that if disturbing factors are removed it would be possible to use the time from insemination to the first segmentation of an egg of Arbacia as a thermometer on the basis of the table on page 295. Facts of this character should dispose of the idea that the organism as a whole does not react with that degree of machine-like precision which we find in the realm of physics and chemistry. Such an idea could only arise from the fact that biologists have not been in the habit of looking for quantitative laws, chiefly, perhaps, because the difficulties due to disturbing secondary factors were too great. The worker in physics knows that in order to discover the laws of a

phenomenon all the disturbing factors which might influence the result must first be removed. When the biologist works with an organism as a whole he is rarely able to accomplish this since the various disturbing influences, being inseparable from the life of the organism, can often not be entirely removed. In this case the biologist must look for an organism in which by chance this elimination of secondary conditions is possible. The following example may serve as an illustration of this rather important point in biological work. Although all normal human beings have about the same temperature, yet if the heartbeats of a large number of healthy human beings are measured the rate is found to vary enormously. Thus v. Korosy found among soldiers under the most favourable and most constant conditions of observations — the soldiers were examined early in the morning before rising — variations in the rate of heart-beat between 42 and 1 08. In view of this fact, those opposed to the idea that the organism as a whole obeys purely physicochemical laws might find it preposterous to imagine that the rate of heart-beat could be used as a thermometer. Yet if we observe the influence of temperature on the rate of the heart-beat of a large number of embryos of the fish Fundulus, while the embryos are still in the egg, we find that at the same temperature each heart beats at the same rate, the deviations being only slight and such as the fluctuating variations would

demand.1 This constancy is so great that the rate of heart -beat of these embryos could in fact be used as a rough thermometer. The influence of temperature upon the rate of heart-beat is completely reversible so that when we measure the rate for increasing as well as for decreasing temperatures we get approximately the same values as the following table shows. Time Required for Nineteen Heart-beats in the Embryo of Fundulus Why does each embryo have the same rate of heartbeat at the same temperature in contradistinction to the enormous variability of the same rate in man? The answer is, on account of the elimination of all secondary disturbing factors. In the embryo of Fundulus the heart-beat is a function almost if not exclu-

sively of two variables, the mass of enzymes for the chemical reactions underlying the heart-beat and the temperature. By inheritance the mass of enzymes is approximately the same and in this way all the embryos beat at the same rate (within the limits of the fluctuating variation) at the same temperature. This identity exists, however, only as long as the embryo is relatively quiet in the egg. As soon as the embryo begins to move this equality disappears since the motion influences the heart-beat and the motility of different embryos differs.

In man the number of disturbing factors is so great that no equality of the rate for the same temperature can be expected. Differences in emotions or the internal secretions following the emotions, differences in previous diseases and their after-effects, differences in metabolism, differences in the use of narcotics or drugs, and differences in activity are only some of the number of variables which enter. 4. As stated above the temperature influences practically all life phenomena in a similar characteristic way, e. g., the production of CO2 in seeds1 and the assimilation of C02 by green plants.2 The writer would not be surprised if even the aberrations in the colour of butterflies under the influence of temperature

turned out to be connected with the temperature coefficient. 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 o° to i° 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 seen clearly 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:

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

physicochemical point of view, we must realize 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.t hydrolyses) causes or furnishes the material for a second reaction or group of reactions (b) (e. g., oxidations). We know that the temperature coefficient for physiological processes varies slightly at various parts of the scale; as a rule it is higher near o° and lower near 30°. But we know also that the temperature coefficients do not vary equally for 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 Cstwald has produced experimentally, through variation of temperature, dimorphism of form in Daphnia. 5. Next or equal in importance with the temperature is the nature of the medium in which the cells are living. It has often been pointed out that the marine animals and the cells of the body of metazoic animals are surrounded by a medium of similar constitution, the sea water and the blood or lymph, both media being salt solutions differing in concentration but containing the three salts NaCl, KC1, and CaCl2 in about the same relative concentration, namely 100 molecules NaCl : 2.2 molecules of KC1 : 1.5 molecules of CaCl2. This has suggested to some authors the poetical dream that our home was once the ocean, but we cannot test the idea since unfortunately we cannot experiit with the past. Plants, unicellular fresh-water algae, and bacteria do not demand such a medium for their existence.

Herbst had shown that when sea-urchin larvae were raised in a medium in which only one of the constituents of the sea water was lacking (not only NaCl, KC1, or CaCl2, but also Na2S04, NaHC03, or Na2HP04), the eggs could not develop into plutei; from which he concluded that every constituent of the sea water was necessary. This would indicate a case of extreme adaptation to all the minutiae of the external medium. for this purpose, namely, the eggs of the marine fish Fundulus, gave altogether different results. The eggs of this marine fish develop naturally in sea water but they develop just as well in fresh or in distilled water, and the young fish when they are made to hatch in distilled water will continue to live in this medium. This proves that these eggs require none of the salts of the sea water for their development. When these eggs are put immediately after fertilization into a pure solution of NaCl of that concentration in which this salt exists in the sea water practically all the eggs die without forming an embryo; but if a small quantity of CaCl2 is added every egg is able to form one, and these embryos will develop into fish and the latter will hatch. This led the writer to the conclusion that these fish (and perhaps marine animals in general) need the Ca of the sea water only to counteract the injurious effects which a pure NaCl solution has if it is present in too high a concentration. 1 When we raise the eggs in a pure NaCl solution of a concentration ^rm/8 practically every egg will develop; and even in a m/4 or 3/8 m many or some eggs will form embryos without adding Ca ; it may be that a trace of Ca present in the membrane of the egg may suffice to counterbalance the injurious action of a weak salt solution.

The concentration of the NaCl in the sea water at Woods Hole (where these experiments were made) is about m/2, and as soon as this concentration of NaCl is reached the eggs are all killed as a rule before they can form an embryo, unless a small but definite amount of Ca is added. It was found that the eggs can be raised in much higher concentrations of NaCl, but in that case more Ca must be added. The following table gives the minimal amount of CaCl2 which must be added in order to allow fifty per cent, of the eggs to form embryos. (The eggs were put into the solution an hour or two after fertilization.)

This indicates that the quantity of CaCl2 required to counteract the injurious effects of a pure solution of NaCl increases approximately in proportion to the square of the concentration of the NaCl solution.1 The reader will notice that the eggs can survive and develop in a solution of three times the concentration of sea water, provided enough Ca is added. It was found also that not only Ca but a large number of other bivalent metals were able to counteract the injurious action of an excessive NaCl solution; namely Mg, Sr, Ba, Mn, Co, Zn, Pb, and Fe;2 only Hg and Cu could not be used since they are themselves too toxic. The antagonistic efficiency of the bivalent cations other than Ca was, however, smaller than that of Ca. The following table gives the highest concentration of NaCl solution in which the newly fertilized eggs of Fundulus can still form an embryo.3

On the other hand it was seen that in all the chlorides with a univalent cation, LiCl, KC1, RbCl, CsCl, NH4C1, the eggs could form embryos up to a certain concentration of the salt; but that this concentration could be raised by the addition of Ca. In short it turned out that the injurious action of the pure solution of any chloride (or any other anion) with a univalent metal could be counteracted to a considerable extent by the addition of small quantities of a salt with a bivalent metal. It was also found in the early experiments of the writer that the bivalent or polyvalent anions had no such antagonistic effect upon the injurious action of the salts with a univalent cation.

We therefore see that what at first sight appeared in the experiments of Herbst a necessity, namely, the presence of each constituent of the sea water, turns out as a special case of a more general law; the salts with univalent ions are injurious if their concentration exceeds a certain limit and this injurious action is diminished by a trace of a salt with a bivalent cation. eggs of the sea urchin? Before we answer this question, we wish to enter upon the discussion of the nature of the injurious action of a pure NaCl solution of a certain concentration and of the annihilation of this action by the addition of a small quantity of Ca. The writer suggested in 1905 that the injurious action of a pure NaCl solution consisted in rendering the membrane of the egg permeable for NaCl, whereby the germ inside the membrane is killed; while the addition of a small amount of Ca (or any other bivalent metal) prevents the diffusion of Na into the egg, x possibly, as T. B. Robertson2 suggested, by forming a precipitate with some constituent of the membrane, whereby the latter becomes more impermeable. The correctness of this idea can be demonstrated in the following way. When eggs of Fundulus, which are three or four days old and contain an embryo, are put into a test-tube containing 3 m NaCl they will float on this solution for about three or four hours; after that they will sink to the bottom. Before this happens the egg will shrink and when it ceases to float the embryo is usually dead. This is intelligible on the assumption that the NaCl solution entered the egg, increased its specific gravity so that it could not float any longer and killed the embryo. When we add, however, I c. c. 10/8 m CaCl2 to 50 c.c. 3 m NaCl the eggs will float, the

heart will continue to beat normally and the embryo will continue to develop for three days or more, because the calcium prevents the NaCl from entering into the egg.1 For if we put a newly hatched embryo into 50 c.c. 3 m NaCl+i c.c. 10/8 m CaCl2 it will die almost instantly; hence the membrane must have acted for three or more days as a shield which prevented the NaCl from diffusing into the egg in the presence of CaCl2. The same experiments cannot be demonstrated in the sea-urchin egg, first, because it can live neither in distilled water nor in very dilute nor very concentrated solutions; and second, because it is not separated as is the germ of the Fundulus egg from the surrounding solution by a membrane which is under proper conditions practically impermeable for water and salts.

Nevertheless it can be shown that the results at which we arrived in our experiments on Fundulus are of a general application. Osterhout2 has shown that plants which grow in the soil or in fresh water are readily killed by a pure NaCl solution of a certain concentration, while they can resist the same concentration of NaCl if some CaCl2 is added. Wo. Ostwald3 has shown the same for a species of Daphnia. We, therefore, come to the conclusion that the injurious

action following an alteration in the constitution of the sea water is in some of the cases due to an increase in the permeability of the membranes of the cell, whereby substances can diffuse into the cell which when the proper balance prevails cannot diffuse. For this balance the ratio of the concentration of the salts with univalent cation Na and K over those with bivalent 6. The importance of this quotient appears in the so-called "behaviour" of marine animals. We have mentioned the newly hatched larvae of the barnacle in connection with heliotropism. These larvae swim in a trough of normal sea water at the surface, being either strongly positively or negatively heliotropic. They collect as a rule in two dense clusters, one at the window and one at the room side of the dish. If such animals are put into a solution of NaCl+ KC1 (in the proportion in which these salts exist in the sea water), they will fall to the bottom unable to rise to the surface. They will, however, rise to the surface and swim energetically to or from the window if a certain quantity of any of the chlorides of a bivalent metal, Mg, Ca, or Sr, is added, but these movements will last only a few minutes when only one of these three salts is added; and then the animals will fall to the bottom again. If, however, two salts, e. g., MgCl2

and CaCl2, are added the animals will stay permanently at the surface and react to light as they would have done in normal sea water. These animals also can resist comparatively large changes in the concentration of the sea water, and it seemed of interest to find out cMgCl2+CaCl2' allowed all the animals to swim at the surface, had a constant value. The MgQ2+CaCl2 solution was 3/8 m and contained the two metals in the proportion in which they exist in the sea water; namely, n.8 molecules MgCl2 to 1.5 molecules CaQ2. The next table gives the result.1 Since these experiments lasted a day or more each, usually two different concentrations of NaCl+KCl of the ratio 1:2 or 1:4 were compared in one experiment.

remains very nearly constant with varying concentrations of QNa+K. In former experiments on jellyfish the writer had shown that there exists an antagonism between Mg and Cax, and this observation was subsequently confirmed by Meltzer and Auer2 for mammals. It was observed that in a solution of NaCl+KCl+MgCl2 the larvae of the barnacle were also not able to remain at the surface for more than a few minutes, while an addition of some CaCl2 made them swim permanently at the surface. Various quantities of MgCl2 were added to a mixture of m/4 or m/2 NaCl+KCl, to find