Forced Movements, Tropisms, and Animal Conduct

The importance of stereotropism in animals was first pointed out by the experiments of Dewitz on the sperma¬ tozoa of the cockroach.^®®. He noticed that when a drop of salt solution containing the spermatozoa was put under a cover glass resting on low supports on a slide, the spermatozoa collect at the solid surfaces of the slide and cover glass, while the liquid between remains free from spermatozoa. When a small glass bead is put into the liquid the spermatozoa will also swim on the surface of the bead, never leaving it again. Dewitz is of the opin¬ ion that this stereotropism is of assistance in securing the entrance of a spermatozoon into the egg. The egg of the cockroach is rather large and the spermatozoon can enter it only through a micropyle. When the egg is laid it passes by the duct of the seminal pouch in which the female keeps the sperm after copulation. On passing the duct some spermatozoa reach the egg. Dewitz points out that these cannot leave the surface of the egg any more but are compelled to move incessantly on the surface of the egg until one of the spermatozoa by chance gets into the micropyle.

It is an! important fact that different organs of the same organism react differently. We have already men¬ tioned the tendency of starfish or flatworms to right them¬ selves, i.e., their ventral surface is positively their dorsal negatively stereotropic. The stolons of hydroids stick to solid bodies, wMle tbe polyps bend and continue to grow away at right angles from solid bodies with which they come in contact. Thus the stem of Tubularia mesem- bryanthemum, a marine hydroid, grows in a straight line. When such stems, after their polyp is cut off, are put with one end in sand, the free end forms a new poljp and the stem continues to grow in a vertical direction upward. ’When, however, the stem is put near the glass wall as soon as the polyp grows out it bends away from

Fig. 40.—The regenerating polyp of Tubularia when in contact with the glass wall of an aquarium bends at right angles to the glass wall. the sO'lid wall, and the stem will now continue to grow at right angles to the vertical wall (Fig. 40). This phenomenon raises the question whether or not the law of chemical mass action underlies phenomena of stereotropism. We have seen that this law dominates the phenomena of heliotropism, inasmuch as the Bunsen- Eoscoe law is the expression of the influence of light on the mass of the photochemical reaction product. We have also been able to show that in the case of the geo¬ tropic curvature of Bryophyllum the mass of the apical

leaf determines the rate of geotropieal curvature of a horizontally placed stem. The only way in which the mass of the leaf could have such an. influence is through the mass of substances it sends into the stem, so that this case of geotropism is a function of mass ^tion. There are indications that the way contact with a solid in¬ fluences the behavior of living matter is also through the influence on the rate of certain chemical reactions. The writer observed that the stolons of a hydroid, Aglao- phenia, have a tendency to adhere to solid surfaces and not to leave them any more if they once reach them, and that as soon as such a stolon reaches a solid surface, e.g., a piece of a glass slide, its growth is accelerated con¬ siderably. It was very astonishing to notice how much more rapid the growth of roots of Aglaophenia was when they were in contact with a solid body than when they grew in sea water. The rate of growth is the function of a chemical mass action (Loeb ).

1. When we create a center of diffusion in water or in air we may theoretically expect orienting effects. Thus when a fine capillary tube containing a solution of a salt, e.g., sodium malate, is put into a drop of water containing motile organisms, and the right side of an organism is turned to the source of diffusion, the diffusing molecules wiU collect in increasing concentration on that side. On the left side of the organism, no such increase in the con¬ centration of molecules will occur. If now the molecules collecting on the right of the organism in increasing den¬ sity are able to produce some chemical or some concen¬ tration chain effect, the two sides of the organism will be acted upon unequally and the tension of the symmetrical motile organs will no longer he the same. As a conse¬ quence the organism will turn until the mass of molecules or ions striking the organism in the unit of time will be the same for both sides. These effects only take place when the organism is close to the opening of the capillary tube, since the diffusion from the tube is slow.

It is obvious, however, that it is difficult to provide experimental conditions which give exact chemotropic reactions. First of all, if the diffusion is rapid the differ¬ ences in concentration of the effective chemotropic sub¬ stance on two sides of an organism are too slight to result in a turning movement. A second condition which is liable to vitiate the result are the unavoidable convection cur¬ rents due to changes or differences of temperature. In

order to get clear results a method must be used which prevents a rapid diffusion of the substance; and, more¬ over, the current of diffusion must be confined to an almost straight line. It is possible that Pfeffer’s method satisfies this condition.^^^. He introduced the sub¬ stance to be tested for its chemotropic effect into a capil¬ lary tube, the end of which was then sealed. The other end was pushed into a drop of water containing the sus¬ pension of the organisms whose chemotropism was under investigation. From this capillary the diffusion was ex¬ tremely slow. Moreover, the current of diffusion was approximately linear at the orifice. Hence the test for the existence of positive chemotropism was perhaps pos¬ sible. When an organism, struck sidewise by the line of diffusion near the opening of the capillary tube, turns toward the tube going into it, some probability of positive chemotropism exists; and when all the organisms coming near the orifice of the tube are thus compelled to go into it, the probability may become certainty, provided that the substance used does not paralyze the organism and therefore act as a trap, allowing the organisms to come in but not to go out. The capillary tubes used were of 10 to 15 mm. length and of a width of about 0.1 mm. Pfeffer and his pupils found that the spermatozoa of ferns go in large numbers into a capillary tube containing sodium malate in a concentration of 0.01 per cent, (a solu¬ tion ten times as diluted is still slightly active). This effect of the malate is specific in this case and this indi¬ cates that either a definite chemical action of the malate ion or a specific permeability of the organism for it is the source of the chemotropism. Such specific chemotropic effects are not rare, since Pfeffer found that Bacterivm termo and Spirillum undula are positively chemotropic

to a liquid containing 0.001 per cent, of peptone or of meat extract. It is stated that cholera bacilli are strongly attracted by potato sap. Pfeffer found also that the sper¬ matozoa of certain mosses are positively chemotropic to cane sugar solution in dilutions of 0.1 per cent. Pfeffer’s work preceded the discovery of electrolytic dissociation, and his pupils Buller®* and Shibata^®® made some of the additions required by the theory, na,mely, that it is the malate anion which acts in the case of the spermatozoa of the ferns, and that when the anion is offered in the form of malic acid the H ion counteracts the effect of the malate anion.

Shibata made extensive experiments on the chemotrop- ism of the spermatozoa of Isoetes *®® which he found posi¬ tively chemotropic for the malate anion, and also for the succinate, tartrate, and fumarate anion, when offered in the form of their neutral salts. The anion of the stereo- isomere of fumaric acid, namely of maleic acid, was with¬ out effect. This indicates a high degree of specificity of these reactions. Neutral sodium malate acted best hi dilutions from m/100 to m/1000, but some action could still be discovered in m/20,000 solutions.

When malic acid was used no positive chemotropism could be discovered in solutions of m/100 or above on account of the contrary effect of the hydrogen ion, and the spermatozoa of Isoetes did not even go into capillary tubes containing m/1000 malic acid. WTien any acid other than malic was added to sodium malate the motion of the spermatozoa into the tube was prevented, even a m/6000 HCl solution still had such an effect. Shibata studied especially the mode by which the spermatozoa are oriented chemotropically by malates and found that the reaction consists always in a turning- of

the axis of tlie body of the spermatozoa toward the capil¬ lary tube eontaining malates or succinates, as the tropism theory demands. When the capillary tube and the surrounding medium contain the same solute for which the organisms are posi¬ tively chemotropic, they will not go into the tube unless the concentration in the tube is a definite multiple of the concentration of the outside solution. Thus Pfeffer found that the concentration of sodium malate in the capillary must be at least thirty times as great as in the outside solu¬ tion to induce the spermatozoa of fern to move into it, and in the case of Bacterium termo the solution of meat ex¬ tract in the tube had to be at least four times as great as the outside solution. In the case of Isoetes spermatozoa Shibata found the ratio of about 400 to 1. This constancy of the ratio is known as Weber’s law, which therefore holds for chemotropic phenomena.

Lidforss found with the aid of Pfeifer’s method that the spermatozoa of Marchantia are positively chemo- tropic to certain proteins, especially egg albumin, vitellin from the egg yolk, hemoglobin, and mucin of the sub¬ maxillary gland; blood albumin, casein, and Icgumin wore less effective. The lowest concentration for hemoglobin solutions and for egg albumin was 0.001 per cent. 1 It may also be stated that Lidforss found a chemo¬ tropic effect of proteins upon the direction of growth of pollen tubes.^®

Bruchmann®! found that the spermatozoa of Lyco¬ podium were positively chemotropic to the watery extract in which pieces of the prothallium had been boiled. Pfef¬ fer’s capillary method was used. They showed also posi¬ tive chemotropism to the citrate anion. Thus, sodium citrate was efficient in a 0.1 to 0.5 per cent, solution. The ' lower limit was a little above a 0.001 per cent, solution. Tbe effect of the free citric acid was a mixed one since the spermatozoa were negative to H ions and positive to the citrate anion. Instead of being able to use a 0.1 per cent, solution, as in the case of the sodium salt, a 0.01 per cent, solution was the highest concentration to which they were positively chemotropic. This means that the hydrogen ion of citric acid solutions above m/1000 repel the spermatozoa, while when solutions of m/2000 or below are used the hydrogen ion effect no longer in¬ hibits the positive effect of the citrate anion. In addition the validity of Weber’s law could be demonstrated. The spermatozoa were indifferent to malates, oxalates, and many other salts, as well as to sugar and proteins.

2. While all the botanical observers, from Buffer on, had found that tbe hydrogen ion has only a preventive effect upon the positive chemotropism of lower organisms, Jennings tried to show that acids have a positive effect, especially when in low concentrations.®®® But his con¬ centrations are not quite as low as he seems to assume, since a 1/50 per cent, (m/180) HOI solution, toward which he believes to have proven positive chemotropism of Para- mcecia, is a deadly concentration.®' Jennings’s interest in the problem was aroused by a phenomenon of aggregation, not infrequently found in the suspensions of infusorians.

It is well known that when certain infusoria are left undisturbed they do not remain scattered, but gather in more or less dense group.s. Thus, if they are mounted on a slide in a thin layer of water, soon dense aggravations will be formed in certain areas, while the remainder of the iThe cells of the stomach resist a much higher concentration of HCl but this is an exception. Infusorians, flsh, and organisms in. general are killed in a short time in m/180 HCl or in a much lower concentration of acid. Thus Pundulus does not live more than one hour in m/3000 HCl or HNO,. (Loeb, J., and Wasteneys, H., Biochem Z., 1911, xxxiii, 489; 1912, xxxix, 167.)

slide will be nearly deserted. One of the first investigators to describe this phenomenon was Pfefier. He observed its occurrence in Glaucoma scintUlanSy and less markedly in Colpidium colpoda, Stylonychia mytilus, and Paramcecium. Pf effer was inclined to believe that these aggregations were due, partly at least, to a contact stimulus, resulting from a striking of the organisms against small solid bodies, and especially against each This conclusion of Pfeffer may after all be correct, since it has been shown that sea water containing jelly from the egg of a sea urchin causes spermatozoa to stick together for some time when they impinge upon each other. This agglutination no longer occurs when the spermatozoa are immobilized. Jennings came to the con¬ clusion that these aggregations of infusorians are due to the fact that they can go into a weak concentration of acid, while they cannot escape from such a weak concen¬ tration; and since Parcmuecia themselves produce COj he assumed that the COj produced by themselves acts as a center of attraction for other Paramcecia. In order to prove this he used the following method:

The organisms were studied in a thin layer of water, by mounting them on a slide covered with a large cover glass supported near its ends by slender glass rods. Their reactions were tested by introducing with a capillary pipette a drop of the substance in question beneath the cover glass, or in some cases by allowing it to diffuse inward from the side of the cover glass^^o Thus Jennings introduced a drop of 1/50 per cent. (m/180) HCl on a slide containing ChUomonas. Very soon a somewhat denser ring of these individuals was formed around the drop (Fig. 41). A 1/50 per cent. HCl solution paralyzes (and soon kills) ChUomonas or Para- mecia and hence the surface of the drop must act like a trap into which the organisms will stea^ly swim, with¬ out being able to swim back. This will naturally increase

the density of organisms around the drop and may give rise to a ring formation around a high concentration of HCl although the organisms are not positive to the acid. Jennings found, however, that when such organisms are in a drop of weak acids which do not paralyze the organ¬ isms quickly, e.g., 1/50 per cent, acetic or in CO 2 solutions, they become negative to the surrounding neutral medium (HjO or hay infusion) and stay in the acid. He, therefore, assumes that the organisms are positive to weak acid, and

Fig. 41.—Reaction of Chilomonas to a drop of 1/50 per cent. HCI. o, preparation immediately after the introduction of the drop (no organisms either within or gathered about the drop), b, the same preparation a few minutes later. (After Jennings.) negative to strong acid as well as to their natural neutral or faintly alkaline medium. This negativity to their natural surroundings when in weak acid as well as to strong acid when in weak acid Jennings does not interpret in terms of the tropism theory, and in this he is probably correct. He interprets both phenomena as a trap action due to the asymmetry of certain infusorians; a sudden change in the concentration of a solution causes a reverse of the stroke of their cilia by which the organism is driven back. When the old nor¬ mal stroke of the cilia is resumed the direction of the locomotion is changed on account of the asymmetrical arrangement of the cilia. This happens when the organ¬ isms go from weak into strong acid or from weak acid into

a neutral medium. In this way a collection of the organ¬ isms at the surface of a drop of acid may be brought about. This phenomenon is not tropistic in the strict sense of the word, and as a matter of fact Paramcecium is not positively chemotropic to acid of any strength. Barratt^^ investigated the chemotropism of Para- maecia for varying concentrations of different acids with Fxo. 42.—Method of proving that Paramcecia are not positive to acids of low concentration.

Pfeifer’s method of capillary tubes, counting the number of individuals going into the tube containing acid and comparing it with the number going simultaneously into a control tube containing only distilled water free from CO 2 (Fig. 42).^ The acids used varied from 0.001 N to 0.0001 N. The results were unequivocal. Toward solu¬ tions of 0.001 N the Paramcecia are negative and possibly h In addition two other controls acco-mpitiiiedl the test, namely, one tube containing hay infusion (the natural medium of the organisms) and one alkali.

also slightly negative to acids as weak as 0.0001 N. In no case, not even with the weakest acid, was it possible to prove the existence of positive chemotropism for acid (or base). The number of Paramacia which went into a tube containing, e.g., 0.00002 N acid, was on the average not greater than that which went into the control tubes. The tubes were sufficiently wide so that the Paramecia could and did move into the tubes. Barratt, therefore, concludes that acids have only a repelling action upon ParamcBcia which, however, diminishes or disappears when the hydrogen ion concentration approaches that of distilled water.

The observations of Barratt contradict the statement that Paramcecia are positive to weak acid. We have seen that when spermatozoa or swarmspores are positive to malates this can be elegantly shown by Barratt’s method. The same method has shown that when even a trace of acid is added to the neutral malates this positivity disappears. By testing systematically all concentrations of different acids within the range to be considered, Barratt found no trace of any positivity to or any trap action: by weak acid for Parcmescia. It may be true, however, that when the organisms are in very dilute acid neutral or faintly alka¬ line water repels them in the way described by Jennings.

Barratt states also that there is nothing to support Jen- nings’s assertion that the CO^ given off by the Paramcecia causes the aggregation in their natural medium, since they are not positive to low concentrations of hydrogen ions. The natural aggregations of iiafusorians may be due, as Pfeffer suggested, to transitdry agglutinations when Paramcecia impinge upon each other, and the sticki¬ ness or tendency to agglutinate may possibly be increased

by certain substances produced and excreted by the organ¬ isms themselves, e.g., GO^. 3. The results obtained with the spermatozoa of ferns and mosses by Pfeffer and other botanists led some authors to the tacit assumption that the spermatozoa of animals were positively chemotropic toward substances contained in of secreted by the eggs of the same species. Some accepted this assumption without test, others made tests which they considered adequate but which seem doubtful, and it may be of some interest to discuss the subject, since far-reaching conclusions might be based on these experiments. Pfeifer’s method of testing for chemotropism with the aid of the capillary tube has proved satisfactory and the application of this method has shown that the spermatozoa of certain animals, e.g., of sea urchins, are not chemotropic toward substances contained in or given off by the egg. Thus BuUer, who had worked in Pfeffer’s laboratory on the chemotropism of the sper¬ matozoa of ferns, investigated carefuUy and extensively the question whether or not the spermatozoa of echino- derms are positively chemotropic for egg substances.®® His results were entirely negative. Thoroughly washed, ripe unfertilized eggs of Arbacia (Naples) were put into a small volume of sea water for from 2 to 12 hours.

Capillary glass tubes, about 12 mm. long and 0.1 to 0.3 mm. internal diameter, and closed at one end, were then half filled with the (super¬ natant) sea water (which had oontained the eggs) by means of an air pump. The tubes were then introduced into a large open drop of sea water, in which freshj highly motile spermatozoa were swimming. If the eggs excrete an attracting substance it was argued that it should be present in the tubes, and the spermatozoa should collect there. . . . No attraction into the tube could be observed. Except for a surface- contact phenomenon to be further discussed, they went in and out with indifference. Apparently, therefore, the water which had contained the eggs exercised no directive stimulus on the spermatozoa whatever.

I then attempted to find some substance which could give a chemo- taetic stimulus to spermatozoa. The substances tested were such as are known to give a directive chemical stimulus to many protozoa, the sper¬ matozoa of ferns, pollen-tubes, etc. The following solutions were tried by the capillary tube method: distilled water; meat extract 1 per cent.; KNOs 10 per cent., 2 per cent.; NaCl 5.8, 2.9, 0.58 per cent.; K 2 malate 1, 0.1 per cent.; asparagin 1 per cent.; glycerine 5 per cent.; grape sugar 18, 9, 4.5, 2.25 per cent.; peptone 1 per cent.; alcohol 50, 25,10 per cent.; diastase 1 per cent; oxalic acid 0.9, 0.09, 0.009 per cent.; nitric acid 1, 0.1, 0.01 per cent.

No definite chemotactic reaction—neither attraction nor repulsion— was observed in any case. Into tubes containing the weaker solutions the spermatozoa went in and out with apparent indifference. . . . On coming into contact with strong acid solutions (oxalic acid 0.9, 0.09 per cent.; nitric acid 1, 0.1 per cent.) the spermatozoa were killed^ and thus formed slight collections. They were thus not able to avoid acids by means of a negative chemotactic reaction.^

other authors, e.g., Dewitz and the writer, have also reached the conclusion that the egg of the sea urchin con¬ tains no substance for which the spermatozoon of the same species is positively chemotropic, and that Buller’s conclusions that positive chemotropism plays no role in the entrance of the spermatozoon of sea urchins into the egg is correct. F, Lillie has recently expressed the opposite view, namely that the egg of the sea urchin contains a substance to which the spermatozoa axe positively chemotropic and to which he gave the name “fertilizin.” He first tried Pfeffer’s correct method with capillary tubes with negative result, just as Bnller and the rest of the obser¬ vers. Instead of concluding that the spermatozoa are not chemotropic he discarded the method and used Jen¬ nings ’s method, stating that it gives “incomparably more delicate results than Pfeffer’s method of using capillary tubes” (p. 533). Lillie found with this method that the spermatozoa of Arbacia are positively chemotropic to

H 2 SO 4 of a concentration as Mgh as N/10 and that they are never negatively chemotropic, not even to the highest concentrations of the strongest acid. It seems to the writer that Lillie’s observations are more naturally ex¬ plained on the assumption that when an acid is sufiSciently strong and concentrated, e.g., N/10 HNO 3 or IlgSO^, it will paralyze and kill the spermatozoa, and that when a drop of such acid is introduced in sea water containing spermatozoa, a somewhat denser ring of the organisms will be formed around the surface of the drop on account of this action of the acid.