The Psychic Life of Micro-organisms: A Study in Experimental Psychology

those of the bee, in the seminal reservoir of the female, remain alive for several years. The seminal elements of mammifers live for quite some time in the genital passages of the female. Balbiani has found living spermatozoids in the ducts of a she-rabbit twenty hours after coition. Ed. vanBeneden, Benecke, Eimer, Fries, have observed that the sperm retains its properties in the uterus of bats for several months. Another remarkable circumstance is, that the copulation of the two sexual elements is not without analogy to the copulation of the two animals from which they originated. The spermatozoid and the ovule, to some extent, repeat on a small scale what the two in-, dividuals perform in their larger sphere. Thus, it is the spermatozoid that, in its capacity of male element, goes in quest of the female. It possesses, in view of the journeys it has to make, organs of locomotion that are lacking in the female and are useless to it. The spermatozoid of man and of a great number of mammifers is equipped with a long tail, the end of which describes a circular conical movement, which together with its rotation about its axis, determines the forward motion of the spermatozoid. The same mode of progression is seen in the zoospores of Algae and in Mastigophores, which are armed with flagella; the movements of the spermatozoid have been not improperly compared to those of a Flagellate.

Other spermatozoids like those of the Triton and Axolotl are provided with a different kind of locomo tive apparatus; it consists of an undulatory membrane that acts like a real fin; the spermatozoid moves forward without turning about on its axis. There has been much discussion as to the nature of the forces that account for the movements of the fecundative elements. The early investigators that concerned themselves with the study of animalcula, naturally attributed to them spontaneous and voluntary movement. Since the spermatozoid has been regarded as nothing else than an histological element, endosmotic, hygroscopic and like actions have been accepted in explanation. M. Balbiani, from whom we have taken the foregoing details, declares that explanations of this character are none at all; for, upon ultimate analysis, all kinds of motion may be reduced to a chemical or physical action — sarcodic or ciliary movement just as much as voluntary movement. " For my part," our scientist adds, "I believe that the spermatozoids do not move about blindly but that they act in obedience to a kind of internal impulsion, to a sort of volition which directs them towards a definite object."* The experiments of M. Balbiani have shown that with weak solutions of ether and chloroform the movements of the spermatozoids may be moderated and made to cease so slowly that the latter are yet able to fecundate the ovules.

In fine, the spermatic element, in directing itself toward the ovule to be fecundated, is animated by the' same sexual instinct that directs the parent organism towards its female. In the higher animals, the movements of the spermatozoid that is endeavoring to reach the female exhibit a peculiar character, which it is important to emphasize: these movements do not appear to be directly provoked by an exterior object, as those of micro-organisms are; the spermatozoid endeavors to reach an ovule which is frequently situated a great distance away; this is the case particularly

with animals that fecundate internally, with birds and mammifers. The place of fecundation is still imperfectly known. Coste at one time accepted the theory that the spermatozoid and ovule met in the ovary. Fecundation probably takes place in the fore part of the oviduct. It has little to do with our purpose, however, to solve this delicate question precisely. A fact that is important to mention in a genera] way is the length of road the spermatozoid has to traverse before coming up with the ovule.

Let us now follow the spermatozoid in its journey to the ovule. It is known that the road it has to traverse is, in certain instances, extremely long. Thus, in the hen the oviduct measures 60 centimeters, and in large mammifers the passages have a length of from 25 to 30 centimeters. We might ask ourselves how such frail and minute creatures come by a power of locomotion great enough to enable them to traverse so long a path. But observation discloses the fact that they are able to overcome obstacles quite out of proportion to their size. Henle has seen spermatozoids carry along with them masses of crystals ten times larger than themselves, without appreciably lessening their speed. F. A. Pouchet has seen them carry bunches of from eight to ten blood-globules. M. Balbiani has attested the same fact. These globules, which have fastened themselves about the head of the spermatozoid, have each a volume double that of the head. Now, according to Welcker, the weight, of a globule of human blood is 0.00008 of a milligramme: allowing that the spermatozoid has the same weight, we may then say that it is able to carry burdens four or five times heavier than itself.

able circumstance here; there are also involutions and intricacies in the path to be followed in reaching the ovule. In this connection an interesting observation has been made upon the silk-worm. " At the moment of conjugation the male deposits its seminal fluid in a special sac, the copulatory sac. The day following, this sac, which was distended by the sperm, is completely flaccid, and nearly all the spermatozoids have traveled out into another sac, which opens into the oviduct opposite the first one, and there they wait to fecundate the ovules as they pass by. Now, the walls of the copulatory sac have no contractile power, and the passage of the spermatozoids from one sac into the other can be attributed only to a spontaneous movement. Further, a fact that well seems to verify this, is, that there still remains in the copulatory sac a few misformed seminal elements, deprived of the power of locomotion." *

Let us now note what happens at the moment when spermatozoid and ovule come in contact with one another. The successive phenomena then taking place have been carefully studied by Fol in his work upon the starfish {Asterias glacialis'}. The ovule has no enveloping membrane; it it is covered about only by a mucous layer, soft and flaky. The spermatozoids come up in great numbers and push forward into this layer; at this point they are all brought to a halt and become entangled among each other with the exception pf one, which, more speedy in its movements, outstrips the others and arrives within a short distance of the surface of the vitellus (or protoplasm of the ovule). At that moment, and before any contact whatever, there results a curious phenomenon of attraction between the

ovule and the spermatozoid; the peripheral substance of the ovule is seen to lift itself up in front of the sper-* matazoid in the shape of a minute protuberance; this protuberance, at first, has a rounded shape, then it grows thinner and forms a point which advances towards the spermatozoid; this point is called the cone of attraction (see Fig. 8). The head of the spermatozoid fastens itself upon the c/W*1 Iv9r\ cone> which seems to draw it into its intfb j o* terior. The tail of the spermatozoid does not appear to enter into the interior of «y.v.0«o-.v.D .;•. the ovule and take part in the process of • ••;?:•;- fecundation, which consists simply in the Fig. S.-A small fusion of the head of the spermatozoid

latter enwraps itself in an envelope, to protect itself against the other male elements. It appears, in fact, to be well settled that the penetration into the vitellus of several spermatozoids marks the beginning of an adverse change: the subsequent segmentation of the ovule is irregular, and development ceases. The membrane in which the fecundated ovule of the Asterias glacialis infolds itself, is formed by a condensation of the peripheral layer of the vitellus; the condensation starts from about the point where the spermatozoid penetrated, and gradually spreads over the whole surface of the ovule; the formation of this protective membrane is so rapidly effected, that access to the ovule is barred against spermatozoids who might be only a few seconds behind the first one.

just as among all animals; it is the most agile and the stoutest spermatozoid that first penetrates the ovule and effects fecundation. The laws of selection, thoroughly developed by Darwin, do not only apply to individuals; they apply also to sexual elements. We are unable to follow the successive modifications suffered by the head of the spermatozoid after its entrance into the ovule; we may state simply, that the head presents the appearance of a radiate figure, of a diminutive sun advancing towards the female nucleus. At the same moment, the female nucleus appears affected and puts itself in motion towards the spermatic nucleus. The two nuclei soon come almost within contact, and it is in particular the female nucleus that then plays the active part. It is disturbed by incessant movements and every moment changes its form; it thrusts out prolongations towards the male nucleus, and one of these prolongations fastens itself upon the latter, presenting at the end a minute depression in the shape of a cup, which receives the male nucleus; and the two nuclei, while executing active movements, fuse into one another. In this manner the first nucleus of segmentation is created.

Selenka has furnished interesting chronological data as to the time of appearance of the different phenomena. The time is in each case taken from the moment of artificial fecundation. After a lapse of five minutes, the spermatozoid has forced an entrance into the ovule.* At the expiration of ten minutes (that is, five minutes after entrance), it has reached the centre of the ovule. At twelve minutes, the female nucleus has put itself in motion to meet the spermatic nucle-

* M. Balbiani, Cours sur lafecondation, passim. Journal de Rficrographze Vol. III. 1879. us. Finally, at the twentieth minute, the two nuclei have united. In the psychical history of animal fecundation as just given, there are many gaps: the history of vegetable fecundation will fill several of these. The simplest forms of sexual reproduction in vegetables are those where the male and female cellules are quite the same and advance to meet each other equally; thus possessing not only the same form, but also the same properties. In a small Alga bearing the name of Ulothrix serrata, the interior of certain cellules divides into two parts, which separate, then come together again and mingle anew into a minute mass which, when set at liberty, reproduces the plant entire. In other species the inside divides into small naked cellules, which are first set at liberty and for some time move briskly about in the water by means of cilia with which they are provided, before fusing into, one another. These cellules are called zoospores. The differentiation is further marked in certain species, the zoospores of which have neither the same form nor the same properties. Some leave their positions to go to meet the others: these are the male cellules, the antherozoids; others make no movement at all and limit their role to that of waiting: these are the zoospores. Similarly, in an Alga bearing the name of Spharoplea annulina, there are found two kinds of filaments, brown and green. In the green filaments the protoplasm of certain cellules breaks up into a definite number of ovoid bodies which remain immobile; in the interim, the cellules of the brown filaments liberate mobile spores provided with two flagella: these spores, veritable male cellules, ply briskly about in the water and then proceed to fix themselves to the green fila-

merits, the cellules of which are pierced by pores; through these orifices they penetrate into the cellules and fuse with the immobile ovoid bodies, which are nothing else than zoospores. The psychical phenomena attending this mode of conjugation may be still more complicated, as shown by the observation that Berthold has made upon the conjugation of the zoospores of the Ectocarpus siliculosus. The Ectocarpus belongs to a group of algae characterized by the presence of mobile spores which reproduce the plant. These zoospores are little pearshaped cellules, of which the tapering end is colorless, and the rounded end shows a brownish-green coloration, which is due to the presence of an extensive chromatophore; at the edge of the chromatophore a deep depression is sharply marked, which appears to be an eye. Every zoospore is equipped, in addition, with two flagella, which rise from the same point of the lateral skirt of the anterior extremity of the body; one of these flagella points forwards and the other backwards. When the zoospores are set at liberty and begin to swim about in the water, they pass each other by unnoticed. The female cellule does not draw about her the male cellules, from which, moreover, it differs by no Fig. 9. —Sexual remorphological mark. But at a given

production of the Ectocarpus siliculosus. Difmoment the female zoospore becomes ferent stages of the female zoospore while distinguished from the male cellules rest (after Berthold). by passing into a state of rest; where- to, the base of the anterior flagellum, which is laterally inserted, proceeds to blend with the anterior part of the body with the effect that the flagellum appears to rise from the extremity; during the same time, it contracts, and presents at its free end a slight protuberance, which allows the zoospore to fix itself upon an immobile point; as to the rear flagellum, it slips back upon the posterior part of the body which it encompasses, and finally disappears.— When the female zoospore has become motionless, the male zoospores, hitherto indifferent, are seen to make towards it and to surround it in a half-circle; the number of zoospores that thus meet, is quite considerable; it frequently exceeds a hundred (fig. 10). They let their second flagellum float loosely behind them, while they all direct their anterior filament towards the female cellule; this filament they draw back and forth over the mFi§. 10. -sexual reproducbody of the female cellule; they

losus. Female zoospore surperform upon it real acts of feeldently to provoke in the female zoospore a genital excitation, as what follows will prove. It happens at times that several of the male zoospores quit the ranks and make off; they are immediately replaced by others who employ their filaments in a ^^ like manner, to stroke <fj£J|) the female. Finally, upon the expiration of a certain time, Fig. ii. - Sexual reproduction of the Ecto- One of the ZOOSpOreS carpus siliculosus. Successive stages of the 1OQ,,OC 4-1-,^ V,o1f rircopulation of a female zoospore with one of the leaves

the female. The two zoospores unite; after having presented the series of changes marked in the figure, — when the fusion is complete, — the female cellule loses its fixatory filament and the little zygote, the result of the fusion, is set free. When the male zoospore is obliged to go a long distance to reach the female zoospore, it has been thought that the latter secretes a substance which acts upon the male cellule as a chemical excitant and which marks out for him the direction to follow. The supposition is quite probable; it was suggested by Strasburger, who had shown that the spermatozoids of the Marchantia polymorpha are attracted by the substance that issues from the archegoniuni. It will only be necessary to assist at an experiment of artificial fecundation with fish-spawn, in order to come to the same opinion. The sperm introduced into the liquid preparation does not spread about homogeneously in all directions; the spermatozoids are observed to whirl about the ovules in great masses; it must be supposed, further, that there is some excitation of an unknown nature which attracts the spermatozoid towards the micropyle, for this minute opening, of which the diameter is scarcely that of the head of a spermatozoid, is the only orifice through which the male element can enter into the ovule to fecundate it.

These ingenious opinions have been latterly confirmed by the very interesting experiments of M. Pfeffer, professor at the University of Tubingen, upon the movements of spermatozoids.* His investigations had to do principally with the spermatozoids of cryptograms. M. Pfeffer discovered that certain chemical substances have the property of attracting these spermatozoids. * Pfeffer, Untersuchungen aus dem botanischen Institut zu Tubingen, Vol. I. Leipzig, 1884, p. 363.

The manner of conducting the experiment is as follows. A solution of the substance to be experimented upon is placed in small capillary tubes with a light-aperture of from five to seven hundredths of a millimeter wide. These capillary tubes dip into a watch-crystal covered with a liquid, wherein quantities of spermatozoids have been placed. Under these circumstances currents of diffusion are soon set up between the tube and the liquid in the watch-crystal, and when the substance experimented upon is the proper one, the spermatozoids are seen to follow the currents of diffusion and to penetrate into the tube.

-The substance exerting such attraction varies with the plants. The author began by experimenting upon the spermatozoids of certain ferns (Adi ant urn cuneatntn). After a great many fruitless trials, one substance, and one only, proved to be effective: namely, a solution of malic acid or malate. It is to be presumed, then, that, in the organic kingdom, malic acid must be the substance acting as a chemical excitation upon the spermatozoids of ferns and guiding them towards the female cellule.

According to the hypothesis of Pfeffer, the actual process takes place in the following manner. The spore of a fern, falling upon humid ground, germinates and gives birth to a green cordate slip, the prothallium, upon which are developed the male organs or antheridia, and the female organs or archegonia. At a certain moment, elongate cellules, spirally twisted and extremely mobile, issue from the antheridium: these are the spermatozoids. They are equipped with vibratile cilia, by the help of which they are able to start in search of the female cellule. At the same instant, the female organ, the archegonium, opens and

emits a mucilaginous substance, which must contain malic acid or a malate, for these compounds are the particular excitatory substance of the fern-spermatozoids. Thanks to a drop of dew that falls upon the prothallium, the spermatozoids swim around and approach the female ovule, which attracts them by acting upon them with the malic acid. A confirmation of this hypothesis is primarily the fact, that all substances tested, with the exception of malic acid and malates, remained completely inactive; another proqf is, that malic acid is found in prothallium-decoctions of the Pteris serrulata and of the Ad- iantum capillus veneris; another proof still, is the circumstance that malic acid is largely diffused throughout the vegetable kingdom.

The author has made, in this connection, a series of very curious experiments upon the degree of concentration necessary to attract the spermatozoids. The lower limit at which attraction begins, is found in a solution containing malic acid in the proportion of one to 1000 parts. This the author has designated by a favorite word of the Germans: Reiz-Schwelle, or, in other words, the threshold of excitation. When the solution in the watch-crystal contains one part malic acid to every thousand parts, in order to make the spermatozoids pass from the watch-crystal into the tube, the solution held in the tube must be thirty times as strong, or 30 x i-iooo = 3-100. If the liquid in the watch-crystal contains one part malic acid to every hundred parts, similarly the solution of the tube must be thirty times as strong, that is to say three tenths.

The author justly compares the result of these experiments with the law laid down by Weber, which M. Delbceuf has happily formulated as follows: "The slightest difference capable of being felt between two excitations of the same sort is due to an actual difference that increases proportionally with the excitations themselves." Thus, in order to tell that one weight is heavier than another, it must be heavier than the other by a fractional difference which varies from one third to one fifth according to the individual, be the original weight what it may. For example, to a weight of three grammes, in order that a difference may be made perceptible, we must add one third of three grammes or one gramme. To four grammes we must add one third of four grammes, or one and one third grammes, etc.*

According to Pfeffer, the application of Weber's law to his experiments is so exact that, when the solution of the tube is only twenty times stronger than that of the watch-crystal, the spermatozoids remain unaffected. Furthermore, the application of the law is not disturbed by changes of temperature varying within certain limits. Thus, down to a temperature of 4. 5° (41° Fahr.) the spermatozoids remain sensible to a concentration of liquid thirty times as strong as that in which they are.

Basing his calculations upon these experiments, the author has succeeded in determining the probable quantity of malic acid that must be contained in the archegonium. This quantity is probably in the proportion of three tenths. The spermatozoids of the Selaginella are likewise wise attracted by malic acid and the malates. As regards the Marciliaceae, the specific substance has not been discovered. The same failure, also, in the case of the Hepaticae. The author concludes from thisy that the substance operating in these two cases can be little diffused throughout the vegetable kingdom.

For the spermatozoids of the Funaria hygrometrica (Confervae), the operative substance is cane-sugar. No other attracts them. The spermatozoids remain unaffected even by substances bearing the closest analogies with cane-sugar. We will cite, by way of example, fruit-sugar or levulose, grape-sugar or glucose, glycogen, manna, milk-sugar, etc.; these substances exert no attraction upon the movements of the spermatozoids, whereas cane-sugar exercises an attraction so powerful that the capillary tube becomes at once crammed with them. The excitation first induces in the spermatozoid a movement of direction: the body is brought into a position enabling it to reach the tube by movement in a straight line. The same phenomenon has been observed by Strasburger in the case of Algae zoospores; when these minute beings are attracted by a chemical or luminous excitation, the first thing that happens is the directing of the body towards the attracting -source.

A solution of one in one thousand parts is sufficiently concentrated to draw the spermatozoids of Mosses into the capillary tubes. The ''• threshold of excitation" for them, accordingly, is the same as for the spermatozoids of ferns. Furthermore, Weber's law is in this instance again verified; only, in order to have the chemical excitation produce a different attraction, it must be stronger than the first in the proportion of 50 to 100. In the experiments upon the spermatozoids of ferns the ratio is a little smaller; being only 30 to 100.

whether, by increasing the degree of concentration, a point would not be reached where attraction would change to repulsion; he has not made the experiment, but he has noticed that great numbers of spermatozoids still penetrate into the tube "when containing a solution in the proportion of 15 to 100, notwithstanding the fact that they there meet a speedy death. The general conclusion to be derived from these numerous experiments is, first, that the spermatozoids are sensible to certain chemical excitations, and consequently, that in every group of plants there exists a special substance acting the part of a specific excitant towards the spermatozoids. The author does not hesitate to regard the spermatozoids as a physiological re-agent of such substances, allowing feeble traces of the same to be detected in a liquid solution. He thus comes to form a spermatozoi-d test, which is not without analogy with the Bacteria test, invented by En- gelmann. An application of the test is the following: a decoction of herbs having presented the property of attracting the spermatozoids of Mosses, the author concluded that the decoction must contain canesugar.

It would be of the highest importance to know what is the seat of the phenomena of the life of relation in the bodies of Micro-organisms. We have seen that Micro-organisms are the equivalent of a simple cellule, composed, according to the classic plan, of a protoplasm, of a cellular nucleus, and of an. enveloping membrane. Each of these elements plays a part of special importance in the vital phenomena of these beings. Long since, scientists have attributed movement, sensibility, and the prehension of foods, to the protoplasm. This was the result of direct observation. While observing an Amoeba, for example, the protoplasm is seen to undergo modifications of form and to throw out pseudopods, either for the purpose of effecting a change of position, or to seize alimentary substances. The protoplasm, accordingly, seems to be the sole agent of all these phenomena. Likewise, the vibratile cilia of the Ciliates, which are at once organs of motion, prehension, and touch; the suckers of the Acinetinidae, which are special organs of prehension, are nothing else than outward expansions of the protoplasm proper.

As regards the enveloping membrane, the same cannot discharge any psychical function: firstly, because it is a product of protoplasmic secretion; and, secondly, because it is wanting in many Protozoans and even in many animalculaquite high in point of organization that, despite their nudity, exhibit marks of psychic life just as complex as those observed in Infusoria having a cuticle. The part acted by the nucleus does not so clearly manifest itself to direct observation; it executes no movements in the ordinary conditions of life; it remains motionless in the centre of the animal's body, surrounded on all sides by the protoplasm; unlike the latter, it is not in direct contact with the outside world.