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Jenkinson JW. Experimental Embryology. (1909) Claredon Press, Oxford.

Jenkinson (1909): 1 Introductory | 2 Cell-Division and Growth | 3 External Factors | 4 Internal Factors | 5 Driesch’s Theories - General Conclusions | 6 Appendices
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Chapter III External Factors

1. Gravitation

In the large majority of cases there is no definite relation between the vertical and either the axis of the egg, the planes of its segmentation furrows, or the position of the development of the embryo in it. Thus the eggs of insects are laid with the axis making any angle with the vertical, and the same may be said of Crustacean ova. In such eggs as develop freely in the sea (some Mollnsca, for example, Polyehaeta, Coelenterata, Ctenophora) the axis and the planes of segmentation undergo a perpetual change of position, and Oscar Hertwig has shown that in the eggs of Echinoderms there is no necessary fixed relation between the direction of the planes of segmentation and the vertical. In these cases it is clear that the features of development referred to cannot depend upon the force of gravitation.

There are, however, instances in which it seems possible that the directions of the planes of segmentation—bearing as they do a constant relation to the axis of the egg-—may depend upon gravity, since the axis is normally vertical. It was Emil Pfliiger who in 1883 first brought forward experimental evidence to show that this was indeed the case.

It is well known that the yolk of the hen’s egg always turns over so that the germinal disc is uppermost, and the egg of the Frog, free to rotate inside its jelly membrane, invariably takes up a position with the black pole uppermost, the white pole below.

This property of the Frog-’s ovum is exhibited alike by the ovarian, the coelomic, the uterine, and the freshly laid egg, by the living egg and the dead egg, by the whole egg, and by portions that contain both kinds of egg-substance, the yolk and the cytoplasm, as Roux showed by floating eggs or fragments in a medium of the same specific gravity. It is simply due to the fact that in the spherical telolecithal egg the heavier yolk is placed mainly on one side, while on the other the lighter protoplasm is more abundant, the yolk granules far smaller and more sparse. The distribution of these two substances determines indeed the axis about which the egg has a ‘ rotation structure ’ or is radially symmetrical. The symmetry is further marked by the disposition of the pigment and the position of the nucleus. The pigment is placed in a thick superficial layer in the protoplasmic portion, it extends over rather more, sometimes considerably more, than a hemisphere, for there is much variation in this respect, and its boundary is a circle whose plane is at right angles to the egg-axis—the line which passes through the centre of the egg, the centre of the pigmented portion or animal pole and the centre of the unpigmented portion or vegetative pole. There is also an axial, less-deeply pigmented plug in the animal hemisphere. The nucleus is placed axially, b11t excentrically, very much nearer the animal than the vegetative pole, in a pigment-free spot or ‘ fovea germinativa ’.

The egg is invested with a layer of jelly (muein), inside which it becomes eventually free to rotate. This, however, is not possible when the egg is first laid, for the jelly is at that time closely adherent to it. In water, however, the jelly swells up, and a narrow cavity is formed in about three hours between it and the egg, and the egg then turns over until its axis is vertical. The formation of the cavity is much more rapid if fertilization (insemination) has taken place; in this case the egg turns over in half an hour. The rapid formation of the perivitelline fluid is the first effect of insemination, and is due to some substance secreted by or accompanying the sperm, since the spermatozoon does not reach the egg for another quarter of an hour (Oscar Hertwig). A second effect is an alteration in the viscidity or cohesion of the egg-contents ; for while in the ovarian or uterine egg no alteration occurs apparently in the disposition of yolk, cytoplasm, and pigment, although the egg-axis may make any angle with the vertical, such an alteration is undoubtedly produced by gravitation (see below) after fertilization has occurred. Another effect noted by Roux is that fertilized (not merely inseminated) eggs turn over more rapidly in a medium of like specific gravity than do unfertilized.

The most important first result of fertilization is, however, the replacement of the radial by a bilateral symmetry. About two or three hours after insemination a certain portion of the border of the pigmented area, crescentic in shape and extending; over about half its periphery, becomes grey by retreat of the pigment into the interior (Roux). The egg can now be divided into similar halves by only one plane, the plane of bilateral symmetry, which includes the axis and the middle of this grey crescent (Fig. 43).

Fig. 43. — Formation of the grey crescent in the Frog's egg (IE. femparuriu). A, B from the side; 0, D from the vegetative pole. In A, C there is no crescent, in B, D a. part of the border of the pigmented area has become grey.

The middle of the grey crescent is always diametrically opposite to the point of entry of the sperm (Roux and Schulze) ; the crescent has hence been held by Roux to be directly caused by that entrance.

The plane of symmetry, as we shall see in another connexion, becomes in most cases the sagittal plane of the embryo, since the dorsal lip of the blastopore arises in the region of the grey crescent. This side becomes the dorsal side of the embryo, while the animal pole marks, approximately, the anterior end.

By complete disappearance of the pigment the grey crescent becomes added to the white vegetative area of the egg.

The foregoing account applies in particular to Rana temporaria and R. fusca; I{.11aZusm'n appears to be similar, but in R. escaZenta it is stated that the egg—axis is eventually not vertical but oblique (Fig. 44). It seems, however, doubtful whether this obliquity is not rather apparent than real. The grey crescent has apparently not been recognized as such—-the pigment is not so deep as in the other species—but included, nevertheless, in the white area, with the result that the centre of this, the definitive

Fig. 44. — Egg of Rana esculenta after fertilization, in its normal position with the axis oblique (‘?). A, from the side; B, from above; an’, egg-axis ; mm, plane of first furrow. (After Korsehelt and Heider.) white area, has been confounded with the centre of the original unpigmented area or vegetative pole of the vertical egg-axis. (Compare Fig. 44 A with Fig. 43 B.)

As is well known, the planes of division during the first few regular phases of segmentation bear a perfectly definite relation to the axis. The first two, at right angles to one another, are meridional and therefore also vertical, the third furrows are parallel to the equator, therefore also horizontal; the furrows of the fourth phase are again meridional, and hence vertical, those of the fifth once more latitudinal and horizontal. It is this obvious relation of the planes of cleavage to the direction of gravity which has raised the question whether there is not a causal connexion between the two, the question which Pfliiger attempted to answer by experiments, performed, however, not on the eggs of the Frog but on those of a Toad, Bombinator igneus.

The close adherence of the unlaid egg to the glutinous jelly, which in its turn could be easily fixed to some object, provided a simple method of keeping the egg in any desired position. The eggs were removed from the uterus, attached with the axis at various angles to the vertical to watch glasses and fertilized with just enough sperm-water to allow of development, but not enough to permit of the formation of the pcrivitelline space and rotation of the egg into the normal position with the axis vertical. In these forcibly inverted eggs it was found that the furrows of segmentation bore the same relation to the vertical as in the normal egg; that is to say, the first was vertical, the second vertical and at right angles to the first, the third horizontal and nearer the upper pole, whatever the inclination of the egg-axis to the vertical, except in the extreme case where the white pole was exactly uppermost (180° of inversion), when segmentation did not occur at all. There was, however, no definite relation between the plane of the first (and therefore of subsequent) furrows and the original axis of the egg; the angle between this axis and the plane of the first furrow, as also that between the first furrow and the plane including the original and the actual vertical axes of the egg, might, it was found, have any value.

Except in a few cases, and where the white area was nearly exactly uppermost, these eggs gave rise to normal embryos. The upper smaller cells divided more rapidly than the lower ones, whether pigmented or unpigmented, and the blastula stage was reached; the dorsal lip of the blastopore appeared on one side a little below the (actual) equator, and the lower surface was covered over by the blastoporic fold in the ordinary Way. Only in the failure of the whole egg to rotate after the closure of the blastopore (owing to the close adherence of the jelly) and in the irregular pigmentation (according to the original degree of inclination) did these embryos differ from the normal. One other point is worthy of notice. In the majority of cases the dorsal lip of the blastopore, marking the sagittal plane, appeared on the unpigmented side and lay in the plane including the original, now inclined, and the actual axis, or vertical line of intersection of the first two furrows. While, therefore, the cleavage planes are definitely related to the vertical but not to the original axis of the egg, the median plane of the embryo appears to be jointly determined by both.

From these experiments Pfliiger drew remarkable and farreachingconclusions. He conceived of the eggas beingmeridionally polarized, composed of a large number of rows of molecules placed meridional] y with regard to the original egg-axis. Each row is equivalent developmentally to every other row, but within the limits of each the molecules are of difierent value, since one end, for example, is anterior, the other posterior. .Which of these equipotential rows shall lie in the sagittal plane of the embryo is decided by gravity, and by gravity alone. Similarly the vertical direction of the first two furrows, the horizontal direction of the third, is due to the operation of some general, though at present unknown, law, in accordance with which ‘ (lie Schwerkraft die Organisation beherrseht ’. 1

The original structure of the egg, on the other hand, has no definite relation either to segmentation or to the symmetry of the embryo, except, of course, in so far as the original axis together with the actual vertical axis determines its sagittal plane, the white side its dorsal side.

‘. . . das befruchtete Ei gar keine wescntliche Beziehung zu der spiiteren Organisation des 'l‘hieres besitzt, sowenig als die Sehneeflocke in einer wesentliehen Beziehung zu der Grosse und Gestalt der Lawine steht, die unter Umstiinden aus ihr sich entwickclt. Dass aus dem Keime immer dasselbe entsteht, kommt daher, dass er immer unter dieselben iiusseren Bedingungen gebracht ist.’ 2

It will certainly be agreed that so sweeping and revolutionary a dogma as this is in need of very substantial support; and though the facts as stated by Pfliiger are incontrovertible, as the repetition of his experiments has shown, it is unfortunate that he did not also take into consideration the internal changes occurring in his forcibly inverted eggs. The deficiency has been made good by Born. Like Pfliiger, Born found that in the

’ 1. C. i11fl'a, XXxii- p. 24. 3 I. c. infra, xxxii. p. 64.

forcibly inverted eggs the cleavage planes had the normal relation to the vertical, but not to the egg-axis; he observed, however, that the first furrow was usually in or at right angles to the streaming meridian, the plane, that is, including ‘the original and the secondary vertical axes. The recent examination of 215 cases by the writer has shown that the first furrow tends to lie either in, or at right angles to, or at an angle of 45° to this plane. Subsequent development was normal, and the sagittal plane coincided with the streaming meridian. The dorsal lip appeared on the white side, which is thus anterior (antc1'o(l0rsa.l). The examination of sections, however, showed that in these

FIG. 45.-—Seetions through forcibly inverted Frog's eggs. In A the egg has just been inverted, in B the streaming of protoplasm upwards and yolk downwards has begun. Both sections are in the streaming meridian or gravitation symmetry plane, including both the axis (a a’) and the vertical. bD, pigmented animal protoplasm; wD, unpigmented vegetative yolk. ct, animal pole; a’, vegetative pole; 1', pigment-free clear area; A-, nucleus; 1), superficial pigment. (After Born, from Korsehelt and Heider.)

inverted eggs there had been a redistribution of the contents, the heavy yolk sinking to the lower side, the lighter protoplasm, with the pigment and the nucleus, and the spermatozoon rising to the upper side (Fig. 45). The movement is rotatory, the cytoplasm and yolk ascending and descending in opposite directions ; and it also takes place naturally parallel to, and in a similar manner on each side of, the plane in which the primary and secondary axes lie, hence known as the streaming meridian. That end of this plane towards which the protoplasm moves in its ascent, the end, that is, marked by the primary vegetative pole, is anterior (more correctly, anterodorsal), for it is here that the dorsal lip of the blastopore appears; the opposite end is posterior (more correctly, postero-ventral).

The pigment moves with the cytoplasm; it is, however, unable to completely displace that yolk which remains at the upper surface in consequence of the greater viscosity of the superficial rind, and here a ‘white plate’ or ‘grey patch’ is formed. Similarly, at the lower surface the pigment is not necessarily wholly displaced by the descending yolk.

There is one special case that may be noticed. When the inversion is complete (l80°) the yolk flows radially and peripherally away from the upper pole while the cytoplasm ascends in the axis.

Born’s observations make it perfectly clear that gravity rearranges the eontents of these inverted eggs, and so confers upon them a secondary structure like that of the normal, and symmetrical about a secondary axis which is likewise vertical. To this secondary axis the direction of elongation of the karyokinetic spindles, and consequently the cleavage planes, bears the same relation as in the normal egg; and there is certainly no more need to explain these directions by reference to gravity, to suppose, in fact, a causal connexion between the two, in the one case than in the other. The planes, indeed, may fall where they do simply because the mitotic figures elongate in the direction of least resistance (Pfluger) or (Oscar Hertwig) in that of the greatest protoplasmic mass, or may be related, in some similar way, to the structure of the egg alone.

The point can only be determined by an experiment in which the directive influence of gravity is eliminated. This experiment has been made by Roux. The eggs were fastened in small vessels, at distances of from one to eight centimetres from the centre, to a wheel rotating continually about a horizontal axis, but so slowly (one revolution in from one to two minutes) that the centrifugal force developed was insullicient to make the eggs turn with the white pole outwards, and therefore negligible. The direction of the force exerted by gravity upon them from moment to moment was thus not constant. Of the eggs some were free to rotate inside their jelly, others were fixed. To anticipate the objection that the plane of rotation, the plane of the wheel, is constant, a third set were packed loosely in test-tubes, and so able to roll over one another in all directions as they fell from one end of the tube to the other with each revolution. The first furrow appeared in all these eggs at the normal time and it was meridional, as in the normal egg; similarly the second was meridional, the third latitudinal; but the egg-axes exhibited no definite relation either to the vertical or to the plane of the wheel. The eggs were allowed to continue their development on the apparatus, and gave rise to normal tadpoles.

From this experiment Roux drew the conclusion that it is not gravity which determines the direction of the planes of cleavage, and that gravity is not an indispensable necessity for the normal development of the egg of the Frog.

Incontrovcrtible though this conclusion appears to be on the evidence, it has nevertheless been disputed by certain embryologists, Sehulze and Moszcowski, the controversy between whom and Roux upon the subject has new extended over many years.

Schulze has urged (1) that eggs placed on such a machine do not develop normally, and that the rotation of the eggs in their jelly exactly compensates for the rotation of the wheel. With regard to the latter point Roux has replied that on this supposition the egg‘-axes ought to be, at any moment, vertical, which is not the case. To the first objection it is a sufficient answer that not only Roux, but subsequent investigators (Morgan, Kathariner and the present writer) have been able to produce normal tadpoles from such rotated eggs.

It may be noticed here that Kathariner has repeated Roux’s experiment with a slight variation. The eggs were kept constantly rotating, not in one but in an indefinite number of planes by a stream of air-bubbles passing through a glass vessel filled with water. Development was normal. This result does not differ materially from that obtained by Roux with the test-tube eggs referred to above, which has indeed been also independently corroborated by Morgan.

The criticisms of Moszcowski take a different form. This author urges that gravity always exercises an influence upon the egg in determining the bilateral symmetry of both egg and embryo. The grey crescent which appears soon after fertilization and is regarded by Roux as a direct effect of this process, is supposed by Moszcowski to be produced by the action of gravity upon the egg-contents during the short interval before the perivitellinc space is formed and the egg able to turn over, to be comparable, in fact, to the grey area or white plate described by Born in his forcibly inverted eggs. Every normal egg, therefore, has a ‘gravitation’ plane of symmetry which later on becomes, as in inverted eggs, the median plane of the embryo ; nor are the eggs on the rotatory apparatus exempt, for it is held that the work of gravity can be accomplished on them even in the few moments before they are placed on the machine.

With regard to the latter point both Katharincr and Morgan have demonstrated that eggs kept in a state of perpetual rotation in all directions, from the very moment of insemination develop into perfectly normal, bilaterally symmetrical embryos, while Roux has replied to the first part of the criticism by pointing out that the grey area observed by Moszcowski was not the normal grey crescent produced by the entering spermatozoon, but the ‘white plate’ of Born due to the incomplete rearrangement of yolk and cytoplasm in an egg which had been quite unintentionally prevented from assuming its normal position. The grey crescent, indeed, Roux argues, could not possibly be due to gravity in a normal egg, for it does not appear until some time after the axis has become vertical.

There seems, therefore, to be little room for doubt that Roux’s original contention, that gravity does not determine the symmetry of the egg and embryo in the Frog, is correct, although it remains a result of considerable importance that this external factor may be made artificially to induce a bilaterality in the egg which is sullieiently strong to persist as the symmetry of the embryo.

There is one other matter of interest in this connexion. It is obvious, and has been experimentally shown by Oscar Hertwig, that a centrifugal force can replace gravity. On a. wheel rotated with sufficient velocity the eggs turn with their axes radial, their white poles outermost. If the velocity is great enough (145 revolutions a minute, radius from 24- to 32 cm.) the yolk is driven inside the egg towards the vegetative pole, and the distinction between it and the protoplasm accentuated. The segmentation of such eggs is mcroblastic ; a cap of small cells is formed, a blastederm, resting upon an undivided, though nucleated, yolk, and these yolk-nuclei are large and irregular, resembling the giant nuclei of the large-yolked eggs of Elasmobranchs and other forms (Fig. 46). An experimental confirmation is

Fig. 46 — S8gm6nt9:ti911 Of We thus afforded of Balfour’s hypothesis egg under the Influence thesis if 'v' rd on comparative

, P11 0).‘: d I)

of a centrifugal force (from KorSchelt and Heldelu after 0- Hert- grounds, that it is on the varying

wi ). The egg consists of a blastogerm and an undivided yolk quantity of yolk that differences (yolk-syncytium): Ich,blastocoel; in the segmentation of eggs prim, yolk-nuclei; d, yolk. marily depend_

If removed from the centrifuge in time, such eggs may continue to develop, though they frequently give rise to monstrosities (Spina bifizla).


G. BORN. Ueber den Einfluss der Schwerc auf das Frosehei, Arch. mikr. Auat. xxiv, 1885.

0. HERTWIG. Welchen Einfluss iibt die Schwerkraft auf die Teilung der Zellen ‘P Jen. Zeitschr. xviii, 1885.

0. HERTWIG. Ueber einige am befruchteten Froschei durch Centrifugalkraft hervorgerufene Mechanomorphosen, S.-B. lci1m'g. preuss. All-ad. Wiss. Berlin, 1897.

L. KATHARINER. Ueber die bedingte Unabliiingigkeit der Entwicklung des polar differenzirten Eies von der Schwerkraft, Arch. Em. Mech. xii, 1901.

L. KATHARINER. Weitere Versuche fiber die Sclbstdifferelizirung dcs Froschcies, Arch. Ent. Mech. xiv, 1902.

F. KEIBEL. Bemerkungen zu Roux’s Aufsatz ‘ Das Niclitiietliigsein der Schwerkraft fiir die Entwicklung des Froscheies ', Amzf. Anz. xxi, 1902.

T. H. MORGAN. The dispensability of gravity in the development of the Toad’s egg, Auat. Anz. xxi, 1902.

T. H. MORGAN. The dispensability of the constant action of gravity and of a. centrifugal force in the development of the Toad‘s egg, Anat. Anz. xxv, 1904.

M. MOSZCOWSKI. Ueber den Einfluss der Schwerkraft auf die Entstehung und Erhaltung der bilateralen Symmetric des Froscheies, Arch. milcr. Anal. 11:, 1902.

M. MOSZCOWSKI. Zur Analysis der Schwerkraftswirkung auf die Entwicklung des Froscheies, Arch. milcr. Amzt. lxi, 1903.

E. P1«‘LiiG1«:R. Ueber den Einfluss der Schwerkraft auf die Teilung der Zellcn, Pfliigeris Amh. xxxi, xxxii, xxxiv, 1883.

W. Roux. Ueber die Entwicklung des Froseheies bei Aufhebung der riehtenden Wirkung dcr Schwerc, Breslau drtz. Zeitschu, 1884 ; also Ges. Abh. 19.

W. RoUx. Bemerkung zu O. Schu1ze's Arbeit iiber die Nothwendigkeit, etc., Arch. Ent. Mech. ix, 1900.

W. RoUx. Das Nichtnothigsein der Schwerkraft fiir die Entwicklung des Froscheies, Arch. Ent. Mech. xiv, 1902.

W. ROUx. Ueber die Ursachen der Bestimmung der Hauptrichtungen des Embryo im Froschei, Auat. Auz. xxiii, 1903.

O. SCHULZE. Ueber die unbedingte Abhiingigkeit normaler tierischer Gestaltung von der Wirkung der Schwerkraft, Verh. Anat. Ges. viii, 1894.

O. SCIIULZE. Ueber die Nothwendigkeit der freien Entwicklung des Embryo, Arch. 1m'kr. Amtt. lv, 1900.

O. SCHULZE. Ueber das crste Auftreten der bilateralen Symmetric im Verlauf der Entwicklung, Arch. milrr. Anal. lv, 1900.

2. Mechanical Agitation

The necessity of perpetual and violent agitation for the very numerous pelagic ova which are ordinarily exposed to the stress of wind and weather is well known to every zoologist who has attempted to rear such forms in an aquarium, and need not be further insisted on.’

There are also other eggs which require a small amount of movement. The Hen turns her eggs every day, and the operation has to be artificially performed in an incubator. Its omission leads to serious consequences, for, as Dareste has shown, the allantois sticks to and ruptures the yolk-sac in unturned eggs, the ruptured yolk-sac cannot be withdrawn into the abdomen, and the Chick cannot hatch out. Death may ens11e at an early stage.

A violent agitation of the IIen’s egg, on the other hand, is equally fatal.

Dareste subjected the unincubated eggs to violent shocks at the rate of 27 a second for varying periods (from %' hour to 1 hour). The percentage of monstrosities observed after three or four days of incubation was very high indeed, except when the eggs were placed vertically with the blunt pole uppermost, the blastoderm therefore resting against the shell

  • It seems probable that the principal value of the mechanical agita

tion to the larvae is to prevent the Diatoms and Algae, of which their food consists, from sinking to the bottom.

Marcacci has exposed the eggs, inside the incubator, to continual rotation for 48 hours. The eggs were fastened to horizontal and vertical wheels rotating 40, 80, and 60 times a minute. At the last mentioned rate of revolution, the direction of rotation was reversed half-way through the experiment.

Many of the eggs actually hatched out, but the chickens were feeble and liable to disease, and exhibited malformations of the muscles or skeleton. Others, however, died before hatching, in some cases at an early stage, and death seems to have been due to rupture of the vitelline membrane; this was always fatal. The vertical motion was, on the whole, more harmful than the horizontal, owing to the perpetual see-saw.

It may be noted here that Féré has succeeded in producing retardation and abnormality of development in the Chick by means of short exposures to sound-vibrations.

Mathews has shown that mechanical agitation —violent shaking in a test-tube—is sufficient to provoke development (artificial parthenogenesis) of the unfertilized eggs in As/arias, but not in /lrlacia (see, however, below, p. 124).


C. DARESTE. Rccherches sur la production des monstruosités par les secousses imprimées aux ueufs de poules, Comptcs Rendus, xcvi, 1883.

C. DARESTE. Sur le role physiologique du retournement dcs oeufs pendant l‘incubation, Comptes Remlus, c, 1885.

C. DARESTE. Non velles rccherches concern-ant Pinfluence des secousses sur le gei-me de 1'wuf de la. poule pendant la période qui sépare la pontc de la mise en incubation, Comptes Remlus, ci, 1885.

C. DARESTE. Note sur 1‘évolution de l’embryon de la poule soumis pendant Pincubation in. un mouvenient de rotation continu, Comptes Ilumlus, cxv, 1892.

C. F1~':R£':. Note sur les differences des effets des vibrations mécaniques sur l'évo1ution de l’embryon de poulet suivant 1‘époque oil elles agissent, C. R. Soc. Biol. (10) i, 1894.

C. Ffiné. Note sur Pincubation dc l'oeuf dc poule dans la position verticalc, C. R. Soc. Biol. (10) iv, 1897.

A. MARCACCI. Influence du mouvement sur le développement (les ceufs de poule, Arch. Ital. Biol. xi, 1888.

A. P. MATHEWS. Artificial parthenogenesis produced by mechanical agitation, Amer. Jomw. Phys. vi, 1901-2. 91

3. Electricity and Magnetism

An external agent, to which all eggs are inevitably exposed, is the natural magnetism of the earth. No evidence has, however, as yet been brought forward that this agent exercises any directive influence upon them, although their development may be distorted by excessive exposure to it.

Thus Windle placed a number of Hens’ eggs between the poles of a large horse-shoe magnet. Over 50 ‘A of these, when incubated, gave rise to abnormalities, the area vasculosa being affected in most cases. No relation could be detected between the position of the egg in the magnetic field and the kind of monstrosity produced.

In the case of Trout ova similarly treated a very high deathratc was observed, but this was attributed by the experimenter to the action of the electric currents set up by the running water between the poles of the magnet. VVealc electric currents had less effect.

Silkworms’ eggs, however, suffered no harm.

The effects of the electric current upon the eggs of Amphibia and Birds were tested by some of the older observers. Rusconi, Lombardini, and Fasola all found that the development of the Frog's egg could be accelerated by weak currents. Lombardini produced monstrosities in the ease of the Chick by this method. More modern experiments are due to VVindle, Dareste, Rossi, and Roux.

Windle observed a fairly high death-rate amongst Trout eggs exposed to the action of the current. Dareste has found a large percentage of monsters among embryos developed from Hens’ eggs subjected for from one to three minutes to the electric spark (12 cm. long from Bonnetty’s machine, 3-35 cm. long from a Rhumkorif coil). Development was, however, normal in the case of eggs placed for an hour in a Tesla’s solenoid traversed by a discharge of 500,000 periods a second. Rossi employed a continuous current passing through the eggs (of Salm/lamb-iua perspicilla/a) in the direction of the axis. Both yolk and pigment became aggregated at the animal pole, leading to the formation there of a grey raised area surrounded more or less completely by a furrow. When segmentation occurred the first two blastomeres were unequal and detached; the vegetative hemisphere was hardly segmented at all in later sta.ges, the previous divisions having disappeared. The nuclei were affected in various ways, and the directions of the cleavage spindles altered. The capacity for resistance to these evil effects was noticed to increase as development advanced.

The polar area produced in these experiments recalls the polar areas observed by Roux in Frogs’ eggs exposed to a horizontal current, at right angles, therefore, to the axis. Alternating currents of 50 and 100 volts were employed. The eggs were fertilized two or three hours before the commencement of the experiment. In from fifteen to thirty seconds after exposure two polar areas appeared in each egg. The polar areas were turned towards the electrodes. They were marked, dotted in various ways, and flocked with white extruded drops of yolk, and separated by furrows from a middle or ‘equatorial’ zone, the width of which varied directly with the distance of the egg from the electrode, inversely with the strength of the current and the duration of exposure.

Unfertilized ova were found to react in the same way. So also eggs in which segmentation had begun, and in those cases where the furrow cut the equatorial zone obliquely, the two halves of the latter turned away from one another.

The polar areas appear too in eggs which are exposed in the ‘ morula ’ stage, each cell having in addition a polar area of its own. The latter, however, do not appear in enfeebled eggs, but only the former.

In the gastrula and later stages the reaction occurs, but less markedly.

None of the eggs which have been exposed to the current develops any further. They stick to the jelly, and consequently lose their power of rotation.

Similar results were obtained by the use of the continuous current (43 volts), but the anodic and the kathodie areas usually differed from one another in certain details.

It is important to notice that neither in these experiments, nor in another in which the eggs were placed inside a glass tube surrounded by a coil, could any definite relation be satisfactorily made out between the direction of the first furrow and that of the current. Indeed, though intrinsically interesting, the experiments throw no particular light upon the problem of development. Rather should they be classed with the investigations of Verworn and others upon the behaviour of Protozoa in the electric current, investigationswhich promise to contribute to the understanding of the structure and movements of living substanee. It may be noted here that Roux has himself produced these polar areas on such structures as the heart and gall-bladder of the Frog and other vertebrates.


C. DARESTE. Rechcrches sur l‘iniluence de l’électi-icité sur 1'4.’-volution dc l‘embr_yon de la poule, Comples Bemlus, cxxi, 1895.

U. ROSSI. Sull‘ azione dell‘ elettricita nello sviluppo dclle uova (legli Anfibi, Arch. Ent. Mech. iv, 1897.

W. RoUx. Ueber die morphologisehe Polarisation von Eiern uml Embryonen durch den electrischen Strom, sowie fiber die Wirkung des eleetrischen Stroms auf die Riehtung (ler elsten Teilung des Eies, S’.-B. Icais. Alrud. IViss. Wien, ci, 1891, also Ges. Abh. 25.

B. C. A. WINDLE. On certain early ma.lformn.tions of the embryo, Jouru. Aunt. and Phys. xxvii, 1892-3.

B. C. A. W1NDLE. The effects of electricity and magnetism on development, Journ. Aunt. and Phys. xxix, 1895.

4. Light

As Roux pointed out long ago in the case of the Frog, light exercises no directive influence upon the development of the ovum. Blane, indeed, has attempted to prove that the direction of the embryonic axis in the egg of the Hen may be made to depend upon the direction of the incident light-rays, but the experiments are hardly conclusive. The method employed was to blaeken the shell of the horizontally placed egg with the exception of one spot to right or left of the blastoderm. On this spot a beam of light was kept directed during incubation. In some cases, but not in all, the embryonic axis was found to deviate from its normal position at right angles to the long axis of the shell. Further, the head of the embryo might be turned towards or away from the source of light. There was no relation between the amplitude of the deviation and the length of the exposure.

Nor are the processes of growth and differentiation necessarily affected in any way at all by the presence or absence of light, or by the kind of light to which the eggs are subjected.

Thus Driesch, who has experimented with the eggs of I9’o/Mme, P/auorbis, and Rana, maintains that neither red, yellow, green, blue, nor violet light has the slightest eifect upon the eggs during the early stages of segmentation and gastrulation, in what he calls the organ-forming period of development; and Loeb has asserted that the development of the embryos of the lish Fzmthzlus is as rapid in darkness as in the light, except that on the yolk-sac (not in the embryo) far fewer pigment-forming cells are produced.

Yung, on the other hand, has brought forward evidence to show that in later stages, at any rate, the embryos of the Frog react difierently to lights of various wave-lengths, some of which are harmful, others, apparently, beneficial.

Yung obtained his colours from solutions of fuchsin (red), potassium bichromate (yellow), nickel nitrate (green), bleu de Lyon (blue), and viole de Parme (violet). The colours, it may be noticed, are not absolutely monochromatic.

Freshly laid eggs of Rana tem17omria were placed under the influence of these lights. After one month, samples of the tadpoles were measured, with the following result in millimetres : TABLE IX Red. Yellow. Green. Blue. Violet. White. Length 2158 25-91 18-83 26-83 29-66 25-75 Breadth 4-83 5-58 4-16 5-75 6-83 5-25

The mortality in the green light was great. After two months the dimensions were as follows :—


Red. Yellow. Green. Blue. Violet. White. Length 26-25 31-83 All ‘ 33-50 41-30 31-00 Breadth 6-00 7-50 dead. 8-00 10-16 7-33

All the tadpoles in the red light eventually died. White and yellow light gave the greatest number of perfect frogs, but, as will be seen, those in the violet were larger. They were, however, less differentiated, for they did not acquire their hind legs so soon as did those in the white light. It may be mentioned, however, that when the tadpoles reared under these conditions are replaced in ordinary light and starved, those from the violet exhibit a. greater power of resistance.

Experiments with Rana ésczzlenta gave the same result. In this case the effect of darkness was also tried and found to be distinctly unfavourable. Thus after one month the lengths in darkness and white light were respectively 19-66 mm. and 23-10 mm., the breadths 4-66 mm. and 5-50 mm. ,- after two months the difference was intensified, the lengths being 21-50 mm. and 32-16 mm. , the breadths 7-16 mm. and 7-66 mm. The deathrate in the dark was exceedingly high.

The eggs and embryos of the Trout were likewise found by Yung to be highly sensitive to green and red light, while the larvae reared in violet hatched out rather more quickly than those from yellow, blue, or white light.

In an experiment on the eggs of I/inmaca stayizalie, due to the same investigator, the effect is measured by the time required for the young to hatch out, as the following table shows :—


Light. Time to hatching in days.

Red . . . . . . 36

Yellow . . . . . . 25

Green . . The heart is formed, then death occurs. Blue . . . . . . 19

Violet . . . . . . 17

White . . . . . . 27

Dark . . . . . . 33

Green light is evidently fatal; development is retarded in red light, less so in darkness ; yellow has about the same clfcet as white light, while there is a considerable acceleration in blue and violet.

The relative effect produced by the various lights is as in the preceding experiments.

The results obtained by Vernon for Eehinoid larvae are, however, not quite consonant with this, as may be seen in the table (Table XII), where the colours are arranged in the order of the efiect they produce. It will be observed that yellow is more harmful than red, while green exerts about the same effect as blue (copper sulphate). The author states, however, that in two other experiments the larvae were entirely killed off by the green light though developing perfectly in the white. He also adds that in violet light no development was possible owing to the swarms of bacteria.


Percentage change of size.

Semi-darkness . . . . . + 2-5 ‘ Absolute darkness . . . . . — 1-3*

Blue (copper sulphate) . . . . -46

Green . . . . . . . -4-8

Red . . . . . . . -6-9

Blue (bleu de Lyon) . . . . —-7-4

Yellow . . . . — 8-9

  • Almost within the limits of experimental error.

In the Pluteus Vernon found that both the oral and the aboral arm-length decreased in darkness, green and blue (bleu de Lyon) lights, while blue (copper sulphate), yellow, and red light exerted little influence on this magnitude.

It only remains to be added here that Blanc and Féré have brought forward some not very satisfactory evidence to show that white light is favourable to the development of the Chick. Féré has also stated that red and orange lights are more harmful than white, while violet has about the same elfect. The experiments are, however, vitiated by the fact that the eggs were not turned over.


L. BLANO. Note sur l‘influence de la lumiere sur l'o1-ientation de l'embryon dans l’oeuf de poule, C. R. Soc. Biol. (9) iv, 1892.

L. BLANC. Note sur les effets tératogéniques de la lumiere blanche sur l’oeuf de poule, C. R. Soc. Biol. (9) iv, 1892.

H. Dnmscn. Entwicklungsmechanische Studien II, Zeitschr. wiss. Zool. liii, 1892.

C. Feat}. Note sur l'influence de la lnmiere blanche et de la lumiere colorée sur l’ineuba.tion des oeufs de poule, C’. R. Soc. Biol. (9) v, 1893.

'1‘. LIST. Ueber den Einfluss des Lichtes auf die Ablagerung von Pigment, Arch. Ent. Mech. viii, 1899.

J. LOEB. A contribution to the physiology of coloration in animals, Journ. Morph. viii, 1893.

J. Lorna. Ueber den Einfluss des Lichtes auf die Organbildung bei Thieren, Pfl12ger's Arch. lxiii, 1896. III. 4 LIGHT 97

H. M. VERNON. The effect of environment on'the development of Echinoderni larvae: an experimental enquiry into the causes of variation,

Phil. Trans. Roy. Soc. clxxxvi, 1895. E. YUNG. De 1’influence des milieux physiques sur les étres vivants,

Arch. Zool. Exp. et G'e'n. vii, 1878. E. YUNG. De Pinfiuence des lumieres colorécs sur le développement

des aniinaux, Mitt. Zoo]. Stat. Neapel, ii, 1881.

5. Heat

As is very well known, those activities by which every organism maintains its specific form can only be carried on within certain definite limits of temperature. So also a certain degree of heat is necessary for the due performance of the functions of growth and difierentiation ; above or below certain limits—more or less definite for each organism, but varying in different organisms—-development is unduly accelerated or retarded, or brought to a standstill, while its form is frequently distorted as well.

To Oscar Hertwig we are indebted for a careful inquiry into the conditions of temperature under which the development of the Frog-’s egg takes place.

In the case of Ifanaflcsces Hertwig has found the cardinal temperature-points to be as follows :—The normal is about 15°—16° C.; above this up to 20°—22° C. development is accelerated without being otherwise altered ; this temperature is therefore the optimum (Fig. 51). Above this point the form of development is altered, a11d at such a high temperature as 30° C. death follows very quickly. At low temperatures (6°-1° C.) there is considerable retardation, and at the zero-point a complete cessation of segmentation ; the eggs are often permanently injured.

At the high temperatures referred to—-from 23° C. upwards— it is the yolk—cells which are primarily affected. At from 29-6‘’ to 27-5" the yolk is unable to divide, though it is nucleated, and segmentation is confined to the animal hemisphere, and soon ceases even there (Fig. 47). At 26-5° the first furrow indeed passes through the yolk, but subsequent segmentation is mereblastic, with the resulting formation of a cap of cells or blastoderm lying upon and separated by a segmentation cavity from the nucleated yolk. The eggs then die. At lower temperatures25°-23°—the yolk is also affected, and many eggs die in the ‘ morula’ stage; such as do survive give rise to distortions or monstrosities (Figs. 48, 49). The injury to the yolk interferes with the proper closure of the blastopore ,- there is consequently a. large, persistent yolk-plug surrounded by a thickened blasteporic rim into which the separated halves of the medullary plate and notoehord are differentiated (spina bifida) (Fig. 50). The

Flo. 47.——Meridional sections of eggs of Rmmfusm developed (A) at 29-5° 0., (B) 26-5“ 0. Five hours ten minutes after fertilization. 7;, nuclei; p, pigment.

Fig. 48. — Meridiona.l sections of eggs of Rama fusca developed at ._‘2 -5 C. Qne day after fertilization. /.-, nuclei; kk, blastocoel; 2, cells imbedded in unsegmented yolk.

Fig. 49. — Abnorma1 embryos of Rauafusca, produced by heat. A, embryo two days old developed at a. temperature of 24° C. ; 13, embryo three days old, reared at a. temperature of about 25° 0. br, brain; y.p, yo1k—plug; Lb, tail-bud ; t,ta.1l; s, sucker ; g, gill.

FIG. 50.- Two transverse sections through the embryo shown in Fig. 49 A. A, passes through the blastopore and yolk-plug; B, through the anterior end. (I, yolk-plug ; m.p, medullary plate; ch, notochord; mk, mesoblast.

front end of the arehenteron is, however, normally developed if the temperature is not too high, and in this case the anterior portion of the nervous system and notochord are undivided ; posteriorly, however, their right and left halves diverge round the blastepore, and are continued into the halves of the double tail when the latter is formed. Gill slits, protovertebrae, striated muscle—fibres, the pronephros and its duct, and

Fig. 51. Melidi0m1section

the tail {in may all be differentiated. of an egg of Rum, f,,_m,, de_

The development of the organs Veloped at 9: t01"Pe1‘°«t“1‘6 0f f t] t .d f t] b d . f_ 22° C. Six hours fifty minutes 0 1e W0 S‘ es 0 1° 0 Y 1s 1c‘ after fertilization. kh, blaste quently unequal. coel.

At low temperatures segmentation and the closure of the blastopore take place very slowly, and at ()° cease altogether. The eggs are not, however, dead, but will resume their development when replaced under ordinary temperature conditions. They show abnormalities, however, due to injury of the yolk;

Morgan has similarly found that the fertilized (not, however, the unfertilized) eggs of an American species (If. palust/'18) which ve been subjected to a temperature of 1° C. and then allowed elop under normal circumstances exhibit spina bifida and

lze has also observed these abnormalities as the result of e cold. On one point, however, this author is not quite ement with O. Hertwig, for he states that eggs and s exposed to 0° in various stages do continue to develop, thld of course very slowly. Thus, in the ease of eggs exposed sl8§ after fertilization, the blastula stage was only reached in days, while a month elapsed before the bl-astopore was formed. ie and Knowlton, however, state that in la’. rireecens and .4m0l_y.s*to1/ta liqrinum segmentation is totally inhibited at 0°. In another species of Frog (If. esculenla) which spawns much later in the year——in May and J une——the cardinal points were found by Ilertwig to be much higher, and the eggs endured a temperature of 33° C. without injury. They are, in fact, acclimatized to a higher temperature, and it is very interesting to notice that Davenport and Castle have succeeded in artificially acclimatizing the eggs of another Amphibian (Bag/‘o le7zli_qz'/zoszw) to a considerable degree of heat. Eggs were reared at 15° C. and 24°—25° C. After four weeks the heat rigor temperature was 40° C. for the former, 43-2° for the latter; and in another experiment the temperature was raised to 43-3’ by allowing the eggs to develop at 33°—34.«° for seventeen days.

A similar lowering of the minimum seems to have been observed by Lillie and Knowlton in the case of /lmM_y&lo1/za tigrimzi/I. In this form, which spawns much earlier than Rana vi7'¢m.v, there is considerably less retardation of development at 4°.

That temperature markedly influences the rate of development, or, as Hertwig puts it, that the quantity of developmental work performed per unit of time is a function of the temperature, is abundantly clear, and is well shown in the annexed diagrams (from Hertwig), in which the curves show the times taken to reach various stages at various temperatures (Figs. 52, 53).

It will be seen that as the temperature sinks the rate of development, or rather of differentiation, decreases, but at an The rate of growth, however, may increase at an increasing (or decrease at a) decreasing) rate, as Lillie and Knowlton found

FIG. 52.— Curves showing development of the Frog (Ramtfusca). The absture in degrees Centigrade, the ordinates the

each of the stages I to ]X. I

dullnry folds closed, sucker

VII, operculum beginning; VIII, opercnlum closing; IX, rudiments of hind legs. (After 0. Hertw 98.)

the effect of temperature upon the rate of cuss e the tempera» <11) uired to reach

and gills; VI,

metail fin ;

increasing rate. Lillie and Knowlton have made the same observation for the species investigated by them.

for the tadpoles of Rana viresccns and Bufo lenliginoxzw. The same authors state that at low temperatures (below 3° in the case of the Frog, below 6° in the case of the Toad) growth was altogether inhibited, while at 2° there was an actual shortening in length in the ease of the Frog tadpole, due, it is suggested, to a diminution in the turgor of the cells.

The cardinal points have also been determined for the lIen’s egg. According to Kaestner normal development occurs only

FIG. 53.—Efl'ect of teinperaturc upon the growth of the tadpole of the frog (Rana fusca). A, B, developed at a temperature of 14-5"--15" (1.; A, two days old, circular blastopore (Stage I in Fig. 52) ; B, three days old (Stage II in Fig. 52) ; C, 1), developed at a temperature of 20° C. ; 6', three days old (Stage V in Fig. 52); D, four days old (Stage VI in Fig. 52). (From Minot, 1907, after 0. Ilertwig, 1898.) between 95° and 102° F. (35° and 39°C.). The maximum, the temperature above which the embryo dies, is 43° C.; the minimum, at which development stands still, 28" C. Edwards, however, fixes the minimum or physiological zero at 20°—21” C., for, as the annexed diagram shows (Fig. 54-),development may continue between 20° and 29°, though it is, of course, very much retarded.

Edwards has further made the highly interesting observation

FIG. 54.— The index of; development (percentage of normal development) for the egg of the Hen at tenrperatures varying from 20°C. to 30-75"G. (After Edwards, 1902.)

FIG. 55.—Growth of the blastoderm of the Hen’s egg independently of the appearance of the primitive streak, at low temperatures. (After Edwards, 1902.)

that at tl1e low temperatures in question growth may occur without differentiation (Fig. 55). Thus in one series of experiments at 24-5° for six days the blastoderm increased in diameter from 4-4 mm. (the average diameter of the blastoderm in unineubated eggs) to 6-9 mm. The primitive streak was, however, not formed.

A temporary exposure to low temperatures often inflicts a permanent injury on the egg and leads to malformations. Kaestner, by subjecting the eggs at many different stages to temperatures of 15°—25°, 10° and 5°, has discovered that the capacity of resistance decreases as development proceeds (though not with absolute regularity). Thus the maximum exposure to 21° consonant with subsequent normal development was 192 hours for embryos of six hours, 96 hours for embryos of one day, 7 2.’ hours for embryos of two to six days, 4-9 hours for embryos of eight (lays, and 24 hours for embryos of 20 days.

At these low temperatures development is stated to be completely arrested, though the heart never ceases beating, irregularly and convulsively. The cooling process may be repeated over and over again without altering the capacity for future growth and diiferentiation, or reducing or increasing the maximum capacity of endurance of cold.

Malformations, as stated above, are of frequent occurrence, b.ut only in those cases in which the embryo has been exposed in an early stage, during the first two or three days of incubation, and only after long exposures.

The Inedullary folds may remain unformed anteriorly, the two halves of the heart may remain widely separate, the head amnion fold may be absent and abnormal gill slits be formed; the heart and blood—vessels are often enormously distended, and hacmorrhages are frequent. Kaestner attributes these monstrosities not directly to the cold but to the pressure of the blastoderm against the shell, for in the cooled eggs, owing to some change in the specific gravity of the albumen or yolk, the latter rises up ; if the egg is placed with the blunt end uppermost, so that the embryo is pressed against the shcll—membrane, no monsters are produced.

Mitrophanow is another observer who has utilized low temperatures to cause malformations. High temperatures also give rise to abnormalities accompanied by acceleration (Féré, Mitrephanow).

The effects of extremes of heat and cold upon the ova and embryos of certain Invertebrates have been studied by Drieseh, the brothers Hertwig, Vernon, Sala, and Greeley.

FIG. .")6.—-The effect of heat upon the segmentation of the Echinoid egg. a, b, c, (1, four successive stages in the segmentation of the same egg of I9'¢'hinus; e, f, two successive stages in the division of the same egg of Sphaercchinus. (After Driesch, 1893.)

FIG. 57.— Suppression of cell-, but not of nuclear, division by heat (E:-hbms). (After Driesch, 1893.)

The first-named observed that by subjecting the fertilized ova of Sp/zaerec/aiizus to a temperature of 30°—3l° (the normal is 19°) development was accelerated and segmentation abnormal (Fig. 56).

After the first furrow—though not after subsequent divisions—— the blastomeres separated and sometimes remained apart, a fact which provided a means of watching the independent development of the first two blastomeres. After the four-celled stage the direction of division became irregular, one spindle being perpendicular, instead of parallel, to the other three, or two perpendicular to two, or all irregular; in the next phase the formation of micromeres was partially or wholly suppressed. Nevertheless these abnormally segmented eggs produced perfectly normal Plutei.

It is also possible for nuclear division to continue while celldivision is suppressed, as a result of exposure to high temperatures (Drieseh) (Fig. 57).

Fig. 58. ~ The effect of heat upon the development of S'pl1mu'e¢-/u'nu.s_r/ranulm'z's. a,exogu.strul-a; b, exog.1.strula.,wit.l1 tripartite gut; r, Pluteus, with tripartite gut; (d) Anenterion, with stoinodaeum, but no gut. (After Driesch, 1895.)

By exposing the blastulae to the same high temperature Driesch brought about a very interesting malformation, an Aneuterion (Fig. 58). The arehenteron was formed and constricted into the normal three portions, but it was evaginated instead of invaginated. Later on it shrank up and disappeared; the rest of the embryo, however, became a Pluteus, with a stomodaeum.

Vernon finds the optimum temperature for Echinoid larvae to be from 17-5° to 21«5° C. Exposure to high or low temperatures after fertilization, either for longer or shorter intervals or continuously, produced a decrease in body-length of the Plutei. The arm-length, however, increased with increasing temperature.

Vernon has also made the most interesting observation that the variability alters with the temperature. Eight-day larvae were measured, and the mean variability (Galton’s Q) of the body-length was found to be at 16° to 18°, 22-2, at 18° to 20°, 26-3, at 20° to 22°, 24-8, and at 22° to 24°, 24-0. Thus the variability is greatest at the temperature most favourable for development, and conversely.

It is also possible for the cell processes that occur during fertilization itself to be seriously affected by heat and cold, as the researches of O. and R. Hertwig have shown.

Moderate exposure (twenty minutes) of the eggs of Sta-ony_ylo— cenlrotmr to a temperature of 31° C. so weakens the cytoplasm that many spermatozoa are enabled to enter. Each sperm forms its own aster, and these combine with one another to form various irregular mitotic figures (triasters, tetrasters, and so on). The segmentation of such eggs is very irregular. With longer exposures the cones of entrance become feebly developed and the asters are not formed, while the numerous sperm-nuclei remain unaltered. Greater heat—over 40° C.— prevents the entrance of the spermatozoa altogether.

This pathological polysperm y may also be produced by cold; in this case also excessive exposure prevents the formation of the vitelline membrane, the cones of entrance, and the sperm-asters, while the spermatozoa remain in the peripheral layer of the egg. The eifect of a low temperature on eggs which have already been normally fertilized is seen in the reduction of the astral rays and spindle fibres, though not of the spheres, and in the thickening and irregular aggregation of the chromosomes. At a normal temperature the achromatic figure reappears.

Very similar phenomena have been described by Sala. in A.9carz'8. This author kept the eggs (the females, that is, containing eggs in all stages of development) at low temperatures ——-from 3° to 8° C.—for from half an hour to five hours and longer. The eifect of a short exposure to a very low tem108 EXTERNAL FACTORS III. 5

perature is not so harmful as a longer exposure to a less degree of cold. The processes of maturation and fertilization were both abnormal. Granules of chromatin took the place of the tetrads and were unequally distributed to the spindle-poles ; or, if the chromosomes (tetrads) had been normally formed before the commencement of the experiment, their division was irregular, in extreme cases all passing to one pole and into the first polar body. Again, the formation of the polar bodies might be suppressed altogether, or abnormal, the second only being formed, or both as one, or the first polar body might be as large as the egg itself. 'l‘hc achromatic figure was also deformed, the spindle being split at one or both poles (pseudotriaster, pseudo-tetraster), and centrosomes appeared instead of the usual centrosomal granules. The cytoplasm became granular, the vitelline membrane was not formed, two or more eggs frequently fused together. Polyspermy, with consequent multiplication of asters and eentrosomes, was very noticeable, aml, in fertilization stages, a separate pronueleus may be formed from each female chromosome, or fragment of a chromosome.

Closely connected with the cytoplasmic effects brought about by these temperature changes is the phenomenon of artificial parthenogenesis, produced by Morgan and Greeley in Ar/mcia and .»1stc7-ins by lowering the tempe1'at111'e of the sea—water to the freezing-point. Greeley has also shown that a lowering of the temperature, like the raising of the osmotic pressure, results in a withdrawal of water, the cause to which, as is well known, Loeb attributed the development of unfertilized ova in his experiments.

Greeley has shown that by the combination cl‘ a low temperature with a chemical reagent :1. higher percentage of swimming blastulac can be obtained.


C. B. DAVENPORT and W. E. CASTLE. Studies in lnorphogencsis; III. On the acelilnatization of organisms to high temperatures, Arch. Em. Mesh. ii, 1896.

H. DRIESCH. Entwicklungsmeeh. Stud. IV: Experimentelle Veranderung des Typus der Furehung und ihre Folgen (Wirkungen von Warmezufuhr und Druck), Zeilschr. u-r'.vs. Zool. lv, 1893. III. 5 HEAT 109

H. DRIEscH. Entwicklungsmech. Stud. VII: Exogastrula und Anonteria, Mirth. Zool. Stat. Neapel, xi, 1895.

C. L. EDWARDS. The physiological zero and the index of development for the egg of the domestic fowl, Gallus do;msti('u.s‘, Amer. Journ.

Phys. vi, 1901-2. _

A. W. GREELEY. On the efl‘ect of variations in the temperature upon the process of artificial parthenogenesis, Biol. Bull. iv, 1903.

O. HERTWIG. Ueber den Einfluss verschiedener Temper-aturen auf die Entwicklung der Froscheier, S.-I3. loom’;/. preuss. Al.-ml. Wis.»-. Berlin, 1896.

0. HER'rwI(}. Ueber den Einfluss cler Temperatur auf die Entwicklung von Rana fusca und esculcnta, Arch. mikr. Anat. li, 1898.

S. KAESTNER. Ueber kiinstliche Kitlteruhe von Hiihncreiern im Verlauf der Bebriitung, Arch. Anat. Phys. (Anna), 1895.

S. KAESTNER. Ueber die Unterbrechung der Bebriitung von Hiihnereiern a.1s Methode zur Erzeugung von Missbildungen, Verh. Amrt. GeseIIsr'h.,

1896. F. R. LILLIE and E. P. KNOWLTON. On the effect of tempemture on

the development of animals, Zool. Bull. i, 1898. L. SALA. Experimentelle Untersuchung fiber die Reifung und Befruehtung der Eier bei Ascaris megalocephala, Arch. milcr. Anat. xliv, 1895. O. SCHULZE. Ueber die Einwirkung niederer Temperatur auf die

Entwickelung des Frosches, I, Anat. Anz. x, 1895. O. SCHULZE. Ueber die Einwirkung niederer Temperatur auf die

Entwickelung des Frosches, II, Anat. Am. xvi, 1899.

6. Atmospheric Pressure. The Respiration of the Embryo

The respiratory exchange, which is so characteristic a function of adult organisms, is a necessity for the embryo also, and in some cases can be detected in very early stages indeed.

In the case of the Chick this need of oxygen is shown by the arrest or distortion of development, or the death of the embryo when the egg is placed in too confined a space, or when the shell is varnished, wholly or above only (Mitrophanow, Féré), though a coat of varnish on the lower side has no effect according to the latter author; or again, when the egg is placed in an atmosphere of hydrogen, or when the pressure of the ordinary

atmosphere is reduced (Griaeomini). Giaeomini found that at a pressure of about 600 mm. the embryos were small and abnormal in respect of the medullary tube and amnion; the optic vesicles and cranial flexure were absent, and there were serious disturbances in the area vasculosa, where, though the blood islands were present, the capillaries were either not formed or failed to reach the embryo. No haemoglobin was produced. Embryos exposed at a later stage (four days) nearly all died in two days of asphyxia, the blood being dark red and haemorrhages numerous. That these efieets were due not to the reduced pressure but to the want of oxygen was shown by the complete normality of embryos reared in an atmosphere of pure oxygen at the same pressure (except in certain characters always exhibited by such embryos ; see below).

Similar methods may be employed to demonstrate the necessity of oxygen for the Frog’s egg, a necessity which is indeed patent to any one who has observed the inferior development, accompanied by spina bifida and open blastopore (Morgan) of the eggs in the middle of a mass of spawn.

Thus, according to Rauber, development is retarded at a pressure of % atmosphere of ordinary air, and the mortality high, while at pressures of 7}; or 7,1 atmosphere death very rapidly ensues. As a result of four days’ exposure to pure hydrogen or nitrogen (ordinary air from which the oxygen had been removed) Samassa observed retarded segmentation, and subsequently irregularities in development of the type already referred to. Carbon dioxide produced irregular segmentation and death in twenty hours.

Godlewski’s experiments are perhaps more thorough. The eggs subjected to ordinary air at a greatly reduced pressure (2 .mm.), as well as those kept in thoroughly boiled water, segmented but little, and cell-division was confined to the animal hemisphere. In an atmosphere of pure oxygen at the same low pressure, however, development was, in many cases at least, neither retarded nor abnormal. Further experiments with pure oxygen, pure hydrogen, and an atmosphere composed of oxygen and carbon dioxide in equal parts, gave the same result, as the subjoined table shows (Table XIII). It is also clear that the absence of oxygen makes itself felt almost from the beginning, while pure oxygen accelerates development.


~__ 1- Oxygen and : Hours Oxygen. Hydrogen. Carbon Controls. Dioxide. 3 First furrow in No furrow M-‘ No furrow seine 3% All but one with One - hall‘ Most with first first furrow with first furrow ' furrow , 4 All but one 4 cells The same, 8 All with 2 cells 2 cells ‘J3 5 All with 4 cells Most with 4 g Most with 21 cells; ce s 4» a few Wltl 4 17% Blastomeres smaller Blastomeres a, Normal than in controls smallerthan 3; in controls o 22} Blastomeres very Segmenta.- 7* Normal small tion ceases _ 47 Blastopore closed White) hemisphere visi le 73 Mcdullary folds Blastopore closed

This method has given similar results for the eggs of the fishes Cte7z0la6¢'u.s and Fmzriulus (Loeb). One or two points are, however, worthy of especial notice.

The former develops at the surface of the sea, and is more sensitive to a lack of oxygen than the latter, the segmentation of whose egg will indeed continue for twenty-four hours in pure hydrogen, though an embryo is never formed. In Cleuolabrus, on the other hand, segmentation never advances further than the eight-celled stage, and the cell-boundaries already formed subsequently disappear, though they can be restored on removal to pure oxygen. In Fvmrlulus the capacity for enduring a lack of oxygen decreases (or the need of oxygen increases) with the progress of development; the fatal exposure for a newly fertilized egg is four days, for a newly formed embryo thirtytwo hours, for an embryo with the circulation established twenty-four hours, and for the newly hatched larva. shorter still. Carbon dioxide is quickly fatal to both species.

The lack of oxygen has also a noteworthy eifect on the pigment cells which are found, especially round about the bloodvessels, on the yolk-sac of Fzmrlulus. These pigment cells are of two kinds, black and red, and when the embryo is deprived of oxygen the former disappear, the latter diminish only a little.

It has been noticed elsewhere that this pigment is less abundantly formed in darkness than in light, and Loeb has suggested that light may promote oxidation.

The ova of Echinoids also require oxygen from the beginning of their development (Loeb). Without this element segmentation is impossible, or, if segmentation has already begun before they are deprived of it, the blastomeres swell up and fuse. According to Lyon the eggs of Arbacia are only sensitive to a want of oxygen for from fifteen to twenty minutes after fertilization. Vernon has shown that water saturated with carbon dioxide and mixed in the proportion of 20 % or more with sea-water is fatal to the development of these forms.

Exact quantitative determinations of the oxygen absorbed and the carbon dioxide excreted have been made by Godlewski for the Frog and by Pott and Preyer for the Chick. The results are shown in the tables annexed (Tables XIV, XV).


Showing the result of one experiment on the respiration of the Frog's egg (Godlewski).

Day? anger A111ourz1llI;‘ilnpg§|;.air‘i)I(p:a‘:é;e:')f24 hrs. femhzatlom 0 absorbed. CO, excreted. 0-03908

1 __ 2 0-4502 0-0995 3 0-7033 0-2131 4 1-0539 0-4193

It thus appears to be the very general rule that the egg begins to respire at an early age. There is a case, however, A.s-crmlv, in which not only can the egg endure an atmosphere of nitrogen or carbon dioxide or nitric oxide for prolonged periods and still develop, but is actually killed by pure oxygen (at 2-} atmospheres) (Samassa). The adult worm, of course, is an endoparasite, and Bungc has shown that it can manage to produce carbon dioxide though denied access to free oxygen.

The eifect of pure oxygen has also been tried on various embryos. In such an atmosphere (at ordinary pressure) the development of the chick is normal, except that skin, allantois, limbs and amniotic fluid are all very red with oxyhaemoglobin; an excessive amount of carbon dioxide is produced. The amount of this gas excreted by the undeveioped though incubated egg in pure oxygen is, however, less than in air.


Showing the oxygen a.bso1-bed and carbon dioxide excreted by the Hen’s egg during incubation. (After Pott and Preyer, from Preyer, Spez.

Phys.) Days of Amount in grammes per 24 hrs. of iIl0l1')Mi0l1- O absorbed. CO, excreted.

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  • Pulinonatry respiration begins.

According to Samassa. and Rauber the development of the Frog-’s egg in pure oxygen is normal, but Godlewski states, as we have seen, that it is somewhat accelerated. At a pressure of 2% atmospheres, however, segmentation is arrested and death ensues (Samassa).

When the tadpoles, newly hatched, are exposed to its influence, the hyoid becomes immensely thickened and the branchial chamber completely closed; the internal gills weak (Rauber), and the same author states that ‘ gastrulae’ subjected to three atmospheres of ordinary air had their development temporarily arrested, while later embryos, in the stage of the medullary folds, became small and immobile in air at twice the atmospheric pressure. This result seems to be due to pressure, not to the oxygen.

In the foregoing the general necessity of respiration for the life of the developing organism has alone been taken into consideration, but it should not be forgotten that oxygen may exert a stimulus on some part, the response to wl1ich results in a process of diiferentiation. Thus His has suggested that the growth of the blastoderm over the yolk is oxygenotropic, and IIerbst that the migration of the blastoderm-forming cells to the surface in Arthropod ova, and the migration of spiculeforming cells in Echinoid larvae are cases of definite reaction to oxygenotactic stimuli. Loeb, we may note, has found that the regeneration of the head of ’l'ubularia will only take place when the stem is supplied with fresh water, and the same author has suggested that the accumulation of the pigment cells round

the blood-vessels on the yolk-sac of Fmztlulus is also an oxygenotaxis.


C. F1’-Jmé. Note sur l‘inllueuce des entluits particls sur l'ine-ubation dc l’u.:ul' do poule, C. If. Soc-. Biol. (10) i, 1894.

C. GIACOMINI. Influence de 1‘ iarifié sur le dévcloppemcnt dc 1'ueufde poule, Arch. Ital. Biol. xxii, 1895.

E. GODLEWSKI. Die Einwirkung des Sauerstolfes auf die Entwicklung von Rana tempormia, Arch. Eut. Mesh. xi, 1901.

C. HERBST. Ueber die Bedeutung der Reizphysiologie fiir die cauale Auffassung von Vorgangen in der thierischen Ontogenese, Biol. Cmtralbl. xiv, xv, 1894, 1895.

J. LOEB. Ueber die relative Empfindlichkeit von Froschembryonen

gegen Sauerstoifmangel und Wasserentziehung in verschiedenen Entwicklungsstadien, Pflfigefs Arch. lv, 1894. III. 6 ATMOSPHERIC PRESSURE 1 15

J. LOEB. Untersuehungen fiber die physiologischen Wirkungen des Sauerstoffmangels, P_fliige)"s Arch. lxii, 1896.

P. MITROPHANOW. Einfluss der veritnderten Respirationsbedingungen auf die erste Entwiekelung des Hfilmerembryos, Arch. Ent. Meclt. x, 1900.

R. Po'r'r. Versuche iibcr die Respiration dcs Hiihner-Embryo in einer Sauerstoffatinesphiire, If/h'iger’s 47071. xxxi, 1883.

R. Po'r'r und W. PREYER. Ueber den Gaswechsel und die chemischen Veri‘r_nderungen des Hiihnereies withrend der Bebrutung, Pflz'iger's Arch. xxvii, 1882.

W. PREYER. Spezielle Physiologic dos Embryo, Leipzig, 1885.

A. RAUBER. Ucber den Einfluss dcrTen1pc1-atur, des atinospliarischen Druckes nnd vcrschiedener Stoffc auf die Entwicklung thierischer Eier, S.-B. Nrmuf. Gas. Leipzig, x, 1883.

H. SAMASSA. Ueber die étusseren Entwicklungsbedingungen der Eier von Rana tomporaria, Verh. ])eutsch. Zeal. (v'cs. vi, 1896.

7. Osmotic Pressure. The Role of Water in Growth

That growth seems to depend in many cases on the absorption of water or a watery fluid—in the swelling of the Echinoderm blastula, for example, or the enlargement of the Mammalian blastoeyst——has been noticed by several observers; in a. few instances experimental proof has been given of the relation between the two.

Although, as is very well known, the Hen’s egg loses weight daily throughout incubation by loss of water, this loss is due almost entirely to the slow evaporation of the albumen, and a humid atmosphere is necessary for development, as Pott and Preyer have found. Féré’s experiments with eggs incubated in desiccators demonstrated, during later stages, a slight reta.rdation accompanied by abnormalities and a high death-rate; in earlier stages, up to about the fourth day, there was on the contrary an acceleration of development.

Davenport has shown for tadpoles of various Amphibia (Amblystoma, Toads, Frogs) that increase in weight is very largely due to increase in weight of water. Known numbers of tadpoles, from which superficial water had first been carefully removed, were placed over sulphuric acid in a. desiccator. Repeated weighings were made until a constant minimum was reached. The results are set forth in the accompanying table and figure (Table XVI, Fig. 59). It will be seen that the percentage of water rises very rapidly in the first fortnight, from 56 % to 96 %, then decreases slightly, afterwards becoming nearly constant.


Showing the rate of absorption of water by 'l‘u.«lpoles (after Davenport). Days after hatching. Percentage of water. 56

FIG. 59.- Curve showing change in percentage of water in Frog tadpoles from the first to the eighty-fourth day after hatching. Abscissae, days; ordinates, percentages. (After Davenport, from Korschelt and Heider.)

A different, and a less satisfactory, method has been employed by Loeb, hypertonic solutions being used to prevent the absorption of water. While the newly fertilized eggs of Fimdulus developed as normally in fresh water as in sea-water, only a blastoderm with occasionally a dwarf embryo was formed in a 5 % solution of sodium chloride in sea-water, and segmentation was arrested in the thirty-two-celled stage when the concentration of the salt was raised to 10 %. Older eggs were, however, far less sensitive, and after three or four days the embryos could he placed directly in a. 276 % solution without arresting their development, though the heart heat more slowly and differentiation was less rapid.

FIG. 60. — A and 0, formation of ex-ovatcs in the egg of Arba('1'(( by dilution of the sea-water; ls, nucleus; m, egg-membrane; B and 1), hlastulae formed from A and C; If becomes constricted into two blastulae, each of which gives rise to a. Pluteus; D produces a single Pluteus. (After Loch, from Kolschelt and lleider.)

The eggs can nevertheless be aeelimatized to the salt. Removed from the 10% solution after the thirty-two cells had been formed to ordinary sea-water for eighteen hours, they were capable, when once more replaced in the strong solution, of giving rise to embryos which lived for a considerable time.

Similar experiments made on Arbacia showed that though cell-division is suppressed in the hypertonie solution (2 Z sodium 118 EXTERNAL FACTORS III. 7

chloride) nuclear division continues all the same, for when returned to seaewater the eggs divided at once into as many cells as had in the meantime been formed in tlie controls, a,result confirmed by Morgan.

That the normal egg is in a condition of osmotic equilibrium with the sea-water is further shown by its behaviour in seawater diluted to twice its volume ; in this experiment the egg

Fig. 61.— Variations in the semnentation of Iu'¢-}u'nu.s- microtubm-culalns produced by dilution of the sea-water. a, tetrahedral four-eel] stage; II, eight cells, three premature niieromeres; 0, eight cells, two precocious microineres ; (I, the same egg afterthe next division, the precociolls micromeres have divided unequally, two normal micromeres have been formed. (After Driesch, 1895.)

(of /lrl/acia) absorbs water, swells and bursts its membrane and so produces a large ex-ovate which may develop independently of the rest of the ovum (Loeb) when replaced under ordinary conditions (Fig. 60). Driesch has produced irregularities of segmentation by the same means (Fig. 61).

Although, therefore, it seems reasonable to suppose that in the cases just quoted the observed eifects really are due to the increased osmotic pressure of the medium and consequent withdrawal of water from, or prevention of imbibition of water by, the eggs, the weak point of the experiment, and of all such experiments, is our ignorance of the extent to which the ova or embryos are permeable to the substance employed, since the osmotic effect, or withdrawal of water, will obviously vary inversely with the permeability. The neglect of this possibly disturbing factor has indeed led in some cases to quite unwarrantable conclusions.

In 1895 O. Hertwig showed that certain abnormalities could be produced by growing the eggs of the Frog (IF. /‘urea and

FIG. 62.—Three sodium-chloride embryos of Rana fusca. df, yolkplug; hp, brain; Ici, gills; .9, margin of epidermic layer of ectoderm; sch, tail; m-, lip of blastopore, (After 0. Hertwig, from Korschelt and Heider.)

esculenta) and of the Axolotl in a solution of common salt. In stronger solutions (1 % to 0-8 %) segmentation was confined to the animal hemisphere, though nuclear division went on in the yolk. Weaker solutions (0-6 %) allowed of further, but distorted, development; the yolk-cells were unable to move beneath the lip of the blastopore, so that the latter remained open with a persistent yolk-plug, and the mcdullary folds failed to close in the region of the brain, a condition recalling the abnormalities known in Human and Comparative Teratology as Hemicrania and Aneneephaly (Figs. 62, 65). The exposed region of the brain underwent a grey deg'enera.tion with disintegration of the epithelium. Other organs were, however, normally formed, the front end of the gut by iiivagination, the notochord and mesoderin, protovertebrae, heart, pronephros, auditory vesicles, optic vesicles, infnndibulum, and liver, until the embryo died. The persistence of the yolk-plug has also been induced by Gurwitsch by means of halogen salts (sodium bromide and lithium chloride) and weak solutions of alkaloids (strychnine, caifein, nicotine) (Fig. 63), by C. B. Wilson in Hana, C/I0?'0j)/I27’IlS, and A7110/‘I/8/07ll(l F;G,53__Me,-idiom1sec. by means of sodium chloride and Egggruffilffiggélzff Ringei"s solution, and by Morgan ‘with 1,, blagtocoel. (After Gm-. Various lithium salts; and Bataillon, Witflchs from Korscllelt and who has used isotonic solutions of caneHeider.) . . sugar, sodium chloride, and a large number of other salts for the purpose, claims that in this case the results produced depend upon the osmotic pressure alone, and are therefore due to a withdrawal of water from the developing embryo.

Yig. 64. — Sections of Frogs‘ eggs grown in solutions of, A, annnoniuin iodide (1-5%), and, B, urea (2-37,). In both cases segmentation is iiieroblastie, although in A there are a few lai'::,e. divisions in the yolk. In B the multinueleate cell masses of the animal hcniispliere protrude above the surface. The nuclei are large, lobed, and lioiiiogeneously clf'_Iroinal;ic in both cases. (Ammonia is probably present in the solution 0 urea.

Recent experiments made by the author do not, however, bear out this conclusion. In the first place, it is to be observed that isotonic solutions (isotonic with a O-625 % NaCl solution) do not produce the same, but markedly difierent effects. Some solutions arrest development at an early stage (during segmentation (Fig. 64), gastrulation, or the formation of the medullary folds); in others development proceeds but is distorted, the medullary folds remaining open in whole or in part, and the yolk-plug uncovered, or either of these malformations may occur without the other; in one case (dextrose) development is quite normal in form but very considerably retarded, while finally in urea development is normal both in form and rate (Figs. 66, 67). No legitimate deductions can be made from these experiments, how

FIG. 65.— Frog embryos grown in a -625% solution of sodium chloride. A and B after five days, 0 and D after six days. In all the yolk-plug is fully exposed. In A the medullary groove is wholly open, in B and C it is closed behind, in D it is closed throughout. ever, until the permeabilities of the tissues to these solutions are ascertained. The tadpole requires water (Davenport), and the degree of shrinkage of the tadpoles in these solutions affords a means of determining the question ; it appears that they are perfectl y permeable to urea, more or less impermeable to cane-sugar, dextrose, and sodium chloride, the sllrinkage being rather greater in the first than in the other two. On the assumption that the permeabilities of the embryo are the same as those of the tadpole, it follows that the greater effect produced on the former by sodium chloride than by cane-sugar, or, still more, than by dextrose, cannot be set down to the osmotic pressure of the solution alone, a. result which is further corroborated by the constancy in the relative toxicities of the bases and the acids in the case of the monobasic salts. The observed deformities are therefore to be attributed to some other—chcmical or physical——property of the solutions, though what this is is not known.‘ It may be added that in Gurwitseh's experiments the concentrations of the alkaloids employed were certainly far below those which would be isotonic with a -625% solution of sodium chloride. It also follows that during the closure of the blastopore the Frog’s eggdoes not need to absorb water from the outside; it may, in fact, be exposed to a very considerable degree of desiccation at this period without interfering in the least with the closure of the blastopore or of the medullary folds, a result which is all the more surprising in that the newly hatched tadpole imbibes water at so rapid a rate.

FIG. 66.— Frog embryos grown in isotonic solutions of, A, sodium chloride (-625%); B, cane-sugar (6-6%); 0, dextrose (3-4%); and, D, urea. (1-14%). In A the medullary folds are closed but the blasto ore open; in B the medullary groove 18 open but the blastopore close ; in C development is normal, but retarded ; in D development is normal, both in form and rate, though the embryos die soon after the stage shown in the figure.

FIG. 67.— A. Longitudinal section of a Frog embryo grown in a -45% solution of lithium chloride. The medullary groove is open, except in front and behind. The notochord is bent in several ilaces and the gut roof much crumpled. B. Longitudinal section of a. ‘rog embryo grown in a. 6-6% solution of cane-sugar. The medullary groove is open, except in front, the cells in its floor degenerating. The gut roof is incomplete in part and there is an evident neurenteric canal.

The experiments which have hitherto been considered relate to the need of water for normal development. There are, however, certain processes for which not the absorption, but, on the contrary, the abstraction, or at least the local abstraction, of water appears to be essential, the phenomena, namely, of fertilization. Cytologists have observed that the entrance cone and funnel, the mechanism by which the spermatozoon is swept into the interior of the egg, appear to be aggregations of a watery substance about the aerosome or apical body, and that the sperm sphere and aster are similarly due to the withdrawal of water by the centrosome in the middle-piece from the cytoplasm ,- in other words, that the stimulus whereby the spermatozoon restores to the egg its lost power of cell-division is essentially a process of local dehydration.

This inference is substantiated by the familiar experiments of Loeb, who has succeeded in rearing normal larvae from the unfertilized eggs of Echinoderms and certain worms by temporary immersion in certain solutions. In his earlier experiments he found that a mixture in equal parts of a 2,9 a solution of magnesium chloride and sea-water produced more Plutci than any other solution tried, and hence believed the result to be specific and attributable to the magnesium ion. Later, however, this artificial parthenogeuesis was successfully brought about by various isotonic solutions (chlorides of sodium, potassium and calcium, potassium bromide, nitrate and sulphate, cane-sugar and others). The increased osmotic pressure was, therefore, considered to be the cause of the phenomenon, and it was suggested that in ordinary fertilization the spermatozoon introduces a substance which has a higher osmotic pressure than, and is therefore able to withdraw water from, the egg.

  • In this view Stockard, as a result of experiments on Fmululus, concurs

(Arch. Em. Mach. xxiii, 1907, and Journ. Exp. Zool. iv, 1907).

Hunter has also shown that sea-water concentrated to 7 0% of its volume is sufficient to bring about the result. It must still be remembered that the permeabilities of the ova to the various solutions are not known; Sollmann, indeed, has proved the secondary swelling after the primary shrinkage of many eggs in hypertonie solutions, which must therefore enter and cause the dissociation of the cytoplasm.

Further, Delage has, as a matter of fact, denied that the increased osmotic pressure is solely responsible for the results. The French zoologist succeeded in making the ova develop in solutions hypertonie to sea-water, but found that isotonic solutions of different chlorides or mixtures of chlorides did not all give the same percentage of larvae. He holds, therefore, that other factors are involved. Other methods, as noticed elsewhere, are low temperatures and mechanical agitation.

Fischer has successfully demonstrated the phenomenon in the Chaetopods, Nereis and A7221;/zzhile, Bullot in 01)/(elm, and Bataillon in Vertebrates (lfamz. _/usca and I’eh-omyzon j/laueri); but in this last case segmentation did not continue for very long and the processes of nuclear division were highly irregular. An attempt made by Gies to incite development (of Echinoids) by means of extracts of spermatozoa was unsuccessful.

Although in brilliancy of conception and completeness of execution Loeb’s experiments are certainly pre-eminent over those of any other investigator, it should not be forgotten that about the same time Morgan had succeeded in inducing asters, and even the beginnings of segmentation, in the unfertili7.ed ova of sea-urchins and some other forms by the use of salts and other substances, and that the way for all recent work was really paved by the original labours of (). and R. Hertwig, to be described in the next section.

Loeb did not undertake an examination of the cytological changes, but Wilson has shown that ordinary nuclear division occurs with asters and centrosomes: a primary radiation centring in the nucleus first appears; this then fades away, and a definite aster with a centrosome is formed just to one side of the nucleus; this divides to form the first amphiaster (cleavage-spindle). Asters also arise independently of the nucleus in the cytoplasm (cytasters) ; these contain centrosomes, and may divide, and the cytoplasm divide round them. The part played by the cytasters in development is, however, insignificant; their activity soon comes to an end. The number of chromosomes is one-half the normal number. This latter statement is confirmed by Morgan, but denied by Delage, who asserts that, as in egg fragments enucleated and subsequently fertilized, the half number becomes doubled.


E. BATAILLON. La pression osmotique et les grands problemes de la Biologie, Ar:-71. Ent. Mach. xi, 1901.

E. BATAILLON. Etudes expérimentales sur l‘évo1ution des Am-' phibiens, Arch. Em. Mecli. xii, 1901.

C. B. DAVENPORT. The rele of water in growth, Proc. Boston Soc. Nat. Hist. xxviii, 1897-8.

C. F1’«:Iu':. Note sur l’influence de la désliydratation sur le développement de Pembryon de poulet, C’. R. Soc. Biol. (10) i, 1894.

A. GURWITSCH. Ueber die formative Wirkung des verandertcn chemischen Mediums auf die embryonale Entwicklung, Arch. Eur. Mach. iii, 1896.

O. HERTWIG. Die Entwicklung des Froscheis unter clem Einfluss sehwiieherer und stéirkerer Kochsalzlesungen, Arch. milcr. Anat. xliv, 1895.

O. HERTWIG. Die experimentelle Erzeugung thierischer Missbild— ungen, Fcstsclzr. Gegenbaur, Leipzig, 1896.

J. W. J ENKINSON. On the effect of certain solutions upon the development of the Frog's egg, Arch. Ent. Mach. xxi, 1906.

J. LOEB. Investigations in physiological morphology, Journ. Morph. vii, 1892.

J. LOEB. Ueber die relative Empfindlichkeit von Fischembryonen gegen Sauerstotfmangel und Wasserentziehung in verschiedenen Entwicklungsstadien, Pflfiger’.-c Arch. 1v, 1894.

J. LOEB. Ueber eine einfache Methode zwei oder mehr zusammengewaehsener Embryonen aus einem Ei hervorzubringen, P_/iiige;-'3 Arch. lv, 1894.


E. BATAILLON. Nouveaux essais do parthénogénése expérimentale chez les Vertébrés inférieurs, Arch. Ent. Mech. xviii, 1904.

G. BULLOT. Artificial parthenogenesis and regular segmentation in an Annelid (Ophdia), Arch. Ent. Mech. xviii, 1904.

Y. DELAGE. Etudes sur la mérogonie, Arch. Z001. E.I:p. et Gén. (3), vii, 1899.

Y. DELAGE. Etudes expérimentales sur la maturation cytoplasmique 126 EXTERNAL FACTORS III. 7

et sur la parthénogénese artifieiclle chez les Echinodermes, Arch. Zool. Exp. et Gén. (3) ix, 1901.

M. A. FISCHER. Further experiments on artificial parthenogenesis in Annelids, Amer. Journ. Phys. vii, 1902.

W. J. Gms. Do spermatozoa contain an enzyme having the power of causing the development of mature ova ? Amer. Joum. Phys. vi, 1901-2.

A. W. GREELEY. Artificial parthenogencsis in the star-fish produced by lowering the temperature, Amer. Journ. Phys. vi, 1901-2.

A. W. GREELEY. On the analogy between the effects of loss of water and lowering of temperature, Amer. Journ. Phys. vi, 1901-2.

A. W. GREELEY. On the effect of variations in the temperature upon the process of artificial parthenogenesis, Biol. Bull. iv, 1903.

R. HERTWIG. Ueber die Entwicklung des unbcfruchteten Seeigeleies, Fesischr. Geyenbaur, Leipzig, 1896.

S. J. HUNTER. On the production of artificial parthenogenesis in Arbacia by the use of sea-water concentrated by evaporation, AlII('I'. Journ. Phys. vi, 1901-2.

J. W. JENKINSON. Observations on the maturation and fertilization of the egg of the Axolotl, Quart. Jam-n. Min-. Sci. xlviii, 1904.

K. KOSTANECKI. Ueber die Veriinderungen im Inneren des unter dem Einfluss von K01-Gemischen kfinstlich-parthenogenetisch sich entwickelnden Eis von Mactra, Bull. Inlrrn. Acad. Sci. Cracovie, 1904-5.

J. LOEB. On the nature of the process of fertilization and the artificial production of normal larvae (Plutei) from the unfertilized eggs of the sea-urchin (two papers), Amer. Journ. Phys. iii, 1899-1900.

J. LOEB. Further experiments on artificial parthenogem-sis, and the nature of the process of fertilization, Ame)‘. Journ. Phys. iv, 1900-1.

J. LOEB. Experiments on artificial parthenogenesis in Annelids (Chaetopterus), Amer. Journ. Phys. iv, 1900-1.

A. P. MATHEWS. Artificial parthenogenesis produced by mechanical agitation, Amer. Journ. Phys. vi, 1901-2.

T. H. MORGAN. The fertilization of non-nucleated fragments of Echinoderm eggs, Arch. Ent. Mech. ii, 1895-6.

'1‘. H. MORGAN. The production of artificial astrospheres, Arch. Em. Mech. iii, 1896.

T. II. MORGAN. The action of salt solutions on the unfertilized and fertilized eggs of Arbaciu, Arch. Ent. Mach. viii, 1899.

T. H. MORGAN. Further studies in the action of salt solutions and other agents on the eggs of Arbacia, Arch. Em‘. Me('h. x, 1900.

E. B. WILSON. A cytological study of artificial parthenogcncsis in sea-urchin eggs, Arch. Em. Mech. xii, 1901.

8. The Chemical Composition of the Medium

By means of solutions of alkaloids and other substances the brothers Hertwig have been able to incite very remarkable cytological changes in the eggs of sea.-urchins (Stronyylocemfrotus).

The effects of nicotine are perhaps the most striking (Fig. 68, a-e). Various solutions—-1 % and less of a concentrated extract——were allowed to act upon the egg for difierent lengths of time (five to fifty minutes) before fertilization; the ova. were then replaced in sea-water and fertilized. The cytoplasm is so paralysed by the poison that the normal vitelline membrane cannot be formed and consequently many spermatozoa enter. In such eggs segmentation does not occur in the ordinary fashion by successive binary divisions, but many small cells are simultaneously formed. The resulting blastulae are abnormal, the segmentation cavity being filled with a solid granular mass (Stereoblastulac), and very few reach the Pluteus stage. The irregularities of segmentation are due to the complex mitotic figures and divisions which polyspermy entails. One, two, three or more of the spermatozoa fuse with the female pronucleus ; each has its own aster, which divides into two. Hence the most complex nuclear figures are formed.

FIG. 68.~ The effect of alkaloids and other poisons on the processes of fertilization and nuclear division in the egg of the sea—urchin, Sh'ong_:/Iocentrotus lividus. (After R. and 0. Hcrtwig, 1887.)

a. The egg was exposed to nicotine (one drop in 200 c.c. of seawater) for ten minutes, and then fertilized; drawn fifteen minutes later.

b, c. The same for fifteen minutes; drawn after one and a half hours.

d. The same for ten minutes; drawn after three hours, ten minutes.

2. The same; drawn after three hours. Only part of the complex figure is shown; the remainder lies in another plane.

f, g, h. Exposed to a 0-05% solution of quinine for twenty minutes one and a half hours after fertilization; drawn from one to two hours later.

is. 1-5, male pronucleus, 6, female pronucleus. Exposed to chloral (05%) one minute after fertilization; fixed after 150 minutes.

I, m. Chloral 06% one minute after fertilization; fixed after six hours. Male and female pronuclei reconstructed and metamorphosing, in m the ‘fan ’ form with commencing division.

11,0. Placed in chloral 0-5% five minutes after fertilization ; preserved after ninety minutes.

n. Female pronucleus (four-rayed rosette), and male pronucleus .

(three-rayed rosette). 0. Fusion of pronuclei.

p. The same. Female pronucleus in the pseudo-tetraster forms.

In the case where two sperm-nuclei unite with the eggnucleus a tetraster is formed, that is four asters united by spindles in a. square or rhombus, or a triaster with an odd aster united to one angle of the system. The chromosomes are grouped in the equators of the four, or three, united spindles, as the case may be, and the egg divides simultaneously into four, or three.

The arrangement becomes still more involved when there are other sperms, whether these fuse with the female pronuclcus or not. Each amphiaster is united by one pole to the tri-, tetra-, or polyaster developed round the combination nucleus, or to the poles of other amphiasters; in one case there were nineteen spindles in all, not, of course, all in one plane. Each centrosphere receives half the chromosomes of the spindle attached to it, and each cell, when division occurs, contains one or more nuclei.

Hydrochlorate of morphine will produce similar effects, but only with longer exposures—a 0-4 % solution for from two to five hours. Strychnine, however, is poisonous in very weak doses (-005 % to -25 ‘Z), and quite short exposures are sufficient to call forth marked results. Other solutions successfully tried were chloral hydrate (from 0-2 % to 0-5 % for from one to four and a half hours), cocaine (from 0-025 Z to 1 ‘Z for five minutes), and sulphate of quinine (-05 % for ten minutes). In quinine (-05 % for thirty minutes) and chloral (o5 %) the entrance cone was small and no asters were formed, from which the

Hertwigs argue that the contractility of the cytoplasm is impaired in these solutions. Chloroform dissolved in sea-water has the very interesting property of stimulating—without the addition of spermatozoa—the formation and separation of the vitelline membrane. The male generative cells are also sensitive to the action of these alkaloids, but not necessarily in the same measure. They can resist, for example, the influence of a solution of nicotine, which is ten times as strong as one necessary to evoke pathological changes in the ova. Though chloral hydrate (0-5 %) and quinine (0-05 %) are both temporarily fatal to the motility of the spermatozoa, sea-water restores the capacity for fertilization. Strychnine (0-O1 %) and morphine (0-5 %) are without elfect.

In the experiments just described the abnormalities seem to be directly due to the initial paralysis of the egg by the reagent and consequent polyspermy.

Should, however, the egg have been first normally fertilized, the irregularities produced by the subsequent action of the poison are, though well marked, not of the same kind, for in this case the vitelline membrane has already been formed and only one spermatozoon has gained admittance. Chloral hydrate (Fig. 68, /:—p) was employed for ten minutes and at varying intervals after insemination (one, one and a half, five and fifteen minutes). Exposure to the solution very shortly after insemination first retards the progress of the sperm-head and the formation of its aster, and when later on the chromosomes are formed they lie heaped together in the centre of an achromatic figure described as a pseudo-tri- or pseudo-tetraster. This consists of three or four conical groups of fibres, the bases resting on, and the fibres connected to, the chromosomes, the apices outwardly directed and sometimes with, sometimes without, asters; in any case, however, they are not united by spindles, as is the ease in the complex figures observed in polyspermy. Isolated asters are also to be seen in the cytoplasm, and, which is perhaps more remarkable, the female chromosomes are themselves the centre of a unipolar (fan-shaped) or multipolar apparatus of the same kind. The reader will not fail to notice the similarity to the phenomena occurring in artificial parthenogenesis.

Should the pronuelei unite—-which is only possible before these pseudasters have been developed, if the eggs have been subjected to the action of the poison immediately (one minute) after fertilization——the conjugation nucleus itself becomes the focus of a similar system. In eggs poisoned after a longer interval (fifteen minutes) the male and female pseudasters may them selves unite. The nucleus—or nuclei—divide irregularly, the chromosomes

passing in unequal numbers to the poles of the figure. The several pseudasters and isolated asters, with which nuclei may possibly become secondarily associated, may be united by clear streaks of protoplasm, thus giving rise to a dendritie figure. Simultaneous and unequal division of the whole ovum follows.

Should the spermaster have already been developed-fifteen minutes after insemination—it degcnerates. The subsequent changes comprise the formation of multipolar figures and irregular cell-division.

In later stages—when fertilization has been completed and segmentation is about to beg-in—the ova are almost or quite indiflerent to nicotine, strychnine, and morphine ; but chloral (0-5 %) destroys the asters which are already in existence and brings about a reconstitution of the combination nucleus with subsequent formation of a tetraster and quadruple division. In future mitoses, however, the spindles are bipolar. Cocaine and quinine (-05 %) (Fig. 68,./'—/1) have the same effect.

The importance of these experiments does not require to be emphasized. Not only do they throw a valuable light on the possible causes of those pathological mitoses that occur in malignant growths, they also contribute very greatly to the understanding of the normal processes of fertilization and karyokinesis.

Thus from the failure of the asters to appear in eggs treated with chloral before fertilization the brothers Hertwig argue that the contraetility of the cytoplasm is diminished by this substance, and from the failure of the pronuelei to unite in eggs which have been immersed in the solution shortly after fertilization they suggest that it is the contractility of the ovum which normally brings about the union of the pronuelei. Since, however, both male and female nuclei are able to divide, this division must be normally incited, not by their union with one

«another, but by the separate action of the cytoplasm on each, a view which is fully borne out by the phenomena. of artificial parthenogenesis and merogony (the development of fertilized enueleate egg fragments), whatever interpretation may eventually be put on the ‘ contractility ’ of the cytoplasm.‘

Another alkaloid which exerts an injurious influence on the ova of Echinoderms is atropine, the sulphate of which retards and dwarfs the development of /lxlericts and /lrbacia (Mathews). Pilocarpine, on the contrary, has an accelerating efiect, a result attributed by Mathews to its activity as an oxidizer, while atropine is regarded as a reducing agent, the property to which Loeb has also assigned the value of potassium cyanide in prolonging the life of unfertilized ova. The eggs of sea-urchins, when once laid, are only capable of fertilization and development within a certain definite limit of time, after the expiration of which they degenerate and die ,- after twenty-four hours, for example, they are only able, when fertilized, to reach the gastrula stage, and after thirty-two hours even fertilization is hardly possible. By treatment with an appropriate solution of potassium cyanide this limit may be considerably postponed. In the most successful series of experiments the ova were first

placed in a solution of KCN 7;?) in sea-water, and then

removed successively every twenty-four hours to

7!: 7! 2500’ 3000' lengths of time, then removed to pure sea-water and fertilized. As the table shows (Table XVII), segmentation was still possible after 168 hours’ sojourn in the solution, but the greatest number of Plutei was obtained after only 66 hours’ stay.

It was also shown that better results could be obtained with artificial parthenogenesis if the ova were first kept in the cyanide solution. Loeb points out that in the higher animals

74 7! 1400’ 2000’ In the last solution they were kept for various

‘ Strictly speaking, only the division of the male chromosomes can be regarded as being stimulated by the egg cytoplasm. What exactly it is which excites the female nucleus to divide is not at all clear.

the efiects of this substance are due to its inhibition of oxidation; that this is the real cause of the prolongation of the life of the eggs is shown by the fact that when kept in‘ an atmosphere of hydrogen for thirty-eight hours they were still capable of being fertilized and developing intoswimming larvae.


Showing the effect of exposures of various length of Sea-urchin

eggs to a solution of KCN§(%—0- (After Loeb.) Length of exposure in hours. Result.

66 80 7; Plutei, vitelline membrane formed 90 30 ‘Z, Plutei, no vitelline membrane formed 99%‘ 20 Z Plutei) n 91 n n

112 Less than 20 Z Plutei

I20 Gastrulae, but no Plutei

139 A few blastulae

140 Blastulae, not swimming

Eight-celled stage only

Simultaneous lowering of the temperature to the freezing-point enhanced the value of the cyanide treatment.

In later stages, however, immediately after fertilization and subsequently, the action of potassium cyanide is by no means beneficial ,- at this time, as we know, oxygen is a necessity (see above, p. 112); and Lyon has shown that the moment at which the ova are particularly sensitive to both KCN and the lack of oxygen is the same, about fifteen minutes after insemination.

Chemical agents are also able to incite irregularities of growth and abnormalities in later stages of development.

In a long series of experiments Féré has shown that monstrosities can be produced by exposing the Hen’s egg to the unfavourable influence of a large variety of substances. Vapours of ether, alcohol, essential oils, nicotine, mercury, and phosphorus, injections of alkaloids such as morphine, nicotine, strychnine, and others, of bacterial toxines (those of tubercle, diphtheria), of peptones, dextrose, glycerine, several alcohols, certain salts (KBr, III. 8 CHEMICAL COMPOSITION 133

KI, S1-Brz), are all baneful, retarding and distorting the embryo to a. greater or less extent. Ammonia, it may be noted, is fatal at once.

It has already been shown (p. 123) that the malformations induced by sodium chloride in Amphibian embryos are to be set down to some other property than the osmotic pressure of the solution, and it is here only necessary to advert to

Fm. (S9.-—Gane-sugar‘ (6-6 2,). Two stages in the forma.tion of the notochord from the whole thickness of the roof of the archcnteron in the Frog.

Dextrose (3-4 7,). Secondary degeneration of the gut roof and ventral part of notochord.

some of the more interesting effects occurring in particular solutions.

Although the more poisonous salts (e.g. LiI, CaCl2, SrBr2, and others) inhibit altogether the formation of the blastoporic fold, a cause which normally assists in its production—the proliferation of small cells in the roof of the segmentation cavity--may continue to operate, with the result that that mof is thickened and thrown into puckers and folds. 134 EXTERNAL FACTORS III. 8

Again, the notochord may be formed from the whole thickness of the arehenteric roof (cane-sugar)recalling the mode of its development in Urodela and Petromyzon (Fig. 69); the solid medullary tube observed in potassium chloride and other salts reminds one of the rudiment of the nervous system in Teleostei and others, while the mode of closure of the medullary tube in, for example, some of the magnesium salts resembles that observed in Am];/ziomas ; the formation of notochordal tissue from the wall of the neural tube and the roof of the arehenteron (Fig. 70) in strong solutions of urea (1-17' % to 1-56 %) shows that the prospective potentialities of these organs are not yet fixed, while the development of an optic cup without a lens in urea, sodium chloride, and sodium bromide demonstrates that the formation of the former is independent of that of the latter of these two parts of the eye.

FIG. 70. - Fornmtion of vaeuolated notochordal tissue in the medullary tube of the Frog embryo under the influence of urea (1-6%). Underneath the notochord is the subnotochordal rod.

The grey degeneration of the exposed part of the medullary plate (due to the distribution of the pigment throughout the cell—body), the protrusion of cells (‘framboisia’ of Roux), and disintegration of the epithelium which is so characteristic in III. 8 CHEMICAL COMPOSITION 135

many of these solutions (cane-sugar, NaCl, LiCl, MgCl2, MgSO,), have been noticed by many observers (Roux, Hertwig, Morgan, Bataillon). All the more violent solutions attack the yolk-granules. In some cases the effect produced appears to be specific; thus in lithium salts the ectoderm is often pitted and wrinkled before any degeneration appears in the nervous system, and in ammonia salts, which are highly poisonous, the nuclei are much enlarged, lobed, highly chromatic, and homogeneous. The very similar appearance of the nuclei (Fig. 64) in those stronger solutions of urea which arrest development in an early stage suggests that ‘the ammonia set free is the toxic agent in this case. In solution isotonic with -625 % NaCl urea permits of normal development up to a certain point, when the embryos die.

In this connexion it is interesting to notice that Moore has found that sodium sulphate will act as an antidote to the poisonous effect of sodium chloride on tadpoles. Thus the average length of life of tadpoles in a 3 NaCl solution was four and a quarter days, but was prolonged to twenty-one days by adding from 4% to 8 X of Na2SO4. The poisonousness of sodium chloride, sodium nitrate, calcium nitrate, and magnesium chloride to Fmululus embryos and the value of other salts as antidotes has been shown by Loeb, while Lillie has noted that sodium is fatal but magnesium and calcium beneficial to the ciliary movement of /lrem.'c0/a larvae, a result first obtained by Loeb for the Plutei of Ea/u'7m3; the muscular contractions of the larva, on the other hand, are inhibited wholly by magnesium, partly by calcium, while sodium is necessary for their continuanee. In an artificial solution which combines the three elements in the proper proportions normal development is possible. The nature of the part played by the ions——-whether toxic or antitoxic— is, however, a very open question.

Arguing from the fact that the evil eifects of such salts as sodium chloride and nitrate may be counteracted by calcium and magnesium salts, Loeb has suggested that toxicity and antitoxicity are functions of valency, and also of electrical charge, since it is further stated that toxicity increases with the valency of the anion, antitoxicity with that of the cation. Ions of the same valency are not, however, necessarily equally antitoxic (Loeb and Gies, Lillie, Mathews), and sodium sulphate, as we have seen, may act as an antidote to the chloride (Moore). Mathews has accordingly sought for the cause of toxicity in another physical property, the decomposition tension of the salt, and has certainly succeeded in showing that the poisononsness of solutions to the eggs of Fzmrlulzw varies inversely with the decomposition tension, and that a similar relation holds good in certain other cases.

Lillie argues that a physiologically balanced solution is necessary, one in which the electrolytes are in a state of chemical equilibrium with the necessary ion-proteid compounds in the tissues. Solutions which only contain some of these substances, or solutions (for example, non-electrolytes) which contain none, are poisonous, because they permit of the outward diffusion of the needful ions.

It must be pointed out, however, that this explanation will not fit the cases where the embryo develops perfectly well in fresh water (lllzmtlulus) or in distilled water (the Frog), and that some other reason must be found for the poisonous effect of cane-sugar upon the latter. The whole question, however, is one which belongs more properly to the province of pharmacology.

Poisonous although these salts are, the embryo can still be acclimatized to them. C. B. Wilson placed the unsegmented eggs of Amlalyertoma, Rana, and 0/102-op/zilzcs in a 0-05 % solution of sodium chloride; after twenty-four hours they were removed to 0-1 %, and then successively to stronger solutions by increments of 0-1% until 10% was reached, a concentration which quickly causes death under ordinary circumstances. In this case, however, development was normal, and the larvae hatched out and lived for some time.

The distortions of development which solutions of salts and other substances call forth in Amphibian embryos find a parallel in the malformations which Herbst has produced in Eehinoderm larvae (I976/dmw, Sp/zaerec/ainus) by similar means; as in the former case, the results were at first assigned to the increased osmotic pressure of the media.

When potassium salts are added to the sea-wa.ter—for example, a 7 % solution in sea-water of a 3-7 % Solution of K01 in tap-water——the egg gives rise to a Pluteus in which, though the gut is, as normally, tripartite, the skeleton is rudimentary and the arms suppressed (Fig. 71). Herbst suggests that the suppression of the arms is" due to the absence of a. stimulus normally exerted by the skeletal spicules. These abnormal forms may fuse together to form double monsters.

Such ‘ potassium ’ larvae are developed in sodium salts, but lithium has a more pronounced elfect (Figs. 72, 73). In this case

FIG. 71. - Potassium larvae of Echinoids. a. Potassium larva of Sphaerechinus (1860 c.c. sea-wate1'+ 140 c.c. 3-7% KNO3). There is no skeleton. The rut is tripartite, and the mouth surrounded by the ciliated ring. I), c. otassium larvae of .E(‘h’i1‘l'llS (20% of 3% K01). Note the buttonshaped apical tuft of cilia, and, in c, the secondarily evaginated archenteron. (After Herbst, 1893.)

the blastula becomes constricted into two portions, a thin-walled gastrula wall provided with long cilia, and a thick-walled archenteron, which may be muscular and mobile, and is thickly covered with short cilia. The arehenteron has, in fact, failed to invaginate, and the larva is an ‘ Exogastrula ’. Occasionally there is an attempt at invagination at the end of the archenteric portion, and, after temporary exposure, the invaginated part may be divided into three, and a mouth formed. All the parts of the gut, however, remain in the same straight line. A middle section may be formed by further constriction of the archenteron (Ea/aimzs) or of the gastrula wall (Sp/zaerec/liuus). Double monsters sometimes arise by fusion of these larvae by their archentera.

A skeleton is not usually developed; if present it is abnormalin position, the spicules being placed near the animal pole and

FIG. 72.———Lithium larvae of Splmerechin us _qrmmlw'is. a. Larva. partially constricted into gastrula wall and archentcric portions, the former with lon , the latter with short cilia. (980 c.c. sea-wa.ter+2O c.c. 3-7% LiCl). b. imilar larva. to the last, but a neck or connecting piece has been formed from the ectodermal portion. 0, (1. Progressive diminution of the ectodermal gastrula. wall portion with increase in the quantity of Li.

the arms of the Pluteus formed under their influence near the mouth instead of by the side of the anus, in the number of the spicules, and consequently the number of arms (three, four, or

five, instead of two), and in the number of their radii (four or five, instead of three).

The gastrula wall is often smaller than the archenteron, and, as the strength of the solution is increased, becomes still further reduced, until nothing of it is left but a small button at the

F10. 73.—~a. Larva with three skeletal spicules, and a. ‘ cell-rosette ’ at the end of the archenteron. Ir. Larva with skeleton—-—more than three spicu1es—and arms developed. The neck is invaginated into the ectodermal portion, the gut tripartite. c. Five-armed Plutcus with five skeletal rods. The gut is normally invaginatecl and tripartite. d. Larva. of Echirms microtuberculatus. There is a neck, and the gut is partly invaginated. In the blastocoel are aggregations of mesenchyme and pigment cells. (After Herbst, 1895.)

animal pole, which only indicates its real character by the longcilia which it carries. Such larvae Herbst terms ‘Holoentoblastia’. This nearly complete suppression of the ectodermal region can, however, only be realized when the salt is allowed to act at a stage in the blastula when the difierentiation into the two primary layers is already beginning. Should the embryos be removed before this stage is reached, after twentyfour hours’ exposure to the solution, only ‘Exogastrulae’, not ‘ Holoentoblastia,’ can be obtained. Should, on the other hand, older blastulae, or gastrulae, or Plutei be placed in the solutions, they die without showing any signs of the characteristic abnormal development. From the fact that equimolecular solutions of monobasie lithium salts produced like effects (such solutions, it must be observed, are also chemically equivalent), Herbst concluded at first that the osmotic pressure was responsible for the abnormalities; but the permanent after-effects of temporary immersion just referred to subsequently convinced him that the ova were permeable to the lithium ions to which he now attributes the specific nature of the monstrosity. He suggests further that they act upon the endoderm cells by increasing their absorptive activity and their power of cell-division, while at the same time they inhibit the functions of those mesenchyme cells which are devoted to the formation of the skeleton.

As in other monstrosities, there is an alteration in the prospective potentialities of cells, elements which would normally be ectodermal becoming converted into endoderm, and additional mesenchyme cells being involved in the secretion of skeletal spicules.

It is only by lithium salts that the typical ‘ Holoentoblastia’ can be, produced ; but Exogastrulac can be reared in others, in sodium butyrate, for example; in this solution a stomodaeum is formed, but is, like the arehenteron, everted. Even lithium, however, is powerless to cause the ‘ holoentoblastic ’ reduction of the gastrula wall in the larvae of Aaterias, although exogastrulation "may, but need not, occur. A characteristic deformity is the absence of the pre-oral region, and the elevation of the mouth on a sort of hypostome. In Amp/ti0.'D7(8 and Ascidians it is impossible to obtain even exogastrulae by these methods. It is evident, therefore, that the specific morphological reaction depends not only on the nature of the substance employed, but also on the constitution of the reacting organism.

Herbst has not omitted to point out the significance of theseand indeed of a1l—monstrosities for the theory of the origin of those larger, discontinuous variations known as ‘sports’, or, in more modern phraseology, ‘mutations’; and Vernon has been able to show statistically that the degree of continuous variation may also be altered by changes in the chemical environment.

In all the foregoing experiments the effect is observed of the addition of some chemical substance to the medium in which the embryo is placed. We have now to consider a very remarkable series of investigations, for whose planning and execution we are indebted to the genius of Curt Herbst, investigations in which substances which are present in the normal environment of the larva are omitted, and an insight thus gained into the part they play, if any, in the normal development of the organism. Herbst has indeed succeeded in demonstrating in the most conclusive manner the necessity to the sea-urchin egg for the normal performance of this or that phase of developmental function of a large number of the elements present in sea-water.

The sea-water at Naples, where Herbst carried out his work, has the following composition:~

N aCl . . . . . 3 %

KCI . . . . . - 7 %

MgCl2 . . . . . ~32 %

Mg-S04 . . . . . -26 %

CaSO4 . . . . . "-1 % CaHl.’O4



Fe2CO Si



It may be said at once that silicon, bromine, and iodine are unnecessary, and that, though earlier experiments led Herbst to believe that phosphorus and iron were essential, he has since assured himself that phosphorus is certainly, and iron probably,

in small quantities.

not. All the other elements, however, can only be omitted under penalty of retardation, abnormality, or death (Figs. 74 A and B).

The method employed was a simple one. A series of artificial sea-waters was made up, from which, one by one, each of‘ the elements was omitted, another being substituted in its place. Care was taken to make these artificial media approximately

FIG. 74.— The necessity of substances contained in sea-water for the normal development of the larvae of sea-urchins.

a. Without OH. Ciliated stereoblastula of Sphaerechinus. b. KOH has been added. c. Normal blastula of Sphaeiw-hinus. d. Blastula in

a K-free medium. e. Reared in K-free and replaced in sea-water (Sphm=r- '

echinus). f. Larva from a medium devoid of Mg (Sphaerec-himls). g. Echinus Pluteus with tripartite gut, mouth and coelom sacs, but neither skeleton nor arms ; reared without CaC0, or CaSO,. h. Normal Pluteus of Echinus.

isotonic with sea-water, and so exclude a possibly disturbing factor, the alteration of the osmotic pressure. The réle of each of these necessary e1ements—or ions—will be considered separately and in some detail. Sp/2ae7'ec/Iinua and E0/timcx were the forms principally employed.

This is ordinarily provided by Mg-S04 and CaSO4 ; when the fertilized ova are placed in a solution in which MgCl2 is substituted for it (as, for example, in 3 % NaCl+-07% KCI + 5 % MgCl2 + CaHPO4 + CaCO3) then their development is retarded from the blastula stage onwards, the embryos are small

and degenerate without reaching the Pluteus stage (Fig. 74- B). The gut is straight instead of bent, and not divided into the

FIG. 74 B.

a. Normal position of skeletal spicules in Sphaerechimls. b, (1. Abnormal position and number after treatment with S0,-free medium. c. Larva of Echiuu.s- from a S-free solution. e. Pluteus of Sphaerechinus with three fenestrated skeletal arms, instead of two. ’l‘rea.ted with a S0,-free medium and replaced in sea-water. f. Normal Pluteus of Sphaerechinus. (After Herbst, 1897 and 1904.)

usual three parts ; in S11/zaerec/u'm¢s no mouth is formed, the gut is evaginated (Exogastrula). The endoderm is very thick, the cells dark and dense.

The sulphuric acid radiele (sulph-ion) is thus necessary for the proper development of the gut, and necessary from the very beginning, for in embryos which have been kept in S0,,-free water up to the mesenehyme-blastula stage and then replaced in sea-water the alimentary tract is still abnormal.

Deprived of S04, in fact, the gut remains radially symmetrical, and the same must be said of the skeleton. Normally there are two tri-radiate spicules, one to the right, the other to the left of the gut and some little way from it. Without the needful sulphate the spicules become placed near the gut, and may with the growth of the latter be pushed towards the animal pole. The number of spicules may also be diminished or increased to one, three, or four, arranged in a circle round the gut. On timely removal to sea-water, however, a secondary bilateral symmetry may arise by two of these outgrowing the rest and stimulating the development of the typical arms of the Pluteus. It seems that a sulphate is present in the calcareous skeleton of the Pluteus, as there is in that of the adult urchin.

A ciliated circum- oral ring is formed, but is abnormal in its position, at right angles instead of parallel to the long axis of the body. The pigment which should be secreted by the secondary mesenchyme cells (separated off from the inner end of the archenteron) remains in abeyance, and the apical tuft of cilia is hypertrophied. Other processes, however—fertilization, segmentation, and ciliary motion——arc independent of S0,.

The development of eggs which are allowed to remain in ordinary sea-water until the blastula stage is no better, whence Herbst concludes that no S04 is taken up during segmentation. During the early stages of gastrulation, however, they appear to absorb a store of it for future needs, for gastrulae reared in sea-water develop further in the SO,-free solution than do those embryos which have been kept in it since fertilization. S04 is equally necessary for the continued life of the Pluteus and of the Bipinnaria larva of Asterias, and without it the rate of regeneration of the head of Tubularia is retarded and the number of tentacles reduced, until eventually a completely tentaeleless head is evolved.

The necessary sulphate can be, to a certain extent, replaced by a thio-sulphate. The addition, for instance, of -35% Na2S2O,, to the SO,-free solution renders it possible for the larvae to reach the Pluteus stage, though the arms are short, the skeleton small, and the pigment reduced. The larvae die.

Selenium and tellurium are both poisonous in an early stage.

A solution was made up in which the sodium chloride was replaced by sodium formate, the magnesium chloride by magnesium sulphate, the potassium chloride by potassium sulphate ; thus, NaCOOH 3- 5 % + MgSO4 -26 % + MgSO4 -4- % + KZSO4 -12 Z + CaSO4 -I Z + CaHPO4+ CaCO3.

The eggs did not segment, and even when KCl and MgCl2 were used in their ordinary proportions, segmentation did not progress very far. Nor did the substitution of Na2SO4 for NaCOOH give any better results. A considerable amount of chlorine appears therefore to be absolutely necessary for the earliest developmental processes, its function being, Herbst suggests, to transport certain necessary cations, the tissues being possibly more permeable to NaCl than to Na2SO4. Later stages ——blastulae, gastrulae, Plutci—all die in the Cl—free mixture.

Chlorine can be replaced in some measure by bromine. Plutei are formed, though with a distorted skeleton, Tubularia regenerates its head and the eggs of the fish Labrax develop as well as in sea-water. Iodine is, however, poisonous; so also are chloratcs.

iii. Na.

2-96 % of MgCl2 was added to a solution containing the usual amounts of KCl, MgSO4, CaSO4, CaHPO4, and CaCO3. In this the ova indeed segmented, but abnormally, the blastomeres being of unequal size. Death followed; nor was the addition of a certain small amount (-84: %) of NaCl suflicient to save them, though segmentation was normal and traces of an archenteron could be detected; with more NaCl (1-34» %) the gastrula stage was reached. The sodium which is thus necessary in the earliest period is also required later on; without it gastrulation is impossible to eggs which have been reared in sea-water even as far as the mesenchyme—blastula stage.

The part played by sodium is not clearly understood. It is known that it counteracts the evil eifeets of calcium and is necessary for the continuance of muscular contractions. Since calcium is necessary for the cohesion of cells (see below) Herbst opines that sodium may pull them apart ;. its action in that case is capillary.

Sodium cannot possibly be replaced by lithium, potassium, rubidium or caesium, all of which would at the necessary concentrations inevitably be poisonous.

iv. K.

In the artificial solution employed the small quantity (-07 %) of potassium present in sea-water is simply omitted.

Without it scgmentation—except the first few phases-is impossible in Ea/cinzw. Sp/zaerec/limts, however, segments, but the blastocoel is reduced, the cells are opaque and not vacuolated, and the ova, though ciliated, are motionless and die (Fig. 74 A, rl, e).

Later stages are also sensitive to the want of potassium. Blastulae gastrulate, but are shrunken, with short archenteron, and in gastrulae the gut does not divide into three. Plutei, like all the others, die when deprived of it.

In the K-free medium spermatozoa temporarily lose their motility, and such spermatozoa cannot effect fertilization. The fertilization, however, of eggs which have been kept without potassium is possible ; in fact, at this earliest stage, no potassium is absorbed, for eggs fertilized in sea—water develop no further in the K-free solution than do those fertilized and kept continuously in it.

The absence of potassium i11 segmentation leaves its effect upon later stages. Two days’ exposure is not too long to prevent normal development on removal to sea-water, but five days’ exposure causes abnormalities of the skeleton (asymmetrical with several triradiate spicules round the gut) and alimentary canal (no mouth).

De Vries has shown the importance of potassium for the turgor of young plant-cells, and its function here is probably similar, to promote growth, as the subjoined table of measurements shows. Its absence also aifects the rate of development

(Table XVIII).

Potassium can in a measure be replaced by rubidium and caesium. The use of lithium either has no effect or, in larger quantities, produces lithium larvae.

Other forms to which the lack of potassium was found to be fatal were the ova of Asterias and Uotflorfiiza, and the adult III. 8 CHEMICAL COMPOSITION 147

Amp/iioxus. Potassium is also necessary for the contractions of muscles (umbrella. and tentacles of Obrlia).


Showing the effect of potassium upon the growth of Sea.-urchin blastulae. (After Herbst.)

‘Ratio (unit 2 Th mm.) of crossdiameter to long diameter.

After Without K. With K. 18 hours . 24 hours 45 hours . . .

Showing the effect of potassium on the rate of development of

Sea—urchin larvae. (After Herbst.) Artificial sea-water with

After -008 Z KCI. -016 Z KCl. .024 % KC]. 36 hours Small gastrulae. Nearly Plutei. Plutei. 60 hours No mouth ; gut not Small Plutei. Fully formed Plutei. constricted. v. Mg.

' The fertilized ova were placed in a solution which did not

include the -32 % MgCl2 and the -26 Z MgSO4 present in seawater.

Segmentation proceeds normally, but the blastula. is slightly smaller than when magnesium is present (the ratio of the diameters is §§). The skeleton, however, though bilaterally laid down, is retarded and deformed (magnesium is present in the skeleton of the sea-urchin and possibly in that of the Pluteus) and the gut is not properly diiferentiated, not tripartite and without a mouth (Fig. 74 A, f).

When the MgSO4 is replaced by an isotonic quantity of Na2SO4 the results are the same.

In the Mg-free solution cilia cease beating, development is retarded (Table XIX), and, though the spermatozoa retain their motility, the ova are so injured that fertilization is impossible unless they are restored to sea-water. The ova, in fact, seem to have a store of Mg which they lose in the Mg-free mixture. Fertilization can, however, take place without magnesium if the eggs have been kept in sea-water, and such eggs develop, in Mg-free water, to precisely the same extent as those which have been fertilized in sea-water; at this period, therefore, the egg needs no external magnesium. The original store of this element apparently suffices also for the first steps in the formation of the gut; for the lack of it is felt equally in the later stages of its difierentiation and in the first moments of its development; the alimentary canal is as abnormal in those larvae which have been kept without magnesium only up to the time when the mesonchyme and archenteric plate arise as in those which remain in the solution throughout.


Showing the retardation of development of Sea-urchin larvae deprived of magnesium. (After Herbst.) Two solutions were employed, mixed in various proportions; one (1)

has no Mg; the other (2) contains Mg. Solution. Result after 24 hours.

1 Nearly motionless; archentcron formed. 1+ 10 Z of 2 Larger. 1 +20 Z of 2 Larger still. 1 + 30 Z of 2 Larger; gut constricted ; mouth i'or1ned.

1 % of 2 )1 n n n 7) 1  % of 2 H n H :9 n 2 H H n 7) n

It is, in fact, only after the gastrula stage that magnesium is absorbed. Eggs, blastulae and young gastrulae, reared in seawater develop far worse when placed in the Mg—free liquid than do gastrulae.

The formation of pigment and the contractility of muscles remain unaifected by the absence of Mg.

In Mg-free media tl1e blastomeres of .1.s-/erizw fall apart and the adult medusae of Obeliu degenerate.

vi. Ca.

The calcium salts present in normal sea-water are C-aCO_.,, Ca2SO4, CaHPO4, and Ca3(PO,)2.

When the carbonate only is absent the blastulae are crumpled and opaque; a few gastrul-ate but are markedly abnormal, the ciliated ring being crumpled, the gut flattened on the oral side and the skeleton absent (Fig. 74 A, g). Should the skeleton have already been formed when the larvae are exposed it becomes dissolved. When the sulphate only is omitted (magnesium sulphate being present) development is still inferior to the normal, inferior even to development in the presence of CaSO, but in the absence of MgSO4 ; CaSO4 may be replaced by CaCl2. It seems, therefore, that the sulphate is necessary as a calcium salt.

FIG. 75.——a—r. Separation of the blastomeres of Echimts microtubermr Iulus in a medium containing NaCl, 3-07%, KCI, 0-08%, MgSo,, 0-66%, MgHPO, and FeCO3, but no Ca. Note the radially striate border, which is the altered uniting membrane. (1. lilastuln disintegrating in the same medium. (After Herbst, 1900.)

If only the phosphate (Ca3(PO4)2) is present the egg dies during segmentation, though, if the other phosphorus compound is substituted for it, the effect is the same as when the carbonate alone is omitted.

Should, however, all calcium salts be removed the result is

more serious still (Fig. 75). The blastomeres are unable to 150 EXTERNAL FACTORS III. 8

cohere, and separate as fast as division takes place, swimming about independently for a time, and then dying. The same phenomenon is witnessed when later stages are placed in such a. calcium-free mixture.

On removal to sea-water division continues without separation, and should the egg membrane still be intact all the cells unite and a whole larva is formed. Even should the egg membrane be lost, a reunion of the cells is always possible so long as they remain in contact with one another. The separation is due to a. change in the surface-tension of the cells; a visible change takes place, in fact, in the superficial layer which covers and unites the blastomeres ; it becomes ill-defined and radially striated.

The lack of calcium also aifects the rate of development, and causes shrinkage, but leaves karyokinesis, ciliary motion, and pigment formation unaltered.

Calcium is not replaceable by magnesium, strontium, or barium.

vii. CO3.

As has just been pointed out, calcium carbonate is necessary for the due formation of the skeleton, although a beginning may be made without it.‘

Whether the crumpling of the larva, due to diminution of internal osmotic pressure, which is observed in the absence of calcium carbonate is attributable to the lack of CO3 or the lack of the hydroxyl ion is difiicult to determine, since, as Herbst points out, a carbonate necessarily introduces OH, while the latter can convert into carbonates the CO2 of the atmosphere and of respiration.

The alkalinity of the sea-water——reckoned by the number of free hydroxyl ions—is provided by the calcium carbonate and calcium hydrogenphosphate. By the omission of these a solution —-neutral to litmus—may be obtained in which the ova give rise to thick-walled, opaque blastulae with granular contents, ciliated but motionless, and doomed to eventual degeneration and death (Fig. 74 A, a, 6). Very occasionally a gastrula with a short gut is formed.

1 CaCO,, is necessary for the formation of the skeleton of the larva of the sponge Sgcandra selosa (0. Maas, S.-B. Ges. Mozph. Phys. Miinchen, xx, 1905). In a medium devoid of all calcium salts the Amphiblastulae fall to pieces.

When the blastulae are immersed in the solution they give rise to small, opaque gastrulae.

A certain degree of alkalinity is necessary for fertilization. The spermatozoa are less sensitive to a want of alkalinity, more sensitive to excessive alkalinity than the ova.

By the addition of a small amount of sodium hydrate to the neutral medium development is accelerated, but an increase of the alkalinity of ordinary sea-water is unfavourable. Loeb, on the other hand, has found that the addition of from -006 % to -008 % sodium hydrate to sea-water accelerates the development of Aréacia.

The formation of pigment and the vibration of the cilia are other processes which depend on the presence of the hydroxyl ion. Plutei die without it and their skeleton is dissolved.

The function of the ion does not appear to be to neutralize any acids produced by the tissues, for these give a neutral reaction even in OH-free media.

Since aeration improves the development of eggs in these media, and the more so if the air is deprived of its carbon dioxide, Herbst has concluded that one function of the OH ion is to neutralize the CO2 and allow of the formation of the necessary carbonates. Another function is possibly, as Loeb suggested, the acceleration of processes of oxidation.

The experiments which we have been considering are unique of their kind, and it is impossible to exaggerate their importance. For, whatever may be the ultimate explanation of the facts, there can be no doubt whatever that the most complete demonstration has been given of the absolute necessity of many of the elements occurring in ordinary sea-water, its normal environment, for the proper growth and differentiation of the larva of the sea-urchin. Nor is this all. Some of the substances are necessary for one part or phase of development, some for another, some from the very beginning, others only later on. Thus potassium, magnesium, and a certain degree of alkalinity are essential for fertilization, chlorine and sodium for segmentation, calcium for the adequate cohesion of the blastomeres, potassium, calcium and the hydroxyl ion for securing; the internal osmotic pressure necessary for growth, while without the sulph-ion and magnesium the due differentiation of the alimentary tract and the proper formation of the skeleton cannot occur; the secretion of pigment depends on the presence of some sulphate and alkalinity, the skeleton requires calcium carbonate, cilia will only beat in an alkaline medium containing potassium and magnesium, and muscles will only contract when potassium and calcium are there.

The part played by each substance is therefore specific; for some particular part of the morphogenetie process it is indispensable. Not, of course, independently of internal factors, but in co-operation with them, it does, in fact, determine the production of organic form; and the relation between the embryo and the environment in which it develops is in this case, at any rate, of the closest and most intimate kind.


C. B. DAVENPORT and H. V. NEAL. Studies in morphogenesis. V. On the aeclimatization of organims to poisonous chemical substances, Arch. Ent. Meek. ii, 1896.

C. Fr':R£:. A series of papers on the effect of various chemical bodies on the development of the Chick, C‘. R. Soc. Biol. (9) v, 1893—1iii, 1901.

C. W. GREENE. On the relation of the inorganic salts of blood to the automatic activity of a strip of ventricular muscle, Amer. Jom-n. 1’h_:/s. ii, 1898-9.

C. HERBST. Experimentelle Untersuchungen fiber den Einfluss der veranderten chemischen Zusammensetzung des umgebenden Mediums auf die Entwickelung der Tiere: I. Versuche an Seeigcleiern, Zeitsrlz. wise. Zool. lv, 1893.

C. HERBST. Experimentelle Untersuehungen fiber den Einfluss der veriinderten chemischen Zusammensetzung des umgebenden Mediums auf die Entwickelung der ’l‘iere: II. Weitcrns fiber die morphologischo Wirkung der Lithiumsalzc und ihre thcorctischc Bcdcutung, Miil. Sim‘. Zool. Neapel, xi, 1895.

C. HERBST. Experimentelle Untersuchungen iiber den Einfluss der veranderten chemischen Zusammensetzung des umgebenden Mediums auf die Entwickelung der Ticre: III-VI, Arch. Ent. Meek. ii, 1896.

C. HERBST. Ueber die zur Entwickelung der Seeigellarven nothwendigen anorganichen Stoffe, ihre Rolle und ihre Vertretbarkeit: I. Die zur Entwicklung nothwendigen anorganischen Stoffe, Arch. Ent. Mach. v, 1897. III. 8 CHEMICAL COMPOSITION 153

C. HERBST. Ueber zwei Fehlerquellen beim Nachweis der Unentbehrlichkeit vom Phosphor und Eisen fiir die Entwickelung der Seeigellarven, Arch. Em. Me¢:h. vii, 1898.

C. H ERBST. Ueber (las Auseinandcrgehen von Furchungs- und Gewebezellen in kalkfreiem Medium, Arch. 1911!. Ma-h. ix, 1900.

C. IIERBS1‘. Ueber die zur Entwickelung der Seeigellarven nothwendigen anorganischen Stoife‘, ihre Rolle und ihre Vertretbarkeit: II. Die Vertretbarkeit der nothwendigen Stoffe dutch andere itlmlicher chemischer Natur, Arch. Ent. Mech xi, 1901.

C. UERBST. Ueber die zur Entwiekelung der Seeigellarven nothwcndigen anorganischen Stoffe, ihre Rolle und ihre Vertretbarkeit: III. Die Rolle der nothwendigen anorganisclien Stoffe, Arch. Em. Mm-h. xvii, 1904.

0. and R. HERTWIG. Ueber den Bet'ruchtungs- und Teilungsvorgung des tierischen Eies unter dem Einfluss ilusserer Agentien, Jen. Zeifsclw. xx, 1887.

O. HERTWIG. Experimentelle Studien am tierisehen Ei vor, wiihrend und naeh der Befruchtung, Jm. Zeflschr. xxiv, 1890.

W. H. HOWELL. On the relation of the blood to the automaticity and sequence of the heartbeat, A mer. Joum. Pk;/s. ii, 1898-9.

W. II. HOWELL. An analysis of the influence of the sodium, potassium, and calcium salts of the blood on the automatic contractions of heartmuscle, Amer. Journ. Pk;/s. vi, 1901-2.

R. IRVINE and G. SIMS WOODHEAD. The secretion of carbonate of lime by animals, Proc. lfoy. Soc. Etlinbmy/h, xvi, 1889.

R. S. LILLIE. On differences in the effects of various salt-solutions on ciliary and on muscular movements in Arenicolu larvae, Amer. Joum. Phys. v, 1901.

R. S. LILLIE. On the effects of various solutions on ciliary and muscular movement in the larvae of Arcml-ola. and 1’oIy_(/ordius, Amer. Journ. Phys. vii, 1902.

R. S. LILLIE. The relation of ions to ciliary movements, Amer. Journ. Ph_1/.s-. x, 19034.

D. J. LINGLE. The action of ions on ventricular muscle, Amer. Journ. Plays. iv, 1900-1.

F. S. LOCKE. On a supposed action of distilled water as such on certain animal organisms, Jam-n. Pity.»-. xviii, 1895.

.T. LOEB. Ueber den Einfluss von Alkalion und Siiuren anf die ombryonale Entwickelung uud (las Wachsthum, Arch. Enf. Mach. vii, 1893.

J. LOEB. On ion-proteid compounds and their role in the mechanics of life-phenomena: I. The poisonous character of a pure NaCl solution, Amer. Journ. Phys. iii, 1899-1900.

J. LOEB. On the different effect of ions upon myogenic and neurogenic rhythmical contractions and upon embryonic and muscular tissue, Amer. Journ. Phys. iii, 1899-1900.

J. LOEB. The toxic and anti-toxic effects of ions as a function of their valency and possibly their electrical charge, Amer. Journ. Phys. vi, 1901-2.

J. LOEB and W. H. LEWIS. On the prolongation of the life of the unfertilized eggs of sea-urchins by potassium cyanide, Amer. Journ. Phys. vi, 1901~2. J. LOEB and W. J. GIES. Weitere Unteisuchungen fiber die entgiftenden Ionenwirkungen und die Rolle der Kationen bei diesen Vorgangen, Pfliiye:-‘s Arch. xciii, 1903.

E. P. LYON. The eifects of potassium cyanide and of lack of oxygen upon the fertilized eggs and the embryos of the sea-urchin (Arbaeia pzmctulata), Amer. Journ. Phys. vii, 1902.

A. P. MATHEWS. The action of pilocarpine and atropine on the embryos of the star-fish and the sea-urchin, Amer. Journ. Phys. vi, 1901-2.

A. P. MATHEWS. The relation between solution tension, atomic volume, and the physiological action of the elements, Amer. Journ. Phys. x, 1903-4.

A. P. MATHEWS. The toxic and anti-toxic action of salts, Amer. Joum. Phys. xii, 1904-5.

A. P. MATHEWS. The nature of chemical and electrical stimulation: I. The physiological action of an ion depends upon its electrical state and its electrical stability, Amer. Journ. Phys. xi, 1904. II. The tension co-efficient of salts and the precipitation of colloids by electrolytes, Amer. Journ. 1’hys. xiv, 1905.

S. S. MAXWELL and J. C. HILL. Note upon the effect of calcium and of free oxygen upon rhythmic contraction, Amer. Jom-n. Phys. vii, 1902.

S. S. MAXWELL. The effect of salt-solutions on ciliary activity, Amer. Journ. Phys. xiii, 1905.

H. MCGUIGAN. The relation between the decomposition-tension of salts and their anti—fermentative properties, Amer. Journ. Phys. x, 1903-4.

A. MOORE. Further evidence of the poisonous effects of a pure NaCl solution, Amer. Journ. Phys. iv, 1900—l.

A. Moons. The effect of ions on the contractions of the lymph hearts of the Frog, Amer. Journ. Phys. v, 1901.

A. Moomz. On the effects of solutions of various electrolytes and non-conductors upon rigor mortis and heat rigor, Amer. Jom-n. Phys. vii, 1902.

A. MOORE. On the power of Na,S0, to neutralize the ill effects of’ NaCl, Amer. Journ. Phys. vii, 1902.

T. H. MORGAN. The action of salt solutions on the unfertilized and fertilized eggs of'Arbac1'a and of other animals, Arch. Ent. Mech. viii, 1899.

T. H. MORGAN. Further studies on the action of salt-solutions and of other agents on the eggs of Arbacia, Arch. Ent. Mech. x, 1900.

T. H. MORGAN. The relation between normal and abnormal development of the embryo of the Frog as determined by the effect of lithium chloride in solution, Arch. Ent. Mech. xvi, 1903. III. 8 CHEMICAL COMPOSITION 155

H. NEILSON. Further experiments on the antitoxic effects of ions, Amer. Joum. Phys. vii, 1902.

S. R1NG1=.n and A. G. PH!-JAR. The influence of saline media. on the tadpole, Jam-n. Phys. xvii, 1894-5.

J. RITCHIE. The relation of chemical composition to germicidal action, Trans. Path. Soc. 1, 1899.

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T. SOLLMANN. Structural changes of ova in anisotonic ‘solutions and saponin, Amer. Journ. Phys. xii, 1904-5.

C. B. WILSON. Experiments on the early development of the Amphibian embryo under the influence of Ringer and salt solutions, Arch. Ent. Mech. v, 1897.

W. D. ZOETI-IOUT. The effects of potassium and calcium ions on striated muscle, Amer. Jom-n. Phys. vii, 1902.

9. Summary

In the numerous experiments which we have been considering the efiect is observed upon the development of the embryo of certain alterations in the constitution of that embryo’s normal environment. Either some factor which is not usually present is added to the environment, or else some factor which is customarily found there is altered by increase or decrease, or removed altogether.

In some cases development remains undisturbed by this treatment, in others it may be merely generally retarded or accelerated, in others again it may be altered not merely in rate but in form, with the production of an abnormality or monstrosity, and if its eifeet is too intolerable the death of the embryo may ensue.

Throwing light as they do on the causes of the formation of natural monsters, such experiments are no doubt of the highest interest from a general teratological point of view. The mere possibility of the occurrence of such malformations is, however, itself a fact of the deepest morphological significance. A monster is an organism in which the development of some part or parts has either exceeded or fallen short of its normal limitations, and any such phenomenon points indubitably to a certain mutual independence of the parts in the growth and differentiation of the organism; while some pursue their normal course, others deviate from it. It follows that when such deformations are due to changes in the external conditions the parts are not equally sensitive to the unusual influence to which they are exposed. Thus in the Frog embryos which exhibit persistent yolk-plugs and open brains when grown in solutions of various kin(ls, the yolk and the medullary folds are alone susceptible to the action of the poison, other parts are unaffected and continue their development as though under normal circumstances: or, again, a sea—urchin passes through the early stages of segmentation and gastrulation unchanged when placed in a sea-water from which magnesium has been removed, but the subsequent differentiation of the gut and the formation of the skeleton are abnormal ; magnesium is necessary for these, though not for the earlier processes. A means is thus afforded of watching the behaviour of one or more parts independently of others, as, for example, of the animal cells in the gastrulation of the Frog's egg when the yolk-cellspare injured, and the most valuable information contributed, often quite unexpectedly, to our understanding of the events of normal ontogeny.

Quite apart from this such experiments have already contributed, and will probably contribute still more in the future, to the study of variation. Between conspicuous monstrosities and those milder abnormalities which are termed ‘sports ’ or ‘ mutations ’ there is every intermediate gradation, just as there is, on the other hand, no sharply defined limit between these discontinuous and those far smaller continuous variations to which the term has been often exclusively applied. The embryo is particularly sensitive to a change in its environment and reacts to such change by a variation in its form of greater or less degree. And not only that ; as Vernon has shown, these changes can produce also an alteration in the variability of the species ; and so provide greater opportunities for the operation of natural selection.

At the same time teratolugy is not the main inquiry with which the experimental emhryologist is concerned. The problem that confronts him is to determine the part played by each factor of the external environment in the processes of normal, specific growth and differentiation, and for the solution of this problem only those experiments, of course, are of avail in which such factors are either altered or removed.

By this means, as we have seen, it has been shown that a certain constitution of the physical environment, fixed within certain limits, is needful for the embryo; to these conditions it is closely adapted; those limits it can only transgress under pain of abnormality or death.

Every factor, or nearly every factor, is necessary for this or that phase or part of the process, some for the whole. Light of a certain wave-length will accelerate development, light of another kind, or in some instances darkness, will retard it, or stop it altogether; a certain degree of heat is indispensable ; oxygen is required for respiration, water for growth ; some eggs demand constant agitation, others comparative rest; fertilization, or segmentation, or gastrulation, or some one or other of the later phases of development may depend absolutely on the presence of some particular chemical element ; remove the factor in question, whatever it may be, and that particular process will not occur, and the specific, typical end which is reached in normal development will not be attained. Nevertheless, the achievement of this end does not depend wholly upon extrinsic forces, for the ovum is no completely homogeneous ‘isotropic’ substance in which the complex circumstances of its environment conspire to produce heterogeneity and coherence. There is no evidence that any physical factor exerts a directive influence sufficient of itself to determine any part of the whole specific cfiect, although this may happen under extraordinary conditions, as when gravity impresses a bilateral symmetry upon the compulsorily upturned egg of the Frog, and so determines the median plane of the embryo.

Intimately bound up though these external conditions are with the proper conduct of the whole series of events whereby the organism comes gradually to resemble the parents that gave it birth, they can only operate in conjunction with internal factors which must be sought for not only in the initial structure and constitution of the germ-cells, but in the mutual interactions of the developing parts.

Jenkinson (1909): 1 Introductory | 2 Cell-Division and Growth | 3 External Factors | 4 Internal Factors | 5 Driesch’s Theories - General Conclusions | 6 Appendices

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