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AeA TIN Ad.
OF
EXPERIMENTAL ZOOLOGY
EDITED BY
WILLIAM K. BROOKS FRANK R. LILLIE
Johns Hopkins University University of Chicago
WILLIAM E. CASTLE JACQUES LOEB
Harvard University University of California
EDWIN G. CONKLIN THOMAS H. MORGAN
University of Pennsylvania Columbia University
CHARLES B. DAVENPORT GEORGE H. PARKER
Carnegie Institution Harvard University
HERBERT S. JENNINGS CHARLES O. WHITMAN
Johns Hopkins University University of Chicago
EDMUND B. WILSON, Columbia University
AND
ROSS G. HARRISON
Johns Hopkins University
Manacine EpIToR
VOLUME III
an
THE JOURNAL OF EXPERIMENTAL ZOOLOGY
| BALTIMORE
1906
CONTENTS
No. 1—February, 1906
Epmunp B. WILson
Studies on Chromosomes. III. The Sexual Differences of the Chromosome
Groups in Hemiptera, with Some Considerations of the Determination
and imhentance Gt ex. With ix PAPtTes cae koe teh se ne leks oo bye I
Davin D. WHITNEY
An Examination of the Effects of Mechanical Shocks and Vibrations upon
theatre of Mevelopment of Pertiltzed (Peps. oma a) .o0 ss os oa 41
Joun W. Scortr
Morphology of the Parthenogenetic Development of Amphitrite. With
Houmwaresandslive Hiotices ther Cbs aes ois ss ve 2 eye aes 49
Cuartes R. SrocKaRD
The Development of the Fundulus Heteroclitus in Solutions of Lithium
Chlorid, with Appendix on its Development in Fresh Water. With
Liner Md SEER TeS aera ee tea neareh ie ea ea Rm ee F 99
E. A. ANDREWS
Partial Regeneration of the Sperm-Receptacle in Crayfish. With Eleven
PUIG SmI Sen eSNG. OS CA sed 4: mi ncke cs «antenna as wat cap eaysro ie aut 121
A. J. GOLDFARB
Experimental Study of Light as a Factor inthe Regeneration of Hydroids. 129
No. 2—July, 1906
FRANK R. LILLie
Observations and Experiments Concerning the Elementary Phenomena
of Embryonic Development in Chztopterus. With One Plate and
Beveney-ciomtenipiresimit hed) Gxt r.. ..\.. 26: e.ciem sles at terete sree cick acts 153
Littran V. Morcan
Regeneration of Grafted Pieces of Planarians. With Seventeen Figures . 269
Cuas. W. Harcitr
Experiments on the Behavior of Tubicolous Annelids. With Three
CRIES SY OSE AAAS 9S SC a era IRR Bae Sem a ee eS ans ea 295
IsapEL McCracken
Inheritance of Dichromatism in Lina and Gastroidea ...............-. 321
No. 3—September, 1906
Oris P. DELLINGER
Locomotion of Amcebze and Allied Forms. With Two Plates and
‘Twenty-nine Figures m the ext 2.1.2... ee hee ee ee ge 337
S. O. Mast
Light Reactions in Lower Organisms. I. Stentor Cceruleus. With Six
Bipures |. 2: ali sceg mee ie heey ecein Bin > im oe wins ot ie elcl aed eee 359
G. H. ParKER
The Influence of Light and Heat on the Movement of the Melanophore
Pigment, especially in Lizards. With Three Figures ............... 401
AtFrrep G. Mayer AND CAROLINE G. SOULE
Some Reactions\ot Caterpillars amd: Moths)... 21... ocisjereie lessee ie 415
H. S. JENNINGS
Modifiability in Behavior. II. Factors Determining Direction and Charac-
ter of Movement.in the Earthworm. .2...0. 02. sachs #gee ss -eker 435
No. 4—December, 1906
T. H. Morcan
The Physiology of Regeneration. With Seven Figures ................ 457
Hydranth Formation and Polarity in Tubularia............-...-..--.- 501
D. H. Tennent anp M. J. HoGue
Studies on the Development of the Starfish Egg. With Five Plates. ..... 517
GeorGE L. STREETER
Some Experiments on the Developing Ear Vesicle of the Tadpole with
Relation to Equilibration. With Twelve Figures .................. 543
C. M. Cuiip
The Relation Between Functional Regulation and Form-Regulation .... 559
W. N. Now in
Study of the Spermatogenesis of Coptocycla Aurichalcea and Coptocycla
Guttata, with Especial Reference to the Problem of Sex-Determination.
With Two'Plates 50 4 ciie aot ayaa tere aoe tence Oo ees ae 583
Victor E. EMMEL
Torsion and Other Transitional Phenomena in the Regeneration of the
Cheliped of the Lobster (Homarus Americanus). With Two Plates .... 603
Eurayia V. WALLING
The Influences of Gases and Temperature on the Cardiac and Respira-
tory Movements in the Grasshopper a2q-4-.2c2 a. eee eee 621
STUDIES ON CHROMOSOMES
Ill. THE SEXUAL DIFFERENCES OF THE CHROMOSOME-
GROUPS IN HEMIPTERA, WITH SOME CONSIDERA-
TIONS ON THE DETERMINATION AND INHERI-
ANGE \OF SEX
EDMUND B. WILSON
Wirth Six Ficures
Since the time of Henking’s able paper on the spermatogenesis
of Pyrrochoris (’g1), it has been known that in certain Hemiptera,
and in some other insects, a dimorphism exists in the nuclear con-
stitution of the spermatozoa, one-half of them containing the so-
called “accessory” or “heterotropic” chromosome, while in the
other half this chromosome is lacking. ‘The meaning of this fact
has hitherto remained undetermined. McClung in 1902 devel-
oped an hypothesis of sex-production based on the conjecture that
the heterotropic chromosome is a sex-determinant, and more
specifically that spermatozoa containing this chromosome produce
males, for the very obvious, yet fallacious, reason that it 1s present
in the male. This hypothesis was based simply on the fact
that the spermatozoa are of two numerically equal classes, like
the sexes of the adults; and it was apparently overthrown by
subsequent observation. ‘The hypothesis implied that the cells
of the female must contain one chromosome less than those of
the male; and although McClung did not specifically place his
assumption in this form, he considered it extremely improbable
that the accessory chromosome, or “any such element,” is present
in the egg. Sutton (02) believed that he had found a
confirmation of this in the grasshopper Brachystola, where he
showed that the number in the male (spermatogonia) 1s twenty-
three, and stated that in the female (odgomia and _follicle-
Journat or ExperIMENTAL Zoétocy, Vor. 11, No. 1.
2 Edmund B. Wilson
cells) the number is twenty-two, supporting this statement by a
single figure (op. cit., Fig. 11). Sutton was, however, able to
examine only a very few of the female groups, and the object is
an unfavorable one as compared with the Hemiptera, owing to
the less compact form of the chromosomes. McClung’s hypothe-
sis seemed to be rendered completely untenable by the later obser-
vations of Montgomery on Anasa (04), and of Gross on Syromas-
tes ('04), both these authors describing and clearly figuring the
same number of chromosomes (twenty-two) in the male and the.
female cells. Gross and Wallace (’05) were thus independently
led to the conclusion that only one of the two classes of spermat-
ozoa was functional, namely, that in which the heterotropic
chromosome is present. Those of the other class were assumed
to degenerate after the fashion of polar bodies.
I am now able to bring forward decisive proof that the appar-
ently adverse evidence brought forward by Montgomery and |
Gross was based on errors of observation, and that the sexes in
Hemiptera of this type do in fact show a constant difference in
the number of chromosomes. As far as these animals are con-
cerned, however, McClung’s conjecture as to the mode of fertili-
zation proves to have been the reverse of the truth; for it is the
female, not the male, that possesses the additional chromosome,
as I have determined beyond all doubt in four genera, namely,
Anasa, Alydus, Harmostes and Protenor. The facts leave no
doubt that both forms of spermatozoa are functional; that all of
the eggs possess the same number of chromosomes; that all con-
tain the homologue, or maternal mate, of the accessory or hetero-
tropic chromosome of the male; and that fertilization by sper-
matozoa that possess this chromosome produces females, while
males are produced upon fertilization by spermatozoa that do
not possess it.
A second type of dimorphism of the nuclei of the spermatozoa
was made known in the first of these studies. In this type all
of the spermatozoa contain the same number of chromosomes, but
half of them contain a large “idiochromosome” and the other
half a corresponding small one. | was led in that paper to suggest
the possibility that the idiochromosomes might play a definite
Studies on Chromosomes 2
role in sex-production, but could at that time produce no evidence
in support of the suggestion. I have now the evidence to show
that this suggestion was in accordance with the facts; for in at
least four genera, Lygzeus, Euschistus, Coenus and Podisus, both
sexes show the same number of chromosomes, but the small
idiochromosome is present only in the male. Somewhat earlier,
and independently, Stevens (05) determined a precisely similar
fact in the case of a beetle, Tenebrio, which indicates that the
phenomenon is of wide occurrence in the insects. ‘These results
confirm the correctness of my conclusion that the heterotropic
or “‘accessory” chromosome has become unpaired in the male
sex through the disappearance in that sex of its mate, and give a
complete explanation of the fact that in forms possessing the
heterotropic chromosome the male number is odd and one less
than the female number. I believe that these facts may give
_ the basis for a general theory of sex-production.
I. DESCRIPTIVE
A. General Character of the Chromosome-grou ps
In two preceding papers (Wilson, ’05, 1; 05, 3,) (where due
acknowledgment is made to previous observers in this field)
I have described in some detail the general nature of the chro-
mosomes in these insects. For such an investigation as the present
one, the Hemiptera present peculiar advantages, owing above all
to the short and regular form of the chromosomes, and the relative
lack of crowding in the equatorial plate. | have employed almost
exclusively Flemming’s strong fluid as a fixative, staining the
sections with iron-hematoxylin and extracting until the cytoplasm
is nearly or quite colorless. “The best preparations thus obtained
leave nothing to be desired in point of brilliancy and clearness,
and show the chromosomes with a distinctness that is hardly
exaggerated by the black and white figures here reproduced.
The very large number of sections now at my disposal (including
all those of Paulmier and a still greater number of new prepara-
tions of my own) has enabled me in the case of nearly every
species to examine numerous division-figures (of which only the
4 Edmund B. Wilson
best have been selected for illustration) and to satisfy myself thor-
oughly of the constancy of the relations as described. Everyone
familiar with such objects will, however, realize that in regard to
such matters as the arrangement and size-differences of the
chromosomes certain apparent variations appear that are due to
slight differences in the form and position of the chromosomes,
and to the various degrees of foreshortening thus caused. This
introduces a slight error, into both the observations and the draw-
ings, that can hardly be avoided. A second source of error lies
in the degree of extraction, which produces surprising variations
in the apparent size of the chromosomes—I have found, for
instance, that by successive extraction the chromosomes may be
reduced almost to one-half their original apparent size, and the
smaller chromosomes may thus be caused almost to disappear from
view. Camera drawings at successive stages of the extraction show,
however, that the relative sizes of the chromosomes remain sub-
stantially unchanged, and the comparison of the same object after
a shorter and a longer extraction has thus, in a number of cases,
given a more certain result than could otherwise have been
obtained. I have, whenever it was possible, figured different
stages of the same species from the same slide, so as to avoid the
error due to different degrees of extraction; but this is not always
possible, since as a rule longer extraction is required to give a
perfectly clear view of the spermatogonial groups than 1s desirable
for the spermatocyte-divisions. For the comparison of the two
sexes, different slides must of course be used, and to this is due,
I am sure, some of the size-differences between the odgonial and
spermatogonial groups that appear in the figures. —
Making all due allowance for the sources of error mentioned,
it remains perfectly clear that the chromosomes in each species
show among themselves constant and characteristic size-differ-
ences; and further, that with the special exceptions in the male
described beyond, the chromosomes of the unreduced groups
(1. e., those of the o6gonia and spermatogonia) may be paired off,
two by two, to form equal or symmetrical pairs. ‘The pairing of
the chromosomes is most evident in the case of especially small
chromosomes (such as the m-chromosomes of Anasa, Alydus,
Studies on Chromosomes 5
Harmostes, etc., or the small pair of ordinary chromosomes of
Coenus and Euschistus, described beyond) or especially large ones
such as the largest pair in Alydus, and in some of the species of
Euschistus. Those of intermediate size are also obviously paired
in some of the forms (e. g., in Protenor, Fig. 1); but in many ot
the species the several pairs are not sufficiently marked in size to
admit of certain recognition. Nevertheless, a comparative study
of many species has convinced me of the correctness of the con-
clusion, first indicated by Montgomery (’01) and afterward more
fully worked out by Sutton (02), that all the chromosomes (again
with the special exceptions referred to above) may be thus paired,
and that the chromosome-group as a whole includes two parallel
series of chromosomes that undoubtedly represent respectively
the descendants of those that originally are brought together in
the union of the gametes. This is very clearly brought out by
making camera drawings of the chromosomes, and arranging
them as nearly as practicable in pairs of equal size. “This arrange-
ment conspicuously shows the sexual differences, as may be seen
by a comparison of Figs. 2, / and 4 (Anasa) and 5,c and g (Lygzus).
There is, of course, a large error to be allowed for in the series
as thus arranged, and no pretense to complete accuracy in the
selection of the members of most of the pairs can be made.
Nevertheless, when all due allowance for differences of form,
foreshortening and the like is made, the fact that such a double
series exists is unmistakable. When it is borne in mind that the
spermatid-nuclei in each case contain a single series of chromo-
somes showing the same size-relations (c}. for instance, Figs. 1, ),
Ed. 92,.4,d,€; 3,4, ¢,],; 4, b,j, d, b), it becomes in a high depree
probable that the corresponding pairs of the somatic groups con-
sist each of a paternal and a maternal member, in accordance
with Montgomery’s original and fundamental assumption (’01).
As may be seen bya comparison of the figures, the members of
each pair when in their natural position, do not as a rule lie in
juxtaposition but may occupy any relative position. Only at
the period of synapsis do they actually couple, two by two, to
form the bivalents whose members are subsequently separated
by the reducing division.
6 Edmund B. Wilson
In order to give a wider basis of comparison | have given new
figures of the chromosome-groups of nearly all the species,
even in the case of forms already figured in my preceding papers.
Since the idiochromosomes or the heterotropic chromosome form
the distinctive differential between the nuclei of the two sexes, I
shall in the descriptive part of this paper call them the “ differen-
tial chromosomes. ”’
B. First T ype. Forms Possessing an “Accessory” or Hetero-
tropic Chromosome
As stated above, I have compared the males and females in
respect to the chromosome-groups in four genera, selecting for
this purpose the most available cells, which are the dividing
oogonia and ovarian follicle-cells in the female, the spermato-
gonia and investing cells of the testis-cysts in the male. ‘The
general result is the same in all, but owing to the conspicuous
size-difference of the chromosomes in Protenor, this form gives
the most obvious and striking evidence.!
a. Protenor belfragei
Montgomery (or) first made known the general character of
the chromosome-groups in this interesting species, showing that
the spermatogonial groups show an odd number, thirteen, that
the heterotropic chromosome (Montgomery’s “chromosome x’’)
is immediately recognizable by its enormous size—tt 1s fully twice
the size of the largest of the other chromosomes—and that it is
unpaired (though he considered it a bivalent). My own observa-
tion confirms his description in every point, except that I have
never seen this chromosome transversely constricted into two
halves. ‘The first glance at a good preparation of the spermat-
ogonial metaphase, as seen in polar view, shows this huge chro-
’There can be no doubt of the identification of the follicle-cells; but there is some uncertainty regard-
ing the cells here called odgonia, since they are from the undifferentiated region of the ovary in which the
distinction between odgonia and follicle-cells cannot be made out. It is therefore quite possible that some
of the groups here described as odgonia may be from very young follicle-cells or nutritive cells; but this
does not affect the main result.
Studies on Chromosomes Z
mosomed a sa long worm-shaped body obviously without a mate,
(fis. 1, dj). The remaining twelve chromosomes may _ be
grouped in symmetrical pairs (indicated by numbers ebony,
y ee °*e
a h (fs
=
/
oO
Ss
Ficure 1
Protenor belfragei—a, Anaphase of second spermatocyte-division; 5, c, sister groups, from the
same spindle, polar view, second spermatocyte-division; d, e, f, spermatogonial groups; g, h, groups
from immature ovaries, probably odgonia; i, group from dividing follicle-cell.
d, e), though the members of each pair may occupy any relative
position. Of these six pairs, one (2, 2) is always much larger than
the others, its members being approximately half the size of the
1All the figures are drawn to the same scale. In all, # denotes the heterotropic chromosome, i the
idiochromosomes (large and small in some cases lettered J and 1 respectively), m the paired micro-
chromosomes, and s the smallest pair of ordinary chromosomes.
8 Edmund B. Wilson
heterotropic. A second pair (3, 3) may usually be distinguished
as the next largest, and a third pair (7, 7) as the smallest, though
this is not always obvious. [This pair probably correspond
to the “m-chromosomes” of my preceding paper. ‘The remain-
ing three pairs are of nearly equal size, though sometimes they
clearly show a progressively graded series as in Fig. 1, d, e. In
synapsis the six paired chromosomes become coupled, as usual,
to form six corresponding bivalents, while the large chromosome
remains as an unpaired univalent. During the whole growth-
period of the spermatocytes this chromosome remains in a con-
densed spheroidal state, forming a very large chromosome-
nucleolus. In the prophases of the first division it again elongates
and divides longitudinally in this division. Each secondary
spermatocyte accordingly receives seven chromosomes. In the
second division six of these (the products of the bivalents) again
divide equally, while the seventh (the large chromosome) passes
undivided to one pole (Fig. 1, a). One-half of the spermatid
nuclei accordingly receive six chromosomes, the other half seven,
the additional one being the large heterotropic chromosome
(Fie anh ve)
In the female the chromosome-groups of the dividing odgonia
and follicle-cells appear with a clearness not inferior to that shown
in the spermatogonial groups (Fig. 1, g-z). Itis at once apparent
that in these groups there are two very large chromosomes, equal
in size, in place of the single one that appears in the male, while
the remaining chromosomes show the same relations as in the
male. ‘There are accordingly fourteen chromosomes in all, which
may be equally paired off, two by two, and no chromosome is
without a mate of corresponding size. Since the largest two are
of the same relative size as the single heterotropic chromosome
of the male, it is quite clear that one of them must have been
derived from a spermatozo6n containing this chromosome, while
the other is its maternal mate or homologue.
I have not been able to follow by actual observation the phe-
nomena of reduction, maturation and fertilization in the egg;
but the data are sufhcient to show, with a degree of probability
only short of certainty, what must be the history of the chromo-
Studies on Chromosomes 9
somes in these processes. Since the odgonia contain fourteen
equally paired chromosomes, synapsis in the odcyte must result
in the formation of seven symmetrical bivalents—z. e., seven
couples of equal chromosomes—and each egg after maturation
contains seven univalent chromosomes, one of which is the
maternal representative or mate of the heterotropic chromosome
of the male. ‘This group contains one chromosome of each of the
original pairs, and is precisely similar to the group present in
those spermatozoa that contain the heterotropic chromosome
(Fig. 1, c). Fertilization by such a spermatozoa doubles this
group, giving the condition observed in the female—z. e¢., fourteen
chromosomes equally paired, the largest pair consisting of the
heterotropic chromosome and its maternal mate (J, 7, Fig. 1, g, /).
Fertilization by a spermatozoon that lacks the heterotropic chro-
mosome will give the condition observed in the male, namely,
thirteen chromosomes, of which twelve are equally paired, while
the thirteenth is the large unpaired one which is obviously derived
from the egg. ‘There is therefore no escape from the conclusion
that both forms of spermatozoa are functional, that females are
produced upon fertilization by spermatozoa that contain, and
males upon fertilization by spermatozoa that lack, the hetero-
tropic chromosome. Since the two classes of spermatozoa are
equal in number, fertilization will in the long run produce males
and females in approximately equal numbers.
b. Anasa tristis
A comparison of the nuclei of the two sexes in this species gives
a precisely concordant result, though the size-differences do not
allow of so exact an identification of the differential chromo-
somes. In the preceding study I showed that the number of
chromosomes in the male (spermatogonia) is twenty-one, not
twenty-two as stated by previous observers. Study of the sper-
matogonial metaphase groups shows that twenty of the chro-
mosomes may be equally paired, two by two, while the remaining
one is, of course, without a mate (Fig. 2, e, 7). “The unpaired
heterotropic chromosome is one of three largest chromosomes,
IO Edmund B. Wilson
but which particular one cannot be determined by simple inspec-
tion, since the three are of nearly equal size. In synapsis two
of these large chromosomes unite to form the largest of the ten
bivalents (1, Fig. 2, a) that appear in the first spermatocyte
division. ‘The third, which retains its compact form as a chro-
mosome-nucleus during the growth-period, remains as the univ-
alent heterotropic chromosome (/, Fig. 2, a). ‘The first spermat-
ocyte division accordingly shows eleven chromosomes, ten of
which are bivalent, and one (heterotropic) is univalent. ‘The
distribution of these chromosomes in the maturation-division takes
the usual course, the heterotropic chromosome dividing equally
with the ten bivalents in the first mitosis while its products pass
undivided to one pole of the spindle in the second (Fig. 2, 6).
Half the spermatozoa accordingly receive ten chromosomes, one
of which (z, Fig. 2, c) is larger than the others, and half an exactly
similar group plus the large heterotropic chromosome, or eleven .
meall(Pig.22, 2).
The o6gonial groups show invariably twenty-two chromosomes,
which may be arranged in eleven equal pairs (Fig. 2, g, b). In
place of the three large chromosomes of the spermatogonial
groups appear four Senior chromosomes, forming two equal
pairs. ‘Iwo of these four are obviously the large chromosome,
common to all the spermatozoa, and its maternal mate, while
the other two must be the heterotropic chromosome (derived in
fertilization from the spermatozoon) with its maternal mate.
It is, therefore, clear that all of the matured eggs must contain
eleven chromosomes, that females are produced upon fertilization
by those spermatozoa that contain a similar group—1. e., by those
containing the heterotropic—males upon fertilization by spermat-
ozoa that lack the heterotropic.
The ovarian follicle-cells often show chromosome-groups
identical with those of the odgonia (Fig. 2, 7). Not infrequently,
however, the number of chromosomes is much greater, and the
same is true of the nuclei of the investing cells of the ovary, of
the oviduct and of the fat-body. In the male similar multiple
groups are not uncommon in the interstitial and investing cells
of the testis. Only in a single case have I succeeded in gaining
Studies on Chromosomes rn
“Get eoscos
fs & en
tg
: @eees ode:
Gbacccecee:
O
T
.
9 §
h
% ra a)
J ae ye ie ese" ;
FIGURE 2
Anasa tristis.—a, Metaphase of first spermatocyte-division, in polar view, showing the nine large
bivalents in a ring, the univalent heterotropic chromosome below it, and the m-chromosome bivalent
in the center; b, anaphase of second division; c, d, sister-groups from the same spindle, polar view,
second division (1 the macrochromosome); e, spermatogonial group; f, the same chromosomes arranged
in pairs; g, odgonial group from a larva; h, the same group arranged in pairs; i, spermatogonial group;
j, group from a dividing follicle-cell; k, double group, from a cell toward the periphery of a larval ovary.
12 Edmund B. Wilson
a clear and complete view of such a group; but this one case
suffices to give, with great probability, the explanation of the
increased number of chromosomes. In this case every chro-
mosome of the metaphase group may be clearly seen, and the
number is exactly twice the odgonial number, namely, forty-four
(Fig. 2, k). Careful study clearly shows that this group contains
four microchromosomes and eight macrochromosomes, in each
case twice the number of those present in the odgonia. ‘This
leaves no doubt that in this case all the chromosomes have divided
once without the occurrence of a cytoplasmic division, and makes
it probable that the increase in number in the cells in question 1s
always due to a process of this kind. I have not been able to
obtain faultless preparations of the dividing cells of other tissues,
and can only state that in the ectodermal cells of the larva the
number of chromosomes is approximately the same as in the
oogonia. The multiple chromosome-groups were only observed
in the cells mentioned above, all of which, it may be observed,
are degenerating or highly specialized cells.
c. Alydus pilosulus
Despite the small number of chromosomes ( 2 14, & 13, as in
Protenor) this genus is in some respects less favorable for detailed
analysis than either of the ones described above, for the size of
the heterotropic chromosome does not distinguish it sufficiently from
the other chromosomes to allow of its certain identification in the
spermatogonia. ‘lhe main fact appears, however, as clearly as
in Protenor or Anasa that the female has one more chromosome
than the male.
In polar views of the second spermatocyte-division this species
shows the sister spermatid-groups with great beauty, one having
six chromosomes and one seven (Fig. 3, e, 7). These chromo-
somes show at least five distinguishable sizes that are constant,
namely, (1) a largest; (2) an extremely small one (m-chromo-
some); (3) a second smallest (the heterotropic); (4) a second
largest, and (5) three others intermediate in size between (3) and
(4), one of which is frequently a little larger than the other two.
Studies on Chromosomes t3
The sister groups are practically exact duplicates save for the
heterotropic which varies considerably in appearance as seen from
the pole owing to foreshortening (cj. the side-views given in my
preceding paper). The spermatogonia correspondingly show
always thirteen chromosomes (Fig. 3, a), of which the largest and
the smallest pair are at once distinguishable. Next follow four
chromosomes nearly equal in size, two of them often appreciably
smaller than the other two. Of the remaining five, one must be
the unpaired heterotropic; but, as already stated, it cannot be
positively identified by inspection. Closely similar groups may
be) j
Ge un
G
h
e. <0.
e2®@ @ 6
2
FIGURE 3
Alydus pilosulus.—a, Spermatogonial group; b, group from a dividing investing cell of the testis;
c, odgonial group; d, from a dividing cell of an egg-follicle; e, f, two pairs of sister-groups, each from a
single spindle, anaphase of second spermatocyte-division, in polar view.
occasionally be found in dividing cells of the enveloping cells of
the testis (Fig. 3,0) 0 Whether fran groups occur like those
described in Anasa, I cannot say.
The dividing oégonia and follicle-cells, of which a large number
have been observed, always show fourteen chromosomes that may
be arranged in seven equal pairs (Fig. 3, c, d). As in the sper-
matogonia, the largest and the smallest pair are usually at once
recognizable, and also the four second largest. The remaining
six, of nearly equal size, must of course include the heterotropic
chromosome and its maternal mate.
14 Edmund B. Wilson
d. Harmostes reflexulus
My material of this species is much less abundant than that of
the three preceding, and the preparations are not of the same
excellence. “hey nevertheless show beyond doubt that the num-
bers are here the same as in Protenor and Alydus, viz., thirteen in
the male and fourteen in the female. In my sections of both sexes
the chromosomes appear less regular in contour than in the other
species examined (probably owing to somewhat defective fixation).
They show clearly, however, in both sexes a largest pair and a
smallest (m-chromosomes), as in the other forms.
C. Second Type. Forms Possessing Unequal Idiochromosomes
The sexual differences of these forms have been worked out in
Lygzeus turcicus, five species of Euschistus (variolarius, ictericus,
tristigmus, fissilis and servus), Coenus delius and Podisus spinosus.
In the last named species the unreduced number is sixteen, in the
others fourteen. In all, the number of chromosomes is the same
in both sexes, but while the males show a large and a small idio-
chromosome, the females show two large idiochromosomes that
are equally paired. ‘This difference clearly appears in all the
species examined but is most conspicuous in Euschistus vario-
larius, E. ictericus and Lygzeus turcicus, where the inequality
of the idiochromosomes is most marked. The relative size of
the idiochromosomes varies somewhat (perhaps owing to differ-
ences in the degree of extraction of the dye) but on the whole is
characteristic of the different species, as described below.
In all of the species of Euschistus examined, and in Coenus
delius, a largest and a smallest pair of ordinary chromosomes
(the latter marked s in some of the figures) are readily distinguish-
able. “hese give rise to corresponding large and small biv Alents
in the first mitosis, and are recognizable as single chromosomes
in the spermatid-groups (Figs. 4, 5). The small chromosomes
are in every case smaller than the large idiochromosome, and in
Mineus bioculatus (Fig. 4, p, g) are actually smaller than the small
idiochromosome. It is possible that this pair of chromosomes
Studtes On Chromosomes [5
FIGURE 4
Euschistus, Mineus.—a, E. variolarius, second spermatocyte-division; b, sister-groups, second
e, second spermatocyte-division, E. tristigmus; f, g,
h, i, corresponding views of E. servus;
E. fissilis; p, Mineus bioculatus,
division; c, d, corresponding views of E. servus;
E. variolarius, spermatogonial and odgonial groups respectively ;
j,k, the same, E. ictericus; /, m, the same, E. tristigmus; ,0,the same,
second spermatocyte-division; q, sister-groups, from the same spindle, second division.
16 Edmund B. Wilson
may correspond to the microchromosomes, or m-chromosomes,
that are so characteristic of the first type (7, in Figs. 2, 3).
e. Euschistus
In E. variolarius the inequality of the idiochromosomes (Fig. 4,
a) is greater than in any other of the observed forms excepting
Lygzeus turcicus. ‘The sister spermatid-groups (Fig. 4, 6) consist
in each case of a ring of six ordinary chromosomes with the idio-
chromosome near its center. In the outer ring may be distin-
guished as a rule four or five different sizes of chromosomes, the
largest and smallest (s) being always recognizable, and usually
also a second largest and second smallest. “The large idiochro-
mosome 1s imers distinctly larger than the smallest chromosome
(s) of the outer ring, while the anal idiochromosome is very much
smaller than Bear and in long extracted preparations looks
exactly like a centrosome. ‘The spermatogonial groups corre-
spondingly show seven pairs of chromosomes (Fig. 4, 7), of which
the small idiochromosome, the smallest pair of ordinary chro-
mosomes, and two large pairs are recognizable. “The remaining
seven include three equal pairs, while the seventh is the large
idiochromosome, but it is impossible to identify this chromosome
more nearly. ‘The odgonial groups show fourteen equally paired
chromosomes, as shown in Fig. 4, g; but my preparations do not
show this so well in this species as in the others.
E. ictericus shows a similar spermatogonial group (Fig. 4, 7)
except that the small idiochromosome is relatively a little larger
and the small pair of ordinary chromosomes but slightly smaller
than the others. The odgonial groups (Fig. 4, &, an unusually
open specimen) very clearly show the absence of the small idio-
chromosome, but the equal pairing of the chromosomes is less
obvious than in the following species.
In E. tristigmus (Fig. 4, e, /, m) the small idiochromosome is
relatively much larger than in the foregoing species, while in
E. servus, it is usually a little larger still (Fig. 4, c,d, b). In both
these forms the smallest pair of ordinary chromosomes are at once
recognizable in the spermatogonia (s, Fig. 4, h, /) and the equal
pairing of the others is evident. In E. servus the o6gonial groups
Studies on Chromosomes 17
show the equal pairing of all the chromosomes with equal clear-
ness, the absence of the small idiochromosome being evident
(Fig. 4, 7). The small pair (s) evidently correspond to ‘the small
pair in the male (4, /) and the large idiochromosome-pair must
therefore be represented by one of the larger pairs. Fig. 4, 1, 0,
show the spermatogonial and odgonial groups of E. fissilis, show-
ing the same relations as in E. servus, save that the small pair are
relatively larger.
The above-described species of Euschistus, while agreeing pre-
cisely in the general relations, present individual differences so
marked as to show that even the species of a single genus may be
distinguishable by the chromosome-groups. In this case the
most interesting feature is the series shown in the inequality
of the idiochromosomes, which becomes progressively greater
in the series (1) E. servus, (2) tristigmus, fissilis, (3) ictericus,
(4). variolarius, the inequality in the last case being fully as great
as in Lygeus. I may again mention the fact that in the opposite
direction the genus Brochymena often shows the idiochromosomes
less unequal than in E. servus; in Mineus they are sometimes
of nearly equal size (Fig. 4, p, 7), while in Nezara no inequality
exists. Practically all intermediate conditions are therefore shown
within the limits of a single family between the extreme inequality
shown in E. variolarius and no inequality at all. It is quite clear
from the observations here brought forward that this progressive
differentiation has occurred only in the male sex, as I conjectured
in my first paper.
7. Ccenus delius
The relations in this form are so closely similar to those seen
in Euschistus servus or fissilis, as described above, as hardly to
require separate description. Fig. 5, b, shows the spermatogonial
metaphase- “group; 5, 1, the corresponding odgonial group. Both
these preparations show very clearly the eel pair (s) of ordinary
chromosomes (not so well shown in the figure of the spermat-
ogonial group in my first paper). Here, as in Euschistus, it is
evident that the large idiochromosome is much larger than the
membérs of the small pair.
18 Edmund B. Wilson
. Lygzeus turcicus
S Ve
In this species the inequality of the idiochromosomes is nearly
or quite as great as in Euschistus variolarius, but the differentia-
tion of the chromosome-pairs is less marked than in that species,
and the small pair cannot be distinguished with certainty in any
of the stages. In the spermatogonial groups, accordingly, only
the ‘small idiochromosome is markedly smaller than the others
(Fig. 534 d); and hence its lack of an equal mate is rendered very
conspicuous. In the female the small idiochromosome is absent
as usual and all the chromosomes are equally paired (Fig. 5, 7, ¢).
The idiochromosomes cannot be distinguished from the ordinary
chromosomes.
hb. Podisus spinosus
In this species both sexes show sixteen chromosomes. In the
spermatogonial groups (of which I am now able to give a better
figure than the one in my first paper) the small idiochromosome
appears relatively larger than in any of the foregoing species,
though still not more than half the size of any of the others
(Fig. 5, 7). In the female (follicle-cells, Fig. 5, &) all the chro-
mosomes are equally paired and the small idiochromosome 1s
absent, but owing to the relatively large size of the latter in the
male the chromosome-groups of the two sexes do not show so
obvious a contrast as in the foregoing cases.
Resumé and Conclusions Regarding the Second T ype
In all the forms described under this type the two sexes show
the same number of chromosomes but differ in that the male
groups include a large and a small idiochromosome while the
female groups have two large idiochromosomes of equal size.
This result agrees with that Acad reached by Stevens (’05) in
the case of the beetle Tenebrio, and involves the same conclusions
that she has indicated. Since all the chromosomes of the odgonial
groups are equally paired, it is evident that all the matured eggs
must contain half such a group, one of the chromosomes being
the maternal representative, or mate, of the large idiochromosome
Studies on Chromosomes 19
o mM
FIGURE 5
Lygeus, Coenus, Podisus, Nezara——a, Lygeus turcicus, second spermatocyte-division; 5, sister-
groups, second division; c, d, spermatogonial groups; e, the chromosomes of d arranged in pairs;
f, odgonial group; g, the same in pairs; h,7,Ccenus delius, spermatogonial and follicle-cell groups;
I, m, Nezara hilaris, spermatogonial and odgonial groups respectively.
20 Edmund B. Wilson
of the male. Fertilization of such an egg by a spermatozoon con-
taining the small idiochromosome will produce a group identical
with that occurring in the male; fertilization by one containing
the large idiochromosome will produce the characteristic female
group. This result is thoroughly consistent with that obtained
in the first type; for if the small idiochromosome be supposed to
disappear in the male, the phenomena become in every respect
identical with those occurring in the first type. The large idio-
chromosome is therefore undoubtedly homologous with the
heterotropic chromosome, and the latter owes its unpaired
character to the fact that its former paternal mate has vanished,
as I conjectured in my first paper.
It is further evident that in synapsis, in both sexes, the members
of each chromosome-pair become coupled to form symmetrical
bivalents, except in case of the idiochromosomes of the male.
In this case alone do chromosomes of unequal size couple to form
an asymmetrical bivalent; and it is a consequence of this coupling
that the subsequent distribution allots the small idiochromosome
to one-half of the spermatozoa and the large one to the other half.
D. Third Type. Forms in which the Idiochromosomes are
of Equal Size
Of these forms I have been able to examine only a single case,
namely, that of Nezara hilaris; and in the course of a whole
summer's collecting I obtained but a single female in the proper
stage to show the odgonial divisions. Forttnately both ovaries
show a considerable number of division-figures which demonstrate
the facts with perfect clearness.
A particular interest attaches to this form on account of the
fact, described in my first paper, that the idiochromosomes are of
equal size and hence give no visible differential between the two
classes of spermatozoa. ‘This form gives therefore a test case con-
cerning my general conclusion that the differentiation of the
idiochromosomes has occurred only in the male; for since these
chromosomes are here alike in all the spermatozoa, it might with
some plausibility be assumed that the differentiation had in this
Studies on Chromosomes 21
species taken place in the female. ‘The facts conclusively show
that such is not the case.
The spermatogonial groups (Fig. 5, /) show fourteen chromo-
somes, all of which may be symmetrically paired. ‘The smallest
pair, 7, 7, (as | showed in my first paper) are the idiochromosomes
as is shown by their characteristic behavior during the growth-
period and in the maturation-divisions. In synapsis the twelve
larger chromosomes couple to form six bivalents, while the idio-
chromosomes divide as separate univalents in the first spermat-
ocyte-division. Their products then conjugate as usual to form
the idiochromosome-dyad, which differs from all the forms hitherto
observed in being composed of two equal members. All the
spermatid-nuclei are accordingly exactly similar in appearance
and no visible dimorphism exists (cf. Fig. 4 of my first paper,
Wilson, ’05, 1). We should accordingly expect to find the
odgonial groups exactly similar to the spermatogonial; and such
is clearly shown to be the fact by the preparations, the odgonial
groups showing fourteen equally paired chromosomes among
which the idiochromosomes are readily recognizable by their
small size (Fig. 5, 7).
In this case, therefore, alone among all those examined, no
visible differences are shown by the nuclei of the two sexes.
One pair of the chromosomes are, however, different in nature
from the others, as is shown by their different behavior in the
male in the growth-period and in synapsis; and it is quite clear
that the two members of this pair are always assigned to different
spermatozoa. In respect to this chromosome, therefore, the
spermatozoa fall into two classes as truly as the other forms,
though they cannot be distinguished by the eye. It is hardly
necessary to point out how important this case is in giving a firm
basis of comparison with the more usual forms in which, if we can
trust the existing accounts, all of the functional spermatozoa are
exactly alike in appearance, and no sexual differences of the chro-
mosome-groups are apparent.
993) Edmund B. Wilson
Ek. The Differential Chromosomes in the Synaptic and Growth-
periods
I will now briefly consider a very marked difference between
the sexes in respect to the behavior of the differential chromosomes
during the contraction-phase of synapsis and the succeeding early
erowth-period.t' In the male, as was fully described in my last
paper, both the heterotropic chromosome and the idiochromo-
somes condense early in the growth-period (usually as early as the
contraction-phase of synapsis) to form rounded, condensed,
intensely-staining chromosome-nuclei. In this condition they
persist throughout the whole growth-period of the spermatocyte,
without ever assuming the looser texture and more elongate form
of the other chromosomes. In the earlier part of this period they
are as a rule closely associated with a large pale plasmosome, but
later become separated from it.
In the female no trace of such a chromosome-nucleus can be
found in the contraction-figure of the synaptic period. My best
preparations of this stage are from the ovaries of the larval Anasa,
which show a distinct synaptic zone of oocytes intervening between
the zone of multiplication and the growth-zone; but I have
observed the same condition in the ovaries of recently emerged
adults of Harmostes, Alydus, Euschistus, Coenus and Podisus.
In all these forms the contraction-figure is very similar to that of
the spermatocytes, the chromosomes being in the form of deeply
staining, ragged, and apparently longitudinally split loops that
are crowded into a spheroidal mass toward the center or one side
of the nucleus and surrounded by a large clear space. ‘The nuclei
at this time occasionally show one or two small deeply-staining
nucleolus-like bodies (probably plasmosomes); but these are
much smaller than the chromosome-nuclei of the spermatocytes
at this period, and in many of the nuclei are absent. ‘The contrast
between these nuclei and those of the male at the corresponding
period is so striking as to be at once apparent. In later stages the
chromosomes spread through the nuclear cavity, become looser
in texture and finally give rise to a fine reticular structure. In
1A fuller presentation of observations on these phenomena is reserved for a subsequent paper.
Studies on Chromosomes 23
these stages a variable number of deeply-staining nucleoli make
their appearance; but their true nature can only be determined
positively when the whole ovarian life of the egg has been followed
and the process of maturation observed. I can, therefore, only
state that no chromosome-nucleolus is present in the contraction
period of synapsis, or in the early growth-period; and even though
‘it be present in later stages, which I think is very doubtful, a wide
difference between the sexes would still exist in respect to the
earlier period.
F. General Resumé
The foregoing results may be given a general formulation as
follows: If m be the unreduced number of chromosomes in the
female, the matured eggs in all cases contain half this number (").
The males are of three types. In the first, one of the chromo-
somes (the heterotropic or “accessory’’) is without a mate, and
the unreduced number is accordingly one less than that of the
female. Half the spermatozoa possess, and half lack, the hetero-
tropic chromosome, the first class having the same number as the
matured eggs (%), the second class one less (?—1). Inthe second
type the male has the same number of chromosomes as the female,
but possesses one large and one small idiochromosome while the
female possesses two large ones. In maturation half the spermat-
ozoa receive the small and half the large idiochromosome. ‘The
third type differs from the second in that the idiochromosomes are
of equal size in both sexes, and no visible differences exist between
the two classes of spermatozoa or the somatic groups of the two
sexes. Designating the large and small idiochromosomes as [
and 7 respectively, the relations in fertilization and sex-production
are as follows:
TYPE I
(Protenor, Anasa, Atypus, HarMostes)
Egg % + spermatozoén % (including heterotropic) = (female).
Egg % + spermatozoén 7 — 1 (heterotropic lacking) = n — 1 (male).
TYPE II
(Lycarus, Euscuistus, Cornus, Popisus)
Egg ® (including I) + spermatozoén ® (including I) = n (including IZ) (female).
Egg * (including I) + spermatozoén ” (including i) = n (including Ji) (male).
24 Edmund B. Wilson
TYPE WT
(Nezara)
Egg ™ + spermatozoén ” = n (male or female, including in each case two equal idiochromosomes),
These relations are graphically shown in the following diagram
(Fig. 6) in which the differential chromosomes are black and the .
ordinary ones unshaded (only two pairs of the latter shown). For
the sake of simplicity only the final result of synapsis (second
column) and the ensuing process of reduction (third column) are
shown, without regard to variations of detail. “The matured eggs
(ov) are represented with a single polar body (the result of the
reduction-division) which is greatly exaggerated in size. The
female-producing and male-producing spermatozoa (sp) are
lettered a and b respectively. It will be evident from an inspection
of this diagram that the second type may readily be derived from
the third, and the first from the second by the reduction (second
type) and final disappearance (first type) of one of the differential
chromosomes. ‘This I believe to represent the actual relations
of the three types.
il GENERAL.
In recent years evidence has steadily accumulated to strengthen
the view that the general basis of sex-production is given by a
predetermination existing at least as early as the fertilized egg,
but there is a wide divergence of opinion in regard to the condi-
tions pre€xisting in the gametes prior to their union.!
The fact that in some organisms (such as Dinophilus, Hyda-
tina or Phylloxera) the unfertilized eggs, sometimes even in the
ovary, are visibly distinguishable as male- -producing and female-
producing forms, has led a number of recent writers to deny that
the spermatozo6n can play any part in sex-determination. Beard,
for example, asserts that “The male gamete, the spermatozo6n,
has and can have absolutely no influence in determining the sex
‘The general question of sex-determination, with its literature, has within the past five years
been so ably and thoroughly reviewed by Cuénot, Strasburger, Beard, von Lenhossék, O. Schultze
and others, that I shall here limit myself in the main to an analysis of the new observations brought
forward,
ee
Studies on Chromosomes 25
OGGoNnIA FERTILIZATION
AND SE — ~~
SPERMATOGONIA Gametes Spermatozoa Zygotes
‘a
|
es
=
we)
ay
@|>
I|
IT Lygaeus
®:
LF Nez ava
4
@ ©
=
26 Edmund B. Wilson
of the offspring” (02, p. 712); and a similar conclusion, though
less dogmatically stated, is reached in the general reviews of
Lenhossék (’03) and O. Schultze (03). The opposite view that
the spermatozoon alone is concerned in sex-determination (which
like the preceding one, 1s of very ancient origin) has, however, been
maintained by some recent writers, for instance, Block (whose
work I know only from Cuénot’s review) and McClung, as already
mentioned|. On the other hand, both Cuénot (’g9) and Stras-
burger (00) in their able reviews, have argued that both gametes
may be concerned in sex-determination; and the last named
author urged the view, afterward recognized as probable by
Bateson and developed in detail by Castle ('03), that sex-produc-
tion takes place in accordance with the Mendelian principles of
inheritance.
The observations here brought forward, together with those of
Stevens on Tenebrio, establish the predestination (in a descriptive
sense) of two classes of spermatozoa, equal in number, as male-
producing and female-producing forms. “Though indistinguish-
able to the eye in their mature state, these two classes differ visibly
in nuclear constitution at the time of their formation; and since
this occurs in the same order of insects as Phylloxera, where the
eggs are visibly distinguishable (by their size) as male-producing
and female-producing forms, it is evident that a substantial basis
now exists for the views expressed by Cuénot and Strasburger, and
for the Mendelian interpretation of sex-production worked out
by Castle. Whether in the Hemiptera that form the subject of
this paper the eggs are, like the spermatozoa, predestined as male-
producing and female-producing forms can at present be a matter
of inference only. I have not been able to distinguish such classes
by their size, and the data show, almost with certainty, that if
they exist they do not exhibit any visible nuclear differences
like those present*in the spermatozoa. But this gives no ground
for denying their existence. No visible nuclear dimorphism of
1“By exclusion then, it would seem that the determination of this difference (the sexual one) is
reposed in the male element’’ (McClung, *o2, p. 78). McClung nevertheless maintained the exist-
ence of a selective power on the part of the egg such that “the condition of the ovum determines
which sort of spermatozoén shall be allowed entrance into the egg substance”’ (op. cit., p. 76).
Studies on Chromosomes 27
the spermatozoa exists in Nezara, yet this condition is con-
nected, by an almost continuous series of intermediate forms,
with one in which a conspicuous difference of nuclear con-
stitution is to be seen. It seems hardly open to doubt that
sex-production conforms to the same essential type throughout
this series. At least a possibility is thus established that in
organisms generally both eggs and spermatozoa may be pre-
destined as male-producing and female-producing forms, whether
they are visibly different or not. In any case, it is evident
that in the Hemiptera the chromosome-combination characteristic
of each sex is established by union of the gametes and is a result
of fertilization by one or the other of the two forms of spermatozoa.
Sex must therefore already be predetermined in the fertilized egg,
and it is difficult to conceive how it could subsequently be altered
in these animals by conditions external to the egg or embryo.
Since the idiochromosomes or heterotropic chromosomes form the
distinctive differential between the nuclei of the two sexes, it is
obvious that these chromosomes are definitely coordinated with
the sexual characters. We must therefore critically inquire into
the causal relation between sex-production and the chromosomes,
of which this codrdination is an expression.
That sex-production may be interpreted as the result of a
Mendelian segregation, transmission and dominance of the sexual
characters has been shown by Castle (’03). ‘The history of the
differential chromosomes in synapsis and reduction evidently
affords a concrete basis for such an interpretation in the terms of
the Sutton-Boveri chromosome-theory. Analysis of the facts now
known will, however, show even more clearly than the more
general considerations adduced by Castle, that this interpretation
is only admissible under the assumption that a selective fertiliza-
tion occurs, such that eggs containing the female-determinant are
fertilized only by spermatozoa containing the male-determinant
and vice versa. Until I had read Cuénot’s recent interesting
paper (’05) on the breeds of mice and their combinations, the
necessity for making this assumption seemed to me an almost
fatal difficulty in the way of the interpretation, but if Cuénot’s
conclusions be well founded the a@ prior: objections to such a
28 Edmund B. Wilson
selective fertilization are in large measure set aside. I therefore
think that the possibility of a SVisadelen interpretation of sex-
production should be carefully examined, though as will be shown,
an alternative interpretation is possible.
I. In such an examination the distinction between sex-deter-
mination and sex-inheritance should be clearly drawn; for it is
well known that each sex may contain factors capable of pro-
ducing the characters of the opposite sex, and it may well be that
the patency or latency of the sexual characters is determined by
factors quite distinct from those concerned with their transmission
from parent to offspring. For the purpose of analysis it will,
however, be convenient to speak of the idiochromosomes or their
homologues as “sex-determinants,”’ this term being understood
to mean that these chromosomes are the bearers of the male and
female qualities (or the factors essential to the production of these
qualities) respectively. [hey may also be designated (whenever
it 1s desirable to avoid circumlocution) as sex-chromosomes or
“oonochromosomes.” As a basis of discussion the Mendelian
interpretation may be taken to postulate, further, that the two
sex-chromosomes, which couple in synapsis and are subsequently
disjoined by the reducing division, are respectiv ely male-determi-
nants and female-determinants in the sense just indicated. The
most convenient approach to the question is offered by the hetero-
tropic chromosome, since its unpaired condition in one sex renders
its mode of transmission more clearly obvious than that of the
idiochromosomes. ‘The facts (especially as observed in Protenor)
clearly prove that this chromosome alternates between the sexes
in successive generations, passing from the male to the female in
the production of females, and from the female to the male in the
production of males (Fig. 6). The important bearing of this
on both sex-inheritance and sex-determination will appear beyond.
Since the heterotropic chromosome is without a fellow in the
male it must, if it be a sex-determinant at all, be the male-determi-
nant, which exerts its effect uninfluenced by association with a
female-determinant. But since the spermatozoa that contain
1Cf. Watase, ’92.
Studies on Chromosomes 29
this chromosome produce only females, it must be assumed that
the maternal mate or fellow, with which it becomes associated on
entering the egg, is a dominant female-determinant. Further,
since sills Hone from fertilization by spermatozoa that do not
contain the heterotropic chromosome, the latter must in male-
producing eggs be derived from the egg-nucleus (c7. the diagram,
Fig. 6). [he general interpretation, ‘therefore, must include the
assumption that there are two kinds of eggs (presumably in
approximately equal numbers) that contain respectively the male-
and the female-determinant,' and that the former are fertilized only
by spermatozoa that lack the heterotropic chromosome (7. ¢., the
male determinant) and vice versa,’ giving the combinations (1m)j
(female) and m—(male). Such a selective fertilization 1s there-
fore a sine qua non of the assumption that the heterotropic chro-
mosome is a specific sex-determinant.
A nearly similar, though somewhat more complex, result follows
in the case of the idiochromosomes. In respect to sex-production
the large idiochromosome 1s identical with the heterotropic chro-
mosome, and the morphological evidence is nearly or quite
decisive that the heterotropic chromosome is actually a large
idiochromosome, the smaller mate of which has disappeared.
The small idiochromosome may therefore be regarded as a
disappearing, or even vestigial, female-determinant that 1s recessive
to its larger fellow (the male-determinant); and its reduction in
size may plausibly be regarded as an atrophy resulting from its
invariably recessive nature (this chromosome being strictly con-
fined to the male). Precisely as in case of the heterotropic
chromosome, the large idiochromosome of the male (male-
determinant) must be derived in fertilization from the egg-nucleus
(Fig. 6); and, as before, it must be assumed that eggs that contain
this chromosome are fertilized only by spermatozoa that contain
the small idiochromosome, those that contain the female-determi-
1This would follow from the coupling of the two sex-chromosomes in synapsis to form the bivalent
(m)f, and its division in such a way as to leave in the egg either the male- or the female-determinant
indifferently.
Otherwise the combinations mm or f— might result, which is contrary to observation, since the sex-
chromosomes are in this type never paired in the male or unpaired in the female.
30 Edmund B. Wilson
nant only by spermatozoa containing the large idiochromosome.
In this type, accordingly, it is clear that the large idiochromosome
(like the heterotropic chromosome to which it corresponds) passes
alternately from one sex to the other, while the small one never
enters the female; and this would remain true even did selective fer-
tilization not occur (Fig. 6). “The same interpretation may finally
be extended to Nezara, where the idiochromosomes are of equal size
in both sexes, the relations of dominance being the same as before.
The two vital points in this result are first, the assumption of
selective fertilization, and second the relations of dominance and
recession in the two sexes. As regards the first point, until the
appearance of Cuénot’s paper, referred to above, almost no
definite evidence had been produced of an infertility between
particular classes of gametes in the same species; though it has
long been known that many plants are in a greater or less degree
infertile to their own pollen, and an analogous fact has been more
recently demonstrated in Ciona by Castle (’96) and Morgan (’04).
Correns (’02), in his study of hybrid maize, was led to suggest
that in this case there might be a somewhat diminished fertility
between the gametes bearing the recessive character (thus account-
ing for a relative deficiency of extracted recessives in the second
generation of crosses, F,). In studying the breeds of mice
Cuénot has found it impossible to obtain pure or homozygous yellow
forms. Yellow mice are invariably heterozygotes (the yellow
being dominant over gray, black or brown) and when crossed
with a pure race of a different color (e. g., gray) give the typical
Mendelian result, yellow and gray offspring appearing in equal
numbers. ‘This proves that a complete Mendelian disjunction
of the yellow and gray determinants takes place in maturation.
When yellow mice of known constitution (e. g.. Y(G)) are paired
with like forms, the first offspring include pure gray forms (ex-
tracted recessives) slightly in excess of the normal ratio of 25 per
cent., and yellow forms; but contrary to the Mendelian expec-
tation the latter, when paired with one another, never give pure
dominants (YY), but again produce pure grays (GG) and
heterozygous yellows (Y(G)). Cuénot therefore concludes that
although complete segregation of both the gray and yellow
Studies on Chromosomes 31
characters takes place in the gamete-formation, and the resulting
yellow-bearing gametes unite freely with those bearing the reces-
sive color, they do not unite with each other: “Ceux-ci (the
yellow heterozygotes) forment bien des gametes de valeur C] ou
AJ, mais ces gametes ne peuvent pas s’unir les uns aux autres pour
donner des zygotes ayant les formules CJCJ, AJAJ ou CJAJ;
par autre, ils s’unissent facilement a tous les autres gametes que
jai essayés pour former avec eux des heterozygotes mono- ou
dihybrides” (op. cit., p. cxxx). This conclusion is sustained by
the fact that the combination Y(G) x Y(G) (CYCG x CYCG in
Cuénot’s terminology) produces a relative deficiency of yellows
in the offspring, as is to be expected.’ In pairing Y(G) with
Y(G), accordingly, the Y-bearing spermatozoa unite only with
the G-bearing eggs, and vice versa, which is exactly analogous
to the selective fertilization assumed in case of the sex-bearing
gametes. Perhaps it may be possible to find a different expla-
nation of the facts; but if Cuénot’s interpretation be well-founded
the case goes far to remove the scepticism which I think one must
otherwise feel in regard to a selective fertilization of the gametes
in sex-production.
An examination of the question of dominance involved in the
Mendelian interpretation leads to some interesting conclusions.
In forms possessing unequal idiochromosomes the sexual formulas
would be for the female (mm) f and for the male m (f) (f being the
small idiochromosome). Applying the same interpretation to
Nezara, where the idiochromosomes are of equal size, the corre-
sponding formulas are (m) f and m (f), giving the gametes (7m),
j, m and (f). Assuming likewise a selective fertilization the facts
would be:
Eccs SPERMATOZOA
(m) +. (f) = (m) (f), producing a male, m/(f).
i 3p = mf, producing a female (m) f.
1The deficiency, though constant, is very slight. Cuénot himself seems to consider this a difficulty,
but I believe a very simple explanation may be given. With equal numbers of the gametes of both sexes
the ratio of yellows to grays should be two to one, instead of three to one as in the typical Mendelian case
(since the class YY is missing). If, however, the spermatozoa be in large excess, as they undoubtedly
are, all or nearly all the Y-bearing eggs will be fertilized by G-bearing spermatozoa, and vice versa, thus
bringing the ratio of yellows (Y(G)) to grays (GG) more or less nearly up to three to one.
32 Edmund B. Wilson
Now it is clear that if the relations of the chromosomes to sex-
production be the same here as in the second type, the chromo-
some m must alternate in successive generations between the male
and the female (like the large idiochromosome or the heterotropic
chromosome to which it corresponds), and hence also shows an
alternation of dominance, being dominant in the former sex and
recessive in the latter. If, therefore, dominance and recession
be inherent in the chromosomes, there must be such a relation
between them that m is always dominant to the chromosome (f)
of the male, and always recessive to the chromosome 7 of the
female, and that the latter two chromosomes (/ and (/)) are never
interchanged between the sexes. ‘This last assumption 1s not so
improbable as it may at first sight appear; for in the second type
it 1s certain, as already pointed out, that the small idiochromo-
some ((f) under the general assumption) never enters the female,
while the large idiochromosome, m, like the heterotropic, alter-
nates between the two sexes in successive generations.
A strict Mendelian interpretation of Speduc ae may unques-
tionably, I think, be constructed upon the foregoing assumptions.
But an interesting suggestion for a somewhat modified Mendelian
interpretation is given by the possibility that the dominance of
the sex-chromosomes is determined by extrinsic factors, namely,
by conditions in the protoplasm of the zygote. If this were the
case it is evident that the idiochromosomes could not be considered
as sex-determinants in the strict sense of the word. ‘The determi-
nation of sex would in this case be due to factors preéxisting in
one or both of the gametes, irrespective of the sex-chromosomes,
and the latter could only be considered as a means by which the
sex-characters are transmitted or inherited. ‘The possibility is
here clearly offered that either or both forms of gametes may be
predetermined as males or females (or at least male-producing
and female-producing) prior to fertilization and irrespective of the
chromosomes; and thus an interpretation of the ordinary forms
of gametes would be reached in harmony with such cases as
Dinophilus and other forms in which male-producing and female-
producing eggs are distinguishable in size prior to fertilization.
Such an interpretation would further be perfectly consistent with
Studies on Chromosomes
5 33
the modification of sex-production in some cases by external condi-
tions, and with the production of both males and females in
parthenogenesis (though this may be otherwise explicable); and
it might also give the explanation of selective fertilization.
II. It has not been my intention to advocate the foregoing
interpretation, but only to set forth as clearly as possible, ihe as-
sumptions that it inv olves. It is nevertheless my opinion that the
analysis places no insuperable obstacles in its way, and that,
however dominance be determined, the Mendelian interpretation
may in fact give the true solution of the problem. I have, how-
ever, endeavored to seek for a different interpretation that may
escape the necessity for assuming a selective fertilization; and
although I have to offer nothing more than suggestions, some of
which undoubtedly encounter serious dithculties, I shall make
them in the hope that they may afford some clue to further inquiry.
Some of these suggestions are equally applicable to the Mendelian
interpretation considered above, but for the purpose of discussion
this interpretation may for the time be laid aside.
It seems possible that the differential chromosomes may per-
forma definite and special function in sex-production without being
in themselves specifically male-determining and female-determin-
ing or even qualitativ ely different save in Te degree of their special
activity (whatever be its nature). ‘his suggestion is given by
the fact that the presence of one heterotropic root toet or
large idiochromosome is associated with the production of a
male, while if two such chromosomes are present a female is
produced. ‘This very obviously suggests that the same kind of
activity that produces a male will if reinforced or intensified
produce a female; and with this would accord the production
of males from unfertilized eggs, and females from fertilized ones,
in the case of the bee. In these cases the decisive factor may be
a merely quantitative difference of chromatin between the two
sexes. But it is obvious that such a difference cannot give the
basis for a general explanation, since in Nezara, and presumably
in many other organisms, both the number of chromosomes and
the quantity of chromatin is the same in both sexes. And yet
34 Edmund B. Wilson
the existence of a quantitative difference in some cases raises the
question whether it is not the result or expression of some more
deeply lying nuclear difference which may still be present in those
cases where no quantitative difference exists. I find it altogether
incredible that two animals as nearly related as Nezara and
Euschistus should differ fundamentally in the relation of the
chromosomes to sex-production; and if there is any reason to
conclude that sex-determination is effected by the idiochromo-
somes (or by the combination of which they form a part) in the
case where they are visibly different, | cannot avoid the belief
that this conclusion applies with equal reason to the case in which
they appear to the eye alike in all the spermatozoa. It therefore
seems to me an admissible hypothesis that a physiological or
functional factor may be present that differentiates the spermat-
ozoa into male-producing and female-producing forms irrespec-
tive of the size of the differential chromosomes; and further,
that the morphological difference that has arisen in some forms
may have been a consequence of such an antecedent functional
difference. If we could assume for instance that the differential
chromosome-pair in the male includes a more active and a less
active member (the latter having in many cases become, reduced
in size or even having entirely disappeared) the suggestion might
be greatly extended in application. Under this assumption the
facts might receive a general formulation in the statement that
the association of two more active chromosomes of this class
produces a female, while the association of a more active and a
less active one (or the absence of the latter, as in case of the hetero-
tropic chromosome) produces a male. Reduction of the less
active member to form a-small idiochromosome would introduce
a quantitative difference of chromatin as well as a qualitative one.
Its complete disappearance in the male, leaving only the active
member as the heterotropic chromosome, would reduce the
difference to a merely quantitative one. The assumption of
such a physiological difference is admittedly a purely specula-
tive construction, and may seem 4a priori very improbable. But
from the a priori point of view it would seem equally improbable
that a morphological dimorphism of the spermatozoa, affecting
Studies on Chromosomes 35
OW
only one pair of the chromosomes, should have arisen; yet this
is an observed fact. I therefore think the suggestion is worthy
of serious consideration. If it could be adeered the necessity
of selective fertilization would be avoided, for the observed results
would follow from the fertilization of any egg by any spermatozoon.
But even if in accordance with fact the suggestion is still
obviously incapable of direct application to cases in which sex
is determined independently of fertilization—for instance, sex-
production in parthenogenetic development or in hermaphrodites,
and in forms (such as Dinophilus) where male-producing and
female-producing eggs are distinguishable in size before fertiliza-
tion. It is possible that these cases may be explicable (under either
general interpretation) as a result of some forms of differential
distribution of the chromosomes occurring at the time of the for-
mation of the polar bodies (parthenogenesis) or at some earlier
period in the cell-lineage of the germ-cells; and this possibility
should of course be tested by a close cytological study of the facts.
On the other hand, there is nothing in the facts to negative the
assumption that in some cases the chromosome-combination,
established at fertilization, may be in something like a balanced
state that is capable of modification by conditions external to the
nucleus (as already suggested in the case of dominance).
Boveri's interesting observations on the dispermic eggs of
Ascaris (’04) have given direct evidence that the chromosomes
react to their cytoplasmic surroundings; and the same fact is
even more clearly shown by the difference of behavior of the
differential chromosomes in the two sexes of Hemiptera during the
synaptic and growth-periods. Hence, even though a preéstab-
lished basis of sex-determination be given in such a physiological
dimorphism of the spermatozoa as i have suggested, the sex of
the fertilized eggs may in many cases be only a matter of greater
or less ciediap oats: and not an immutable predetermination.
The nuclei, and hence the primordial germ-cells, may in such
cases be in a state of approximate equilibrium, and still retain the
power of response to varying conditions in the cellular environ-
ment. The production of eggs or spermatozoa in hermaphro-
dites may thus be explicable as a result of greater or less nuclear
36 Edmund B. Wilson
activity in the two cases, incited by intra-cellular conditions that
are external to the chromosome-groups; and a similar explana-
tion may apply to the related case of the formation of visibly
different female-producing and male-producing eggs in the same
organism.
It would not, [ think, be profitable to speculate further in regard
to these special cases, but I have wished to indicate that a hypo-
thesis of sex-production which recognizes in some cases a fixed
predetermination in the chromosome-groups of the fertilized egg
is not inconsistent with the control of sex-production in other
cases by conditions external to the nucleus. The constant
chromosomal differences of the sexes existing in many Hemiptera,
therefore, by no means preclude experiments on the modification
or control of sex-production.
I have intentionally excluded from the foregoing suggestions
any discussion of the specific nature of the activities of the differen-
tial chromosomes, since we are almost wholly ignorant of the
functions of chromosomes in general. But although we here
enter upon still more debatable ground, I think we should not
hesitate to consider such possibilities in this direction as the facts
may suggest.
One of the principal, or at least most obvious, differences
between the germ-cells of the two sexes is their great contrast in
constructive activity, evinced by the enormous growth of the
primary oocyte as compared with the primary spermatocyte.
This growth of the odcyte involves the production of a mass of
protoplasm (including under this term the yolk or metaplasm as
well as the active protoplasm) thousands of times the bulk of the
spermatocyte; and although the latter also increases noticeably
in size during the growth-period, the accumulation of proto-
plasm is almost insignificant as compared with that which takes
place in the female. Now, as described above, the idiochromo-
somes and heterotropic chromosome remain during this period in
the male in a relatively passive condition as compared with the
other chromosomes, while this is not the case in the female. “The
thought cannot be avoided that there is a definite causal connec-
tion between the greater activity of these chromosomes in the
Studies on Chromosomes 27
odcytes and the great preponderance of constructive activity in
these cells; and it is especially this coincidence that leads me to
the general surmise that one of the important physiological
differences (I do not say the only one), between the chromosome-
groups of the two sexes, may be one of constructive activity.
I have elsewhere (The Cell, Chapter VII) reviewed at some
length the evidence pointing toward the conclusion that the
nucleus (more specifically, the chromatin) 1s especially concerned
with the constructive processes of cell metabolism; and while I no
longer hold the view that the nucleus can be considered as the
actual formative center of the cell, it still seems to me very
probable that the formative processes are directly or indirectly
under its control, as has been advocated by many students of
cell-physiology. If this view be well-founded, the facts observed
in Hemiptera give a very definite and concrete basis for assuming
a greater constructive activity in the cells of the female generally,
which reaches a climax in the growth-period of the odcyte.’ It
seems possible that some of the specific differentiations that take
place in the later history of the germ-cells may be directly trace-
able to the primary difference in the growth-process. It is well
known that the young odcytes and spermatocytes show a very
close similarity, not only in size but also in many details of struc-
ture. [he enormous accumulation of cytoplasm in the odcyte
as compared with the spermatocyte leaves the latter with a great
relative excess of the kinoplasmic or archoplasmic material in which
the most characteristic differentiations of the spermatozoa—such as
the acrosome, middle-piece, axial filament and tail-envelopes—
take their origin. Perhaps a direct causal relation here exists.
1This suggestion recalls the theory developed by Geddes and Thomson, in their well known work on
the ‘‘ Evolution of Sex,” that “the female is the outcome and expression of relatively preponderant anabo-
lism, and the male of relatively preponderant katabolism” (of. cit., revised ed., 1901, p. 140). As de-
deloped by these authors, this theory has always seemed to me to have too vague and general a character
to have much practical value, though it expresses a certain physiological contrast between the sexes that
undoubtedly exists. My suggestion is only remotely connected with that theory, since it refers the differ-
entiation of the sexes to a functional difference that preéxists in the cells of the male, and involves no
contrasted processes of anabolism and katabolism. Nevertheless, the observations here brought forward
may harmonize with that side of the theory which lays stress on the preponderant constructive activity of
the female cells.
38 Edmund B. Wilson
III. Though I have found it convenient to consider the two
foregoing interpretations separately, they evidently have many
points of agreement, and perhaps may be reduced to a common
basis. Both assign to the differential chromosomes a specific
function in sex-production, both recognize the possibility of a
determination of sex (as opposed to its transmission), by con-
ditions external to the chromosome-groups, and both assume,
in one sex, a specific difference in the sex-chromosomes, followed
by a Mendelian disjunction in the formation of the gametes.
The essential point in which the second interpretation diverges
from the first is that the sex-chromosomes are not conceived as
bearing the male or female qualities respectively but as differing
only in the degree of their activity, and this difference is assumed
to exist in the male only (owing to the relation of fertilization
to sex-production). It must be admitted that each interpreta-
tion involves a considerable element of pure conjecture, and that
each includes assumptions which without additional data must
be considered.as sericus difficulties. The principal one involved
in the first interpretation is the assumption of selective fertiliza-
tion; but if this assumption be granted I believe that it may give
an adequate solution of the problem of sex-production in the sexual
reproduction of divecious organisms. ‘The second interpreta-
tion avoids this difficulty; it may explain the primary difference
between the gametes of the two sexes, the latency of female
characters in the male, and the development of such secondary
female characters as may be regarded as an exaggeration or inten-
sification of corresponding characters in the male. It seems con-
spicuously to fail to explain the reverse case of characters that
are more highly developed in the male; and to many this will
doubtless appear a fatal difficulty. But we are still ignorant of
the action and reaction of the chromosomes on the cytoplasm and
on one another, and have but a vague speculative notion of the
relations that determine patency and latency in development.
Additional data will therefore be required, I think, to show
whether the difficulty in question is a fatal one, and in what meas-
ure either of the two general interpretations that have been con-
sidered may approach the truth. ‘The positive result of the
Studies on Chromosomes 39
observations of Stevens and myself is to demonstrate the existence
of a constant and definite correlation between the chromosomes
and the sexual characters, which is visibly expressed in the relations
of a single pair of chromosomes. ‘These relations unquestionably
afford a concrete basis for an interpretation of sex-production
that assumes a Mendelian segregation and transmission of the
sex-characters and to this extent they accord with the general
assumption of Castle. The validity of both this and the alterna-
tive interpretation suggested must be tested by further inquiry.
Zodlogical Laboratory of Columbia University,
December 8, 1905.
WORKS CITED
Beard, JOHN, ’02.—The Determination of Sex in Animal Development. Fischer,
Jena.
Bovert, I. H., ’04.—Protoplasmadifferenzierung als auslosender Faktor ftir Kern-
verschiedenheit. Sitzungsber. der Physikal.-med. Ges. Wirz-
burg, 1904.
CastLe, W. E., ’96.—The Early Embryology of Ciona intestinalis. Bull. Mus.
Comp. Zool., xxvii.
’03.—The Heredity of Sex.. Jbid., xl, 4.
Correns, C., ’02.—Scheinbare Ausnahme von der Mendel’schen Spaltungsregel
fur Bastarde. Ber. d. deutschen Bot. Ges., xx.
Cuénot, L., ’99.—Sur la détermination du sexe chez les animaux. Bull. Sci. de
la France et de la Belgique, xxxii, v, 1.
’05.—Les races pures et leurs combinaisons chez les souris. Arch. Zool.
Exp. et Gén. (4), i11, Notes et Revue, No. 7.
Gross, J., °04.—Die Spermatogenese von Syromastes marginatus. Zool. Jahrb.,
Anat. und Ontog., xx, 3.
HenkKING, H., ’91.—Ueber Spermatogenese und deren Beziehung zur Eientwick-
lung be: Pyrrochoris apterus. Zeitschr. Wiss. Zool., li.
Lennosskk, M. v., ’03.—Das Problem der geschlechtsbestimmenden Ursachen.
Fischer, Jena.
McCiune, C. E., ’02.—The Accessory Chromosome. Sex-determinant/ Biol.
Ball? ant, 2:
Montcomery, T. H., ’o01.—A Study of the Germ Cells of Metazoa. ‘Trans.
Am Phil Soc:, xx:
*o4.—Some Observations and Considerations on the Maturation Phenom-
ena of the Germ-cells. Biol. Bull., vi, 3.
40 Edmund B. Wilson
Morcan, T. H., ’04.—Self-fertilization Induced by Artificial Means. Jour.
Exps Zool. a).
ScHULTZE, O., ’03.—Zur Frage von den geschlechtsbildenden Ursachen. Arch.
mik. Anat., Ixiil.
Stevens, N. M., ’05.—Studies in Spermatogenesis with especial Reference to the
“Accessory Chromosome.” Publication No. 36, Carnegie Insti-
tution of Washington, Sept., 1905.
STRASBURGER, E., 1900.—Versuche mit didcischen Pflanzen in Ricksicht auf
Geschlechtsverteilung. Biol. Centralbl., xx, 20-24.
Sutton, W. S., ’02—On the Morphology of the Chromosome-group in Brachy-
stola magna. Biol. Bull., iv, 1.
Wa tace, L. B., ’05—The Spermatogenesis of the Spider. Biol. Bull., viii, 3.
Watasg, S., ’92.—On the Phenomena of Sex-differentiation. Journ. of Mor-
phology, vi, 3.
Witson, E. B., ’05, 1.—Studies on Chromosomes. I. The Behavior of the Idio-
chromosomes in Hemiptera. Journ. Exp. Zool., ii, 3.
*05, 2—The Chromosomes in Relation to the Determination of Sex in
Insects. Science, xxii, 564, Oct. 20, 1905.
’05,2. Studies on Chromosomes. II. The Paired Microchromosomes,
Idiochromosomes and Heterotropic Chromosomes in Hemiptera.
Journ. Exp. Zool., 1, 4.
AN EXAMINATION OF THE EFFECTS OF MECHANI-
CAL SHOCKS AND VIBRATIONS UPON THE RATE
OF DEVELOPMENT OF FERTILIZED EGGS
BY
DAVID DE WHEL NEY
It has been shown in recent years by several investigators that
mechanical shocks and vibrations may start the development of
unfertilized eggs of certain animals.’ It has also been stated by
Meltzer? that the early development of fertilized eggs of the sea
urchin is greatly accelerated when the eggs are subjected to me-
chanical shocks and vibrations. Furthermore, Mathews and
Whitcher® believe they have obtained results which show that such
influences may cause the embryos of the sea urchin to be either
larger or smaller than those of the control eggs, or abnormal in
shape, or to develop more slowly than normal eggs.
Assuming these results to be well founded, I undertook, during
the summer of 1905, some experiments to determine the influence
of shaking at different stages of development. In order to exclude
the obvious possibility, that the results of Meltzer, and perhaps
those of Mathews and Whitcher, were due to differences in tem-
perature, care was taken to make this factor the same in the shaken
eggs and in the control. Insomecasesthis was done by means ofa
water jacket around bothsetsofeggs. Itsoon became apparent that
no difference at all im the rate occurred when this precaution was
carried out; so that the work resolved itself into testing the claims
of these investigators respecting the influence of shock on the
early stages of development.
At the suggestion and under the kindly supervision of Prof.
‘Mathews: Amer. Jour. Physiol., rgo1, vi, p. 142. Fisher: Amer. Jour. Physiol., 1902, vii,
p- 301.
"Meltzer: Amer. Jour. Physiol., 1903, ix, p. 245.
*Mathews and Whitcher: Amer. Jour. Physiol., 1903, viii, p. 301.
Journat or Exprrimentat Zodiocy, Vor. m1, No. 1.
42 David D. Whitney
T. H. Morgan, I have studied the influence of mechanical agita-
tion and vibration upon the fertilized eggs of Arbacia, Asterias,
Fundulus heteroclitus and Ctenolabrus.
Of the fertilized eggs of Arbacia twenty-five lots from six
individuals were shaken from one to forty-five minutes. Some
were shaken by hand and others by means of a small hot-air
engine. [he eggs were placed in a test-tube half full of sea water,
which in some cases contained small pebbles about the size of an
ordinary pin head; in other cases water alone was present. The
tube was then shaken back and forth one hundred to three hundred
times per minute.
Six other lots from two other individuals were placed in a thick
walled test-tube which swung vertically, and, by the use of the
small engine, the bottom of the tube was made to strike quite hard
against the side of a board from one hundred and fifty to four
hundred times a minute.
In all cases the temperature of the control was the same as that
of the shaken eggs. When the engine was employed for shaking,
the test-tube was kept under running sea water, but when shaken by
hand it was wrapped up in wet cloths. ‘The eggs of the controls
were also kept under similar conditions. When taken from the
test-tube, the eggs were all kept at room temperature, 23°-26° C.
Thus the temperature of the shaken and non-shaken eggs was
always exactly the same.
After fertilization, the eggs were usually allowed to stand three
to five minutes before using. In some cases, however, they were
shaken immediately after fertilization. In other cases they were
allowed to stand from five to twenty-five minutes.
In all these thirty-one sets of shaken eggs from eight individuals
no acceleration of cleavage was noted in the earlier stages, nor
were the plutei ever larger than those of the controls. In many
cases the majority of the embryos of the shaken eggs were small
and abnormal in shape, and were swimming upon the bottom of
the dish, thus corroborating some of Mathews’ and Whitcher’s
observations. ‘This was especially true if the eggs were shaken in
the test-tube with the small pebbles. Usually a few large plutei
were found in both the controls and the shaken lots; but in no one
Effects of Mechanical Shocks upon Development 43
of these lots did there seem to be more large plutei than in the
other.
The following experiments will illustrate the general character
of the results obtained from Arbacia:
Experiment I. “fuly 1r—Temperature of sea water 23° C.; of
room 25°C. All lots of eggs, after fertilization, were kept at 23°
C. for forty-five minutes, and were then placed in finger bowls of
water at room temperature.
Lot A. Control. Great care taken in handling the eggs so as
to prevent any jarring or shaking.
Lot B. Three minutes after fertilization the eggs were placed
in a test-tube the bottom of which struck for forty-five minutes
against the side of a wooden box two hundred and seventy-five to
three hundred and fifty times a minute.
Lot C. ‘Ten minutes after fertilization. Shaken five minutes
back and forth two hundred and fifty times a minute in a test-tube
half full of water plus some small pebbles.
Lot D. Immediately after fertilization. Shaken five minutes
in same manner as lot C.
The early stages and the plutei of lots B, C and D were like
those of the control.
Experiment II. “fuly 1r.—Conditions and results the same as
in I.
Experiment III. “fuly 12.—Yemperature the same as in [
and JT.
Lot A. Control.
Lot B. Shaken thirty minutes back and forth two hundred and
forty times per minute, in a test-tube half full of water. “The tube
was in a jacket of running water.
Early cleavages and the plutei of B were the same as A.
Experiment VI. “fuly 13.—Temperature of sea water 23° C.;
of room 26° C.
Lot A. Control.
Lot B. Shaken five minutes back and forth two hundred times
a minute in test-tube half full of water plus some small pebbles.
Lot C. Shaken one minute back and forth three hundred
times per minute in same manner as B.
44 David D. Whitney
Early segmentation of all three lots the same.
Fuly 16.—Lot A. Normal plutei swimming through water in
the dish.
Lot B. Normal plutei swimming upon the bottom of the dish.
Lot C. Normal plutei. A few swimming through the water,
and the remainder swimming upon the bottom of the dish.
Experiment VIII. ‘uly 13.—Lot A. Control.
Lot B. Shaken three minutes by hand back and forth three
hundred times per minute in a small vial half full of water plus
small pebbles.
Lot C. Shaken five minutes in same manner as B.
B and C were one-half hour later than A in showing the first
cleavage.
Fuly r7.—Lot A. Normal plutei swimming through water in
a dish.
Lot B. Very few normal plutei. Mostly gastrule, and many
of them abnormal. Both plutei and gastrula were swimming upon
the bottom of the dish.
Lot C. Normal and abnormal plutei swimming upon bottom
of dish.
Experiment XII. “fuly 17,—The sea urchins were kept in an
ice chest at a temperature of 9° C. from five to six hours. ‘Then
the ovaries were quickly removed to water at 9° C. and fertilized
by the sperm that had been shed by the males while in the ice
chest.
Lot A. Control at 9° C. for thirty minutes.
Lot B. Shaken thirty minutes back and forth two hundred
and ten times per minute, in a test-tube half full of water. The
tube was in an ice water bath at 3° C.
Lot C. Shaken one minute back and forth three hundred
times, at a temperature of 11° C. They were allowed to remain
at this temperature for thirty minutes. At the expiration of this
time, the three lots were placed in room temperature of 25° to
262°€-
The early segmentation and normal plutei of the three lots
appeared at the same time respectively.
Some eggs were placed at room temperature—25° to 26° C.—
Effects of Mechanical Shocks upon Development 45
and others from the same lot were put under running sea water
at a temperature of 23° C. ‘The eggs at room temperature devel-
oped in the early segmentation one stage in advance of those
which were under running water.
In all the experiments great care was taken in handling the
control eggs, both before snd after fertilization, so as to prevent
jarring which might influence the rate of development of the con-
trol eggs in such a way that it would be identical with the eggs
intentionally shaken.
Meltzer shook the eggs of Arbacia by hand and also by means
of the piston of a stationary engine. He also placed them in
dishes on the vibrating part of the same engine. He does not
state whether the eggs of the control were kept at the same tem-
perature as those that were being shaken, but only states that the
temperature of the room was slightly higher than the outside
temperature. I have made careful records of the temperature of
the places in the engine house where he performed his experi-
ments. If the conditions then were the same as they are now, all
his results can be explained by the different degrees of tempera-
ture to which the eggs were subjected.
The eggs that he shook by hand in small vials containing small
glass beads developed faster. [he temperature in such a case
would be increased by the friction of the beads and water, and by
the warmth of the hand, in a remarkably short space of time.
I found that the temperature in the test-tube which struck
against a board, when not kept under running water, was raised
two to four degrees if the experiment was continued for fifteen to
twenty minutes.
Meltzer may have avoided all these difficulties and have kept
all the eggs of a series at the same temperature, but he does not
state this; and from what he does say, he seems to have paid little
attention to temperature and consequently concluded that the
acceleration of development which he obtained was due to vibra-
tion and shaking. I find on the contrary, that by keeping all the
eggs of a series at the same temperature no acceleration of develop-
ment can be obtained by subjecting them to a slight or even to a
great amount of mechanical agitation or vibration.
46 David D. Whitney
Fertilized eggs of Asterias subjected to mechanical shocks gave
only negative results. About thirty-five lots from twelve individ-
uals were used. ‘The apparatus employed for producing the
shocks was a thick glass test-tube, the bottom of which struck
against two boards from two hundred to three hundred and fifty
times per minute—the same that was used in connection with the
eggs of Arbacia.
‘The eggs were kept in this tube, which was constantly in motion,
from fifteen minutes to seven hours. Some were subjected to
shock immediately after fertilization, and others remained undis-
turbed from five to thirty minutes before being placed in the tube.
‘The temperature was kept uniform in all lots of eggs of the same
experiment.
When many of the eggs had reached the eight-cell stage, about
two hundred of them were selected at random, and the percentage
of the eggs in the various segmentation stages was determined by
counting. ‘The shaken eggs usually varied from I to 3 per cent.
either above or below the percentage of the same stages in the non-
shaken lots. As the percentage varied so slightly, sometimes
above and sometimes below that of the control, I concluded that
the segmentation was neither hastened nor retarded by mechani-
cal shocks.
The eggs of Fundulus heteroclitus were also placed in the test-
tube of this apparatus and subjected to slight and to severe shocks,
from a few minutes to ten hours. About forty lots—from
twenty to twenty-five individuals—were used. As the early
development of these eggs was found to take place normally in a
damp chamber, some of them were placed in a test-tube, the air
of which was kept moist by a piece of wet filter paper, and sub-
jected to shocks. Similar results were obtained from both
methods.
A few lots were placed in a test-tube half filled with water and
the tube was made to move back and forth in a horizontal position
from two hundred to three hundred and fifty times per minute.
In none of these experiments did the early cleavage stages of
the shaken eggs appear earlier than in those of the controls.
As the eggs of Ctenolabrus float upon the surface of the water,
Effects of Mechanical Shocks upon Development 47
the test-tube was inverted, and its upper end made to strike
against boards, as in the former experiments. In some of the
experiments the tube struck against the boards gently, and in
others it struck severely enough to kill many of the eggs. Inter-
mediate shocks were also tried.
About thirty lots—from fifteen to twenty individuals—were
used and were subjected to shocks from five minutes to several
hours. In all these experiments the early cleavages appeared at
the same time both in the controls and in the shaken eggs.
From the foregoing observations it appears that mechanical
shocks and vibrations are not effective in accelerating the early
segmentation of the fertilized eggs of Arbacia, Asterias, Fundulus
and Ctenolabrus.
These observations were made at the Marine Biological Labora-
tory at Wood’s Hole, while occupying one of the tables of the
Carnegie Institution.
MORPHOLOGY OF THE PARTHENOGENETIC
DEVELOPMENT. OF “AMPHITRITE
BY
JOHN W. SCOTT
Wiru Four Piates anp Five Ficures IN THE TEXT
INTRODUCTION
I. General Statement as to Object of Work and Results Obtained
The subject for the following investigation was suggested to
me by Dr. F. R. Lillie early in the summer of 1902. Some recent
statements by Loeb and others, in regard to the effects of certain
salt solutions upon the development oe the eggs of some marine
Annelids, had led Lillie in the previous year to make a series of
observations upon the egg of Cheetopterus. Lillie’s chief purpose
in making such a series of experiments was, “To test what was
the swnineames of cleavage in the egg, and what was the role of
cell division in ‘eclosanente * He arrived at the following general
conclusion: “The process of cell division, as such, is necessary
neither to growth, differentiation, nor to the earliest correlations;
but it is accessory, in Metazoa, to all three as a localizing factor,
often from the earliest stages.” Lillie made the suggestion that
it would be well to test the questions raised in his paper in some
other marine Annelids.
My experiments upon the eggs of Amphitrite, however, have
involved conditions not met in the egg of Chztopterus and
necessarily other questions have come up for solution. In start-
ing out | had in view, among others, the following considerations:
1. Using the methods adopted by previous investigators to pro-
duce artificial parthenogenesis, was it possible to syne ae differen-
1A dissertation submitted to the faculty of the Ogden Graduate School of Science in candidacy for
the degree of Doctor of Philosophy, Department of Zodlogy, The University of Chicago, June, 1904.
Journat or ExperimentaL Zoétocy, Vor. ur, No. 1.
50 ‘fobn W. Scott
tiation without cleavage in the unfertilized eggs of Amphitrite?
2. May such differentiation be produced in the fertilized eggs?
3. Can normal trochophores or normal adult worms be raised
from unfertilized eggs? 4. If so, how are abnormalities appear-
ing in the early stages regulated to produce a normal embryo?
In other words, my object was to learn if possible how this sort
of parthenogenetic development was related to the normal.
From observations on living eggs and from material preserved
during the summer of 1902, I succeeded in demonstrating:
1. That differentiation without cytoplasmic cleavage may occur
in the unfertilized eggs of Amphitrite. (Scarcity of material
prevented testing this question for fertilized eggs.) Cleavage,
when present, is usually abnormal and is always so in the later
stages. 2. Strictly speaking, this kind of development cannot
be termed parthenogenesis, for the differentiation so resulting
never leads either to normal or to abnormal self-sustaining
organisms. However, I shall use in this paper the term partheno-
genesis in a restricted sense to indicate the development that is
initiated in the eggs of Amphitrite by treatment with salt solutions
and by agitation. 3. There is no correlation or regulation of
organs in the later development under the conditions of the experi-
ment. 4. No specific solution was necessary to produce partheno-
genesis as was claimed by Fischer (’02), though some solutions
apparently do not have any effect upon the egg. 5. The egg was
found to be extremely susceptible to agitation or shaking, espe-
cially at certain periods after it had been removed from the body
cavity. 6. It was learned from a study of the preserved eggs
that the parthenogenetic development was closely connected with
the method and extent of the nuclear division and the chromatin
distribution and that much depended upon the ripeness of the egg.
Considering these results, I found it necessary during the fol-
lowing summer to make an examination of the very early
development, both normal and parthenogenetic. In the normal
egg this included a study of the origin and number of chromo-
somes, the maturation, and fertilization, none of which had been
described for Amphitrite. Corresponding stages in the parthen-
ogenetic eggs were also preserved and studied. In general, it
Parthenogenetic Development of Amphitrite 51
has been found that the early development of the fertilized
Amphitrite egg conforms to that of the typical Annelid, while in
the later stages, so far as they were studied, I have confirmed the
observations of Mead (97). With the unfertilized eggs, on the
other hand, the form of development varies considerably under
different conditions. The polar bodies may or may not be
expelled, and when expelled they may be normal or abnormal.
Cleavage likewise may be present or absent, and is always
abnormal in late stages. I have found also that the principal
conditions which produce variations in the form of development
are the state of ripeness of the egg and the kind and strength of
the solution used.
2. Methods
For a normal series the usual method of adding sperm to the
dish of eggs was employed. ‘The care and precautions necessary
in manipulating and handling the unfertilized eggs have been
described in a previous paper. Boveri's picroacetic (dilute) and
Kleinenberg’s picrosulphuric were used as killing fluids, the
former giving the better results. “The eggs were preserved in 80
per cent alcohol until used. ‘
From each lot of eggs whole mounts and sections were prepared.
For whole mounts Conklin’s hematoxylin method gave fairly
satisfactory results. ‘This solution was prepared by adding 20
parts distilled water to 5 parts Delafield’s hematoxylin; to each
5 cc. of this mixture was added one drop of Kleinenberg’s (undi-
luted) picrosulphuric acid. The best results were obtained when
eggs were stained for twenty minutes and afterward washed for
about one hour in frequent changes of 50 per cent alcohol.
Several stains were used for sections, but the best results were
obtained by staining three to five minutes in strong Delafild’s
hematoxylin followed by from thirty to fifty seconds in Orange
G. The iron-alum method proved unsatisfactory on account
of the great amount of yolk present. For the same reason it
was found necessary to cut the sections 7 to IO micra in
thickness.
he ‘fohn W. Scott
NORMAL HISTORY OF THE EGG
I. Periods of Maturation and First Cleavage
I have tried to determine just when and where the germinal
vesicle breaks down under normal conditions; whether this occurs
after the egg is deposited in sea-water or while the egg is still
floating in the body cavity. ‘The latter view is correct as shown by
the following facts: Eggs from the ripest female that I obtained
in two seasons’ work were deposited between 6.15 and 6.50 P. M.,
while I was away from the laboratory. At 6.54 P. M. some of
these eggs were fertilized and a part of the lot was preserved.
All of those preserved were in the metaphase, with a few excep-
tions that showed more advanced development. ‘This could
not have happened if the vesicle breaks down only after the egg is
in the sea-water, for the eggs are deposited a few at a time at each
rhythmic peristalsis of the worm’s body. In other experiments
where the eggs were cut out of the body of the female and left
unfertilized, I have noted that the germinal vesicle may break
down within a few minutes, but frequently does not until several
hours later. In any case a peri-vitelline space forms and the egg
flattens or undergoes collapse at the animal pole, the spindle rests
in metaphase and sperm are very rarely found within the cyto-
plasm until after this phase. In fertilized eggs the first polar
body makes its appearance about ten minutes later. ‘This is
followed after an interval of about eight minutes by the second
polar body. I have seen the second polar globule thrown off
within twenty-four minutes after the eggs were deposited and the
first cleavage occurs eighteen to twenty minutes later. The
periods here given are minimal; they may be much longer and
are affected by ripeness of the egg, fertilization, agitation, certain
chemical agents, and probably other causes.
2. Orientation of the Egg. Origin and Number of the
Ch rormosomes
The rather large germinal vesicle in the ripe egg of Amphitrite
has a slightly eccentric position, near the animal pole. The yolk
Parthenogenetic Development of Amphitrite 53
next the vegetative pole is somewhat denser than it is in the region
above the nucleus. This eccentric position of the germinal
vesicle is present in very immature eggs just after they become
free in the body cavity, when the egg consists simply of a germinal
vesicle and a surrounding thick (ares of cytoplasm in which no
trace of yolk can be fans The nucleolus has no definite position
within the nucleus.
As the egg increases in size by the growth of the nucleus and
the deposition of yolk, the denser portions of the latter substance
are found in the immediate neighborhood of the nucleus; the
yolk found nearer the surface of the eg ge 1s composed of small
granules and there is not so much deposited in the upper side
of the egg as at other places. At this stage dark masses or
patches that present a fiber-like appearance are frequently found
in the cytoplasm; judging from the staining reaction these are
probably portions of the undifferentiated reticulum. ‘The ger-
minal vesicle now shows a definite reticulum with small micro-
meres at the intersections. When the egg is near the mature
condition chromatin granules, or chromomeres, are found collected
in various groups. The reticulum of the nucleus has nearly dis-
appeared. In eggs which seemed to be ripe and in which the
germinal vesicle was on the point of breaking down, chromatin
groups were found present in the nucleus and the chromatin
granules were apparently fusing to form the chromosomes.
Peculiar difficulties have interfered with getting the stages
immediately succeeding those just mentioned, stages that show
the exact origin of the chromosomes and asters. The next stage
found in my preserved material, where the eggs had been deposited
as a result of the rough handling of the female, is represented in
Fig. 1. One distinct aster is present which seems to have arisen
within the nuclear area, and a few radiating fibers point to the
origin of the other aster somewhat nearer the animal pole. ‘The
germinal vesicle has apparently just broken down. A part of
the contracting, fading nucleolus is still visible, and the chro-
mosomes or probably chromomeres are in groups or strings. In
the egg of Fig. 2 both asters are well formed, the chromosomes
are found in scattered groups, and the nucleolus is still intact.
54 ‘fohn W. Scott
The process next observed is the collecting of chromatin to
form the prophase of the first maturation division. While this
is in progress the asters enlarge, the centrospheres make a rapid
growth, and a symmetrical spindle develops and rotates to take
its definitive position. ‘The chromomeres or chromosomes have
become quite numerous and there are strong indications that they
are arranged in eleven groups of four each. In Fig. 3 the clear
area shows the original position of the nucleolus which has dis-
appeared; some of the chromosomes are fusing. In Fig. 4 the
spindle is better formed, the chromomeres are collecting near the
equator, and some show appearances of fusing.
In describing the maturation and fertilization of another
marine annelid, Arenicola, Child figures and describes a “nuclear
cavity.” I have found the same appearance in the eggs of Amphi-
trite but my sections have enabled me to prove that the clear area,
or “cavity,’’ represents the original position of the nucleolus.
The latter in disappearing gradually shrinks (Fig. 1), grows
irregular in shape, and apparently goes into solution leaving a
clear area (Fig. 3).
In early metaphase it is usually possible to count eleven chro-
mosomes in the spindle (Fig. 5). A polar view of the chromo-
somes in the early prophase shows that the chromomeres have
not all fused. “The reduced number of chromosomes I have
found to be eleven; this count was made at a stage shortly after
the expulsion of the second polar globule (Fig. 9). At a later
stage in one of the cleavage cells twenty-two were counted as the
entire number.
3. Nuclear Changes in Maturation
The nuclear phenomena of maturation in Amphitrite are in
general like those of Chztopterus as described by Mead. The
first aster makes its appearance after the germinal vesicle breaks
down and is first seen in a position that represents a lateral por-
tion of the upper hemisphere of the nuclear area. “The second
aster appears a little later, near the surface of the egg, and lies
directly under the animal pole (Fig. 1). Both asters are now
growing rapidly, though the second aster always remains behind
Parthenogenetic Development of Amphitrite 55
the first in size. The spindle forms and the chromosomes arrange
themselves in prophase; but before this process is complete the
spindle swings around, and when the metaphase is reached it has
migrated to the surface where it rests in its definitive radial posi-
tion (Fig. 5). At this time the spindle is long and tapering.
If not disturbed in any way, practically all eggs will remain indefi-
nitely in this condition unless fertilized. But if slight agitation
or certain chemical stimuli be applied further development may
occur. When fertilized other changes make their appearance
within five or six minutes. The chromosomes quickly pass
through the anaphase, and at the time of early telophase the asters
as well as the spindle fibers begin to fade while the spindle itself
has become shorter and barrel-shaped (Fig. 6). In later telo-
phase when the first polar body is being constricted off, the
chromosomes of the inner group have approached still nearer the
surface and remnants of the spindle and inner aster have almost
entirely disappeared (Fig. 7). here are stages just later than
this when no traces of this aster can be discovered. Very soon the
second maturation spindle arises nearly parallel to the surface,
and the chromosomes which have receded a little arrange them-
selves in prophase. Frequently at this time the pole above this
region shows a flattening or slight concavity and as the spindle
turns the chromosomes pass into metaphase. The rest of the
process in throwing off the second polar globule-is similar to that
described for the first. Differences, however, have been
observed. The spindle is smaller than the first and the rays of
the aster remaining in the egg, instead of fading out in telophase,
extend further into the cytoplasm, persisting until a later stage
(Figs. 8 and 9).
4. Relation of Cytoplasmic to Nuclear Phenomena in Maturation
The ripe egg with germinal vesicle intact gives no indication of
the animal pole if judged simply by its shape. But when the
germinal vesicle breaks down a rapid and remarkable change
takes place in the shape of the egg, during which the egg undergoes
collapse or flattening in the polar diameter. Unless fertilization
is accomplished most eggs remain indefinitely in this condition,
56 fobn W. Scott
with the first maturation spindle in metaphase. However one or
both polar bodies may be thrown off and, as Mead has pointed
out, some eggs may pass through the early cleavage stages. ‘The
rapid change just mentioned in the shape of the egg is interesting
when considered in connection with the coincident movement of
the spindle to the surface. “The camera lucida drawings of Text—
Fig. I. Diagrams to show changes in shape of eggs during expulsion of polar globules. Collapse
in the polar diameter to show how first maturation spindle comes to the surface, in A (typical), and B
(rather infrequent). First polar body being expelled in C, egg regaining spherical shape; D, anaphase
of the second maturation spindle, the egg nearly spherical. Camera lucida. Leitz oc. 1, obj. 1-12.
Fig. | show conditions found at this time. ‘The typical condition
is shown in Fig. I, 4, where the polar diameter is to the transverse
diameter about in the proportion of three to five. An extreme but
frequent condition is shown in Fig. I, B. In both figures there is
represented a thin ectoplasmic layer; an oval or elongate area,
marking the region of the germinal vesicle; the spindle in meta-
Parthenogenetic Development of Amphitrite 57
phase occupying a radial position, lying within a differentiated
area in which there is very little yolk; the area in question being
in direct communication with or lying partly within the original
nuclear area.
The explanation of these phenomena is apparently as follows:
After the germinal vesicle breaks down, the nuclear sap begins to
diffuse out through the cytoplasm in which are many yolk oranules,
and staining reactions show that it very soon reaches the surface
of the egg. However, owing to the slightly eccentric position of
the egg nucleus and oye bel also to the structural organization
of the cytoplasm, this material comes to the animal pole of the
ege earlier than to the rest of the surface (Figs. 1 and 2). The
action of the nuclear diffusion seems to render the cytoplasm more
fluid and therefore less viscous, especially at the animal pole, as is
shown by the fact that the cytoplasm usually flows out into and
takes the shape of the inequalities of the enclosing egg membrane.
The result is what we should expect if the nuclear sap were with-
drawn from the nuclear area, 7. ¢., the egg flattens or undergoes
collapse at the animal pole. In this way the surface is carried
down to the region of the first maturation spindle that up to this
time has moved very little or not at all from the place of its origin.
This state of affairs is shown clearly in Text—Fig. I, 4 and B.
The general mass of the cytoplasm now becomes more uni-
formly affected by the nuclear sap, as the stains seem to indi-
cate, and under the influence of surface tension the egg begins
to slowly assume a globular shape; the spindle remains near the
surface and so is carried away from its original position. When
the anaphase is reached the typical condition is shown in Fig. I, C;
the shape of the egg has become a rounded oval.
Leaving out of consideration the causes which separate the
chromosomes into two groups, the idea that the nuclear material
tends to liquefy the cytoplasm suggests a hypothesis which may
possibly explain the formation of the polar globules. When the
chromosomes which are to form the first polar body very nearly
touch the surface of the egg (Fig. 6), a sort of papilla-like projec-
tion pushes out ahead of them; soon these chromosomes sur-
rounded by a small amount of clear cytoplasm are pinched off in
58 fohn W. Scott
the form of a little droplet, the first polar globule. Now this is
just what we should expect if the region immediately surrounding
the chromosomes is rendered more liquid while the general mass
of the egg is undergoing slow contraction in resuming the globular
condition. ‘The formation of the second polar body, which soon
follows the first, may be explained in the same way. Fig. I, D,
shows the second maturation spindle in metaphase. ‘The egg has
apparently resumed its original shape by the time the second polar
body is completely constricted off.
5. Fertilization. Fusion of Germ-nuclet
By the time the second maturation spindle is in anaphase the
sperm head has penetrated one-third to one-half the distance to
the center of the egg. It lies slightly in advance or to one side
of the centrosphere which is beginning to be recognized by its
characteristic appearance. [he sperm head has begun to enlarge
and its more pointed end is directed toward the centrosphere.
During the telophase of the second maturation the male pronu-
cleus (as we may now call the sperm head) continues to enlarge
and to advance toward the center of the egg. ‘There is evidence
to show that it breaks up into a number of chromosomes, each of
which forms a vesicle, and that these vesicles rapidly fuse to form
a larger vesicle. At any rate, two, three, or four lobes or vesicles
are frequently found which fuse at a later period. ‘The sperm
aster is clear cut and growing; the centrosphere which lies in
advance of the pronucleus is still hazy without definite outlines.
Just after the expulsion of the second polar body the male pro-
nucleus has traversed approximately two-thirds of the distance
to the center of the egg and appears as a medium-sized vesicle
with deeply-staining reticulum. ‘There is a rather large vanishing
aster and the centrosphere has increased in size and clearness.
About this time the egg chromosomes begin to swell, while the
small aster, somewhat enlarged and persisting from the matura-
tion spindle, has at its center a clear area that has the appearance
of a centrosphere. By the time the male pronucleus reaches a
region near the center of the egg it takes the form of a clear vesicle
with deep-staining and sometimes diffusely-staining reticulum.
Parthenogenetic Development of Amphitrite 59
The female pronucleus is moving toward the center with each chro-
mosome forming a little vesicle. “Ihe female centrosphere is
small and ill-defined, the male centrosphere is large with well
marked boundaries (Fig. 10). [he male pronucleus increases
in size, but it remains almost stationary near the center
awaiting the approach of the female pronucleus. ‘The female
chromosome vesicles now fuse to form a vesicle which rapidly
enlarges. [he female aster and centrosphere have in the mean-
time entirely disappeared and only traces of the male aster may
be seen. Fig. 11 shows the germ-nuclei in contact, approximately
of the same size and appearance. Soon the limiting walls break
down and the chromatin quickly collects to form the primary
stage of the first cleavage.
6. Cleavage
An early spireme stage of the first cleavage presents a typical
appearance. When the fully-formed tapering spindle takes its
characteristic position it lies in or very nearly in a plane perpen-
dicular to the polar axis slightly nearer the animal pole with the
chromosomes in metaphase (Fig. 12). While the clear-staining
area at each end of the spindle (centrosphere) becomes larger,
the spindle itself becomes broader, blunt at both ends, and
decidedly shorter. In Amphitrite the centrosome, whatever may
be its function, is not a permanent organ of the cell; at least it is
not differentiated at all times by the fixation and stains I have used.
In Fig. 13 is seen the condition of the egg in telophase. The
chromosomes with faint outlines are passing into the vesicular
condition. ‘These collect and fuse in the region of the centro-
sphere to form the resting nucleus of the daughter-cell. The
asters are undergoing degeneration, and the cytoplasmic division
is well under way. Fig. 14 shows the chromatin collecting in the
vesicular nucleus in preparation for the second cleavage; the
asters have already divided.
The cell-lineage of Amphitrite has been described by Mead;
and it is not necessary to describe later nuclear changes, for the
Same processes are repeated as have been described for the first
cleavage. “These processes may be stated briefly as follows: the
60 Fobn W. Scott
chromatin, contained in a clear vesicle with a sparsely-staining —
reticulum, collects into the form of a number of threads or spireme
which breaks up into a number of chromosomes; these collect,
divide and pass into a vesicular condition as they approach the
centrosphere; here their outlines become indistinguishable and
they fuse to form the vesicular nucleus of the daughter-cell.
No yolk lobe is formed in cleavage as is the case with ake egg of
Cheetopterus.
The first cleavage is unequal and in the 4-cell stage one cell,
D, is larger than any of the others. The sagittal plane of the
future embryo passes through this cell and the one diagonally
opposite. ‘he cleavage is now oblique and alternates in direction
up to the 64-cell stage face the germ layers are completely sepa-
rated. At this stage one cell is mesoderm, seven cells are ento-
derm, and the rest ectoderm. The complete invagination of the
mesoderm is shown in Fig. 15. The mesoderm in this egg con-
sists of four cells, one small cell from each large one not being
shown. The nuclei of certain cells are moving away from the
periphery, the first indication of the invagination of the entoderm.
There were 104 nuclei present in this egg
7. Structure of the T rochophore at Various Stages
By the time the 64-cell stage is reached, the primary prototroch
is composed of four separate groups of cells of four cells each;
these become flattened down, never divide again, and soon become
ciliated. ‘he completed prototroch, formed by the addition of
certain other cells, is composed of twenty-five cells which may all
be recognized after the larva begins to elongate. “The paratroch
is composed of four cells differentiated at a comparatively early
period; these persist as a ring of ciliated cells around the body
until the larva has developed five or six metameres. The cilia
are at first numerous and fine; later they become larger, stronger,
and have more vigorous movements. The apical tuft is large and
well developed in the young trochophore but it slowly atrophies
when the body begins to seni. The normal round blastulas
always swim up leu the soins of the dish in which they are
kept; this is due to the location of the cilia, and the presence of a
Parthenogenetic Development of Amphitrite 61
segmentation cavity no doubt plays a part. [he movement at
first is chiefly rotary and gradually increases in rate. When the
trochophore begins to increase in length, the altered shape causes
it to swim in a more or less winding Teal with rapid movements
that are evidently produced by deseutely correlated forces. ‘The
regular shape of the trochophore and the correlated movements
of the symmetrically arranged cilia are the controlling factors of
the path through which it swims.
The shifting of areas in the lower hemisphere—the subumbrella
region—is the chief means by which the one-layered blastula
becomes metamorphosed into the three-layered larva. In this
process the contour of the egg is not very much altered. ‘The
mesoderm and entoderm sink in, filing the segmentation cavity
and diminishing the surface area. ‘The cells of the somatic plate
become thinner and spread over the surface of this region. After
the blastopore closes the subumbrella region elongates rapidly
due to the cleavage of cells just in front of the paratroch. ‘The
formation of metameres, the differentiation of the alimentary
tract, of the mesoderm, of the mucous glands, of the problematical
bodies, and of the chief part of the nervous system need no further
mention for our comparison.
The rate of early development in Amphitrite is quite rapid.
The time required for maturation and first cleavage has been
mentioned. Cell division continues with equal swiftness, cilia
are developed by the time the 64-cell stage is reached, and under
favorable conditions the blastula swims in from four to five hours
after the egg is fertilized. By the time the trochophore is twenty
hours old it is approximately pear-shaped, a stomadeum has
developed, a large enteron is very noticeable, vacuoles are found
in the region of the prototroch, and the brownish-black pigment
of the eye spots is to be seen in the pretrochal region (Fig. 16).
The figure just menticned represents a camera lucida drawing
of a normal, living gastrula twenty-three hours old. Its shape was
slightly altered by pressure of the cover-glass used to hold it in
position. Some of the more characteristic details of structure
are given and in one region the cells lining the enteron are
shown.
62 fobn W. Scott
DEVELOPMENT OF THE UNFERTILIZED EGGS
A. Description of the Living Material
1. Methods Used in Producing Parthenogenesis, and Their
General Effect upon the Eggs
As soon as the worms were brought into the laboratory, each
female was washed thoroughly from one to two minutes in fresh
water to rid it of any chance sperm and then placed in a dish of
sterilized sea-water until ready for use. “The Amphitrite was
usually washed several times in fresh water, though this was found
unnecessary as demonstrated by the control eggs. The body cavity
of the worms was opened in sterilized sea-water by snipping the
thin body-wall over the region of the egg pouches. In this way
very little blood or fluid was mixed with the eggs; they did not de-
velop so well if much superfluous material was in the solution. The
eggs were next placed in the desired solutions, usually at once, but
frequently after various intervals. Care was taken to avoid
shaking or squirting the eggs unless agitation effects were desired.
Many solutions with different dilution and time of exposure
were employed, but the following methods have proved most
successful in producing differentiation in unfertilized eggs of
Amphitrite; the eggs were never all in the same stage of ripeness
and the percentages give the best results found:
No. oF Time PERCENTAGE OF
MeErtHop. Soxgzton (Meraop) Exeroren: EXPoseED. Active SwIMMERs.
2- 5 parts (8 N)Ca(NO3)> + 98-95 parts s.w. Permanently. 25 per cent.
2 s-ro “ ($N)Ca(NOsz)2 + 95-90 “ s. w. 1 hour. I5 per cent.
3 5 “ (@iM)KCl + 95 SoS AW. 1 hour. IO per cent.
4 5 “ (@iM)KNO3 + 95 cSt Wi: 1 hour. 15 per cent.
5 5 “ (4M)CaCle + 95 G gAge 1 hour. 5 per cent.
6 By agitation. 20 per cent.
Of the methods mentioned above the surest and most satis-
factory is number 1, though number 2 is almost as good. The
percentage of swimmers varies in different experiments from
5-25 per cent. The highest percentages of swimming embryos
found in any of my experiments were obtained by using methods
Parthenogenetic Development of Amphitrite 62
>)
3 and 4; in the former 30-40 per cent, in the latter 50 per cent.
However this was from a single experiment and the results given
were due to two causes, for these solutions were used in connection
with the disturbance and agitation incident to handling the eggs
in changing the solutions. Even the results given in he table for
methods 3 and 4 may have been affected sleuly by agitation,
as the control seemed to show. In another paper I have shown
how extremely sensitive the egg of Amphitrite is to agitation.
This is a very important but uncertain method of producing
parthenogenesis in these eggs. Usually not more than 2 or 3 per
cent of the eggs will develop in this way, occasionally 5 to 10 per
cent, and in one case 20 per cent. In the paper mentioned I
have called attention to periods of susceptibility to agitation;
further experiments show that the time of susceptibility to such
a stimulus varies within wide limits, depending chiefly upon tem-
perature and the ripeness of the particular lot of eggs.
A general effect of the calcium nitrate soln | is partially to
inhibit cytoplasmic cleavage and to stimulate a division of the
nucleus. If the eggs are nec ripe when removed from the body
of the female, the. ae solutions do not seem to interfere with
the expulsion of normal polar bodies or the first few cleavages.
In the stronger solutions there is sometimes a tendency for the
eggs to adhere or stick together when in contact, but I have never
found giant embryos. In the potassium chlorid solution cleavage
of cytoplasm is more aided than interfered with, especially if the
eggs have reached the right degree of maturity. Consequently
cleavage of the cytoplasm frequently keeps pace with that of the
nucleus until quite a late stage. ‘The first polar body is not formed
except in very rare cases. ‘The effect on cleavage of the potassium
nitrate solution is much the same as that of the potassium chlorid
solution. The effect of the calcium chlorid is more like that of
the calcium nitrate solution.
The eggs that develop after agitation usually do not form polar
bodies and nearly always are unsegmented. ‘The nuclear divisions
are as extensive and take place in the same manner that they do in
eggs treated by the salt-solutions. Occasionally an egg will show
a considerable amount of very irregular and abnormal cleavage
64 Fobn W. Scott
(Fig. 39). Cleavage of the cytoplasm without previous cleavage
of the nucleus has not been found in developing eggs.
2. Early Stages.
It would be a needless task to undertake the description of
individual experiments where much was repeated again and again
in order to verify results and obtain comparisons of development.
Fig. II. Diagrams to show. early development. Method No.1 used. A, B and C were types
forty-one minutes after beginning the experiment; D, another egg sixty minutes later. Camera draw-
ings from whole mounts. Leitz oc. 1 (15), obj. 7.
Hence a general description will be given. The germinal vesicle
breaks down, sometimes within a few minutes, sometimes several
hours after the eggs are subjected to the solutions. It has been
found that the riper the eggs are, the more rapidly does the ensuing
differentiation take place and the nearer it approaches the normal
development.
ay
Parthenogenetic Development of Amphitrite 65
One or both polar bodies may be normal, or both abnormal, in
both size and appearance; or only one is formed, which is always
abnormal. Most of these abnormal polar bodies can be readily
distinguished in the living egg by their size which varies greatly
as II, III; 44-46). When normal polar bodies are found in
the solutions the time required for this process is also normal.
But it takes longer to complete the process of throwing off the
abnormal polar bodies, and the longer the process the more
abnormal it becomes. When both polar bodies are formed, here
as under normal conditions the egg undergoes flattening or collapse
in the polar diameter. When no polar bodies are thrown off, the
change in shape is never so striking and may not be noticeable
(Figs. 40-41, 55).
Cleavage usually begins within one and one-half hours after
the eggs are placed in the given solution, though I have seen eggs
in the process of cleavage ne forty minutes ene the beginning
of an experiment. ‘The first two blastomeres of the egg are ie
quently abnormal in size and appearance, but a study of the living
material shows that an apparently normal cleavage may continue
for several divisions. In every case ameboid movements, or abortive
attempts at cleavage, occur at this time, even in eggs in which no
distinct cleavage plane is ever formed; the egg often shows two
or three lobes, a condition which is more common in the potassium
chlorid cultures. The cleavage cells of an egg treated with cal-
cium nitrate are always more compact and fit closer together than
the normal, while the potassium chlorid eggs have the opposite
tendency. The different types of cleavage can be best understood
by consulting the diagrams (Figs. II, III; 17, 49, 50, 58-61).
In six to twelve hours the cilia become strong enough to cause the
rotation of the eggs. Up to this time cytoplasmic cleavage, when
present, occasionally keeps pace with the nuclear divisions, but later
it seems to lag behind more and more and often entirely ceases.
Certain unripe eggs after being treated with potassium chlorid
solution tend to break up rapidly into many small spherules
(Fig. IV). Occasionally this effect is produced by some other
solutions if the concentration is sufficient and I have found it
rarely in the controls. “This phenomenon was noticed by Fischer,
who says, “Sometimes the eggs go into the morula stage. The
66 fobn W. Scott
Y Fig. III. Diagrams to show types of early development. From whole mounts, same magnification
as Fig. Il. £, one hundred minutes, and F-#, two hundred and twenty-five minutes after beginning
the experiment. Dotted lines enclose nuclei.
Parthenogenetic Development of Amphitrite 67
origin of the morulas is somewhat obscure. ‘The fact that they
appear in solutions which have at no time contained any black
eggs would suggest that nucleated eggs can, in a very short time,
give rise to morulas without any external signs of cleavage.”
Fischer thus raises the question whether nucleated eggs may not
pass into the “morula”’ stage without any external signs of cleav-
age and so give rise to swimming blastulas or later stages. But
these eggs do not develop, as we shall see. In one of my experi-
ments 95 to gg per cent of the eggs showed this condition. At
11.30 A. M. the eggs were removed from the body of a large female
and placed for one hour in a potassium chlorid solution. Before
they were returned to sterilized sea-water, the germinal vesicle
had broken down in nearly all eggs and a few showed a very small
peri-vitelline space. ‘Thirty minutes later, at I p. M., the eggs
showed protuberances over their surfaces as though cells were
being constricted off. At 2.15 P.M.
nearly all the eggs were in a multi-
spherular condition; when pressed
down slightly with a cover-glass no
segmentation cavity was found and
the central portion of the egg was
still unsegmented. ‘The eggs were
examined at frequent intervals until
8.45 P. M., when the spherules were
not more than half the size of
those found earlier in the afternoon. Fig. IV. A typical multispherular egg
The next morning no swimming six hours old from a potassium chlorid
eges were to be found; many were
apparently bordering on dissolution
and all seemed full of small vacuoles. Sections of these eggs showed
that the germinal vesicle had broken down in most cases and had
diffused out into the cytoplasm before spherulization began.
Very infrequently an ege was found with the germinal vesicle
intact and surrounded by spherules. There was never any
mitotic division of the nucleus. In eggs where the nuclear sap
diffused out into the cytoplasm, there was found a more or less
diffuse achromatin stain in each spherule. It is very evident that
solution. Camera’ drawing. Leitz oc. I
(16), obj. 7.
68 ‘fohn W. Scott
this peculiar change in some eggs is not one of differentiation,
but is due to the effect of the potassium chlorid in altering the
viscosity and lowering the surface tension of the egg-cytoplasm.
Since the spherulization continues for some hours after it begins,
it seems that the cytoplasm may undergo a ripening process,
somewhat as the normal egg must do in order that cleavage into
small cells may occur; and inasmuch at the process of spheruliza-
tion continues longer in eggs where the germinal vesicle has pre-
viously broken down, it indicates that the diffused nuclear sap
may have something to do with this ripening process. ‘Treadwell
(02) when working upon Podarke noticed that some of the control
eggs broke up into a great many “small globules” with no trace
of nuclear division, the nucleus lying in one cell which was usually
larger than the others. He found no ciliated embryos among
these eggs.
3. Description of Swimming Embryos, Eighteen to Twenty-
five Hours Old
At this age the various cultures as a rule show their best develop-
ment, though the swimming eggs frequently show wide differences
in structure and appearance. ‘To illustrate these differences we
may cite a few examples. The egg in Fig. 20, from a potassium
chlorid solution, was found swimming near the surface of the
water about twenty-four hours after the beginning of the experi-
ment. A clear ectoplasmic layer is differentiated, in marked
contrast to the yolk which lies throughout the central portion of
the egg. The cilia projecting from the entire surface are large,
stiff and more strongly developed in certain regions. Segmenta-
tion is extensive; scarcely ever are eggs found with a greater
number of cells than shown here, although the cells are not so
numerous as in the normal egg of fifteen hours. ‘The cell bounda-
ries are not clearly defined, indicating a tendency to fuse, and cer-
tain regions do not show cell structure. Clear spots show the
position of nuclear areas in the cells. “This egg was compressed
slightly under a cover-glass until it could be examined with a high
power, but this treatment failed to reveal the segmentation cavity
which can be easily demonstrated in the same manner in the
Parthenogenetic Development of Amphitrite 69
normal egg (Fig. 16). Vacuoles are sometimes present as shown
in Fig. 20. Another egg, raised under the same conditions and
nearly the same age, was taken from the bottom of the dish where
the great majority of the swimming eggs are found (Fig. 21).
The size and distribution of cilia and the arrangement of cyto-
plasm and yolk are practically the same as in the previous case,
but there is not a trace of segmentation and only two nuclear areas
are found. We may take as another typical example an egg from
a calcium nitrate solution (Fig. 28). Here we find the cilia
arranged irregularly in three distinct groups, a well differentiated
ectoplasmic layer, the yolk pretty evenly distributed throughout
the interior of the egg, traces of segmentation, numerous nuclei,
and some limited patches of brownish pigment.
Eggs as a rule do not show such uniform distribution of cilia
as these examples, or such regular shape (Figs. 22, 23, 26, 29, 39).
The long apical tujt of cilia so characteristic of the normal trocho-
phore is always absent. In shape the eggs may be comparatively
globular, oval, or pear-shaped (Figs. 29, 24, 35); ordinarily these
contours are represented in the active swimmers. On the other
hand abortive cleavage may produce an irregular outline (Figs. 22,
36), and cleavage may stop though nuclear division proceeds.
The number of cells may vary from one to many, and where the
egg is manifestly composed of more than one cell the boundaries
of these cells may be indicated by mere indentations, or by com-
plete cleavage planes; both conditions are found frequently in
the same egg (Fig. 22). The great majority of the swimming
eggs have no true segmentation at all and in these unsegmented
eggs the nuclei are usually very numerous (Figs. 29, 32, 33), but
are sometimes few in number (Fig. 37); the nuclei also differ in
size (Fig. 33). In eggs with considerable cytoplasmic segmenta-
tion there is as a rule one nucleus to each cell though two or three
may be present. In all cases of swimming embryos the ectoplas-
mic layer is pretty well differentiated and this differentiation seems
to bear some relation to the distribution of the cilia. For where
the cilia extend around the entire circumference of the egg there
is a correspondingly well developed ectoplasmic layer, and where
the yolk lies close to the surface the cilia are noticeably absent.
7O Fohn W. Scott
However there may be an ectoplasmic layer where no cilia develop.
The yolk may be broken up into groups in the case of segmenta-
tion or be scattered by nuclear division (Figs. 29, 32), and very
frequently it exhibits a tendency to collect near one side of the
egg, a region which I believe indicates the position of the vegeta-
tive pole.
Another important and significant differentiation at this period
is the development of a boeniek pigment. If one may judge from
color and appearance this pigment is undoubtedly homologous
with the eye spots of the normal embryo. In the normal trocho-
phore this pigment is definitely localized in two small brownish
or reddish-brown spots (Fig. 16). In the parthenogenetic eggs
the pigment is never localized in spots. Occasionally two or
more pigmented areas are found (Figs. 26, 28, 31), but more
frequently the pigment is found in a single diffuse mass scattered
along near one side of the egg (Figs. 32, 35, 37). Rather infre-
quently swimming eggs are facied which have an oblong or blunt
pear-shape; to a casual observer they might not appear dis-
similar to the normal trochophore (Figs. 21, 23, 255 32). ‘There
appears to be such a differentiation in shape in the egg shown in
Fig. 35, but I think the form is due mainly to an early segrega-
tion of cleavage materials (yolk, cytoplasm) in which the cleavage
division had the most prominent effect, this being followed by a
fusion of the blastomeres.
From what has been said about the unsymmetrical distribution
of cilia, the lack of an apical tuft, and the irregular shape of these
swimming structures, it 1s not surprising to find that their swim-
ming movements are distinctly different from those of the normal
trochophore. They do not swim so rapidly, they do not move in
such a definite path, nor do the movements of the cilia appear so
well correlated.
4. Later Stages and Fate of the Parthenogenetic Eggs.
The maximum percentage of swimming eggs is found between
the twelfth and twenty-fifth hours, depending upon the tempera-
ture and the rate of development of a given lot. After the twenty-
fifth hour they die rapidly and in most experiments all are dead
ia
Parthenogenetic Development of Amphitrite 71
at thirty-six or thirty-seven hours, although I have raised them
until over forty-eight hours old. Death is not due to the environ-
ment, for the normal eggs are readily raised under the same con-
ditions. ‘Typical conditions of eggs from the calcium nitrate
solutions are shown in Figs. 29 and 31. In the various cultures
the amount of segmentation tends to diminish in these later
stages, due to fusion, wholly or in part, of adjacent blastomeres.
Frequently, however, eggs retain a condition of definite segmeénta-
tion which was reached at a much earlier period (Fig. 30). Fig. 32
is typical of the oldest eggs that came under my observation.
There is no apparent segmentation of the cytoplasm, a great many
light areas are present some of which may be vacuoles; the yolk
is irregularly distributed, and no pigment can be seen. In the
same experiment quite a large per cent of the eggs were still
swimming at the age of forty-four hours. Some of these were
round, some oblong, some with irregular outlines, and all were
distinctly different from the normal. Later the vacuoles increased
in number.
The fate of the parthenogenetic eggs may be summed up as
follows: Segmentation of the cytoplasm remains until very late
in substantially the same condition that it reached before the
development of the cilia. “Those eggs which have extensive and
strongly developed cilia survive those on which the cilia are
limited in extent or weakly developed; these surviving eggs also
have a shape nearest to the normal. After swimming actively
for some time the cilia do not move so rapidly, apparently shrink
in size and seem to disappear; the protoplasm assumes a lighter
appearance, vacuolization sets in, and just before dissolution the
cells, if present, tend to flow together and the egg as a whole tends
to assume a more roundish shape. ‘The egg-wall breaks down at
some point and soon dissolution is complete. I have seen cilia
still vibrating after the ege-contents began to flow out.
I have mentioned in the first part of this paper the definite
change in shape that occurs in eggs at the time of maturation and
I have referred to the ameboid movements of the cytoplasm that
are found in early cleavage stages of both fertilized and unferti-
lized eggs. In the normal eggs these ameboid movements possess
72 Fobn W. Scott
a definite character and relate to cleavage. Moreover they take
place slowly in all eggs. In eggs with no segmentation these
ameboid movements are very noticeable, for every nuclear division
that takes place is within the limits of one cell and consequently
the whole egg is influenced by each division of the nucleus.
It is for this reason that ameboid movements in the unsegmented
egg are so much more noticeable than in those eggs in which the
cleavage iscomplete. In contrast with these movements of the egg
connected with segmentation phenomena, there are other ameboid
movements of a different
a 6 Cc : :
a kind that are sometimes
a z : ;
< found in the salt-solution
eggs at a comparatively late
Z ¥ £ h stage of differentiation. An
ege is occasionally found
thirty to forty-five hours
after the beginning of the
experiment that shows no
ee segmentation and no cilia-
tion but is undergoing extra-
ordinary ameboid activity.
aves Such an egg has a general
opaque appearance but the
Fig. V. Camera drawings to show rapid ameboid border is clearer than the
movements. From a calcium nitrate solution, forty- rest of the ege. The small
three hours old. Sketches made at one-half minute inter- pseudopodia somet 1 mes
vals, a-h; sketches at one-quarter minute intervals, i—p. form and disappear very
quickly. In Text-Fig. V are shown camera lucida drawings of an
egg of this kind forty-three hours after the beginning of the
experiment. In the first two rows, a—h, are eight successive
sketches made at one-half minute intervals; in the third and
fourth rows, i—p, are sketches made eleven minutes later of the
same egg at one-fourth minute intervals. These drawings do
not show more than half the forms that were taken by this egg
during the time the sketches were being made. The peculiar
differentiation Just described may be correlated I believe, with
processes found in the normal larvas of about the same age. By
Parthenogenetic Development of Amphitrite 73
the time the trochophore is forty hours old it has developed three
or four well-defined body segments. During this development in
structure a physiological differentiation has occurred so that the
larva is now able to make comparatively quick, jerky and wrig-
gling movements. I think without doubt that this is the sort of
differentiation that takes place in these eggs treated with salt-
solutions. ‘The protoplasm presents the same medium-opaque
appearance and the movements possess the same general character.
B. Description of the Preserved Material
1. Early Stages
If the eggs are quite ripe, that is, in a condition to be fertilized,
a study of the sections of unfertilized parthenogenetic eggs dis-
closes no abnormalities until after the expulsion of the polar
bodies. ‘This applies as a rule only to the weaker calcium nitrate
solutions. In strong salt-solutions the process of development is
hindered or considerably modified. Unripe eggs when treated
with any of the solutions always give abnormal results. Since
any given lot of eggs removed from the body cavity usually con-
tains many stages of ripening, there are always many forms of
differentiation in each experiment. Fig. 45 gives a type in which
the polar bodies depart slightly from the normal (c/. Figs. 7, 8, 9).
It will be noticed that the first polar body is slightly larger than
the normal due to additional cytoplasm and that the second polar
body is still further increased in size. The chromosomes in the
second polar body are noticeably ragged in appearance. In
Fig. 44 these differences are still further accentuated. By refer-
ring to Fig. 46 we find a very abnormal type of both polar bodies.
The first polar body is in practically the same condition as the
second polar body of Fig. 44, while the second polar body has
increased enormously in size by the addition of yolk granules.
The state of affairs where only one polar body is formed is
represented fairly well by Fig. 47. The remaining chromosomes
have retreated to near the center of the egg and are in a stringy,
fragmentary, decidedly abnormal condition which probably cor-
responds to a stage just previous to the first cleavage. It will be
74 Fohn W. Scott
noticed that the egg membrane is very thick, a condition which 1s
produced by the calcium nitrate solutions in unripe eggs and which
has served to distinguish certain types of differentiation at a later
period. I have also observed, after the first polar body was
thrown off, the chromosomes of the second polar body lying in an
isolated group in the cytoplasm near the animal pole; the remain-
ing chromosomes retreat to the center of the egg to take part in
certain phenomena of cleavage. Sometimes the chromosomes
of the first polar body are thrown of in a separate group which
lies somewhere near the surface as in Figs 42.
It frequently happens that no polar Redies are expelled (Figs.
40,41). Inthiscase the egg does not collapse in its polar diameter,
the spindle lies in or very near its original position, the chromo-
somes, spindle and asters all show slight abnormalities, and the
stain indicates that the egg is immature. [he chromosomes are
not usually separated permanently by this kind of maturation
process and frequently do not separate at all. Another abnormal
condition of the first maturation spindle is shown in Fig. 43. In
eggs where both polar bodies are expelled the spireme for the
first cleavage is formed in the same manner as under normal
conditions (Figs. 44, 45, 46).
I have mentioned that observations on the living egg sometimes
failed to disclose any abnormal process in Ves Soe cleavage
stages and an examination of the sections shows that the same is
true of the nuclear phenomena. However there is a tendency for
the chromatin division to be fragmentary or unequal and the
astral radiations do not possess the distinct, definite outlines of
the normal condition (Fig. 48). Very rarely there is found a
tripolar mitotic figure and occasionally the first division results in
three nearly aptal cells (Fig. 50). Even when the nuclear division
is approximately normal and complete the cytoplasmic cleavage
is quite often incomplete or abortive. Frequently therefore more
than one nucleus is found within the limits of one cell, confirming
observations made upon the living egg.
The development of eggs ied treated with potassium chlorid
solutions does not begin il after the eggs have been returned to
sea-water. In aoe rare cases the formation of one abnormal
Parthenogenetic Development of Amphitrite 75
polar body has been observed in cultures from this solution. Though
no polar bodies are formed certain peculiar phenomena take
place within the egg at this time, the chromosomes pass into the
vesicular condition and greatly enlarge. he vesicles are less
numerous at first and some have faint outlines, but later there
are from 16 to 22 in each egg. Maturation asters form in the
proper position and a faint spindle may be present, but the spindle-
rays do not seem to have any important influence upon the chro-
mosome vesicles (Figs. 55, 56). Sometimes the spindle and
vesicles move up to a region indicating the position of the animal
pole. [he maturation asters are rather delicate in appearance
and fade out before the chromosome vesicles fuse in preparation
for the first cleavage division. Very commonly the maturation
asters and spindle Ao not form at all, though the chromosome
vesicles enlarge in the manner described Ziel e. Fig. 57 shows
an early condition in the formation of the first cleavage spindle;
the cleavage asters are distinctly different in size and appearance
from the maturation asters just described.
The cleavage phenomena in the potassium chlorid cultures
show more variability than in solutions where the calcium nitrate
salt was used. This variability seems to be correlated with the
fact that an excessive amount of chromatin remains within the
egg. At the first cleavage the amphiaster is similar but distin-
guishable from the normal; the chromatin-division takes place in
the normal manner, but with certain minor irregularities which
for the most part consist in scattered and fragmentary chromatin-
granules or chromosomes (Figs. 58, 61). Later cleavage stages
show more abnormal conditions. Frequently a see aster 1S
found at the first cleavage and three-celled eggs or triple groups
of chromosome vesicles are very much more common in this than
in the calcium solution. I have seen four, five, six, and in one
case, seven asters all taking part in the chromatin division, and in
some of these eggs there was no sign of cytoplasmic cleavage.
In Fig. 61 is shown an egg which may be taken as a type; the part
that each of five asters is taking in the distribution of the chromatin
is readily seen, and one is not surprised to find in later cleavage
that each cell may contain one or many nuclei and that these nucle1
76 ‘fohn W. Scott
may be of various sizes (Figs. 62, 63). When cleavage of the
cytoplasm occurs the nuclear division 1s most nearly normal.
In general a potassium chlorid culture after five or six hours con-
tains a few eggs with many cells having one nucleus each, a great
many more with a smaller number of polynuclear cells more or
less incompletely divided, and some eggs undivided but multi-
nuclear. ‘The unequal size of the nuclei in some of the cells
indicates that the chromosome vesicles in telophase may fuse to
form two vesicles instead of one. I believe that some of the nuclei
may arise in this way though I have made no direct observations
to prove it.
Some interest attaches to the number of chromosome vesicles
to be found in potassium chlorid eggs in the late anaphase and
telophase of the first cleavage. A constant number cannot be
counted owing to the fact that some usually begin to fuse before
others have distinct and definite outlines. In the egg of Fig. 59,
in the left-hand group of vesicles, 18 (+4?) could be counted,
while in the other group 20 (+4!) were found. ‘The usual number
that can be definitely counted is from 15 to 20. At a somewhat
later stage, in an egg where the cleavage of cytoplasm was normal,
eleven vesicles were counted in each of the two cells (Fig. 60).
With these conditions may be compared Fig. 49, which represents
an egg treated with calcium from which both polar bodies have
been expelled; it is interesting to note that we have here Io (+1)
vesicles in one group and 11 in the other (not all shown). In the
calcium nitrate solutions where both polar bodies are formed the
reduced number of chromosomes, eleven, appears again in the
daughter-cells of the first cleavage. This number no doubt con-
tinues in the later cleavage-cells. In the potassium chlorid egg
(Fig. 59), there is evidently the full number of chromosomes in each
of the two cells, and as no polar bodies were expelled this number
can be accounted for on the theory of the individuality of the
chromosome, since these chromosomes had no maturation division;
still the number must have been reduced from 44 to 22 by fusion
before division. But if we take a potassium chlorid egg, like
that shown in Fig. 60, the eleven vesicles of each daughter-cell
indicate a bivalent character retained throughout division; or
Parthenogenetic Development of Amphitrite 77
they have assumed this character after division by fusion in twos,
a view that is improbable from lack of any direct evidence.
Certain difficulties prevent a thorough analysis of these phenomena
but the above conditions may be accepted as typical.
2. Condition of the Eggs as Shown by Sections of Later Stages
In a general way my observations on the living egg are com-
pletely confirmed by a study of the sectioned material, which also
furnishes additional and interesting data. Usually cleavage 1s
more extensive within the egg than at the surface, probably indi-
cating fusion at the surface subsequent to cleavage. Some eggs
are multinuclear without any signs of cytoplasmic cleavage,
and cleavage of both cytoplasm and nucleus falls farther and
farther behind the normal as development proceeds. More
nuclei are uniformly found in the calcium than in the potassium
cultures, especially in the unsegmented egg, but the potassium
chlorid solution shows eggs with more distinct and more complete
cleavage planes. I have been able to discover cilia along the
edges of the prepared material of all such types of eggs as are
represented in my drawings of late stages and there is no doubt
that such eggs represent typical swimmers. ‘The killing fluids
used injured or destroyed most of the cilia, hence no attempt has
been made to figure them in the drawings and such ciliation has
not been found of any benefit in localizing regions of the eggs
One of the most advanced types of cleavage in the potassium
chlorid solution at eight to nine hours is represented by Fig. 64,
and Fig. 65 is a type near the other extreme with very little cleavage.
In the segmented eggs mentioned there were found portions of a
segmentation cavity and evidences of an attempt at invagination
(cf. Fig. 15). In the egg with very little cleavage the nuclei vary
in size and the largest one has evidently not divided for some time
since no nuclei of a similar size were found near it. ‘This fact, that
a nuclear vesicle in an unsegmented egg enlarges extraordinarily
unless mitosis occurs, is in accord with Lillie’s findings for the egg of
Cheetopterus and indicates that the size and growth of the nucleus
are independent of mitotic division. In all developing eggs,
whether segmented or not, the early differentiated ectoplasmic
78 fobn W. Scott
layer is retained. Fig. 51 represents an egg taken at fourteen
hours from a calcium nitrate culture; the cleavage planes are
incomplete and there is a clear attempt at invagination.
No further very important developmental changes occur in most
of the swimming eggs. However, conditions are occasionally
found that throw additional light upon parthenogenetic develop-
ment or upon causes connected with the cessation of development.
Fig. 67 is an egg from a potassium chlorid culture at the age of
twenty hours Shick may be taken as ty pical of the most advanced
differentiation that I have discovered in eggs from this solution,
although other eggs with more cells and more nuclei have been
found. While manifest irregularities are noticeable, the invagi-
nation of the mesoderm is easily made out and is well under way.
Why the development has advanced no further than where it
should have been some twelve. hours earlier, if fertilized, seems
traceable to the following causes: ‘The number of cells is too
small to complete invagination in the normal manner; only small
portions of the segmentation cavity were formed, probably due to
the action of the solution upon the surface tension of the cyto-
plasm and perhaps to slow and imperfect cleavage; and incom-
plete cleavage may also have hindered the invagination. In
another egg 5 the same age from the same solution (Fig. 66), there
was no trace of segmentation, and vacuoles, which characterize
the fertilized eggs at about this age, were found.
The conditions in the calcium solutions at about twenty hours
are also interesting. Unsegmented eggs frequently show a large
number of small nuclei (Fig. 52); this fact verifies observations onthe
living egg. In Fig. 53, the amount of cleavage, the number and
distribution of nuclei, the lack of any segmentation cavity, the
ectoplasmic differentiation, and the remnants of the region of the
germinal vesicle are all characteristic of eggs from this solution at
fis age.
vee rarely I have found an egg that showed a striking
resemblance to the normal, as in Fig. 54. Although this egg is
twenty and one-half hours of age, it is at a stage that it should have
reached twelve hours earlier under normal conditions. The
invagination of the mesoderm is complete; and in each of the
Parthenogenetic Development of Amphitrite 79
two mesoderm cells there has been nuclear division without
division of the cytoplasm. The nuclei of the entoderm cells
(partly shown) are migrating toward the segmentation cavity and
are thus taking the first steps toward the invagination of the ento-
derm. Large vacuoles are found which are not present in ferti-
lized eggs of the same number of cells but are found in fertilized
eggs of the same age. There 1s little essential difference between
this and a normal egg (Fig. 15). In the normal egg the cleavage
of the cytoplasm is a little more definite and complete, the meso-
derm consists of four different cells (two small ones not shown),
and this egg is more symmetrical in shape.
From the study of sections of a few well-developed unfertilized
eggs, it would seem not impossible to raise normal gastrulas and
even later stages from unfertilized eggs. But I have not succeeded
in doing so, though I used the same and even better precautions
than those that gave me success in raising the normal embryos.
THEORETICAL CONSIDERATIONS
She Brief Review of Some Previous Papers
Before taking up the theoretical discussion it 1s desirable to
give a short resumé of some papers relating to the subject under
consideration.
Mead (98, 2) tried the effect of a weak potassium chlorid
sea-water solution upon the unfertilized eggs of Chzetopterus.
He found that the formation of the polar bodies and the yolk lobe
took place in a normal manner and noticed abortive attempts at
cleavage. [he abnormal phenomena began after the matura-
tion mitoses and the reconstruction of the egg nucleus. He came
to two conclusions in regard to the effect produced by the potas-
sium chlorid: “First, it is of the nature of a stimulus, compatible
with the continuance of the normal developmental processes, and
is not of the nature of a poison or an irritant setting up irregular,
abnormal, and inconstant changes; second, the stimulus must be
referred to the specific properties of the salt and not to a change
in the density of the water in which the eggs are placed.” Morgan
(00) repeated and verified Mead’s experiments. By increasing
SO fobn W. Scott
the concentration of the sea-water (NaCl, MgCl,), he found that
development may proceed in much the same manner, but no polar
bodies were extruded. Loeb (01) produced trochophores and
claimed that “increase in the osmotic pressure or the loss of water _
on the part of the egg is the cause of the parthenogenetic develop-
ment of the egg.” He also believed that potassium chlorid pos-
sessed a specific effect upon the eggs of Chzetopterus.
Fischer ('02) succeeded in raising the unfertilized eggs of
Amphitrite to the “trochophore stage”’ by adding a small amount
of calcium salt to sea-water and by agitating mechanically the
sea-water in which the eggs were contained. ‘hough he noticed
that the developing eggs “present a totally different appearance”
from the fertilized eggs, his observations were incomplete and
superficial. In the same paper he states that “the appearance of
the swimming trochophores, the ciliary activity, and the general
behavior of the parthenogenetic larvas is exactly that of the nor-
mally fertilized larvas.”
‘Treadwell (’02) used a potassium chlorid solution with high
osmotic pressure to produce artificial parthenogenesis in Podarke.
No polar bodies were formed; the nuclei were much larger and
stained more intensely than the normal ones; ciliated structures
were produced without cleavage; and he concluded, though his
experiments were “incomplete,” that the cleavage which included
both nucleus and cytoplasm was not normal, 7. e., was not karyo-
kinetic. It seemed “as if one of the cells is to be regarded as a
lobe of protoplasm which contains some of the nuclear material,
and may later become completely divided by a membrane from
the other cell.” It seems probable to me, however, that he was
wrong in this conception.
Recently Bullot (’04) has worked upon the eggs of the annelid
Ophelia with the view of solving the problem of the relation
between differentiation and cleavage. Though his results are
inconclusive they are entitled to a briet mention here. He asks
the question, “Do the swimming blastulas found after ten hours
in the cultures arise from the segmented egg, or do they originate
from the unsegmented eggst”’ By placing shallow dishes under
the field of his microscope, he was able to make camera lucida
Parthenogenetic Development of Amphitrite SI
drawings of individual eggs at various intervals. For one expert-
ment he gives a series of eight drawings the last one made nine
and one-fourth hours after the eggs were placed in the solution.
Out of forty-eight eggs sketched in this series sixteen were swim-
ming at nine and one- Seutthi hours and these had arisen from eggs
that had previously shown segmentation. His drawings show, how-
ever, that the segmentation in these eggs was not always the same;
yet he concludes, “These experiments show conclusively that in
the anneiid of the genus Ophelia which was used in these experi-
ments the parthenogenetic larvas originate from regularly seg-
menting eggs”? (italics mine). ‘This cian 3 is also open to
certain objections. First, he says nothing about the later fate
of the thirty-two eggs which were not swimming at nine and one-
fourth hours. A see of the individual eggs figured by Bullot
shows that the segmentation does not pieced in all at the same
rate and therefore it is unlikely that all should begin swimming
at the same time. In Amphitrite the maximum number of swim-
mers is sometimes not reached until ten hours after the first swim-
ming eggs are seen; the earliest swimming structures always show
more or less segmentation, but some hours later swimming eggs
are found with little or no cleavage. ‘This latter condition is partly
due to fusion of blastomeres but more frequently to the fact that
the egg has not divided or that cleavage has been abortive. It is
also difficult to understand what is meant by the phrase, “regu-
larly segmenting eggs.” He cannot mean the definite, determi-
nate cleavage characteristic of the fertilized Annelid egg, for his
figures show otherwise. From his own experiments Bullot
attempts to cast doubt upon the results obtained by Lillie. It is
readily seen that no weight should-be attached to this objection.
I shall take the privilege of referring to the important paper of
Lillie on Chztopterus (02) as the discussion demands.
2. Effects of the Salt-solutions and of Shaking
There are several reasons why the eggs of Amphitrite are
favorable subjects for experimental studies of this kind. In the
fertilized egg the characteristic changes in shape at the time when
the polar bodies are expelled, the determinate type of cleavage
$2 Fobn W. Scott
resulting in an early segregation of the germ-layers, the rapid
ieiliesanaa, and early appearance of important differentiations,
all furnish favorable points for comparison. The unfertilized
egg responds not only to treatment with certain salt solutions but
is also highly susceptible to mechanical agitation. A specific
salt, calcium nitrate, may be used but the egg develops nearly as
well, especially if slightly agitated, in solutions where the osmotic
pressure has been raised (potassium chlorid, potassium nitrate).
These conditions applied separately to the egg give some important
modifications in development, but the indirect, or general, effect of
any one method is to bring about certain differentiations found in
the fertilized egg. It is not claiming too much then to say that
these differentiations are independent of the particular method
used, 7. ¢., they depend upon the organization of the egg. We may
also speak of the direct or specific effects upon the unfertilized egg
produced by each particular method.
Herbst has shown that cells in calcium-free sea-water tend to
separate from each other, and he (04) regards their tendency to
“Join together” in calcium-containing sea-water as a reversible
coagulation process. ‘This effect of salem nitrate then would
ultimately hinder cleavage and gastrulation as in the eggs of
Amphitrite. But when the osmotic pressure 1s increased either
with the calcium or potassium salt, the polar bodies are no longer
thrown off; the egg does not collapse when the germinal vesicle
breaks down, thus preventing the maturation spindle from getting
to the surface. It is probable that an excessive absorption of water
tends to prevent the collapse of the egg and so prevents the for-
mation of the polar bodies. Herbst (’04) has shown that potas-
sium is important for growth and that it leads to the taking up of
water. The appearance of Amphitrite eggs after treatment with
potassium chlorid is in this way accounted for if we consider the
blastomeres as swelling up with absorbed water (Fig. 17). The
strong solution also destroys, or weakens, some of the processes
of maturation (asters, spindle) and at the same time causes the
chromosomes to pass into a vesicular form and grow to an unusual
size. ‘lhe question arises, Is the suppression of the maturation
asters due to the chemical effect of the salt, or to an increased
Parthenogenetic Development of Amphitrite 83
osmotic pressure? I am inclined to the latter alternative. ‘The
effect is only temporary and this would probably not be the case
if the chemical nature of the egg had been changed. Another
argument in favor of this view is ‘that i in Cheetopterus eggs weak
solutions of potassium chlorid do not hinder the process ae matura-
tion while strong solutions do. ‘There is also evidence to show
that differentiating processes are taking place at the same time
though they may not be expressed in the normal morphological
form; for we find in proper sequence a reconstitution of the egg
nucleus by a fusion of the enlarged chromosome vesicles and the
ordinary phenomena of mitotic cleavage division. Besides I have
shown in an earlier paper that “there are processes of differentia-
tion going on in the unfertilized eggs of Amphitrite which may be
started into activity at definite intervals by mechanical agitation.”
After agitation the unripe eggs do not expel the oalee bodies
for the reasons which have been ruerconed. In ripe eggs shaking
causes such a disturbance in the organization of be cy ‘gplnen:
that cleavage is usually absent, and when present 1s very irregular
(Fig. 39). It is probable that this treatment so coagulates or at
least so alters the protoplasm in the ripe egg that it fails to flatten
in the polar diameter, and the physical conditions are then unfavor-
able for the expulsion of the polar globules.
The action of potassium chlorid on cleavage cells and in producing
spherules may be applied in explanation of certain phenomena
described by Lillie and Treadwell, who worked with strong con-
centrations of this solution. “Treadwell neticed in Podarke eggs,
a great variety of cytoplasmic cleavages. Lillie (02) describes
as a very common occurrence a sort of pseudo-cleavage which was
due to the separation of pseudopodia. But he also found in the
two cell-stage, where the nucleus passed into one cell, that the
direction of the cleavage plane was normal. In other words we
have here two factors at work, one a direct effect of the solution
in altering the viscosity or consistency of the egg cytoplasm, the
other a process connected with the differentiation of the egg.
It seems apparent that pseudo-cleavage and the production of
spherules are independent of cytoplasmic differentiation; these
conditions are the results of a general effect of the salt upon the
84. Fobhn W. Scott
egg protoplasm and have nothing to do with development.
I may add that I have repeated some of Lillie’s experiments, have
confirmed his results for the living egg, and find further support
for this view.
3. Is this Development Parthenogenesis ?
In discussing whether this sort of development should be termed
parthenogenesis or not, this much is clear: we find certain recog-
nizable differentiations that are found normally in fertilized eggs.
In the fertilized egg these differentiations are closely correlated
in time and place, in the unfertilized egg the correlation is less
complete. In the early development of the calcium-treated
unfertilized egg of Amphitrite there are found all the processes (as
indicated by mitotic division, cleavage, cilia, etc.), that are charac-
teristic of normal development. In so far then we may use the
term parthenogenesis, and if we wish to distinguish it from par-
thenogenesis that occurs under normal conditions we may call it
“artificial.” But on the other hand we must consider that
physiologically self-sustaining organisms are not produced; we
must remember that these processes become more and more
divergent as this pseudo-development proceeds and that a normal
larva is never produced even under favorable circumstances.
Therefore we cannot speak of this development as parthenogenesis,
meaning the production of a normal embryo from an unfertilized
egg. [he end result is always abnormal.
4. Relation of Differentiation and Cleavage
We may speak of differentiation as possessing a morphological
or a physiological character, and in a wide sense the problem of
the origin and process of differentiation is the problem of develop-
ment. But in this discussion the term will be used to mean any
specific morphological characters or physiological processes that
are clearly homologous to characters or processes in the develop-
ment of the fertilized egg. ‘The definition has no reference to the
accurate localization of organs and substances or to the correlation
of processes necessary to normal development. Used in this sense
the important differentiations that I have found in the unfertilized
Parthenogenetic Development of Amphitrite 85
egg of Amphitrite are the following: ‘The early development of a
layer of ectoplasm and with it a tendency for the yolk to collect
centrally, the formation of cilia, the development of a brownish-
black pigment, the appearance of vacuoles that are found in the
fertilized egg of the same age, ameboid movements that are con-
nected with cleavage, and ameboid movements at a late stage that
are independent of cleavage. We have seen that these forms
of differentiation are relatively independent of cytoplasmic cleavage
and the expulsion of polar globules. What then 1s the nature of
these differentiations’ ‘The growth of cilia shows that there has
arisen in these regions a newspecific composition of the cytoplasm.
The development of a pigment likewise indicates a chemical com-
position which was not present before. “The rapid ameboid move-
ments occurring at a late period suggests a functional difference
in the cytoplasm, and the slow ameboid movements connected
with cleavage depend upon processes of differentiation which
modify the viscosity of the cytoplasm. The presence of vacuoles
at a definite period after development begins, points to the con-
clusion that they are caused by processes which have their basis
in the organization of the egg. [he same may be said of the
early differentiation of a layer of ectoplasm and the processes
involved in the transformation of the asters and the mitotic
division of the nucleus. In all of these differentiations we find
evidence of preceding morphological organization or chemical
activity, a fact in agreement with the generally accepted idea that
morphological differentiation usually involves and is preceded by
differentiation of a chemical character. “The nature of differentia-
tion therefore depends ultimately upon the organization of the egg,
which has its basis in cytological structure and specific chemical
composition. That the unfertilized egg of Amphitrite gives
evidence of both morphological and physiological organization 1s
shown by the following facts. Polarity is present from a very
early stage and the yolk is deposited in relation to this polarity.
The cytoplasm shows a difference in permeability and the definite
way in which the ripe egg collapses preceding maturation 1s cer-
tainly dependent upon the structure of the egg. But in addition
to these morphological characters we can prove the presence of
86 ‘fobn W. Scott
conditions in the egg which, if disturbed by mechanical agitation, -
lead to certain kinds of differentiation and development. ‘The
organization in many other eggs is well known; for example, the
sea-urchin (Boveri, ’01), Unio (Lillie, ’o1), Crepidula (Conklin,
’02), the Ctenophore (Fischel, ’03), and the Ascidian (Conklin,’03).
These phenomena of differentiation are relatively independent
of each other and, since they are equally independent of cleavage,
Lillie (02) has pointed out that the cleavage, therefore, cannot be
considered the cause of any of these differentiations, and we may
add that the same is true of the processes involved in throwing off
the polar bodies, unless one excepts the fact that the extra amount
of chromatin is conducive to the formation of multipolar spindles.
The development of cilia is relatively independent of nuclear
division, for cilia are found on eggs that have many or very few
nuclei. A similar relation holds for the development of vacuoles.
On the other hand considerable nuclear division seems always to be
present whenever pigment is found. In like manner transforma-
tion of the asters is always accompanied by division of the nucleus,
and both processes are followed by more or less effectual attempts
at cleavage. In regard to the rapid ameboid movements at a
late stage, I have studied such a small number of eggs that it would
be unsafe to draw general conclusions. Whenever observed these
eges had none of the other differentiations mentioned and were
without cleavage, but the cytoplasm possessed a light appearance
which is characteristic of normal development at about the same
age.
It is well known that nuclear without cell division may take
place normally in the eggs of Arthropods. Various methods have
been used with success to produce the same process by artificial
means; we have also seen how the ripeness of the egg determines
whether the two processes shall occur together, the treatment being
the same in each case. ‘The study of enucleated, fertilized egg-
fragments has added further data to this question. The multi-
plication and transformation of asters in such egg-fragments has
been found to occur independent of the nucleus; it has been shown
further that this may be found without (Boveri, ’97), or with
division of the cytoplasm (Zeigler, ’98; Wilson, ’or; Boveri,
Parthenogenetic Development of Amphitrite 87
M. ’03). Boveri (97) and Driesch (’98) have contributed the
additional fact that the rhythm of cleavage in hybrid-fertilized
ege-fragments depends upon the egg-cell and is not controlled by
the sperm. It is probable then that the rhythm of cleavage
depends upon the cytoplasm and not upon the nucleus. Conklin
(02) concluded that the position of the spindle is the result of
movements and stresses in the cytoplasm and that the position of
the spindle and direction of division are functions of the cyto-
plasm. He has also called attention to the importance of cyto-
plasmic movements in causing early differentiations in cell-
divisions. Lillie (98) demonstrated in the ege of Unio that the
size of the cells and the tate and direction of cleavage possess a
prospective significance and he concluded that these phenomena
were explainable, “by the hypothesis of differentiation of the.
cytoplasm into materials of different qualities and positions.”
It will be remembered also that he found cytoplasmic without
nuclear division in the egg of Chaetopterus. ‘Treadwell (’98) in
comparing an egg with equal (Podarke) with anosher with unequal
cleavage (Amphitrite), says, “There can be no question that in
Podarke there is as great differentiation as in any Annelid of the
unequal type.’ Wilson (or) found that the nuclear area may
give rise to a monaster which passes through transformations
parallel to normal division. During telophase the egg frequently
becomes ameboid and later it may actually divide into a number
of irregular masses, one of which contains a nucleus. From this
study of the parthenogenetic egg of Toxopneustes he drew the
conclusion, “that any or all of the asters, whether connected with
the nucleus or not, may operate as centers of cytoplasmic division,
though if unconnected with the nuclear material the activity ordi-
narily goes no further than an abortive attempt to divide.”
Having mentioned some of the facts in regard to these phenomena
let us return once more to the conditions in Amphitrite.
My observations show that the more nearly normal the asters
and the astral radiations are, the nearer the cytoplasmic cleavage
approaches the normal. ‘That is, when the centrospheres develop
into the typical large, clear areas and the astral radiations reach
far into the cytoplasm with straight, strong, and clearly defined
$8 fobn W. Scott
rays, che cytoplasmic cleavage is normal. But when the centro-
sphere and asters are poorly developed, the cleavage of the cyto-
plasm is more or less incomplete or abortive, whether the egg be
fertilized or not. “The same thing may be said in regard to the
relation between abnormalities in the asters and the division of
the chromatin, only in this case the fragmentary and unequal
division of the chromatin is apparent in late metaphase before the
aster has reached its full growth.
From a study of these facts it is seen that the development and
transformation of the asters is the most general of all these phe-
nomena. And since this is the process that invariably precedes
cleavage, we may consider it the expression of the active forces
which so alter the viscosity and surface tension of the egg-cell that
cleavage is the result. ‘his view does not in any way conflict
with the conditions found in Unio and Crepidula, where the place
of cleavage is prearranged in the cytoplasm. Looked at from this
point of view cleavage is an incident correlated with certain
phenomena of differentiation, and its purpose is adaptive to the
needs of the organism. And if we regard the egg as a simple
mosaic, as all recent works seem to indicate, then cleavage is a
tendency to localize or isolate processes of differentiation by
separating them with cell walls. When each new cell wall becomes
established greater differentiation becomes possible. “Uhe form of
cleavage results from organization and differentiations in the egg,
and is not itself a process of differentiation.
5.- Causes of the Cessation of Development
In our description and discussion of the unfertilized egg, we
noted various conditions which hindered or stopped further
development. But the facts given, instead of being causes for no
further development, were in most cases simply a statement of the
conditions within the egg after it had become so abnormal that it
was incapable of regulating or correlating further processes.
Let us see if we can trace these conditions to more immediate
causes. From our understanding of the relation of cleavage to
differentiation, it is easy to see why there is no further develop-
ment in unripe eggs. For in these eggs the viscosity of the cyto-
Parthenogenetic Development of Amphitrite 89
plasm is unfavorable to cleavage and lack of cleavage prevents
the localization of various differentiating processes. Even in the
fertilized eggs where the cytoplasm is not quite ripe we find the
same retarding effects and ultimately the same cessation in develop-
ment. ‘lhe interesting question for us then is, Why does the
development of the unfertilized eggs stop though they are
apparently in a ripe condition? In such eggs polar bodies are
formed in quite the normal manner, the early cleavages have the
same outward appearance and practically the same rhythm as the
fertilized ege. ‘That development ceases is not from a lack of
nutritive material for this is sufficient in quantity and in a condi-
tion suitable for metabolism. It is not from a lack of formative
material for the entire egg is present and the cytoplasm is of a
proper ripeness. But whenever abnormalities appear they are
first found in connection with the asters, and following or accom-
panying them we find unequal and fragmentary divisions of the
chromatin. Abnormal phenomena in all other respects appear
later. It is therefore logical to conclude that the cessation of devel-
opment has some intimate connection with the developmental
processes concerned with the growth and transformation of the
asters, and that the latter process is in close relation with the
periodic nuclear changes. Let us examine still other conditions
found in the egg.
‘The entrance of the sperm certainly causes profound changes in
the cytoplasm since no other sperm then finds it possible to enter
the egg. ‘That the effect of the various salts I have used is not the
same as that of the sperm is proved by the fact that swimmers
may be developed by these methods from eggs that are too unripe
to be fertilized by the sperm. In the one case the sperm is kept
out by the unripe cytoplasm, in the other the solution penetrates
or alters the cytoplasm and causes the germinal vesicle to break
down, thus setting free forces that lead to nuclear division and
other phenomena of development. But the fact that some of
these eggs may be started in development by mechanical agita-
tion indicates that simply a change in the state of the protoplasm
is sufficient to start development. Mathews and Whitcher (’03)
have concluded that, “For artificial parthenogenesis nothing else
go ‘fohn W. Scott
is necessary than that this change be produced.” Nevertheless
we have seen that something else is necessary in order to insure the
development of normal larvas, and that this something has to do
with asters and chromatin. It is probable that agitation in some
way causes the walls of the germinal vesicle to break down and
thus permits reactions between nucleus and cytoplasm.’ Now it
is clear that the effect of the methods I have used to produce
artificial parthenogenesis 1s not to cause a cessation of develop-
ment for the end result is practically the same in each case.
Primarily of course the egg lacks the elements introduced by the
sperm, the most important being the male chromosomes and the
sperm centrosome. But inasmuch as normal mitosis may take
place without the sperm, and on account of the inconstant presence
of a distinguishable centrosome in eggs of Amphitrite, it is evident
that we may neglect these factors so far as the question under con-
sideration is concerned. In any case they can bear only a sub-
sidiary relation to cleavage and differentiation.
Perhaps no other fact is so evident, and at the same time so
little understood, as the reaction of nucleus and cytoplasm in
development. When these relations are solved there is little doubt
that we shall obtain thereby at least some of the causes of mor-
phogenic metabolism. We know that by supplying chemically-
known food constituents we may produce the growth and repro-
duction of the yeast-cell, and in its development it follows ordi-
nary laws of chemical reaction. ‘There are many reasons to
believe that similar processes take place in the animal cell. But
here the chemical complexity hides from us at present the exact
nature of many of the processes of development and differentia-
tion which could be easily understood. All my experiments indi-
cate in a most striking manner the intimate relation that exists
between cytoplasmic and nuclear differentiation; the correlation
in development between these two factors is very complete where a
normal organism results. And inasmuch as the cessation of
development is a culminative process, that is, the abnormalities
appear in successive transformations of the asters and nucleus,
we must look upon the cessation of development as due to incom-
plete reactions between the nucleus and the cytoplasm, each suc-
Parthenogenetic Development of Amphitrite gt
cessive reaction depending in some measure at least upon the
preceding one. In applying this conception to Amphitrite, the
question is still left open as to whether the cessation of develop-
ment is a qualitative or quantitative divergence from the normal
course of events. While it is probably true that the nuclear
material derived from the male is qualitatively different from that
derived from the female, as is shown by inherited characters, so
far as the phenomena found in the unfertilized eggs of Amphitrite
are concerned, the abnormality reveals itself first as a quantitative
and not as a qualitative difference. I refer to eggs where only
half the number of chromosomes are present. Now, where two
chemical substances are combined in definite proportions to pro-
duce a series of chemical phenomena, if only one-half the required
amount of one of these be taken, it is evident that the series of
reactions will be incomplete, and the process will come to an end
sooner than under normal conditions; we may suppose the reac-
tion in the beginning differs simply in quantity from the normal,
and that qualitative differences may possibly appear later due to
reactions set up with the substance found in excess. ‘This con-
ception then applies to the egg treated with calcium nitrate where
both polar bodies are expelled; the first mitotic division does not
differ in kind from the normal but only in the number of chro-
mosomes that undergo splitting at metaphase.
The conditions of my experiments have been such that in ripe
eggs either both or no polar bodies were expelled. If a means
could be devised to retain the chromosomes of the second polar
body within the ripe egg, it would be interesting to note if normal
development occurred. For we should then have the normal
number of chromosomes. In the potassium chlorid more than the
normal number are present; connected with this is a tendency to
form multipolar asters as in double-fertilized eggs, though the
normal number of chromosome vesicles or only half this number
may be present in the telophase of the first cleavage division.
The frequent occurrence of the multipolar asters might be
explained as due to a tendency of the excess of chromatin to collect
in more than one vesicle during the telophase of the preceding
division. Or, it may be a tendency for the chromosomes that
92 Fobhn W. Scott
normally constitute the polar bodies to maintain their individuality
in groups. But in all cases the development stops, and it cannot
always be due to an insufficient amount of chromatin. While the
suggestion of Boveri (02) that the chromosomes are qualitatively
different, may be accepted as a possibility, it seems probable that
in Amphitrite the sperm chromosomes are a means of producing
morphogenic processes which the normal number or half the
normal number of female chromosomes may start but find it
impossible to continue for any length of time. However there is
still the possibility that the strong solution so affects the cyto-
plasm that it is incapable of reacting on the nucleus in the normal
manner.
SUMMARY OF RESULTS
Normal Egg
1. In general, the early development (maturation, fertiliza-
tion) conforms to the typical Annelid type. For cleavage and
later development, so far as my observations go, I have verified
the work of Mead.
2. Under normal conditions the egg is retained in the body
cavity until the first maturation spindle is in metaphase. When
deposited and left undisturbed the egg rests in this condition.
If fertilized the formation of the polar bodies and the ensuing
development follows.
3. The ripe egg shows a distinct polarity as evidenced by the
eccentric position of the germinal vesicle. This polarity is found
before any yolk is laid down in the cytoplasm and the deposition
and arrangement of the yolk is apparently determined in relation
to the nucleus. ‘There is thus indicated a definite structural
organization of the egg at a very early period.
4. Peculiar and perfectly definite cytoplasmic changes occur
in connection with the formation of the polar globules.
5. The reduced number of chromosomes is eleven, the somatic
number twenty-two. The eleven chromosomes found at the
metaphase of the first maturation division are probably derived
from eleven groups of chromomeres (chromosomes) of four each.
Parthenogenetic Development of Amphitrite 93
6. The rate of development is comparatively rapid. ‘The
primary germ lay ers are entirely separated at the 64-cell stage,
and blastulas swim within four to five hours after fertilization.
Unfertilized Eggs
1. Under the conditions of the experiments certain forms of de-
velopment occur with or without cleavage and with or without the
formation of polar bodies in the unfertilized egg of Amphitrite.
Such development takes the form of nuclear ee. the early
differentiation of a layer of cytoplasm, the growth of cilia, the
appearance of vacuoles that are found in the fertilized ege of the
same age, the development of a brownish pigment, the ameboid
movements of the cytoplasm that are connected with cleavage,
the ameboid movements at a later stage of development that
appear entirely independent of cleavage, aad the change in shape
of the egg which in most cases at least 1s connected with incom-
plete, arrested or abortive division of the cytoplasm. ‘The apical
tuft of cilia which is characteristic of trochophores from fertilized
eggs is always absent.
2. [he means employed for producing this development were,
(a) the use of certain salt solutions and (6) some method of
agitating the eggs.
3. he calcium nitrate solution used stimulated the for-
mation of polar bodies, produced nuclear division, and tended to
cause a fusion of the cleavage cells. “The potassium chlorid pre-
vented the formation of the polar bodies, stimulated division of
the nucleus, and where cell division was complete tended to sepa-
rate the blastomeres.
4. Though some eggs would occasionally stick together no
actual fusions took place.
5. Cleavage when present is usually abnormal; it tends to
grow more abnormal as development proceeds and [| have never
seen a perfectly normal type of cleavage at a late stage. The
segmentation cavity is therefore wanting or only partially
developed. ‘There was no cytoplasmic without preceding nuclear
division.
6. The cleavage asters, especially the astral radiations, fre-
94. Fobn W. Scott
quently show imperfections at the first cleavage. [he rays tend
to be weak and fragmentary and do not take such a clear, definite
stain as the normal.
7. Nuclear division is always mitotic. It may be equal at
first but sooner or later becomes unequal and fragmentary. In
the potassium chlorid solutions multipolar mitoses are common;
this is undoubtedly associated with the extra amount of chromatin
retained in the egg. In later stages a nuclear vesicle may enlarge
extraordinarily unless mitosis occurs.
8. Normal polar bodies are expelled in the weaker calcium
nitrate solution if the eggs are in a ripe condition. If the cyto-
plasm is not ripe the egg does not alter its shape; consequently
the maturation spindle though normal in other respects does not
come to the surface and the polar bodies are not expelled. Solu-
tions with high osmotic pressure prevent entirely or permit only
partial formation of very weak asters and spindle that are incom-
petent to cause maturation division. Moreover the chromosomes
swell up into large vesicles at this time.
g. The “morula” of Fischer is in all probability not a form
of development, since no differentiations characteristic of normal
development are ever found.
10. Amphitrite eggs are very susceptible to agitation, especially
at certain periods of development.
11. Results obtained from any experiment depended much
upon the ripeness of the given lot of eggs. The riper the eggs,
the more rapidly does differentiation take place and the nearer
it approaches the normal development.
12, The rate of development is always slower than for the
fertilized eggs. [he germinal vesicle breaks down sometimes
within a few minutes, sometimes several hours after being treated
with the solution. ‘The first swimmers are usually noticed seven
to ten hours after the beginning of the experiment, and ordinarily
the maximum number of swimming eggs is found between the
twelfth and twenty-fifth hours.
13. It has not been found possible to produce physiologically
self-sustaining organisms by these methods. The differentiation
always diverges more and more widely from the normal and con-
= ae
Parthenogenetic Development of Amphitrite 95
sequently we do not find in the late stages any regulation of abnor-
malities or any true correlation.
14. That death is not caused by environment is proved by the
fact that fertilized eggs may be raised under the same conditions.
REFERENCES.
Bovert, M., ’03.—Ueber Mitosen bei einseitiger Chromosomenbindung. Jen.
Zeitsch., Bd. xxx, S. 401.
Boveri, TH., 97.—Zur Physiologie der Kern. und Zellteilung. Sitz.-Ber. Phys.
Med. Ges. z. Wiirzburg.
‘o1.—Die Polaritat von Ovocyte, Ei und Larve des Strongylocentrotus
lividus. Zodl. Jahr. Abthiel. f. Anat., Bd. xiv.
*o2.—Mehrpolige Mitosen als Mittel zur Analyse des Zellkerns. Verh.
d. Phys. med. Gesell. z. Wurzburg, Bd. xxxv,
Buttot, G., ’o4.—Artificial Parthenogenesis and Regular Segmentation in an
Annelid (Ophelia). Archiv fur Entwickelungsm. der Organis-
men, Bd. xviii.
Cuitp, C. M., ’97.—The Maturation and Fertilization of the Egg of Arenicola
marina. ‘Trans. N. Y. Acad. Sci., vol. xvi.
Conkiin, E. G., ’98—Protoplasmic Movement as a Factor of Differentiation.
Biol. Lect., Woods Hole in 1898-99.
’o2.—Karyokinesis and Cytokinesis in the Maturation, Fertilization and
Cleavage of Crepidula and other Gasteropoda. Jour. Acad.
Nat. Sci., Phila., series 2, vol. xi.
°03.—Lecture at Marine Biological Laboratory, 1903. Biol. Bull.,
June, 1904.
Drirscu, H., ’98—Ueber rein-miitterliche Charactere an Bastardlarven von
Echiniden. Archiv f. Entwickelungsm. d. Organismen, Bd. vii.
FiscHEL, A., ’03.—Entwickelung und Organ-Differenzirung. Archiv f. Ent-
wickelungsm. d. Organismen, Bd. xv.
FiscHer, M. H., ’o2—Further Experiments on Artificial Parthenogenesis in
Annelids. Am. Jour. Physiol., vol. vii.
Hersst, C., ’04.—Ueber die zur Entwickelung der Seeigel-larven nothwendigen
anorganischen Stoffe, ihre Rolle und ihre Vertretbarkeit. III.
Teil. Die Rolle der nothwendigen anorganischen Stoffe. Archiv
f. Entwickelungsm., Jan., 1904.
96 Fobn W. Scott
Linu, F. R., ’98.—Adaptation in Cleavage. Biol. Lect. Wood’s Hole, 1898.
°or.—The Organization of the Egg of Unio Based on a Study of the
Maturation, Fertilization and Cleavage. Jour. Morphol., vol. 17.
°o2.—Differentiation without Cleavage in the Egg of the Annelid Chz-
topterus pergamentaceous. Archiv f. Entwickelungsm., Bd. xiv.
Loes, J., °01.—Experiments on Artificial Parthenogenesis in Annelids (Chztop-
terus) and the Nature of the Process of Fertilization. Am. Jour-
Physiol., vol. iv, No. 9.
’02.—Ueber Methoden und Fehlerquellen der Versuche tiber kiinstliche
Parthenogenesis. Archiv f. Entwickelungsm., Bd. xiii.
Loes, J., Fiscuer, M. H., anp Netrtson, H., ’°03.—Weitere Versuche tber kinst-
liche Parthenogenesis. Vorlaufige Mittheilung. Pfliiger’s Archiv.,
Bd. Ixxxvil.
Matuews, A. P., anpD WuitcHer, B. R., ’03.—The Importance of Mechanical
Shock in Protoplasmic Activity. Am. Jour. Physiol., vol. viii.
Meap, A. D.,’97.—The Early Development of Marine Annelids. Jour. Morphol.,
vol. xiii.
’98.—The Origin and Behavior of the Centrosomes in the Annelid Egg.
Jour. Morphol., vol. xiv.
*98.—The Rate of Cell-Division and the Function of the Centrosome.
Biol. Lect. Wood’s Hole, 1896-97.
Morean, T. H., ’95.—Studies of the Partial Larvae of Sphzrechinus. Archiv f.
Entwickelungsm., Bd. 11.
‘oo.— Further Studies on the Action of Salt-solutions and of Other Agents
on the Eggs of Arbacia. Archiv f. Entwickelungsm., Bd. x.
Scott, J. W., °03.—Periods of Susceptibility in the Differentiation of Unfertilized
Eggs of Amphitrite. Biol. Bull., June, 1903.
TREADWELL, A. L., ’98.—Equal and Unequal Cleavage in Annelids. Biol
Lect., Wood’s Hole, 1898.
‘o2.—Notes on the Nature of “Artificial Parthenogenesis” in the Eggs
of Podarke obscura. Biol. Bull., Oct., 1902.
Witson, E. B., ’or.—Experimental Studies in Cytology I. A Cytological Study
of Artificial Parthenogenesis in Sea-urchin Eggs. Archiv f.
Entwickelungsm., Bd. xii.
Parthenogenetic Development of Ampbitrite 97
Witson, E. B., ’o1—02.—II. Some Phenomena of Fertilization and Cell-division
in Etherized Eggs. III. The Effect on Cleavage of Artificial
Obliteration of the First Cleavage-furrow. Archiv. f. Entwick-
elungsm., Bd. xiii.
ZiEGLER, H. E., ’98.—Experimentelle Studien uber die Zelltheilung. I. Archiv
f. Entwickelungsm., Bd. vi.
DESCRIPTION OF FIGURES.
The essential features of all figures were drawn with a camera lucida. The tube length used is
written in (cm.) immediately after the size of the ocular.
Reference letters.—a, Apical tuft of cilia; c, chromomeres (chromosomes); d, egg membrane;
e, brownish pigment; f, female chromosome vesicle (vesicles); g, polar globules; /, layer of ectoplasm;
m, male pronucleus; me, mesoderm cells; 1, nucleus, nuclear areas; nu, nucleolus; p, peri-vitelline
space; pr, prototroch; a, paratroch; r, region affected by nuclear sap; s, segmentation cavity;
v, vacuoles; x, clear spot left by nucleolus; y, yolk granules (yolk).
Prater I.
All figures to illustrate the normal development.
Fig. 1. Unfertilized egg after breaking down of germinal vesicle. Shows collapse of the egg,
origin of asters, formation of chromosomes, dissolution of nucleolus, and a region differentiated by
escape of nuclear sap. Leitz oc. 1 (20), obj. 1-12.
Fig. 2. Unfertilized egg; asters better developed but other conditions perhaps not quite so advanced
asin Fig. 1. Leitz oc. 1 (20), obj. 1-12.
Fig. 3. First maturation spindle forming; nucleolus has dissolved leaving a clear spot. Leitz
oc. 4 (20), obj. 1-12.
Fig. 4. First maturation spindle has rotated and now lies in the polar axis; chromosomes in early
prophase. Leitz oc. 4 (20), obj. 1-12.
Fig. 5. Metaphase of first maturation spindle; peri-vitelline space formed. Leitz oc. 4 (20),
obj. I-12. .
Fig. 6. Late anaphase, first maturation. Leitz oc, 4 (20), obj. 1-12.
Fig. 7. Condition just after expulsion of the first polar body. Leitz oc. 4 (20), obj. 1-12.
Fig. 8. Late anaphase, second maturation spindle. Fifteen minutes after fertilization. Leitz
oc. 4 (20), obj. 1-12.
Fig. 9. Shortly after expulsion of the second polar body. Fifteen minutes after fertilization.
Leitz oc. 4 (20), obj. 1-12.
Fig. 10. Age, after fertilization, twenty minutes. The male pronucleus in central position; female
chromosomes passing into the vesicular condition and retreating toward the center of the egg. Leitz
oc. 1 (18), obj. 1-12.
Fig. 11. The greatly enlarged pronuclei just before fusion. Age, twenty-five minutes. Leitz
oc. 4 (20), obj. 1-12.
Fig. 12. Metaphase of first cleavage division; age thirty minutes. The asters affecting a large
area enclosed within a dotted line. Leitz oc. 1 (18), obj. 1-12.
Fig. 13. Early telophase of first cleavage division. Age, forty-one minutes. Leitz oc. 1 (18),
obj. 1-12.
Fig. 14. Asters dividing for second cleavage division. Age, forty-eight minutes. Leitz oc. 1
(38), obj. 1-12.
Fig. 15. Sagittal, horizontal (nearly) section of early gastrula; cilia not shown. The mesoderm
’ consists of four cells, two small ones not shown in this section. Dotted lines enclose nuclei. Nuclei
of some cells moving centrally, a characteristic of invaginating entoderm. Leitz oc. 3, obj. 1-12.
Fig. 16. Normal gastrula twenty-three hours old; camera drawing from living embryo; only some
of the more characteristic details shown.
THENOGENETIC DEVELOPMENT OF AMPHITRITE. Joun W. Scort. PLATE I.
HE JOURNAL oF ExPERIMENTAL ZoGLoGy, VOL. II.
Pirate II.
All figures, except 33 from a whole mount, are from living material. Figs. 17-22 and 30 are from
eggs treated with potassium chlorid, method 3 of text; Figs. 23-29 and 31-33 are from calcium nitrate
cultures, method 1; 34-38 from calcium chlorid cultures, method 5; 39 from swimming egg produced
by shaking. For the age of an egg, the time after the beginning of the experiment is given.
Fig. 17. Age, four hours and fifty minutes. Loose arrangement of cells; nuclei shown in dotted
lines.
Fig. 18. First one seen to move in this experiment; age, six‘hours and twenty-eight minutes.
Fig. 19. Egg taken from the bottom of the dish; age, nineteen hours. Ectoplasm well differen-
tiated from the yolk.
Fig. 20. Age, twenty-three hours and twelve minutes. Swimming at surface. Unusual amount
of cleavage, one nucleus to each cell; vacuoles present. Type of more active swimmers.
Fig. 21. Age, twenty-three hours and twenty minutes; from bottom of dish. No cleavage; two
large, clear spots (nuclear areas) were to be seen.
Fig. 22. Age, twenty-one hours and twelve minutes. A type intermediate between Figs. 20 and
21; egg composed of four cells, but incompletely divided; cilia on one cell.
Fig. 23. Age, twelve hours. No cleavage; bluntly pear-shaped. Leitz oc. 1, obj. 7.
Fig. 24. Age, twenty-three hours and twenty minutes. Comparatively little cleavage, cells not
completely separated. Leitz oc. 1, obj. 7.
Fig. 25. Age, nineteen hours and thirty minutes. Incomplete cleavage. Leitz oc. 1, ob. 7.
Fig. 26. Age, twenty hours. Common type; no cleavage; several nuclei; brownish pigment in
two regions; moderately active. Leitz oc. 1, obj. 7.
Fig. 27. Age, twenty hours. Four cells; many nuclei in the large cell in which pigment is
developed. Leitz oc. 1, obj. 7.
Fig. 28. Age, twenty-two hours and thirty minutes. Common type; incomplete and probably
partly fused cleavage planes; many nuclei; pigment in four regions. Leitz oc. 1, obj. 7.
Fig. 29. Age, twenty-eight hours. One type of most active swimmers; no cleavage; many nuclei;
pigment in two regions. Leitz oc. 1., obj. 7.
Fig. 30. Age, thirty-seven hours and twenty-five minutes. Comparatively few cells and nuclei,
but these are distinct; from bottom of the dish.
Fig. 31. Age, forty-one hours. Swimming actively; no cleavage, but with an attached yolk lobe;
many nuclei; pigment in four regions. Leitz oc. 1, obj. 7.
Fig. 32. Age, forty-eight hours. Oldest actively swimming embryo found; indications of one
cleavage plane; very many nuclei (comparatively); diffuse pigment in one region. Leitz oc. 3, obj. 5.
Fig. 33. Age, fourteen hours. From stained whole mount; no cleavage; nuclei vary greatly in
size; one or more nucleoli in most of the nuclei. Leitz oc. 1 (15), obj. 1-12.
Fig. 34. Age, nine hours. Intermediate type of cell and nuclear division.
Fig. 35. Age, twenty hours. Much pigment; bluntly pear-shaped, due to early cleavage
conditions.
Fig. 36. Age, twenty hours. From same experiment as preceding; three incompletely divided
cells, with one nuclear area in each.
Fig. 37. Age, twenty-five hours. Very active swimmer; no segmentation; three very large
nuclear areas; much pigment.
Fig. 38. Age, twenty-three hours. No cleavage; considerable nuclear division.
Fig. 39. Produced by shaking. Swimming near surface at about twenty hours.
PA RTHENOGENETIC DEVELOPMENT OF AMPHITRITE. Joun W. Scort.
Stine aye
Ses
17
: Journat oF ExperiMENTAL ZoGroey, VoL. m1.
II.
Prate III.
All figures of this plate are from sections of eggs treated with calcium nitrate. The age is dated
from the time the eggs were put in the solution.
Fig. 40. Unripe egg. Age, fifty-six minutes. No collapse in polar diameter; first maturation
spindle in metaphase, undergoing mitosis without moving to the surface; thick egg membrane charac-
teristic of unripe condition. Leitz oc. 1 (20), obj. I-12.
Fig. 41. Unripeegg. Same age as preceding; in late anaphase.
Fig. 42. Unripeegg. Age, two hours and twenty-six minutes. First maturation spindle in ana-
phase has advanced part way to the surface; unequal division of chromosomes. Leitz oc. 4 (20),
obj. I-12.
Fig. 43. Very unripe egg. Age, three hours and fifteen minutes. Development very slow; very
abnormal division of chromosomes; asters still in position of origin. No polar bodies formed, Figs.
40-43. Leitz oc. 4 (20), obj. 1-12.
Fig. 44. Both polar bodies expelled, each one slightly abnormal; female chromosomes passing
into vesicular condition. Age, forty-one minutes. Leitz oc. 1 (20), obj. 1-12.
Fig. 45. First polar body nearly normal, second polar body too large. Age, twenty-four minutes.
Leitz oc. 4 (20), obj. 1-12.
Fig. 46. Polar bodies abnormal, the second very much enlarged with yolk granules; the female
pronucleus in preparation for first cleavage division. Age, sixty-eight minutes. Leitz oc. 1 (20)
obj. 1-12.
Fig. 47. Unripeegg. First polar body partly expelled; rest of very abnormal chromosomes have
retreated to near center of egg. Age, three hours and fifteen minutes. Leitz oc. 4 (20), obj. 1-12.
Fig. 48. Division of the nucleus without cleavage; no polar bodies. Age, sixty-eight minutes.
Leitz oc. 1 (20), obj. 1-12.
Fig. 49. Polar bodies (not shown); groups of chromosome vesicles removed less than a normal
distance from one another; eleven vesicles in each group; abortive cleavage results. Age, sixty-eight
minutes. Leitz oc. 1 (20), obj. 1-12.
Fig. 50. Three-celled egg (drawn from three sections). Age, one hour and forty minutes. Leitz
oc. I (17), obj. 1-12.
Fig. 51. Typical incomplete cleavage. Age, fourteen hours. Leitz oc. 1 (20), obj. 1-12.
Fig. 52. Typical of condition with no cleavage; many nuclei, which vary greatly in size; some
vacuoles. Age,twenty hours. Leitz oc. 1 (19), obj. 1-12.
Fig. 53. Typical of condition when cleavage is present; few cleavage planes complete; vacuoles.
Age, twenty hours. Leitz oc. 1 (20), obj. 1-12.
Fig. 54. The most advanced and most nearly normal type of cleavage found in my sectioned
material; the mesoderm has invaginated; vacuoles present. Age, twenty hours. Leitz oc. 1 (18)
obj. I-12.
RTHENOGENETIC DEVELOPMENT OF AMPHITRITE. Joun W. Scort. PEALE Lie
di
Pue JourNar or ExpertMentaL Zo6.oey, Vor. 1.
Pirate IV.
Figures of this plate are all from eggs treated with potassium chlorid.
Fig. 55. Condition during maturation; one aster present; chromosomes in vesicular condition
and greatly enlarged; no collapse in polar diameter. Age, one hour and thirty-eight minutes. Leitz
oc. I (18), obj. 1-12.
Fig. 56. Maturation phenomena; both asters and a faint spindle present; egg did not collapse.
Age, one hour and thirty-eight minutes. Leitz oc. 1 (18), obj. 1-12.
Fig. 57. Prophase of first cleavage division. Age, two hours and forty-three minutes. Leitz
oc. 3, obj. I-12.
Fig. 58. Anaphase of first cleavage division. Cleavage in such eggs usually abortive. Age,
two hours and seventeen minutes. Leitz oc. 3, obj. 1-12.
Fig. 59. Telophase of first cleavage divisions; 20 (+4?) chromosome vesicle in the right hand
group, 18 (+4?) inthe left hand group. Cleavage in such eggs is frequently complete. Age, two hours
and twenty-five minutes. Leitz oc. 3, obj. 1-12.
Fig. 60. Two-celled stage; division complete; eleven enlarged chromosome vesicles in each cell.
Probably indicates one origin of multinuclear cells. Age, three hours and fifty-five minutes. Leitz
OC. 3, obj. I-12.
Fig. 61. Multipolar asters; anaphase of the second division. Chromatin division irregular and
unequal. One method of origin of multinuclear cells. Age, three hours and fifty-five minutes. Leitz
0c. 3, obj. I-12.
Fig. 62. Cleavage planes complete; irregular and unequal division of chromatin in one cell.
some cells multinuclear; diffuse chromatin stain near center of egg indicates original position of the
germinal vesicle. Age, four hours and forty-two minutes. Leitz oc. 3, obj. 1-12.
Fig. 63. Several vesicles in each cell; cleavage planes complete. Age, four hours and forty-two
minutes. Leitz oc. 3, obj. 1-12.
Fig. 64. Egg showing typical cleavage. Mitotic figure in one cell; more than one nuclear vesicle
in others; part of segmentation cavity present. Age, eight hours and forty-seven minutes. Leitz
Oc. 3, obj. I-12.
Fig. 65. Egg typical of incomplete cleavage with probably later fusion. Nuclei vary greatly in
size. Age,eight hours. Leitz oc. 3, obj. 1-12.
Fig. 66. Typical of eggs with no cleavage. A few large nuclei; large vacuoles. Age, twenty
hours. Leitz oc. 3, obj. 1-12.
Fig. 67. One of the most advanced types of cleavage found in this solution. Evident invagination
of the mesoderm. Age,twenty hours. Leitz oc. 3, obj. 1-12.
PLATE IV.
PA RTHENOGENETIC DEVELOPMENT OF AMPHITRITE. Joun W. Scorr.
‘Tue Journat or ExperiMentaL Zo6 oey, VOL. Ill.
ear) ‘faces
;
oa
foe DEVELOPMENT OF FUNDULUS HETEROCLI-
Lis aN SOLUDIONS, OF LITHIUM CHLORID,
With, APPENDDSE ON ITS DEVELOPMENT IN
FRESH WATER
BY
CHAREES R. STOCKARD
With NINETEEN Ficures
The eggs of Fundulus heteroclitus are hardy and, therefore,
stand experimental treatment most satisfactorily. Since they
are capable of development in both salt and fresh water it 1s
possible to eliminate all of the physical effects of hypertonic
solutions, for effective solutions of a salt may be applied in fresh
water that have a final pressure lower than that of normal sea
water. Mathews has stated that the eggs of this fish are pecu-
liarly adapted to treatment with solutions because they appear
to be easily penetrated by nearly all sorts of ions, and they are
quite insensitive to variations in osmotic pressure of the solutions.
Whether this statement is absolutely true or not there can be little
doubt of the fact that the ions resulting from LiCl in solution do
penetrate the membranes and give an effect that is certainly not
due to osmotic pressure, since the same result 1s obtained with
solutions of LiCl in both salt and fresh water, the one being hyper-
tonic while the other is a hypotonic solution. Whether or not
the membranes are easily or quickly penetrated is a question
better answered by a study of the results.
The fact that lithium salts produce a marked and really specific
effect on development was observed first in 1893 by Herbst while
studying the eggs of the sea-urchin. The definite type of embryo
thus produced was termed by him the lithium larva. Soon after
this several investigators studied the action of lithium salts on the
development of the frog’s egg and found that it induced decided
effects, yet they failed to show that a distinct type of embryo
Journat or Experimenta Zobtocy, Vor. m1, No. 1.
100 Charles R. Stockard
resulted, although Gurwitsch concluded that his radial larva was
the specific result of the lithium action: Morgan then in 1902
carried on experiments to test the effects of LiCl on frog’s eggs,
and again in the following spring he used a number of lithium salts
along with other solutions for comparison. _ He finally concluded
that lithium produced in the development of the frog’s egg a
typical larva just as Herbst had shown for the sea-urchin. Both
of these workers claimed for similar reasons that the effect was
due to the chemical action of the lithium ion and not to osmotic
or pressure effects; hence the specific character of the embryos.
Loeb, Mathews and others have subjected the eggs of the fish,
Fundulus heteroclitus, to solutions of lithium salts, but only to
observe the physiological effects of the solutions, ignoring the
morphological changes induced.
With these results in view Prof. I. H. Morgan kindly suggested
that I study, from a morphological standpoint, the effects of
lithium salts on the fish’s egg. It is, therefore, a pleasure to avail
myself of this opportunity of expressing my indebtedness to Pro-
fessor Morgan for his kindly advice and criticism throughout the
progress of this work. ‘The experiments were conducted during
the past summer while occupying a table in the United States
Fish Commission Laboratory at Wood’s Hole, and I wish to
thank the authorities of this laboratory, particularly the director,
Dr. F. B. Sumner, for the courtesies extended me while there.
The Fundulus egg differs in its mode of development from both
that of the sea-urchin and the frog, hence it is of interest to note
whether or not an effect such as that produced by lithium is at
all comparable in the three cases. Of course it is fully realized
that no strict embryological comparison should be made among
types of development so different as these, yet one may find little
objection to a comparative study of the chemical effects produced
in the three widely different forms by one and the same element,
lithium. At present I am not in a position to state that the
abnormalities which have been induced in the Fundulus egg by
means of LiCl are specific for lithium, as a number of other salts
must first be experimented with. The abnormalities described
are not exceptional, but general, occurring in as large a per cent.
a
Development of Fundulus in Solutions of LiCl IOI
of the eggs as could be expected. All of the experiments were
repeated frequently—some as often as a dozen times—and always
with constant results.
METHOD
The following treatment and precautions were used throughout
the experiments. It was decided to keep the eggs as nearly as
possible under normal conditions by using solutions of the LiCl
in ordinary sea water, and to run experiments with fresh and
distilled water solutions merely as checks on these. Normal sea
water controls were carried in each experiment and as a further
check eggs were kept developing in ordinary fresh and distilled
water. A series of solutions of LiCl in sea water was prepared
in strengths }n, $n, $n, 4n, 2n, $n, Zn, and normal, a normal
solution of LiCl being equivalent to about a 4.25 per cent. solu-
tion; the eggs were then subjected to these to ascertain the various
effects of different strength solutions. It was found that those
solutions weaker than 3 n produced no apparent effect during the
early stages of deyelopwient. In the ? n, 7 n, and normal colt
tions the eggs showed varying degrees of abnormalities, the ones
in the stronger solutions dying after a few hours. A series was
then prepared between $n and ?n so as to obtain different degrees
of abnormal development. ‘These solutions were 2.62 per cent.,
mee? Pel cent., 3-02 per cent. and 3.22 per cent. LiCl or .62 n,
.66n,.71nand.76n. ‘These four strengths were then used in all
of the experiments. A fresh water series was also prepared and I
found, as Mathews had, that a $n solution was the minimal
poisonous dose, that is, the weakest solution preventing the forma-
tion of an embryo; it will be noted that this is only about one-half
the strength required to affect the eggs when in a sea water solu-
tion of LiCl. A +5 nor 1.48 per cent. LiCl solution in distilled
water was resorted to, though its effect was rather severe, being
about the same as that produced by a 3.22 per cent. LiCl solution
in sea water. Controls were always taken from the same bunch
of eggs as those on which the experiments were performed. It is
quite noticeable that Fundulus eggs withstand solutions many
102 Charles R. Stockard
times stronger than those used to affect the eggs of the sea-urchin
and frog.
RESULTS
When Fundulus eggs are placed in LiCl solutions shortly after
fertilization, or before the two-cell stage, a very constant modifica-
tion in the early stages of development will be noticed. ‘The pro-
toplasmic cap or blastoderm becomes unusually prominent, bulg-
ing up in an arched fashion that is much more marked than in
normal eggs. After reaching about the sixty-four cell stage and
later, a clear bubble-like appearance is noted in the living eggs
below the disc (Figs. 1 and 2). On sectioning and staining the
blastoderms at this stage it is found that the bubble-like appearance
is due to the greatly enlarged condition of the segmentation cavity
(Fig. 13). The central periblast has been forced from its normal
place close below the blastoderm (Fig. 14) and pushed down into
the yolk mass at the same time causing the cap to arch more
decidedly above on account of the strain thus induced about its
periphery. ‘his certainly looks like an osmotic effect but one
fails to see how it could be attributed to such a cause when it is
remembered that the same condition exists both in the sea water
LiCl solution and in the distilled water solution (compare Figs.
1 and 2), the one being hypertonic while the other is hypotonic
and thus in the two cases opposite, and not equal, effects would be
expected.
Lithium chlorid in all cases most obviously delays the develop-
ment (compare Figs. 1, 2, 3 and 4 with 11; 5 and 8 with 6). In
the stronger collicione: eggs as old as forty-three hours, show the
iveeodecnt Still asa paler cap and in these older stages it
presents a most interesting appearance. On examining such
living eggs the blastoderm is found to be greatly raised and in
some cases almost pinched away from the yolk. When one looks
down on the top of this blastoderm it is seen to suggest very
strongly a gastrula of some holoblastic egg such as that of a sea-
urchin or starfish, the center appears thin while the peripheral
curved surface has a much thicker appearance as if the center
showed below it the blastopore and the sides represented the
Development of Fundulus in Solutions of I1Cl 103
folded double wall of the gastrula seen in profile. Stages were
seen in which this “blastopore”’ appeared very large as if invagina-
tion was just beginning, and other stages showed various degrees
of decrease in the size of the blastoporic appearance (Figs. 15 and
16). These eggs finally died, in many cases on account of the
blastodermic edges becoming approximated, thus pinching the
cap entirely away from the yolk. On studying sections of these
peculiar forms it was found that as the segmentation cavity
became abnormally large, thus pushing the blastodermic roof up
into a more decided arch, the edges or periphery of the blasto-
derm were brought closer together at the same time becoming
thicker and showing more cell layers than were seen in the crown
or top of the disc (Figs. 15 and 16). ‘The periblast seems more
loosely connected with the blastoderm than is usually the case;
thus it is readily understood how the latter may finally pinch
away from the yolk mass—compare Figs. 13, 15 and 16. ‘These
figures also make clear the manner in which the blastopore-
like image in the living egg seems to decrease in size—as if a
blastopore was closing; this is merely the visual effect produced
by the decreasing circumference of the blastodermic periphery as
it becomes puckered together like the mouth of a sac. No divid-
ing cells were found in these sections of the blastoderms, and when
it is recalled how abundant such cells usually are in sections of
fish blastoderms at this stage we are struck with the inhibition
or decrease in division rate caused by the LiCl, and hence the slow
rate of growth. Normally a forty-hour egg would have the young
fish well formed. No indication of invagination was shown about
the edges of these blastoderms, but it may be possible that if eggs
in this condition were transferred from the strong solutions back
to sea water that the slight recovery thus induced might cause
them to continue development and perhaps to slightly invaginate
as this appears to be the easiest direction of growth. Lack of
material during the latter part of the season prevented the trial
of such an experiment.
Many eggs die when the blastoderm becomes confined to the
polar region as is indicated above, but in rare cases they survive
and develop to the extent that the blastoderm thins out and
104 Charles R. Stockard
EXPLANATION OF FIGURES.
All were drawn from camera sketches of the living eggs except figures 5, 6, 8 and 9.
Fig. 1. Egg from a normal 55 LiCl distilled water solution when twenty-two hours and twenty
minutes old. sg, Segmentation cavity; od, oil drops.
Fig. 2. When twenty hours old in 3.22 per cent. LiCl in sea water.
Fig. 3. Twenty-three hours old in 2.82 per cent. LiCl in sea water. gr, Germ ring; es, embryonic
shield.
Fig. 4. Twenty-two and one-half hours old in 2.82 per cent. LiCl sea water.
Fig. 5. Nine days old, taken from 3.02 per cent. LiCl when twenty-eight hours old and placed
in pure sea water. h, Head; 7, tail; f, fin.
Fig. 6. Control embryo nine days old. ht, Heart.
Fig. 7. In 2.82 per cent. LiCl fifty-four hours old.
Fig. 8. Embryo with no eyes, nine days old, from 2.82 per cent. LiCl into sea water at thirty-two
hours old. ht, Heart.
Fig. 9. Cauda bifida normal 3 LiCl forty-eight hours. 1, Tail.
Fig. 10. In 2.82 per cent. fifty-four hours old.
Fig. 11. Control twenty-four hours old. em, Embryonic thickening.
Fig. 12. In 3.02 per cent. thirty-one and one-half hours.
Development of Fundulus in Solutions of LiCl 105
106 Charles R. Stockard
flattens slightly, and in some cases forms an embryonic shield in this
polar position. Fig. 18 shows such a shield which, forming in a
cap forty-six hours old, extended no further over the yolk than a
normal high segmentation stage would or than the germ area
shown in Fig. 1 or 2 does. A longitudinal section of this shield
is shown in Fig. 17 and it is seen that the folding and cell arrange-
ments are more or less normal, in this case little or no indication
of the germ ring was found beyond the edges of the shield. Fig. 19
shows a section through a blastoderm with the embryonic shield
thickening well marked. In Figs. 3 and 4, embryos about
twenty-three hours old, the germ area 1s still high up on the yolk,
but the germ ring and embryonic shield have reached a stage of
development commonly seen when the germinal area has descended
about one-third of the way down the yolk. In these eggs the
embryonic area never completely covers the yolk, the blastopore
always remains open and through it the yolk is seen to protrude.
These instances show most clearly that a second effect of lithium
chlorid 1s to prevent the down-growth of protoplasm in the egg.
The embryos that result from the eggs just mentioned are
peculiar, being necessarily short as the embryonic material only
extends part of the way down the yolk. ‘They are also poorly
formed in the head region and the tail seems to dwindle out
suddenly as a small pointed end. Figure 5 shows such a monster
in position on the yolk when nine days old; a normal control
embryo of the same age is shown in Fig. 6. “These short embryos
were cleared and stained and finally studied in sections. ‘They
usually lacked all indications of eyes, either optic vesicles or lens,
but the ears were being formed and the trunk organs were present.
As is seen in the figure the fins are poorly developed.
When weaker solutions of LiCl were used, such as 2.62 per
cent. and 2.82 per cent., the formation of the embryo approached
nearer the normal, but even in the 2.62 per cent. no embryo was
ever developed in an entirely normal way. In all cases the rate
of development was retarded, the heart beat was slower, and the
blood was colorless, seeming to lack the hemoglobin constituent.
‘The latter condition must have lessened the respiratory efficiency to
quite an extent, a fact which would also contribute to their slow
as
Development of Fundulus in Solutions of LiCl 107
rate of development. Embryos in these weaker solutions often
showed cauda bifida (Fig. 9), and others that were more nearly
normal in form had rather irregular and twisted outlines, some
of these having the tail end bent at right angles to the body
axis. [hese malformations of the caudal end are no doubt
due to the slow descent of the protoplasmic mass over the yolk
and in some cases to the entire failure of this mass to completely
enclose the yolk. ‘This causes the embryo to be short, crowded
into less space, and twisted in appearance.
A most noticeable feature in these living embryos was the
apparent absence of eyes (Fig. 8 a nine-day embryo). When one
looks at a batch of developing fish eggs after the embryos have
formed, a most conspicuous feature is the large black eyes on each
egg—the lack of such an appearance is striking in the lithium
embryos—and the entire mass of eggs has, therefore, a pale,
unhealthy look. Sections of the heads of these larvas show that
in many cases no indication whatever of eyes existed, while in
some a small deep-seated poorly-formed eye was found, which,
on account of its probable paleness, was entirely unnoticeable in
the living embryos.
The germinal area in some eggs advanced over the yolk in a
very peculiar manner. Figure 10 shows a thin cellular sac that
extends more than half way down the yolk, constricting it slightly
about the equator and giving the entire egg a rather unusual
shape. At the border of the iyeplecns sac little or no
indication of germ ring or embryonic shield could be distinguished,
though these structures must have existed to some extent since at
later stages short malformed embryos would arise appearing in
life as if their tail ends were entirely lacking. Figure 7 shows a
circular cap which does not extend so far down the yolk as in the
last case, but the very strong line of demarcation which often
exists between yolk and protoplasm is well shown. Figure 12
shows a rare case in which a small part of the yolk is pulled up into
the blastodermic cap as a small polar cone.
The rapidity of action of the LiCl solution, the degree of abnor-
malities and death rate produced by solutions of different strengths
as well as the periods in development at which the solutions seemed
108 Charles R. Stockard
most and least effective may best be treated by referring directly
to notes taken while testing these several questions.
As was mentioned before the rapidity or ease with which metal-
lic ions penetrate the membranes of Fundulus eggs is uncertain.
But the examples cited below seem to show that lithium ions, at
any rate, enter with comparative readiness and produce abnor-
malities which in these experiments, at least, can not be attributed
to other causes. Eggs that were fertilized “dry” and then placed
in sea water LiCl solutions of 2.82 per cent., 3.02 per cent. and
3.22 per cent. showed characteristic lithium effects after a period
of only three hours, while in the eight-cell stage, the blastoderms
bulging up abnormally. ‘The control from the same lot of eggs
failed to show any such modifications. Again on applying a
series of solutions of LiCl in sea water of strengths of 1.59 per cent.,
2.12) per cent... 2/05. per cent, 3:16 per cent. 447 lipericcMnmaina
4.25 per cent. and examining after sixteen hours the eggs in the
three weaker solutions presented a normal appearance, while those
in the three stronger solutions contained a large per cent. of abnor-
mal forms, all in the strongest solution being very abnormal.
The blastoderm was arched and the segmentation cavity appeared
as a bubble below. After being in the solutions forty-eight hours
the 2.65 per cent. lot contained twenty-six per cent. undeveloped
eggs and many abnormally-shaped embryos, some with cauda
bifida and irregular outlines. The 1.59 per cent. and 2.12 per
~ cent. lots also showed malformed embryos though not so many as
in the 2.65 per cent. lot. ‘Thus it is shown that weak solutions
do not affect the development in its early stages though they later
cause the production of deformed embryos. In another experi-
ment eggs were subjected to 2.82 per cent. LiCl when twenty-two
hours old, and others when twenty-five hours old were put into
3.02 per cent. LiCl. When they were all forty-four hours old, the
ones which had been in the stronger solution, though for three
hours less, showed more marked modifications than those in the
weaker. ‘These facts seem to show that LiCl does enter the egg
with sufficient readiness for all experimental purposes.
The degree of effectiveness for the different strength solutions
may be illustrated as follows: When forty-six hours old many eggs
Development of Fundulus in Solutions of LiCl 109
in 2.62 per cent. LiCl show a faint line indicating the embryo, and
there are also a large per cent. of dead eggs present. In the 2.82
per cent. solution at this time there are still more dead ones; the
Fig. 13.
Seventeen hours old in LiC! normal solution. bd, Blastoderm; pb, periblast; sg, seg-
mentation cavity.
Fig.
Fig.
Fig.
Fig.
Fig.
14.
1G
16.
17e
18.
Control ten hours old. sc, Segmentation cavity.
In 3.02 per cent. LiCl forty-three hours old. sc, Segmentation cavity.
In 3.02 per cent. LiCl forty-three hours old. sc, Segmentation cavity.
Longitudinal section embryonic shield from Fig. 18. pb, Periblast.
Embryonic shield in polar cap forty-six hours old in 3.22 per cent. LiCl. es, Embryonic
shield; od, oil drop; e, border of blastoderm.
Fig. 19. Section of polar cap twenty-two hours old in 3.22 per cent. LiCl. es, Embryonic
shield; sc, segmentation cavity.
germ ring has not grown completely over the yolk except in rare
cases, but the embryo is formed in the embryonic shield. . Among
those in 3.02 per cent. none of the blastopores have closed and the
IIO Charles R. Stockard
embryos are all short and abnormal, with a still larger number of
dead eggs. In the 3.22 per cent. LiCl less than half are still alive;
all of the blastopores are unclosed; some of the embryos are fairly
well developed in the embryonic shield, while some of the em-
bryonic caps are still at the upper pole with the embryos forming
in a thickening, like an embryonic shield, which often lies imme-
diately over the pole of the egg (Fig. 18); others have blastoderms
which may finally pinch away from the yolk and die (Figs. 15 and
16).
e another experiment the control embryos are distinctly
formed when fifty-four hours old; while in the 2.62 per cent. LiCl
the blastopores are only about two-thirds closed and embryos are
forming in the embryonic shields. In 2.82 per cent. LiCl blasto-
pores are still wide open; in 3.02 per cent. caps, with slight embry-
onic shields, are present; and in the 3.22 per cent. LiCl all seem
to have stopped developing and many have the blastoderms
broken away from the yolk and floating freely within the egg
membrane.
When the same eggs are sixty-nine hours old forty-five are alive
in the 2.62 per cent. LiCl solution. “The embryos are all well formed,
though a few have failed to close the blastopore. In 2.82 per cent.
LiCl only seven out of thirty-five are still alive, or 20 per cent.,
and only one of these has a definite embryo formed. In 3.02 per
cent. LiCl only five out of fifty-three eggs are living, or 9.5 percent.,
and these are all deformed. Of those in the 3.22 per cent. LiCl
solution sixteen are dead and one is living, or 6 per cent. alive.
In this one the cap is scarcely half way down the yolk.
At ninety-six hours old these eggs are in the following condi-
tion: [he 2.62 per cent. LiCl lot have thirteen out of fourteen still
living, all with well formed embryos though far behind the control
in development. ‘The 2.82 per cent. ones have not fared so well;
only three out of seven are still alive but these, also, have well
formed embryos. ‘The one survivor of the 3.22 per cent. LiCl
solution is now dead, the cap has pulled back off of the yolk,
which on the day before it had half covered, and is now a dead
mass of cells at the upper pole.
The embryos in the 2.62 per cent. LiCl solution when nine days
Development of Fundulus in Solutions of LiCl III
old are far behind the control in development; the heart beat is
only about one-half as fast as normal, and the yolk is badly
shrunken so that the entire embryo swings about freely in the egg
membrane. The 2.82 per cent. LiCl embryos are still alive,
though they also have much shriveled yolks and are otherwise like
the ones in the 2.62 per cent. solution.
Eggs of various ages were subjected to the LiCl solutions to
ascertain whether or not there were periods in development when
they appeared more sensitive than at other times. As a further
means of deciding this question, eggs were also removed at inter-
vals from the LiCl solutions and placed in pure sea water.
Eggs which were sixteen hours old (early germ-ring stages) were
placed in 3.02 per cent. LiCl and when they were forty hours old
they had become very abnormal and many were dead. Eggs put
into 3.02 per cent. LiCl when seventeen hours old are described
thus, when eight days old: The pigment is confined almost alto-
gether to the yolk area and is largely of the black type, little if any of
the brown being present; the blood 1s colorless and the heart beats
slowly; the eyes have little or no color and in most cases seem
absent, and the yolk has decreased greatly in size. Of the eggs
put into 2.82 per cent. LiCl when nineteen hours old almost the
entire lot died, the remaining ones resembling those which had
been in the same solution since fertilization. If put into 2.62 per
cent. LiCl when twenty hours old they appear, when forty-four
hours old, similar to those that have spent their entire life in a
solution of this strength. Those put into 2.82 per cent. when
twenty-two hours old appear at forty-four hours almost normal
though a little behind in their development. ‘The extreme sensi-
tiveness of the nineteen-hour and twenty-hour stages as thus
indicated is noteworthy. Figure 11 shows a normal egg some-
what beyond this age, and it is seen that the germ ring has just
turned the equator of the egg. Other eggs were put into 3.02 per
cent. LiCl when eighteen, nineteen, twenty, twenty-one and
twenty-two hours old, respectively, and kept in this solution for
three days. ‘Then they were transferred to pure sea water again.
Seventeen days later they were still alive but developing very
slowly and seemed far from being ready to hatch while the control
12) Charles R. Stockard
ones had hatched from three to four days previous. ‘Thus it
seems that LiCl leaves a lasting effect on these embryos and that
they are unable to recover, though under normal conditions, for
as long as seventeen days after.
Eggs sixty-five hours old with well-formed embryos were placed
in 3.02 per cent. LiCl solution; twenty-eight hours later on exam-
ining the living eggs no variations from the normal could be
detected; fifty-four hours after being in the solution no change was
observed, though when nine days old they were behind the control
in their development at least two days.
To test the time of greatest sensitiveness in another manner,
eggs were taken from the LiCl solutions after being in them only
thirty minutes, while in the two-cell stage. [he LiCl in this case
left no trace of any effect that could be detected in their later
development. Again eggs taken from 3.02 per cent. LiCl after
six hours treatment were put into sea water, their age being then
seven and three-quarter hours, they appeared normal when
twenty and twenty-four hours old, but when forty-four hours old
they had fallen behind the control in their development. Eggs
were taken from 2.82 per cent. LiCl when fifteen and one-half
hours old, having been in the solution fourteen hours and were
placed in sea water, twenty-four hours later the eggs showed no
improvement over those still in the solution. When examined
after being out of the solution for sixteen days they still showed
little if any recovery. In another case eggs were placed in sea
water after being in 2.82 per cent. LiCl for eighteen and one-half
hours and others in a 3.02 per cent. solution seventeen and three-
quarter hours. When forty-four hours old they were still abnor-
mal but far ahead of those remaining in the lithium solutions.
After being in 3.02 per cent. LiCl for twenty-six and three-quarter
hours eggs were placed in sea water where they failed to recover,
though their death rate was materially lowered; after two days 8g
per cent. still were living while of those left in the solution only
g.5 per cent. remained alive. ‘This lowering of the death rate was
noted in many instances where morphological recovery from the
lithium effect seemed lacking. It appears as though the eggs gain
in power of resistance, or rather resistance is no longer required
Development of Fundulus in Solutions of LiCl 113
after being transferred to the sea water and hence though they
may not recover in one sense, they are better able to survive than
they would be under the depressing effects of the lithium.
During one experiment a very strange result was noted: The
entire series of LiCl solutions failed to give any visible effect for
more than the first day of development, but later the character-
istic effects were manifested and those eggs that were removed
from the solutions and placed in sea water during the day of
apparent non-effectiveness later showed the effects of the LiCl on
their subsequent development in the sea water, although no effect
was observable when they were removed from the lithium solu-
tions.
The above experiments were all repeated several times and the
particular instances cited merely serve as examples to illustrate
the general results obtained. From such responses to the LiCl
treatment one may be justified in drawing the following conclu-
sions.
CONCLUSIONS
The rapidity with which lithium chlorid in solution affects the
egg varies directly as the strength of the solution, eggs being very
slow to manifest the effects when placed in dilute solutions, while
a response was obtained on one occasion within three hours in
rather strong solutions.
The degree of abnormality and the death rate also vary directly as
the strength of the lithium chlorid solutions, striking abnormalities
being found in 3.22 per cent. solutions while similar malformations
in a less marked degree with a much lower death rate were noted
in the 2.62 per cent. solutions.
The lithium chlorid solution affects the development of the embryo
at any stage in which the eggs may be placed init. But the extent
of the abnormalities seem to vary inversely with the age at which
the eggs are subjected to the solution, showing merely a lessened
rate of development with other minor effects if placed in the
solution during late stages.
When eggs are once affected by the lithtum chlorid solution—which
is found to be the case 1f they remain in it as long as six hours—
114 Charles R. Stockard
they are unable to completely recover from this effect even though
they be placed in pure sea water during the remaining period of
their development.
REVIEW OF LITERATURE
Herbst, in ’92, 93 and 96, conducted extensive experiments to
test the action of a large number of salts on the developing eggs
of the sea-urchin. He found that of a number of salts used, those
of the metal lithium produced the most decided results, and
further that with the different salts of lithium the results were the
same. ‘Thus the conclusion was reached that the action of these
salts was not due to their several acid radicals but to their common
lithium ion. Herbst, therefore, stated that the abnormalities
induced were typical lithium effects, not being obtained by the
use of salts of other metals. He used the term “lithium larva”
to designate the peculiar monster that resulted. The effect of
lithium on the sea-urchin’s egg seemed chiefly to cause “exogas-
trulation,” that is, the endoderm instead of becoming infolded by
invagination became really inverted and formed a sac connected
with the ectoderm or outer gastrula wall by a short stalk, thus
producing a three-part larva, two sacs, the gastrula wall and the
primitive gut, joined by a short tube or stalk. From the end
opposite the stalk the primitive gut sac in many instances formed
another small sac representing, as Herbst claimed, the vaso-
peritoneal pouch. ‘The ectoderm sac at times became smaller
and occasionally finally disappeared leaving the entire blastula wall
endodermal. ‘Uhe larva recovered slightly so as to form the cal-
cium framework when removed from the lithium solutions; the
amount of recovery depending upon the length of time it had spent
in the solution. ‘This briefly is the main action as found by Herbst
in three genera of sea-urchins treated with, in all, thirteen salts of
lithium. ‘The features of especial interest to us are the marked
inversion of the layers and the resulting enlarged embryonic
cavities.
Gurwitsch, in ’95 and ’96, treated developing frog’s eggs with
LiCl and other salts, finding that sodium and lithium salts were
most effective. His lithium solutions were very weak (0.3 per
Development of Fundulus in Solutions of LiCl rms
cent., 0.4 per cent. and 0.5 per cent.), only about one-half the
strength required to affect the Fundulus egg when applied in
fresh water, and about one-third or one- sou the concentration
necessary to give a response when used in sea water. Frog’s
eggs under the influence of LiCl showed a retardation in the seg-
mentation of the white area, in rare cases this area failing entirely
to divide. Thus they became marked into upper Sad lower
hemispheres and the cells at the sides of the floor of the segmenta-
tion cavity seemed to push in tending to fill this cavity. * At this
period Gurwitsch claimed that his embryos exhibited a radial
symmetry but Morgan contradicts this and holds that they are
really bilateral although the bilaterality may be less evident than
that shown in the normal egg. Gurwitsch also finds that the
downgrowth of the substance constituting the upper hemisphere
is lacking, as indicated above, and this is without doubt a marked
characteristic of the action of LiCl on the fish’s egg. ‘This also
gives in the two cases an abnormally high position to the develop-
ing embryo and may, along with the failure of the blastopore to
close, cause the malformed caudal areas of many individuals.
Abnormalities in the position of the blastopore and brain, par-
ticularly of the former, were found to be the most decided effects
of the LiCl solutions.
Madame Rondeau-Luzeau conducted an extensive series of
‘experiments to test more especially the action of several chlorids
on development. She found that a double action exists, namely,
a physical action due to “hypertonicity” of the solutions and a
chemical action depending upon the kind of salt employed. Only
in the case of LiCl did the chemical action predominate when weak
solutions were tested. In my work the fact that solutions in both
fresh and sea water were used seems to preclude, in this case, all
chances for the physical or hypertonic effect and leaves the chemi-
cal effect alone responsible for the abnormal development ob-
served. Except in the case of LiCl Mm. Rondeau-Luzeau found
that death was much oftener due to the osmotic pressure of the solu-
tions than to chemical poisoning. ‘The teratological action of the
chlorids appeared to be due to their chemical action in the case
of eggs treated after fertilization. ‘The effects of the solutions
116 Charles R. Stockard
were also found to be more marked at some stages than at others,
particularly so at the time of closure of the blastopore and of the
formation of the medullary folds. LiCl was found to exert a more
powerful chemical action than any other salt tried. “The minimal
amount of this salt that will effect the development of the frog’s
egg may be represented by an osmotic pressure of 169 cm. (0.3 to
0.4 per cent.); while for KCl, it it 405 cm. (1.2 per cent.); for NaCl,
459 cm. (1.9 per cent.); for MgCl,, 485 cm. (1.3 per cent.); and for
CaCl, 484 cm. (1.5 per cent.).
Morgan, in ’02 and ’03, subjected frog’s eggs at various stages
in their development to 0.4 per cent., 0.5 per cent., 0.6 per cent.,
0.7 per cent. and 0.8 per cent. solutions of LiCl. Most of these
solutions would be too weak, even though prepared in fresh water,
to affect Fundulus eggs. Morgan found that in the frog’s egg
LiCl did produce a specific effect which he attributed to the chemi-
cal action of the lithium ion. Delayed development was noted as
being a most obvious condition and this 1s equally true of the fish’s
egg. Egos in two-cell and four-cell stages were more affected
than those in later segmentation, and those beginning to gastru-
late were least affected. Stronger solutions were necessary to
give an equal effect when applied in later stages. Similar facts
~ are also indicated in my notes above. But it was found that the
Fundulus eggs placed in the solutions during cleavage and at the
beginning of gastrulation up to the sixteenth or Sevenreenil hours
were equally affected without regard to the stages at which they
were subjected to the action of the salt. Another very noticeable
effect of this salt was to prevent the downgrowth of protoplasm
from the upper hemisphere of the frog’s egg, as Gurwitsch also
found. ‘This makes gastrulation difficult, so that embryos in the
stronger solutions do not pass beyond this stage. “The absence
of ye downgrowth of the protoplasm in the fish’s egg has been
emphasized above and it will be remembered that in the stronger
solutions the blastoderm folded into a ball upon the upper pole
and finally pinched itself away from the yolk and died. One type
of the frog embryo showed a complete inversion of the layers, the
black area folding down into the yolk cells. The complete accom-
plishment of such a feat by the fish’s egg could hardly be imagined,
ah
Development of Fundulus in Solutions of I1Cl 117
but it seems possible that it may attempt a similar process, when
it is recalled to what extent the periblast area may be forced down
into the yolk when the segmentation cavity becomes unusually
enlarged as it sometimes does. In the frog the black area becomes
more or less spherical and sinks into or is engulfed by the yolk-
cells of the white area. ‘The frog eggs often form a sharp line
between the black and white hemispheres, usually above the
equator. This line, Morgan states, corresponds to the inturned
edge of a circular blastopore whose position at or above the
equator of the egg is explained by the lack of downward move-
ment of the material of the upper hemisphere. ‘The segmenta-
tion cavity of the upper cells was obliterated. ‘This is a very
different state of affairs from that seen in the fish where the seg-
mentation cavity becomes so enormously exaggerated. Many
short and folded embryos were found in later stages, some also
showed cauda bifida; short embryos and forked tails are also
common among the late effects of LiCl on the fish embryo.
After considering these investigations upon the effects of
lithium one is strongly inclined to conclude that the salts of this
metal do exert a specific influence on developing eggs. But I am
not now in the position to state that these effects on the Fundulus
eggs are really specific for lithium, though they have been so
repeatedly and constantly obtained that one is led to believe that
they are at least characteristic of lithium action on eggs of this
form. Other chemical or physical stimuli might possibly pro-
duce similar abnormalities; this could be definitely stated only after
the study has been continued with a large number of salts of
various metals.
SUMMARY
1. Lithium chlorid delays development to a most obvious
degree.
2. Eggs seem equally affected by the solution when placed in it
at any time during cleavage stages; at other times the effects
vary. [hey seem most sensitive about the period when the germ-
ring circles the equator; though they are always affected to a
ereater or less degree; the rate of development is slower, the em-
118 Charles R. Stockard
bryo presents a pale appearance, since the blood lacks color and
the pigment spots are fewer than normal.
3. After having remained for as long a time as six hours in
LiCl solutions of sufficient strengths to cause abnormalities the
eggs are incapable of complete recovery when placed in pure sea
water during the remainder of their dev elopment.
4. In LiCl solutions the blastoderm is usually prevented from
growing downward over the yolk, it therefore bulges up as a cap
on the upper pole of the egg. ‘This cap in the stronger solutions
constricts its border, thus folding its periphery, and finally pinches
itself away from the yolk and dies.
5. The segmentation cavity is enormously enlarged since the
central periblast pushes down unusually far into the yolk mass
while the blastoderm bulges up giving the cavity a more arched
roof.
6. In many eggs the blastoderm never completely encloses the
yolk; thus the blastopore remains open and short, peculiarly
formed, and often cauda bifida embryos result.
7. In the late embryos the heart beats slowly, the eyes often
fail to develop, the blood is colorless and, therefore, appears to
lack hemoglobin. ‘These characters taken with the inability to
recover from the lithium effect, seems to prove without doubt that
such an effect 1s due to chemical, and not to physical, causes. The
fact that similar abnormalities are induced by LiCl solutions pre-
pared with sea water and fresh water, therefore, giving both
hypertonic and hypotonic solutions show further its chemical
rather than its physical, action.
APPENDIX
Notes on the Development of Fundulus heteroclitus in Fresh
W ater
At Cold Spring Harbor, Long Island, in the summer of 1904,
I collected material for comparing the development of the eggs of
Fundulus heteroclitus in fresh water with their development in
sea water, which is their normal medium. I wished also to ascer-
tain whether or not those embryos hatched in fresh water would
Development of Fundulus in Solutions of LiCl 119
show a higher degree of adaptability for living in this medium
than the normally hatched fish. ‘The fresh water at this place,
as indicated by later experiments, evidently contained some sub-
stance that affected the embryos very strangely, as almost with-
out exception the late embryo assumed a peculiarly twisted
position upon the yolk the tail bending up in a circular fashion
and striking the body about half way from the head. Almost all
of these eggs died before hatching and those embryos that lived
to hatch were unable to straighten their bodies and died within
afew hours. ‘The control embryos began hatching three and one-
half days before these fresh water ones.
During the past summer at Woods Hole this experiment was
repeated with the extra precaution of running a distilled water con-
trol. Here the results were entirely different in regard to the form
of the embryo. ‘Those fish that hatched in the fresh and distilled
water exhibited perfectly normal shapes having also occupied the
usual position on the yolk. But again the Giech water embryos
were late in hatching, being in the ailiest case two days behind
the control and some were five days late in coming out.
The fresh and distilled water embryos gave an interesting mor-
tality record for different periods of their development. During
the first ten to twelve days these eggs were about as hardy as those
in the sea water, though from this time until hatching began
they died at a rate of over 5 per cent. per day. In one case where
there were two hundred and twenty-five eggs when hatching
began, thirty-nine, or 172 per cent. died that night and only six
individuals were hatched. During the next twenty-four hours forty-
three, or 23} per cent. died, leaving only one hundred and forty-
three still alive. The following day, or three days after hatching
had begun, only eighteen were alive, 874 per cent. having died that
night, five dying after hatching. None lived in fresh water longer
than ten hours after hatching out. From this it is seen that the fish
become peculiarly sensitive to the unusual medium shortly before
the hatching time and only a few survive to break through the
ege membrane.
The fish that did hatch in the fresh water were certainly no
better fitted to live in this medium than those hatched in sea water.
120 Charles R. Stockard
When embryos that had hatched in fresh water were transferred
directly to either one-half sea and one-half fresh water, or to pure
sea water, they began at once to show quicker movements, and
always lived normally in these media.
Thus it is seen that although the eggs of Fundulus heteroclitus
will develop in an apparently normal fashion in fresh water, so far
as form is concerned, they are slower in hatching. ‘The eggs die
in large numbers during the hatching period, and those that do
hatch are unable to survive unless transferred to sea water. At
this period of late development they probably die from the same
cause that kills the mature fish if they are put into fresh water.
Zodlogical Laboratory, Columbia University,
October 16, 1905.
LITERATURE
Gurwitscu, A., ’95.—Ueber die Einwirkung des Lithionchlorids auf der Entwicke-
lung des Frosch-und Kroteneier (Rana fusca und Bufo vulg.).
Anat. Anz., xi.
*96.—Ueber die formative Wirkung des veranderten chemischen Mediums
auf die embryonale Entwickelung. Arch. f. Entwick., iii.
Hersst, C., ’92.—Experimentelle Untersuchungen tber den Einfluss der veran-
derten chemischen. Zusammensetzung des umgebenden Mediums
auf die Entwickelung der Thiere. I Thiel. Zeits. fiir wissensch.
Zool., iv, 3.
"93.-—Experimentelle Untersuchungen. II Thiel. Mittheil. aus der
Zool. Station zu Naepel.
*96.—Experimentelle Untersuchungen. III, IV, V, und VI Theil.
Arch. f. Entwick., iv.
Martuews, A. P., ’04.—The Relation between Solution Tension, Atomic Volume
and the Physiological Action of the Elements. Am. Jour. Physiol, -
re
Morean, T. H., ’03.—The Relation between Normal and Abnormal Develop-
ment of the Embryo of the Frog, as Determined by the Effects of
Lithium Chlorid in Solution. Arch. f. Entwick., xvi.
Ronpravu-Luzeav, ’02.—Action des Chlorures en Dissolution sur le Développe-
ment des ceufs de Batraciens. Théses prés. Faculté des Sci.
de Paris Univ.
PARTIAL REGENERATION OF THE SPERM-RECEP-
TPAGI te CRAY BEISH
BY
E. A. ANDREWS
Wir ELeven Ficures
In American crayfish of the genus Cambarus there is a small
pocket in the shell of the female that receives sperm from the
male and subsequently frees it when the eggs are laid. ‘This 1s
not found in other crayfishes, and as Cambarus is the most
specialized of the group the sperm-receptacle, or annulus ventralis
as it has been called, seems to be a new acquisition. At the same
time it is used as one of the necessary reproductive organs. From
its character, as a dense part of the shell and from its small size
and protected position, on the ventral side between the legs, it
seems improbable that it has been subjected to very many injuries
in the history of the genus. Yet at the time of shedding all parts
of the shell are soft, and amongst several hundred females exam-
ined there were two cases in which the annulus had been injured,
as if by the claws of some other crayfish pressing against the
shell when soft. However, these were peripheral injuries and
did not necessarily prevent the use of the organ.
That the organ has ever been removed in the history of cray-
fish without the death of the organism seems highly improbable
as we have no knowledge of any enemy that could cut out that
region without destroying the neighboring nerve trunk, at the
least.
To determine what would happen if the organs were removed
twenty-four females were operated upon in May and June, 1904,
so that all, or most all, of the annulus was extirpated. ‘These
were adult females, some of which had laid in April and May.
The telson-rostrum measurements of these were as follows:
Journat or Experimenta Zoétocy, Vor. 1, No. 1.
129 E. A. Andrews
One was 50 mm. long; one 55 mm.; three were 60 mm.; eight
were 65-80 mm.; one 85 mm.; five 100 mm.; four 110 mm., td
one 120 mm. in length.
In July five of these cast their shells; one 100 mm., and four
8o mm. in length. In each case there was something new formed
of the nature of an annulus, but so imperfect as to be of no use as
a sperm receptacle.
To understand these partial regenerations it is necessary to
consider the anatomy of the normal annulus. The part of the
shell so-called has in Cambarus afhnis the form indicated in Fig.
A, which is a ventral, or external view of the annulus from a
female 110 mm. long, decalcified and made translucent. ‘There
are here two prominent elevations or knobs with a groove between,
and a transverse depression posterior. Running across this
depression is a ZAg-Zag crack, represented by a heavy black line.
Following the zig-zag in a general way is a wide dotted streak,
intended to represent the tubular cavity that contains the sperm.
This tubular cavity opens to the exterior by a rounded hole, partly
concealed under the larger of the two knobs; it also opens along its
whole length by the crack represented by the heavy line. The
curved broken lines indicate the very thick, chitinous walls of the
tubular cavity, showing through the translucent shell in this
preparation.
All this, however, is only the shell of the annulus; under the
shell there was a corresponding modification of the epidermis and
connective tissue. ‘The shell fits like a slightly exaggerated mask
over the epidermis. The knobs, depressed area and_thick-
walled tube all have their correspondingly-shaped, underlying
epidermal counterparts, upon which they were made.
The male puts the sperm into the hole at one end of the tube
and it subsequently comes out of the zig-zag crack to meet the
eggs as they pass over the annulus.
A comparative study of several species of Cambarus shows that
the essential part of the annulus is the tubular cavity and that in
one of the lower species this is more evidently a longitudinal
groove or pocket with its edges nearly closed together. Also in the
ontogeny of Cambarus afhnis it is found that the annulus of the
Partial Regeneration of Sperm-Receptacle in Crayfish 123
young larva presents only a single longitudinal depression and
that in successive moults this Becories bent, almost closed, and
associated, with external sculpturing like that of the adult.
The essence of the annulus is thus an inpitting of epidermis
which produces a chitinous pocket, opening only to the exterior.
In extirpating the annulus the shell represented in Fig. A was
cut away from the rest of the crayfish’s shell and conver
dragging with it a variable amount of live epidermis and con-
nective tissue that remained in the cavities of the knobs and
adhering to the walls of the tube and to other parts of the removed
shell. ‘This left in place of the annulus a soft, bleeding, sponge-
like mass which the pressure of contained blood caused to bulge
out, more or less. [he wounded surface soon became covered
over by a layer of material that hardened and turned brown and
then remained as a firm protection until the crayfish cast its shell.
The cast-off shells then showed in place of such an annulus
(Fig. A), which is seen in cast shells from normal crayfish, merely
a rough, brown membrane, continuous all about its edges with
the old shell of the crayfish.
The animals that cast their shells showed new annuli as stiff
bluish plates of the form of an annulus but without its sculpturing
and with very little to represent its pocket, or tube.
The regenerated annuli thus obtained are indicated in Figs. 1,
I24 E. A. Andrews
2, 3, 4, 5, drawn to the same scale as Fig. A and enlarged about
13 diameters. ‘The part in each that seems to represent the essen-
tial sperm pocket is shown in Figs. 1’, 2’, 3’, 4’, 5’, each, magnified
about 100 diameters.
All the regenerated annuli are noticeably short, as in young
crayfish; but the figures fail to give the full surface since these
annuli are not flat but have a large posterior face as well as the
ventral (and anterior) one shown in the figures. ‘The posterior
and ventral faces make a large angle where they meet in an ele-
vated ridge, which ridge is ae posterior boundary of the above
figures. It is near this aie ated ridge that the inpitting of epider-
mis and shell has taken place to form what is like the early stage
in the sperm-pocket in the larva. In the larva also the inpitting
takes place near the posterior edge of the ventral face.
In the largest female that regenerated, 100 mm. long, there was
a peculiar abnormal outgrowth from the posterior face of the new
annulus, Fig. 5. ‘This was a soft protuberance bearing a rounded
lobe and two papillae that were slightly constricted, or jointed,
somewhat suggesting rudimentary limbs. ‘The smaller papilla
was dorsal and is not represented in the hgure. ‘This annulus
was again abnormal in having some ten pits, or infoldings scat-
tered over its ventral surface as well as the median lengthwise pit
that seemed to be homologous with a sperm pocket. On the
posterior, or dorsal, face there were also nearly as many super-
numerary ridges and depressions as upon the ventral face. Most
of these were, in general, transverse, and upon the dorsal face more ©
were upon the right of the median line, while upon the ventral face,
Fig. 5, more were upon the left.
The median pits that are thought to represent the beginnings
of sperm pockets had: the following characters in the five speci-
mens studied.
In the first, Figs. 1 and 1’, there was a very simple lengthwise
groove, or pit, with closed lips and near it elevations that con-
verged as indicated by the lines in Fig. 1. In Fig. 1’ the central
line represents the closed lips of the groove as seen at the surface,
while the cavity of the pit is represented by shading and the very
thick shell walls of the pit are indicated by the broken lines.
Partial Regeneration of Sperm-Receptacle in Crayfish 125
Fic. I Fic. 1’
Fic. 2 Fic. 2
Fic. 3 Fic. 3/
126 E. A. Andrews
Optical sections of this preparation showed that the epidermis
was turned in as a deep groove in which the epidermal cells
abutted against the shell lining the pit.
In another specimen the lengthwise pit was associated with an
anterior depression indicated by the lines in Fig. 2, and with a
sharp transverse ridge suggesting the posterior curved edge of a
normal annulus. The pit, Fig. 2’, opened anteriorly into the
depressed area and its open mouth branched posteriorly right and
left. The bottom of the pit, shown by lighter shading, ended
bluntly posteriorly. “he very thick aeusned shell is indicated
by broken lines indicating its parallel lamellz.
In the third specimen ihe pit was at the posterior edge and, as
in Fig. 3, the preparation showed beneath the shell a wide open
epidermal groove drawn away from the shell, artificially. “The
pit, Fig. 3’, was open along its length, ended sharply in front and
opened out behind between two prominent ridges. ‘The annulus
was much elevated at its posterior edge and from a more posterior
view the above ridges looked not unlike the knobs of a normal
annulus, Fig. A, while posterior to them there was a transverse
ridge similar to that seen in Fig. 2 and again suggesting the pos-
terior rim of a normal annulus.
In specimen four there were two pits close together and some-
what involved with one another, Figs. 4 and 4’. Both were wide
open, ended bluntly at their anterior ends and expanded pos-
teriorly into a wide depression. ‘The more dorsal pit was nearer
to the median plane, the other more to the animal’s left side.
In the fifth specimen, Figs. 5 and 5’, the median pit was much
like that in a very young larva. A depressed area near the pos-
terior edge of the annulus leads forward into a deep groove, the
walls of which are very thick inturned shell. In this specimen
there was a slight pouch on each side where the cavity of the
groove was wider than the open mouth of the groove. In a
vague way these side pouches suggest the “recess”’ of the normal
annulus.
These imperfect annuli, laid bare at the time of shedding,
would doubtless have continued outwardly in that shape until the
next shedding. Whether they would have progressed, during
Partial Regeneration of Sperm-Rece ptacle in Crayfish
Fic 4 Fic. 4’
127
128 E. A. Andrews
successive moults, toward the normal adult form must be deter-
mined by the results of future experiments.
In the normal growth of crayfish the annulus has a new shell
formed over it at each period of shedding and these successive
shells are more and more complex from the early larva to the adult
stage.
These experiments show that when in the adult the shell
over the annulus was removed with more or less of the epidermis
and connective tissue (removed, probably, for the first time in the
history of the organ), a new pit and also a new shell, comparable
to an early larval annulus in complexity, but to an adult annulus
in size, were formed.
Thus there may be a partial restoration, in a few months’ time,
of an organ that is historically new, though essential, and that 1s
moreover a single organ without fellow or metameric homolog
and found only in one sex.
An ability in the crayfish partially to regenerate this special
adult organ, to begin to retrace the ontogeny of this organ, would
seem of use to the species only if it were carried far enough to
complete a functioning sperm receptacle. Experiments may show
this to be the case.
That this special ability could have been evolved by natural
selection seems difficult to conceive since accidental extirpation
is but a remote possibility. ‘The ability to start this organ a
second time in one generation would seem comparable to the
fundamental ability to make the organ again and again in suc-
cessive generations.
November 8, 1905.
ExPERIMENTAL SPUDY OF LIGHT AS A FACTOR IN
THE REGENERATION OF HYDROIDS
BY
A. J. GOLDFARB
In a paper entitled “The Influence of Light on the Develop-
ment of Organs in Animals,” ’95, Loeb states “that light favors
the development of polyps in Eudendrium (ramosum); that no
polyps, or only very few, are developed in the dark.” At the
suggestion of Prof. Morgan, I undertook to determine the mini-
mum amount of light required by this hydroid to regenerate its
polyps. ‘[he experiments were conducted during the summer of
1905 at the Marine Biological Laboratory, at Wood’s Hole, Mass.,
while occupying a room of the Carnegie Institution.
EXPERIMENTS ON EUDENDRIUM RAMOSUM
Preliminary Experiments
Vigorous colonies of Eudendrium ramosum were selected.
These consist of a main stem or stock and its branches. “These
primary branches in turn give rise to secondary and tertiary
branches bearing polyps at their distal ends. ‘These polyps—
and gonads when present—were separately cut off. The decapi-
tated colony was placed in a glass bowl containing about 200 cc.
of normal sea water. These bowls were allowed to remain in the
light for fifteen, thirty, sixty minutes, respectively. At the expira-
tion of each of these periods the bowls were placed in a dark cham-
ber, from which all light was carefully excluded. Other series, of
six bowls each, prepared in the same way were placed in the dark
immediately after the removal of the polyps. After forty-eight
and seventy-two hours, respectively, each series was exposed to
the light as above mentioned.
In the following tables, the numbers indicate the regenerated
hydranths which have free and distinct tentacles.
Tables A, B and C indicate results of these experiments.
JournaL or ExperiMentTAL Zoo ocy, Vor. m1, No. 1.
130 A. Ff. Goldfarb
A
ExPERIMENT BEGUN JULY 14, 1905, 12.30 M.
(Exposed before being placed in dark)
|
Time | |
No. of July 15 | July 16 | July 17 | July 18 | July 19 | July 21
Exposure. | |
I 15 min. ° Tee LO. 8 ° °
2 30 min. fo) 13 22, Seah omen )
3 45 min. | fc) 14 ta) 8 fc) fo)
4 1 hr. 15 min. ° 19 16 7 ° °
5 2 hrs. 15 min. co) TI, 9 21se Meld ia I fo)
6 3 hrs. ° Tie Rta eit °
| |
B
ExPEeRIMENT BEGUN JULY 12, 1905, 12.30 M.
(Exposed after forty-eight hours in dark)
Time | | |
No. of July 13 July 14 | July 15 | July 16 | July 18 | July rg July 21
Exposure. |
I 5 min. ° 27) : 18 20 3 ° °
2 10 min. ° 13 15 18 | I °
3 IO min. ° 18 23 II abe || I °
4 10 min. OM ne | 15 II 2 ° °
5 15 min. ° 27 24 18 7 ° °
6 15 min. fo) 10 10 $e) 3 ° °
Cc
ExPERIMENT BEGUN JULY 13, 1905, 4.30 P. M.
(Exposed after seventy-two hours in dark)
|
Time eee Pee) |e 2
Ne: of Fag fie eee ee ipe |eE | .|
Exposure. ees es aes SS) =
2 et | i rie, aes ee eee ee a nl Ne aie [ee ey ee eee |
I 3 min. ° oP i © 4 4 3 | I ° ° ° °
2 5 min. fo) ° ° 4 3 | 2 | 0 ° ° ° °
3 8 min. ° ° ° 3 3 3 | “2 ° ° o | °
4 10 min. ° I ToeexG 8 6 4 2 2 ie I} 6)
5 15 min. ° 4 | 7 Gl | 5 4 ° I I I
6 25 min. 3 6 | 9 9 5 5 2 fo) ° °
|
Light as a Factor in the Regeneration of A ydroids 131
It appears from these tables, that the brief exposure of three
minutes was probably not the minimum required by this hydroid
for the stimulation of its hydranth formation. It is further to be
observed, in those colonies kept in the dark forty-eight hours or
more after removal of polyps, and then exposed, that regeneration
of hydranths occurred before such exposure. Further, the num-
ber of newly-formed hydranths may or may not be increased after
an exposure.
The appearance of hydranths, formed in the dark, may be
explained: (1) As the result of the exposure from the moment
when the hydranths were removed to the moment when the decapi-
tated colony was placed in the dark. (2) Or, what seemed less
probable, that hydranths were regenerated normally in the dark.
First Cycle
‘To avoid a source of error, all Eudendrium colonies were here-
after prepared as follows: The room was converted into a photo-
graphic dark room. Light was admitted only through a pane of
red glass about six inches square. Particular pains were taken
to keep out all white light.
In the preceding experiments each hydranth was separately
removed and the main stem with all its branches was used. In
the following experiments each primary branch bearing other
branches and hydranths was cut off near its point of attachment
to the main stem or trunk of the colony. The trunks with the
stumps of the primary branches only, were used. The number
of primary branches so removed was noted in each case. The
colonies were separately exposed, then placed in dark chambers.
These were made of double boxes, one within the other, painted
black within, and edges protected by bands of black cloth. One
colony of nearly every series was kept in shaded but continuous
light of the room. ‘This colony will be termed the “Control.”
In series D—more or less typical of the results obtained in three
different series of experiments—the colonies were exposed soon
after removal of the branches. In series E, F and G the colonies
were at once placed in the dark, and exposed one, two and three
days, respectively, after removal of the branches.
132 A. f. Goldfarb
D
EXPERIMENT BEGUN JULY 21, 1905, 2.00 P. M.
(Colonies exposed before being placed in dark)
ee mene Are fapa alate oleiis| al 2) a) /2
Branches of | S| & |e] | =| 2] |B! eB} so) so] so] so] eo] eo
Removed. Exposure. ee Fa eae fee PEN PS EN eA ee en oa iP | Bh)
| | | |
a —— |— |_| — |} —|— |_| |---| || | + —
15 Not exposed 6} ‘9 | 10.|6°|6 1.4 1.3 13) 03" | aa anontontompontnc
15 1 min. light COMIN a Ta Ya RC 20 V LS Pec inl cal edt ey eR eo GaP) ©
15 I min. sunlight Ot 7 | 4 4o) se las hg, hrc) aa eae ae von toaline
15 2 min. light CP I MG Ge ts Ge Wye ey iligy Il 23 to: || 2 | ey || @
15 2 min. sunlight PHO CC GAN ON or 234) see | wi ihes elt |S || © |, ©
15 4 min. light © |) 14 | ora 8, | 9) eOn| sealncilieo | © |.) ||0%]) tao cine
Er
EXPERIMENT BEGUN JULY 22, 1905, 3.00 P. M.
(Colonies exposed to light twenty-one hours after removal of branches)
No. of Time = + a S iS <6 Re 5 = Z aot =
Branches of ee er er te he ea eer oy Safle Tt os
Removed: ||) Exposures.) p23} =) eee tee eee ee
21 | Notexposed | o ° | I I I r 2 I I ° ° °
26 I min. ° 5} 13 9 5 5 4 ° ° ° ° °
28 2 min. ° Bo He 7 8 4 I ° ° ° °
23 4 min © =} 14 13 Se ae | rae” ae 8 6 5 4 4
22 6 min | ° 2 | nS PG 5G} 9 4 I I I ° °
26 8 min | Ou | ae slang 7 9 | I0 4 I ° fo) ° °
22 Control | o ch loys. |) sar 7 7 5 I I ° ° °
1Six buds formed.
Light as a Factor in the Regeneration of H ydroids 133
EXPERIMENT BrGuN JULY 22, 1905, 3.00 P. M.
(Colonies exposed to light forty-eight hours after removal of branches)
F
|
No. of Time of ral ae ww Ss = oo a Goines 3
a aq a a a a a a) | oa) iS
Branches Exposure. > a a > > => => ex alll ees so
e =: = E} = E =) el 3
Removed. a = = = = a | as] <
a Seale = SS Sede —
19 Not exposed fe) fo) I I 2 2 I fo) fe) °
23 I min. ° 8 II 8 II 13 re bell fa ae °
27 2 min. ° 4 7 4 5 4 4 2 | 2 °
24 4 min. ° ° 2 4 4 4 ° ° ° °
21 I min. sunlight fe) ° I 4 3 2 I fo) fo)
20 2 min. sunlight fo) Zl 4 9 II fe) 8 3 fo)
23 Control ° 3 13 14 14 8 7 I 2 8
G
EXPERIMENT BEGUN JULY 22, 1905, 3.00 P. M.
(Colonies exposed to light seventy-two hours after removal of branches)
Nook CP la : : : ; , ea|| ae
ee Tansey ssh) cay Wren ss tee eS? Sr sel ee
ranes<S | __ Exposure. aa Soa ene meee Neb WN ies) Bo) ex lee Batt ade
Removed. | 1S 2,8) 8) 4) 2) 2)2 212
| | |
| | | |
17 Not exposed § oO fo) ° || 5 Gi) 4 2053
19 I min. L 2 4 5 7 6 4 Joi Ch |e
15 2 min. | oO ° ° 4 II 10 || 4 e) || Ye
17 4 min. fo) I 5 3 I 2 3 2 || @
14 | Imin. sunlight | o I ° I 9 12 10 5 2 | 2
17 | 2min.sunlight | o ° 3 9 8 9 6 4 ° °
eee Control once) ° 4 7 7 2 3 apy) °
It will be observed: 1. About forty-eight hours after removal
of the hydranth-bearing branches, new hydranths were formed.
2. These hydranths may appear as follows: (a) Single hydranths
may appear at the oral ends of the branches that have been cut;
(b) single hydranths or group of hydranths may appear at the
aboral end of the main stem or trunk; (c) one of the branches may
greatly elongate, give rise to new secondary and tertiary branches,
each bearing a hydranth; (d) the aboral end of the main stem may
greatly elongate, give rise to many short branches, each bearing a
134 A. “f. Goldfarb
hydranth; (e) any combination of (a), (4), (c), (d), may occur.
. No record was kept of the history of the individual hydranths.
This a Is tobe terretted. (Kor, hy dranths on a given colony may
be regenerated yet will not be recorded if other hydranths on the
same colony should disintegrate or be absorbed. Such occur-
rences are probably rare, anal dowict seriously affect the conclu-
sions. 4. Ihe tables reveal a large degree of variability No
one table can correctly be said to be ty pica in all its details of the
phenomena in any given experiment. [he conclusions are not
based on the ‘ eeical: tables but on all the collected data.
From the foregoing experiments we may draw the following
conclusions:
1. Colonies that had not been exposed to the light during
or after removal of the branches, regenerate their hydranths.
2. The difference in the percentage of hydranths regenerated,
on colonies exposed for brief periods and those not exposed is in
many cases negligible.
3. In most cases, there is but little difference in the percentage
of hydranths regenerated on colonies kept in the dark but exposed
for brief periods, and on the “control”’ colonies.
It seemed desirable to determine under what conditions these
hydroids would thrive best, and to determine if it were possible to
keep all the colonies under practically uniform conditions.
Colonies were prepared as previously mentioned.
(a2) Some were kept in shaded light of the room, cooled by
evaporation from wet cloths; the water in the bowls was not
changed. (b) Conditions the same, but water in bowls changed
daily. (c) Bowls partly immersed in running water. The water
in bowls was not changed. (d) Colonies placed in the direct rays
of the sun. Water in bowls changed daily. (e) The hydranths
were separately cut off, leaving main stem, primary branches and
a large number of secondary and tertiary branches. Water in
bowl was not changed.
This experiment showed that the direct rays of the sun or the
heat caused by them, were injurious; as no polyps were developed
in (d). Colonies from which the hydranths had been separately
Light as a Factor 1n the Regeneration of H1 ydroids ne5
removed in (e) regenerated very few hydranths. Colonies placed
in the diffuse light of the room, with the temperature of the water
lowered by means of wet cloths, and the sea water changed daily,
throve the best, and regenerated the largest number of hydranths.
Hereafter the sea water in all bowls was changed daily or every
other day, and all bowls were covered with wet cloths.
There was the possibility that the pane of red glass admitted
too much light into the dark room, and probably such light was
not monochromatic. Partially to overcome these defects, a large
sheet of red paper—used in photographic work—was so arranged
that it could be made to cover to a greater or less extent the red glass.
This paper covered the red glass completely on bright days, or
uncovered a very narrow slit of the glass on cloudy days.
In the following experiments every precaution was taken to
keep the Eudendrium colonies in the most vigorous condition and
to admit as little red light during observations as circumstances
permitted. “Lables H and I may be considered typical of results
obtained in experiments in which ‘colonies were exposed imme-
diately after or twenty-four hours after removal of branches. But
on observing that hydranths were regenerated in colonies not
exposed to the light, experiments were then undertaken in which
none of the colonies of the series was exposed. ‘Table J is typical
of results thus obtained.
H
ExpPERIMENT BeGcuNn AuG. 14, 1905
(Colonies exposed immediately after removal of branches. Two colonies in each bowl!)
| |
a 4 Time of Sm See (re 1S | | Nl onl eee
ranches : ee lw : 5 : ; : Berl ee
=e Exposure. seieeeleach os kos tos lia | a | ao ae
poets << | S| Seite foe Naas (rec Ubi (acct ba
| = || | = a ee
|
44 | Not exposed fo) if) 16 | 16 19 iy | © 7 6) ke 5
44 5 sec. ° ° aa |G | 9 10 II II ie GY I 9
4 CO 10 sec. ° rae teres aeaer Mes Hees oa Neato allis al aise 0)
42 | % min. ° r || <0 ° ° 2 2B 2 Zu | 4
42 I min. fo) © || © Te ee! 8 12 12 10 4 3
42 5 min. ° fo) Oeil 3 | i 10 | Te) 1) 14 | 3 2
86 Control ° 3 © |) 13 | 282 40) |50) 540." |. |r 3
} |
—- Bs 7 we ‘ite |e os | = ar. | an eke
Memperature -C\,).cn4.502-.--> ZO ZO erShEx9 | 18 | FON zon i2gah lick Wate aleaz
fi a Piece leer:
13 colonies in bowl.
136 A. fF. Goldfarb
I
ExPEeRIMENT Brcun Aue. 8, 1905, 2.00 P. M.
(Colonies exposed twenty-four hours after removal of branches. Three colonies in each bowl)
| | l
Sos raaace ll lees aaa EMEabs ps) oe fo eS
Removed. “i<}/<e/</<ej<a/</<)/<4]/<|</</<\</</<
60 | Not exposed! (osha) 4) @| ON (20) 2 Saar e igi tel ROMEO
60 15 min. OF rom e23h iran Se] 38 il Aes Pie ier fee Pe ac |2|olo
60 30 min. ° I On| DI ase RON) Grae ear I On| erg 20 eo ecmed!
60 6o min. |o] 16/17]! 7 6 6: | (90, 0 | ea ella ig elo Mees
54 3 hours. .-|"0.|'26 | 20 |"14.| 14] 2.] 0! © | ‘o)]\Gor) -o onimralen|eaaiec
54 Control o | 19 | 44 | 47 | 24 | rz | 12 | 13 | 14 | 18 | 17 |23 [21 |17 | 6 | ©
Temperature °C........ —| 25 | 25 | 24 | 21 | 20 | 20 | 20 18 | 19 | 18 |19 20 21 23 |22
ExPERIMENT BeGuNn AUuG. 14, 1905, 4.00 P. M.
(No colonies exposed to light during or after removal of branches. Two colonies in each bow!)
ae a »l¥l}se|lelealele]ele¢ a) leas
Branches bo | a6 do dp do a0 do | a0 oo bo bb
eae ae eed een ee a | |
|
54 fe) 2 5 5 2 I fo) ° ° ° fo)
54 ° 4 13 15 6 4 I ° ° °
54 ° 3 14 14 5 2 ° ° ° ° °
50 ° 7 15 14 II 7 6 6 3 ° I
50 ° 5 8 12 7 6 5 2 I ° °
5° fo) 10 20 14 6 6 3 6 6 I °
Temperature °C. .| 20 20 18 19 | 19 19 20 21 = |), 28 22
From all of the foregoing experiments we may conclude that:
1. Hydranths are regenerated about forty-eight hours after
removal of hydranths or branches.
2. [he largest number of hydranths observed at any one time
is recorded from the second to the fourth day after removal of the
hydranth or branch, or on the first or second day after the appear-
ance of the regenerated hydranths.
'The total number of hydranths and branches removed exceeded 2,391.
Light as a Factor in the Regeneration of H1 ydroids 1237
3. Thereafter there is a gradual decline in the number of
hydranths.
4. Finally no hydranths are recorded on any of the colonies
constituting a series, and the close of the first “cycle” is reached.
It may take from nine to twenty-two days before all the hydranths
of a series are gone. l’he average time is thirteen days.
5. [here seems to be no causal relation between the number
of regenerated hydranths of a colony and the time during which
such colony had been exposed. Colonies exposed for one minute
may form an equal, greater or less per cent. of hydranths than
those colonies exposed for two minutes.
6. In four experiments the per cent. of hydranths formed on
colonies kept in the dark but exposed for brief periods, was equal
to or greater than the per cent. recorded in the control colonies.
In but two experiments was the per cent. less than in the control
colonies.
7. Colonies kept in the dark forty-eight and seventy-two hours,
respectively, after removal of the branches, and then exposed,
regenerate their hydranths before such exposures.
8. Colonies that had at no time during the experiment of
fifteen days been exposed, nevertheless regenerated a great many
hydranths.
om ihe percentage of regenerated hydranths on colonies not
exposed was in four series equal to or greater than in those colonies
exposed for brief periods. In four other series the per cent. was
less than in the colonies that had been exposed. Furthermore,
in two series the percentage of regenerated hydranths in colonies
not exposed was greater than in the “control” colonies.
We may safely conclude that during the first period or cycle of
about thirteen days, darkness does not prevent the regenerative
processes In Eudendrium ramosum jrom taking place. Nor does
darkness necessarily retard the development nor decrease the number
of hydranths formed. On the contrary exposure to the light for
short periods may or may not increase the rate of growth nor the
number of hydranths.
138 A. “f. Goldfarb
Second Cycle
The colonies of Eudendrium ramosum seemed to be, during
this first period of about thirteen days, in a condition not unlike
phototonus in plants, 7. ¢., under the influence of previous exposure
to the light. “In darkness,” Pfeffer’ says, “‘a plant continues to
erow normally as long as it remains in a condition of phototonus,
1. e., so long as the iadnene: of the previous exposure persists.
This period is frequently very prolonged. So long as the plant
remains in a condition of phototonus, . . . small changes of
illumination produce no perceptible effect.”” It seemed not
improbable that the colonies that regenerated hydranths in the ,
dark were under the influence of the light previous to cutting
away the branches.
Acting on this supposition the following experiments were
undertaken. After all the hydranths of a series had disappeared
and the end of the first cycle was reached, 7. e., when the influence
of the previous illumination was gone, all the colonies but one
were subjected to the light for varying periods of time. This one
colony was carefully ene in the danke There was also in nearly
every series one control colony placed in the light.
Tables Ez, K, L and M are typical results obtained in nine
series.
E2
(This Table is continuation of Er)
First No. of Second |
No. | Exposure | Branches Exposure Aug. 5. [ous 6. | Aug. 7. | Aug. 9. | Aug. Io. | Aug.1II.
July 22- \inemovedl> AUue4-
Time. Time.
1 | Not exposed 21 Not exposed ° ° | ° ° °
2 I min. 26 3 min. 2 4 ° °
3) a) 2 min. 28 4 min. o (| fo) ° ° ° °
4 4 min. 23 + min. — | =
5 6 min. 22 I min. 7 5 o | °
6 | 8min. | 26 4 min. oo | Q | I o | ° °
7 | Control | ° ° Ou o | o | °
|
| | | |
Memperaturess Cee see eee et rer ee | = 24 eve || Pex |) BS 25
| |
Pfeffer: “The Baeieen e Plants,” ron Dp sed 1903. Trans. by Alf. J. Ewart.
a
Light as a Factor in the Regeneration of H ydroids 139
K
Date or EXPERIMENT JULY 13, 1905
First Exposure, | Second Exposure, s Pa ES 3 ey eee | a | a
No. July 16. July 27. & = = = = Salad ten aah
: : S| 5)
Time. Time. = = | zk = ul et & 4
I 3 min. Not exposed ° ° ° ° ° fo) °
2 5 min. I min. ° 4 I ° °
3 8 min. 2 min. ° Te || 5 4 I I °
|
4 10 min. 4 min. ° 9 17 17 13 3 ° °
5 15 min. 6 min. 2 GN) aug) 9 rie | 6 2 fo)
6 25 min. I min. sunlight ° 6 | 9 8 2 ° ° fo)
L
| No. of | First Exposure, Second Exposure. Hydranths regenerated at end of
No. Branches Aug. 14.
‘Removed. Time. Time. Date. | 1 day | 2 days |3 days|4 days 5 days
SO = iS Ss SO a (eed ee S| ee
ie ee are Not exposed | Not exposed | Aug. 27 fe) ° ° o | —
ze || 22 | min. | jhymin Aug. 27 ° ° ° o | —
| 21 % min. $ min Aug. 25 ° ° fo) fe) 2
a) | 21 I min. } min Aug. 25 ° 2 2 4 4
5 21 I min. ¢ min Aug. 26 ° I 6 9 =
6 21 5 min. Imin. |Aug.26| o | 0o 2 3 —
7 86 Control 3 I 2 fe)
1More than one colony in each bowl.
M
No. of First Exposure, Second Exposure, Hydranths regenerated after
No. Branches Aug. 8. Aug. 25.
Removed. Time. Time. 1 day |2 days|3 days | 4 days
I 60! Not exposed Not exposed ° ° ° °
2 60! 15 min. 4 min. fo) ° I 3
3 40! 30 min. + min. fo) fo) fo) °
4 40! 1 hour 4 min. ° ° I 2
5 361 3, hours I min fo) fo) I co)
6 56 Control I I 3 9
Memperatune Ce gece ere Nees siesatekea) «,folnle eel eM ajnniece sieieiew 18 18 17 18
1More than one colony in each bowl.
140 A. Ff. Goldfarb
As in the first cycle hydranths are regenerated about forty-eight
hours after exposure.
There is also no causal relation between the time of exposure
and the number of hydranths developed.
Formation of new hydranths occurred with but two exceptions,
on colonies that were exposed, even for such brief periods as five
or ten seconds.
The maximum number of polyps observed at any one time is
recorded on the third or fourth day after exposure, or on the
second day after the appearance of the polyps. This is somewhat
later than in the first cycle.
Thereafter there is a rapid decline in the number of hydranths,
more rapid than during the first cycle.
Finally no more hydranths develop or are observed on any of
the colonies comprising a series. ‘The end of the second cycle 1s
reached. It may take from seven to fourteen days before all the
hydranths are gone. ‘The average is ten days—much shorter than
in the first cycle.
The five colonies not exposed at the beginning of the second
cycle did not regenerate any hydranths during this cycle. In two
colonies two hydranths were formed, however, though colonies
were not exposed.
The control colonies bore no more hydranths by the end and
in some cases by the middle of the second cycle.
From these observations we may infer that after the first cycle,
light was necessary jor the regeneration of the hydranths. Without
light no regeneration of hydranths, or very little, takes place. It 1s
surprising to note what a short exposure—in some cases one-
twelfth minute—was sufficient to start the regenerative processes.
‘These brief exposures, however, may so stimulate the develop-
ment of hydranths that the maximum number of hydranths noted
at any one time during the second cycle is often equal to, or greater
than, the maximum at a during the first cycle. Those colonies
that had not been exposed but hon nevertheless formed new
hydranths during the second cycle, may have been stimulated by
over-exposure to the red light. In one case at least, according to
an observation made at the time, a colony which had not been
Light as a Factor in the Regeneration of 1 ydroids
141
exposed was removed from the bowl. This was thoroughly
cleaned to remove sediment that had collected.
then replaced. All this was done in red light.
The colony was
Two days later
a hydranth appeared on the colony.
Third Cycle
At the end of the second cycle the colonies of four series were
again exposed. By this time several colonies were broken into
two or more pieces, or stolons or branches broken off, or the colony
was otherwise mutilated.
Tables E3 and N give tabulated results.
F3
(This Table is continuation of Et and Ez)
No. of First Second Third
| ail 5 . : Oo : . : . ON) Sele so
N branches| Exposure, | Exposure, | Exposure,| §| | +) Ww) S| LL] 6) Al Go x] al] Hou
oO. Padi tittle tal tn | er aa feted =a ea a | al ala
re- | July 23. | Aug. 4. | Aug. 11. | o5/ oe] wo] eo] so] uo] so) eo] eo| so) eo] eb] eo
: : ; 4) eh Sh S| er sen) Eh ea el ea Sub aye
moved. Time. Time. Time. | aici aici alietiael| ele aieia lea
| |
ee = = eu ee ERE SN | | |e
. . . | | |
1 eer o min. o min 2 min On|ROn MON Se (ia Naaliean eTanO o|O;/oj;0
2 26 I min. + min I min OD igo" |k0) || or |e) | on On| Fonlonlro) to
5 1 mi i 6 | | |
3 | 28 2 min. + min 4 min Oy |) Te ND Wey AI Zee Na |
4 23 4 min. o min } min || Gr) 2 | eC NSae ire | se se |) se ie |] ee Ie
| |
5 | 22 6 min. I min $ min Onl sete 2g) AT ON On| On| tay On Onlin
6 | 26 8 min. 4 min © min On| FONE 2e eres Onl er PON Onion EON ONO
7 | 22 Control @) {S| O||. © | OF) |) Oy) | © |-@ | © 1) ©
y e ae =a ee = se ee 7 ma
fe}
Ilaria “(Con cpoonnceoqsncassuesosseabar 24 |21 |— |20 |20 |18 |19 |18 |19 |20 |22 |23 |22
| |
1. We observe that hydranths may be formed forty-eight or
seventy-two hours after exposure. They may not, however,
appear until four or five days after exposure.
2. [he largest number of hydranths that appeared at any one
time was in four colonies greater, in two colonies equal to, and in
twelve colonies less than, the largest number that appeared at any
time during the preceding cycles.
3. Asin the first two cycles, the maximum number of hydranths
was observed on the second or third day after exposure, or on the
first or second day after the appearance of the hydranths.
142
A. f. Goldfarb
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Light as a Factor in the Regeneration of H ydroids 143
4. [he number of hydranths formed bears no causal relation
to the time of exposure.
5. The number of hydranths declines, but for no apparent
cause may increase in number. So that the third cycle may last
for five days or may last as long as eighteen days.
6. Colonies not exposed at the beginning of the third cycle
may or may not regenerate hydranths. Out of the four colonies
not exposed at the beginning of the third cycle, three had been
exposed at the beginning of each of the preceding cycles.
7. [he control colonies regenerated but one hydranth.
We notice how erratic the response to light has now become.
Hydranths are developed on colonies not exposed at the beginning
of the third cycle. The number of hydranths decreases, finally
all are gone, yet for no apparent cause will increase again. Is it
possible that at the beginning of the third cycle, arbitrarily chosen,
the cycle had not actually come to a close, and the hydranths that
had formed were influenced by the exposure at the beginning of
the second cycle? Or, had the colonies become so sensitive that
the slight exposure to red light necessitated by daily observations
was sufficient to start regeneration? Could the colonies after
a time dispense with light and thrive in the dark, except for slight
red illumination? ‘The evidence is not conclusive in support of
any one of these suppositions.
Fourth Cycle
The evidence is practically identical with that obtained for
third cycle. ‘Table O is typical of results obtained.
Some control colonies also show periodic increase and decrease
in the number of hydranths. There may be two, three or even
four cycles observed in the history of one control colony. Such
cycles are not explicable by a difference of temperature or change
of sea water in the bowls, for these cycles occur apparently inde-
pendently of such changes. A lapse of thirteen or fourteen
days during which no hydranths appear may intervene between
cycles.
A. fF. Goldfarb
144
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Light as a Factor in the Regeneration of A ydrotds 145
Exposure to Continuous Light
The colonies of four series were again exposed to the light as in
the preceding experiments, but no formation of hydranths occurred
even by the fifth day. It seemed not improbable that the colonies
were spent. Did the protracted stay in the dark permanently
injure the hydroids or rather did darkness permanently effect
the regenerative processes in Eudendrium? ‘This could be deter-
mined by permanently exposing the colonies to the light. The
colonies of the four series were removed from their dark chambers
and placed permanently in the light. Before examining the
results we should bear in mind that:
1. Prior to this exposure the colonies bore no hydranths for
periods of three to fifteen days.
2. This exposure occurred from twenty to twenty-five days
after removal of the stems.
3. Many of the colonies were now badly injured or were but
remnants of the original colony.
Tables P and Q are typical of results obtained.
It will be observed that hydranths are regenerated very fre-
quently five days after exposure, though it may take nine and even
twelve days before hydranths make their appearance. This 1s
much longer than was necessary at any other time.
2. The number of hydranths formed is surprisingly large. In
four colonies the maximum number of hydranths equaled, in five
colonies was greater, and in fourteen colonies was less than the
maximum noted on any colony at any other time.
3. [he maximum number of hydranths for any one time was
recorded from the seventh to the tenth day after exposure, or from
the third to the fifth day after the appearance of the hydranths,
much later than during any preceding period. During preceding
cycles, hydranths regenerated by the second or third day after
exposure and decreased in numbers thereafter. In these experi-
ments hydranths were slow in making their appearance, but kept
on regenerating new ones in some cases for the first six days.
4. [he per cent.t of newly-formed hydranths on control colo-
’The per cent. in this and previous cases is obtained by dividing the number of branches removed
into the number of hydranths regenerated in a colony.
A. f. Goldfarb
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148 A. f. Goldfarb
nies 1s exceeded by the per cent. on colonies previously kept in the
dark.
5. The regenerative processes were certainly not impaired by
the almost continuous confinement in the dark.
6. After the first cycle light certainly stimulates the regenera-
tion of hydranths in Eudendrium ramosum.
EXPERIMENTS ON PENNARIA TIARELLA.
The hydroid Pennaria tiarella is found abundantly on the same
piles covered by Eudendrium ramosum. ‘The environment for
both these hydroids is practically the same, although Pennaria
may also be found in exceedingly large numbers attached to
floating eel grass. In the following experiments colonies of Pen-
naria were taken with one exception from the piles of a dock.
Vigorous colonies were chosen. ‘The branches were removed and
the colonies treated in the same way as Eudendrium. A total of
2,672 branches were cut off.
Tables R and S are typical of results.
From these experiments we may conclude that:
1. If colony with its branches and hydranths be kept in the
dark for forty-eight hours or more the hydranths are dropped off
or disintegrated.
2. If these colonies be kept in the dark they do not regenerate
any hydranths.
3. Exposure to daylight or direct sunlight for less than three
hours did not result in the development of hydranths. Hydranths
were formed after an exposure of three, four, four and one-half
and five hours, respectively, only in Experiment R. The hy-
dranths thus formed disappeared after forty-eight hours in the
dark.
4. Exposure to diffuse light or to sunlight for periods varying
from a few minutes to nineteen hours did not result—with the
exceptions noted above—in the formation of hydranths. Ex-
posures for two days invariably induced formation of new hy-
dranths. Probably exposure to brilliant light for not less than
two days is in most cases, needed for regeneration to take place.
5. Confinement in the dark for periods of thirteen to seventeen
149
Light as a Factor in the Regeneration of H ydrotds
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Light as a Factor in the Regeneration of Hydroids 151
days does not permanently prevent regeneration, for, on continu-
ous exposure after such confinement, hydranths are readily
developed in large numbers.
6. Pennaria colonies kept in the dark never developed any
hydranths. Yet these colonies were often literally crowded with
living, thriving colonies of Bryozoa (species undetermined), with
many small campanularian hydroids probably Clytia cylindrica,
and with many young individuals of Eudendrium ramosum.
7. Light is absolutely essential for the normal growth, develop-
ment and regeneration of Pennaria tiarella.
SUMMARY
Eudendrium
In examining the effect of light on regeneration of hydranths in
Eudendrium ramosum, we must distinguish between
1. Colonies kept in the dark but under the influence of
previous illumination
2. Colonies kept in the dark and not under the influence of
previous illumination.
1. These colonies regenerate their hydranths two days after
removal of their branches. Such regeneration is independent of
light. ‘Uhat light is not essential to the regeneration of hydranths
during the first thirteen days (during which influence of previous
illumination obtains) is shown by the fact that, (a) colonies kept
in the dark and not exposed develop a large number of hydranths;
(6) colonies kept in the dark and not exposed may regenerate an
equal or greater per cent. of hydranths than those exposed for
short periods, or those kept in the continuous light, viz., the con-
trol colonies.
2. In colonies not under the influence of previous illumination,
1. e., after the first thirteen days or thereabouts, the response to
light is decidedly different. Such colonies do not develop hy-
dranths or very few hydranths unless exposed. The surprisingly
short exposure of one-twelfth minute may sufhce to start the
regenerative processes, but some exposure is essential.
The illumination prior to removal of branches will stimulate the
152 A. fF. Goldfarb
regeneration of hydranths on colonies kept in the dark, and main-
tain such hydra nths for a long period or cycle of about thirteen
days. [he second cycle is iaied by a brief exposure which
stimulates the colony to renewed activity. Similarly short expo-
sures may rejuvenate the series of colonies during a third and fourth
cycles. ‘The per cent. of hydranths formed at any one time during
the second, third or fourth cycle may be equal to, or greater than,
the per cent. formed at any preceding time.
There is no causal relation between the number of hydranths
formed and the time of exposure, 7. ¢., the per cent. of hydranths
formed after exposure of one-half minute may be greater,
equal to, or less than per cent. formed after exposure of one minute.
The minimum diffuse bright light required to cause hydranths
to regenerate is probably one-twelfth minute. It is not impossible
that under certain conditions a less exposure could bring about
regeneration.
‘The regenerative processes are not impaired by almost continu-
ous confinement in the dark for from twenty to twenty-five days.
On continuous exposure the per cent. of hydranths formed may
equal or exceed that during any preceding time.
Sometime after removal of hydranths, the colonies kept in the
dark, but exposed for brief periods, are stimulated to regenerate
hydranths more readily than those colonies kept in the light, viz.,
the control colonies.
Pennaria
Prolonged darkness is inimical to development and maintenance
of hydranths in colonies of Pennaria tiarella. If kept in the dark
colonies lose their hydranths within forty-eight hours, and do not
develop new ones.
Exposure to brilliant light for three or four hours may stimulate
the development of hydranths, but usually an exposure of not less
than two days is necessary for such development.
As in Eudendrium, confinement in the dark for long periods
does not permanently affect the regenerative powers of these
colonies.
OBSERVATIONS AND EXPERIMENTS CONCERNING
THE ELEMENTARY PHENOMENA OF EMBRY-
ONIC DEVELOPMENT IN CHAXTOPTERUS'
BY
FRANK R. LILLIE
With One Prare anp SEVENTY-EIGHT FiGcurEs IN THE TEXT
il, Ilnnonlnetions 66.40 ocaabatcasch ase bees Coma dd et tOer Gee ec merece rae anerrs a 154
itive, Microscopic Composition of the Protoplasm of the Egg. ..- 2 2.2... ee seo ee 156
fis “lee Inia IER ES ocootad6asucos5t G00 UU OsaN Up EOS DUS EBOUORADoUanoOUoaGeH 156
z. Comparison.of the Living and of the Fixed and Stained Protoplasm................ 160
Lin, “The GUniGihs i Gas ats ehguandar 5 bSanbhuos seb ConeneD Hada eo SnmoeEoneEener Aonoeee 162
Tie (OLR EONS a6nho Acro came dane Gros DSCC ETO CHECn Oat aaa peer mao See sb or 162
aa) Betore| Rupture of the Germinal Vesicle <2 2. -!.--------.---. wisp ysis + eee 162
Goethe se enod om Maturation: and ertilizatiom ~ceiciciy- © 2 1) > = «ole erosion Bere LOT
Gy Action ol, CentrtugalsPorce in’ the Unsepmented Egg... ..).5.5...s.emee se 4: 179
peelsiteraturerandabiscussioneseer eee seeee cce fee es SBA as eee ete ss oo pacdonde 187
a. The Axis of the Ovarian Ovocyte and its Relation to Polarity.................-- 187
peme Rhesbictoplasnuce nay ermer etme sete er tei taleytatciore lates ole aim) © Sin = a olegrereien selene erate 188
GaekesidualiSubstancevot the! Germinal Vesicle: «.5.- << oe + ss... - ever ioe eiacl= sls IgI
ih IPO RRA FON oA o86.o n.56 ce Gouna AO ob nD GOO ee oa ae. 5, ao ation oe rarissoc.2 193
IY, Clleaiyaras auial Diana thatoing os apo qu dbs cso Done DORE a ADE Cees oc coteponcosoGaTdcc™ 194
Tr) Hormative Stufis in the Early, Normal) Development. ........-.2..0.-----+eeneeeese- 195
a. “(Nae ING OLENA esa sso dddadSecdade Hobe SONS BBBMOGMDD 50 - Sneed aauuspoacucac 195
a, Ine Iden or CleANAies soon ctu scmgnadouees Seas o> BBHe > odocesuaroobdbcasrun ac 204,
Eee Distributionvorsoupstancesmusthe: Cellseert sila e- = - tebe) ersbels eteiole lois et 207
Gi, lbatare: Wikia Siemaiers, 5 oo Sia nec can nedde OSD OSERREnT 9 00 Csbr aa Sbacn bod oauobesC 212
2, IDMiterentaeystoa Wiptdevts (CIENT EIS Jo 55. ¢enncce nae Beeeo> oc aac ce uuoBoonddDoUnSoouT 220
ae,» Uinta eleeial( OVA po docents baa Sn OEE cs eon oscars hon ien bona 223
io Ieudhy Saracsuatstrn Ge lNesmeion se SiGe Rep 55 ooo dp oounoeosbadoooDUOnaesor 223
iia COnceming thes Growthyof the Nucleus. cae mmic cas oleiicies core ie ilsinis el 227
fie ateractionvols Nucleuseand | C@ytoplasm oer se erseltele ies ate orate) ere) ole mel =)= lore 230
ive, Water Distribution’ of Formative Stufisst 2.2.2. .-2..cc eens esse tse: 232
Vee Bormationsob Ciliacand, Other Cell-Constituents. ....-..2..<0--2-++eesr sens 236
pee Mon iicleatedmUnusermentedes@va).. . <jueiterseteter a= tele ietelele ele lal orin)s eee = 239
Cupleiteravorerand sl) i1sCUSSIOMs Ae soc « - «3, sperrverra eerste ore sc lejcia(s ls sie isco ereiaysie = eve 243
1 The experimental parts of this study and the observations dealing with the living egg were car-
ried on at the Marine Biological Laboratory of Woods Holl, Mass; the study of the preparations
in the Zcélogical Laboratory of the University of Chicago.
154 Frank R. Lillie
V.'; General Discussions refer. vei éc-iese olcln ne /e, 5 axe esas ghee ale «te a en ae es 246
i. Relation*of Nucleus and!iCytoplasm..«.. .\<sis 6-2. aise ore chee aera renee eee 247
2. Dhe @Oripinal!Diversity of Organization ......0 1.33 ee eee eee eee nee eee 249
3. Properties of the Whole. (Principle of Unity)................- fe A ee 251
A. ithevMosaic Jbheory) of Development... 2) =. 4..9 2 ee eee ee eee eee 255
5. Concerning Wormativey Stulls.t a5 fe = <2 sos « Scere eee eee ee 258
6; Nuclear Specification = 2). Sis eis), ys. wis dae 5 Se cat ot Ae Oe I eee eee 259
7. SUuMMALy Of DISCUSSION 324%. 22 ece--e as ene )etAccerne eee OE Oe Oe 263
I. INTRODUCTION
This paper is based on the study of the egg of a single species of
annelid, Chatopterus pergamentaceus Cuvier, and is a con-
tribution to the study of the elementary phenomena of develop-
ment. I have been able to extend my earlier observations on
differentiation without cleavage (Lillie, ’02) and to demonstrate
homology of regions and cell-constituents between unsegmented
eggs that have undergone differentiation and larve normally
formed. It has been found in the course of the later study that
the homology is dependent upon a very exact localization of ger-
minal areas and specific substances in the unsegmented egg, which
play essentially the same role whether they be divided by cell-
walls or not. The microscopic composition of these substances
and the relations of nuclear activity to differentiation have also
been studied, and it has been necessary to include the ovogenesis,
maturation, fertilization and cell-lineage in order to control the
other data.
In the present paper the attempt will be made to present the
substance of the entire study without too much detail, leaving the
elaboration of certain parts for a later contribution. However, I
shall make no apology for entering into details, because there is
no other explanation of heredity than a complete account of devel-
opment, and one cannot describe even a small part of so complex
a thing without many words, unless one knows in advance what
is essential and what is not.
‘The numerous cytological studies of germ-cells of the last few
years have focused attention especially on the chromosome-
complex, and the remarkably uniform results have established a
series of propositions on so firm a basis that they seem destined
Elementary Phenomena of Embryonic Deve lopme nt 155
to form the working hypothesis of investigations in heredity and
development for a eorsiclesalals period of time. These are: (1)
The constancy in number of the chromosomes in any species.
(2) The persistence of a descendent of each chromosome in every
cell throughout the series of cell-generations. (3) The composi-
tion of the chromosome-complex of the first cleavage-spindle of
corresponding maternal and paternal groups, equal in number and
in all other respects, save individual variation. (4) The biparental
character of the embryonic nuclei, similar to that of the first
cleavage nucleus. (5) That the individual chromosomes of both
the maternal and paternal chromosome-complexes are probably
qualitatively different. (6) That pseudo-reduction (synapsis)
consists in union of corresponding maternal and paternal chromo-
somes. (7) That reduction in the number of chromosomes 1s
effected by separation of the units of such bivalent chromosomes.
Though all of these propositions are not accepted by all cytolo-
gists, yet in the main they have become the current opinions.
Enough, certainly, has been definitely established to put the hypo-
thesis, that the chromosomes constitute the bearers of the heredi-
tary qualities, on a definite basis. Tbe embryologist, therefore,
has his problem defined, and it 1s no less an undertaking than to
derive the entire body of the individual jrom the chromosome-
complex as its germ. This 1s the embryological program. It has
been recognized as such inthe more important recent theories of
development; but hypothesis has far outstripped observation, and
it is remarkable how very few observations have been recorded
establishing definite morphogenic relations between nucleus and
cytoplasm. Yet, if the hypothesis be true, such relations must be
of the very essence of embryonic development.
The observations recorded in this paper have led me to some
very definite opinions as to the relations of nucleus and cytoplasm
in embryonic development. | am well aware that they cover only
a very small part of this embryological program; but I venture to
express the hope in putting them forward that others may be led
to test and to extend them.
Our advance along these lines is very dependent on methods.
The results of this pay could not have been attained without the
156 Frank R. Lillie
methods employed. It is probable that centrifuging and staining
intra vitam will prove to be very generally applicable, and I
believe they will yield valuable results. “The method of analyzing
the developmental processes by suppressing the cleavage without ~
prejudice to other morphogenic activities, has been the most
valuable one on the whole. I believe that it is capable of quite a
wide application even in cases where the differentiation does not
proceed to the formation of cilia. Bastian (04) seems to have
witnessed such a process in rotifers, though, by an incomparable
blunder, he interpreted the unsegmented ciliated ova as a definite
genus of ciliate infusorian. It applies in other annelids than
Cheetopterus as Scott’s (06) and Treadwell’s (02) papers show,
and I think there is every reason to believe that it will prove
available in animals of different phyla.
II. MICROSCOPIC COMPOSITION OF THE PROTOPLASM OF THE EGG
Tn be Living Protoplasm
The egg of Chetopterus is a spheroidal mass of a semifluid,
transparent and homogeneous substance with a large number of
granules of various sizes and optical properties suspended in it;
more fluid droplets occur here and there. ‘The living protoplasm
shows no evidence of a filar, reticular or alveolar structure, except
that granules or droplets may be so closely set in certain places
as to produce the appearance of an emulsion. ‘This, however, is
a secondary character. The primary optical properties of the
protoplasm are those of a transparent colloidal solution, the
particles of which are ultramicroscopic; this may assume the
most various configurations according to the number, size and
arrangement of the suspended, microscopically visible, granules.
The ground substance is a suitable name for the fluid that con-
tains and suspends all the granules and droplets; if all these were
imagined removed it would preserve a faithful semblance of the
egg. ‘Thus it is regarded as forming the external pellicle and as
continuous through the nuclear membrane with the nucleo-
plasm. Its optical properties are the same everywhere like a drop
of water, but, unlike such a drop, it may exhibit different physical
and chemical properties in different parts.
Elementary Phenomena of Embryonic Development 157
The Granules.—The varying microscopic ieee of regions
of the egg is thus due entirely to the granules and droplets sus-
pended in the ground substance. Droplets of a homogeneous
transparent liquid, more fluid than the ground substance, are
especially characteristic of the germinal vesicle and of the pro-
toplasm in the neighborhood of the vegetative pole (Fig. 1). I
have not, however, been able to follow TheiE behavior very fully.
The granules on the other hand are more solid bodies, though i it 1S
pe bable that some are semifluid; certainly all have aang cis al:
density.
By subjecting the living egg to considerable pressure one can
examine it with the highest powers; my observations were made
with a Zeiss 2 mm. homogeneous oil-immersion objective, apert.
130, and compensating oculars 6, 8 and 12. Under such magni-
fications one sees that the protoplasm of any part consists of the
ground substance with granules of two kinds, viz: slightly or non-
refringent granules of ete ely large size, which I shall designate
spherules, and very minute (less * hae I” in diameter) bode
refringent granules of approximately uniform size, the microsomes.
The slveatles vary in size from about 2.25 to about the size of a
microsome.
In the living protoplasm the microsomes and the smaller
spherules are in a constant state of tremulous agitation vibrating
in rapid rhythm with considerable amplitude. ‘The range of these
movements is considerably increased if the egg is agai crushed
so that the protoplasm is broken into small fragments. No one
who has studied these movements, as I have done for hours at a
time, could believe that the microsomes are nodal points of a net-
work, or are connected by filaments as they appear to be in the best
stained sections. One is forced to conclude that they have free-
dom of movement in all directions, 7. ¢., that they are suspended
in a fluid medium which has no filar, reticular or alveolar structure.
The description so far applies to the substance of the germinal
vesicle as aptly as to the cytoplasm; though the spherules of the
germinal vesicle are probably small. droplets in the sense used
above; they certainly differ in many respects from the spherules of
the cytoplasm. But the substance of the germinal vesicle con-
158 Frank R. Lillie
tains as large a proportion of microsomes as does the cytoplasm,
and these cannot be distinguished apart in the living condition,
though they can be differentiated by fixation and staining, as
described below. ‘The germinal vesicle is of course characterized
also by the nucleolus described in Part III; the chromosomes were
not distinguished in the living germinal vesicle. ‘There are also
large spaces filled with fluid (Fig. 1).
Microsomes may occur separately or in conjunction with
spherules; in the latter case a certain number appear to be in-
separably united to the spherules; no matter how much the pro-
toplasm is broken up, the union cannot be dissolved. I am not
sure that this is characteristic of all the various kinds of spherules,
but it certainly is of those of the ectoplasm. There are different
kinds of spherules characteristic of different regions of the egg and
related to different kinds of differentiation. ‘Their differences,
arrangement and properties are considered later.
The relation between microsome and spherule is more or less
problematical. From the fact that spherules may grade down in
size to approximately the dimensions of microsomes, one is tempted
to assume that the spherules are produced by growth of micro-
somes, or by their agglutination and fusion, and the same con-
clusion follows from other facts considered beyond. ‘The micro-
somes would thus be the primitive source of these larger granules;
the spherules a farther step in the processes of differentiation.
According to this view the microsomes are the primitive formed
elements of the cytoplasm. ‘The source of the microsomes them-
selves becomes therefore a vitally important question. There are
three possibilities: (a) That the microsomes arise from smaller
ultramicroscopic particles in the ground substance; (>) that they
are an original cytoplasmic element multiplying by fission; (c)
that their primitive source is the nucleus, in which event they are
to be regarded as chromatin derivatives, for all the formed ele-
ments of the nucleus are chromatin derivatives, or, at least, have
their base in the chromosomes.
I have observed no phenomena that indicate either of the first
two possibilities, but have on the other hand repeatedly observed
the transformation of nuclear microsomes into cytoplasmic micro-
i
Elementary Phenomena of Embryonic Development es
dy . 59
somes in sufficient quantity to supply all the microsomes found in
the egg. In addition to these direct observations several im portant
considerations unite in Support of the hypothesis, t that microsomes
are chromatin particles.
The direct observations concerning the structure and fate of the
residual substance of the germinal vesicle (pp. 172-178) and con-
cerning chromatin distribution in uninucleated unsegmented eggs
that undergo differentiation (pp. 230-232) conclusively prove the
derivation of a large proportion of the microsomes from the nucleus.
Now each daughter nucleus is built up from a particular chromo-
some-complex, and it is a commonplace cytological generalization
that chromosomes arise by the union of chromatin particles (chromo-
meres) of approximately the size of cytomicrosomes; this 1s readily
observed in the egg of Chztopterus. When the nucleus comes to
rest the chromosomes resolve themselves again into these ultimate
formed elements of the chromatin, after passing through specific
form changes. ‘Thus the chromosomes are composed of particles
of about the same size as the cytomicrosomes. ‘The staining
reaction of the chromomere and cytomicrosome is the same, in
certain stages at least; newly formed microsomes take the basic
stain with nearly the same intensity as chromatin particles. In
the ovogenesis of Chztopterus the main source of the microsomes
is the structure described by Mead (’98, pp. 193, 194) as the
paranucleus as follows: “Up to the time when the egg has attained
about two-thirds its full size, only a part of the protoplasm pre-
sents the loose reticular appearance; the rest remains as large
purple masses, which I consider to be equivalent to the Neben-
kerne or paranuclei of various authors (Fig. 1, c, d, 7). These
masses are not homogeneous, but resolve themselves into a cyto-
plasmic network, of which the meshes are much compressed, and
the strands usually parallel with the surface of the nucleus, though
at the periphery of the masses they fray out and become continuous
with the open network which contains the yolk.” I would add
that the paranucleus forms a kind of cap to the germinal vesicle,
from which it probably originates. The reticulum, in’ my
opinion, is an artefact, and the essential thing about the para-
nucleus is that it is composed of a dense aggregation of micro-
160 Frank R. Lillie
somes, that subsequently become distributed to all parts of the
cytoplasm.
These various considerations have convinced me that the larger
proportion of the microsomes are derived from the nucleus. It
seems improbable that there should be any radical difference in the
origin of any, for the morphological characters of the cytomicro-
somes, such as size, density, color and refringibility in life, and
staining reactions, are remarkably constant. I will not deny that
they may multiply outside of the nucleus; but, except in the case
of the centrosome, I know of no evidence in favor of such multi-
plication of bodies resembling microsomes. On the other hand
it is certain that the chromomeres multiply within the nucleus.
If the microsomes are extranuclear chromomeres, as here sug-
gested, the doctrine of the individuality of chromosomes must be
understood, as Hacker has pointed out, not in the sense that the
entire substance of a chromosome is handed on through successive
generations of cells, but only in the sense of persistence of part of
the substance in each cell-generation. The ultimate consequences
of this hypothesis of the microsomes are far-reaching, and need
not be considered here.
2. Comparison of Living, and of the Fixed and Stained
Protoplasm
In general it may be said that the sections give an extraor-
dinarily faithful picture of the protoplasm, with one exception,
viz: the appearance of a reticulum in the ground substance
(Fig. 1). Practically all methods of fixing and staining employed
show this reticulum. I have already mentioned one reason for
supposing it to be an artefact, viz: the rapid ‘‘ Brownian” move-
ments of the very particles (microsomes) that appear in the prepa-
rations to be in the nodes of the reticulum. A second reason for
this belief is the fact that the spherules may be driven through the
cy toplasm i in any direction by centrifugal force without destroying
its capacity for development (Part III, 1,c). A third argument is
found in the formation of pscudopene at certain stages in the
experiments described in Part IV, of such exceeding tenuity that
»
Elementary Phenomena of Embryonic Development 161
an alveolar or reticular structure of the pseudopodial protoplasm
in any way resembling that of the sections is impossible; but such
delicate strands of protoplasm are living and exhibit character-
istic movements, though they grade off into invisibility. When
one adds that no reticulum can be seen by any of the usual devices
in the living protoplasm, and that a similar reticulum has been
demonstrated to be formed in colloid solutions known to be
homogeneous by the usual methods of coagulation employed in
the preservation of protoplasm, it certainly seems much more
reasonable to conclude that the reticulum is an artefact in the
preparations.
The preparations, however, in other respects greatly extend the
observations that it is possible to make on the living protoplasm.
Thus they enable one to distinguish different classes of micro-
somes and spherules by wiser here reaction, to differentiate
the chromatin, and to obtain fixed pictures of stages that pass like
a vision in the living egg. A proper correlation of observations on
the living protoplasm and the fixed and stained sections greatly
enlarges the scope of each and is indispensable for thorough inter-
pretation.
It is not my intention to enter into a discussion of the various
theories of the morphological composition of protoplasm; I desire
only to meet one objection that will certainly occur to most cytolo-
gists, viz: as Wilson (99) expresses it, ‘A continuous series of size
gradations exists from the largest deutoplasm spheres down to the
minutest ‘granules,’ and these bodies and the ‘alveoli,’ which
form the middle terms of the series, arise in a sensibly homogeneous
protoplasm.’ As I have already pointed out, the spherules in the
protoplasm of Chetopterus grade down to the size of microsomes;
it is also true that granules smaller than microsomes occur. Thus
microsomes are terms in a series of sizes. This, however, does
not prove that they may not be specific elements for the size-grada-
tion may be interpreted in various ways. Microsomes are aggrega-
tions of molecules, and it is probable that, if we could trace their
origin morphologically, smaller granules would be recognized as
their precursors. It might, therefore, be better to use some other
term than “microsomes” for the granules in question, but any
162 Frank R. Lillie
more specific term would be open to other objections. “The word
microsome is used in this paper only fora class of granules which
are very numerous, very uniform in size, and with other specific
characters described in the text and illustrated in the figures.
Me BEE STRUCTURE OF SDE SEGG
1. Observations
a. Before Rupture of the Germinal Vesicle
The ovaries of Chztopterus occupy the bases of the enlarged
parapodia in the posterior sexual division of the body, and have
the form of convoluted tubes. The wall of the tubes is made up
for the most part by the ova which extend from the lumen. The
pole of the ovum farthest from the lumen may be called the free
pole and that next the lumen of the ovarian tube, the attached
pole. he epithelial arrangement of the ova remains until they
are full grown, so that there 1s no difficulty in distinguishing free
and attached poles at any stage. It may be stated at once that the
free pole becomes the animal pole of the oosperm, and the attached
pole consequently represents the vegetative pole. Thus it is
possible to trace the germinal areas recognizable in the odsperm
back to an early stage of the ovocyte.
By so doing the so-called period of growth of the ovogenesis
assumes a new significance as a period in development, and it
must be studied from this standpoint. ‘This problem, however,
will be left for later treatment, though certain broad features may
be mentioned from time to time in the present paper.
The following account is based on a study of the living egg and
of sections of material killed in picro-acetic acid, sublimate-acetic,
Flemming’s fluid and Zenker’s fluid; sections from each kind of
material were stained in the following ways: (1) Iron hematoxylin
followed by orange G; (2) thionin and orange G; (3) neutral
gentian (see Bensley ’00). So that at least twelve different
combinations were used, and all gave essentially similar results in
differentiating the structures described. Thus it will not be neces-
Elementary Phenomena of Embryonic Development 163
sary to describe the results of particular methods in detail. Most
of the features described were seen in the living egg and demon-
strated also by experimentai méans.
The cytoplasm and germinal vesicle will be described separately.
1. Cytoplasm.—The main structural feature of the cytoplasm
is its differentiation into two layers, an outer or ectoplasmic and an
inner or endoplasmic.
The ectoplasm (Fig. 1) covers the free hemisphere and ends a
short distance below the equator so that the endoplasm comes to
Fig. 1. Axial section of a full-grown primary ovocyte, which still formed part of the ovarian
epithelium. Note that the ectoplasm is confined to the upper two-thirds of the egg.
A, Animal pole or free pole of ovarian ovocyte; c, chromatin; EF, ectoplasm; e.a., endoplasm a;
e.b., endoplasm b; e.c., endoplasm c; ”, nucleolus; r.s., residual substance of the germinal vesicle;
s.n., sperm nucleus; /’, vegetative pole or attached pole of ovarian ovocyte.
Note.—All figures of sections in this paper were drawn with the camera at a magnification of 1900
diameters (Zeiss comp. oc. 12 and obj. 2 mm. oil immersion). They have been reduced to about 0.6
of original size. Most of the drawings of living eggs were made with the camera at a magnification
of about 400 diameters; these were subsequently redrawn, somewhat enlarged, in ink.
164 Frank R. Lillie
the surface at the vegetative pole. There is sometimes a defect
in the ectoplasm in the center of the free or animal pole. This is
particularly obvious after staining in thionin, for the spherules
remain unstained and the microsomes stain intensely blue; thus
is produced the effect of a blue plug in the center of the free hemi-
sphere. The ectoplasm is covered externally by a very delicate
membrane, and the vegetative pele where the endoplasm comes to
the surface is naked. ‘The constant characteristic of the ecto-
plasm that enables one to follow it throughout the development is
the presence of a large number of spherules of very uniform size,
closely set together so as to produce the effect of a pavement when
one focuses on the surface of the entire egg. ‘These are as large
as the average spherules of the endoplasm, but they differ from
them in many respects: (1) In the living egg they are colorless,
whereas the endoplasmic spherules are yellow in mass; (2) their
position and arrangement are characteristic in each stage; (3) they
always stain differently from the endoplasmic spherules; thus the
osmic acid of Flemming’s fluid leaves them unstained, but stains
the endoplasmic spherules from gray to solid black; a section of
such an egg mounted in balsam appears to have a layer of vacuoles
in the ectoplasm, which close examination shows to be the ecto-
plasmic spherules. After iron hematoxylin and orange G follow-
ing either picro-acetic or sublimate-acetic fixation, the ectoplasmic
spherules are intensely orange, while the larger endoplasmic
spherules are black; this result 1s easily obtained by proper ex-
traction of the iron hematoxylin, which at first stains all spherules
black, but which washes out of the ectoplasmic spherules long
before it does from the larger endoplasmic ones; if one stops the
extraction at this point, and stains in orange G a very clear differ-
entiation of the ectoplasm is obtained. Other methods and stains
differentiate the ectoplasm in different ways, but as | am unable
at present to reason from the microchemical reaction to the chem-
ical nature of the spherules involved, it seems superfluous to
describe the details.
There are from one to three layers of these spherules in the
ectoplasm over the upper hemisphere (Fig. 1). These are em-
bedded in the ground substance which likewise contains micro-
i
+
Elementary Phenomena of Embryonic Development 165
somes external to the spherules as well as between and on them.
The layer of the ground substance external to the ectoplasmic
spherules may be called the surface pellicle; during the maturation
and cleavage this pellicle forms characteristic waves described
later on.
The endoplasm is not so uniform in its composition as the ecto-
plasm. There is, in the first place, a striking difference between
the upper and lower hemispheres (Fig. 1). In the upper hemi-
sphere the protoplasm 1s dense, and contains an aggregation of the
largest spherules, which stain black in the osmic acid or iron
hematoxylin (endoplasm a); these are massed largely toward the
periphery leaving a zone of protoplasm (endoplasm 6); surrounding
the upper half of the germinal vesicle, relatively free from the
spherules. Thus thionin (which is an intense microsomal stain)
shows a deep blue crescentic area surrounding the upper half of
the germinal vesicle, and sending out processes among the spher-
ules to the periphery. At the free pole these processes are strong-
est and may cause an interruption in the ectoplasm, where the
endoplasm comes to the surface. ‘There is a beautiful reticulum
in the cytoplasm where it is not concealed by spherules, and the
microsomes lie in the nodes; I have already given reasons for
believing that the reticulum is an artefact. In the lower hemi-
sphere the protoplasm is vacuolated, and contains smaller spherules
that stain differently from the larger endoplasmic spherules*
(endoplasm c).
Thus the endoplasm comes to the surface at two places, (1)
a small area in the center of the animal pole, where the polar
globules are later formed;? (2) over a large portion of the vegetative
hemisphere where the spermatozo6n usually enters.
2. The Germinal Vesicle—TYhe diameter of the germinal
1 The endoplasmic spherules have collectively a yellow tinge that is very bright in the eggs of some
individuals and relatively pale in others. The spherule-bearing endoplasm is, therefore, often named
in the following pages the yellow endoplasm, or yellow substance.
2T have not always been able to find this ectoplasmic defect in the primary ovocyte prior to the
rupture of the germinal vesicle, and at the best it is relatively slight. But I believe it to be significant,
for it marks the position of a constant exposure of the endoplasm that arises at the time of formation
of the first maturation spindle, and persists throughout the development. The apical flagella arise
from this exposed endoplasm.
166 Frank R. Lillie
vesicle is approximately half that of the entire egg (Fig. 1), and |
thus comprises about one-eighth of the substance of the
latter. Only a small portion of it goes to form the chromo-
somes of the maturation spindles. ‘There is also a large nucleolus.
The remainder is a special substance which I shall call the residual
substance of the germinal vesicle (Fig. 1, r.s.); it plays an impor-
tant role in the developmental processes and can be assembled,
even hours after the germinal vesicle has broken down, by the
action of centrifugal force; thus it is certain that it does not
“intermingle” with the rest of the cytoplasm. It is important,
therefore, to describe carefully the structure of the vesicle.
In the living condition one recognizes easily the membrane and
the large nucleolus; the peripheral part of the vesicle contains
more vacuoles than the central mass and thus appears less dense
(see Figs. 6-16). Apart from the droplets occupying the vacuoles,
the substance consists of an enormous number of microsomes and
small spherules embedded in a homogeneous ground substance.
The chromatin is not separately visible in the living germinal
vesicle.
In sections stained with iron hematoxylin and orange G the
germinal vesicle (Fig. 1) is seen to be occupied by a meshwork of a
few thick strands, bounding vacuoles. The strands are penetrated
through and through with microsomes that stain orange instead
of haematoxylin like the cytomicrosomes. But when the germinal
vesicle breaks down, these microsomes change their staining
reaction to that of cytomicrosomes. With thionin and orange the
cytomicrosomes stain an intense blue and the microsomes of the
germinal vesicle, orange, until the germinal vesicle breaks down,
when they, too, take the blue thionin stain. Thus the staining
reaction sharply differentiates the microsomes of the cytoplasm
and germinal vesicle. The membrane of the germinal vesicle
consists of a thin layer of the ground substance of the nucleus in
which the microsomes take the acid stain, and a thin layer of the
ground substance of the cytoplasm in which the microsomes take
the basic stain.
My study of the nucleolus and chromosomes is incomplete and
I shall defer a detailed description for another occasion. The
Elementary Phenomena of Embryonic Development 167
close association of certain chromosomes with the nucleolus is a
very striking and constant character (see Figs. 1 and 4).
It will be seen, then, that the full grown primary ovocyte is
highly differentiated: (1) Its polarity is expressed by, (a) the con-
stant relation of free and attached poles; (>) the extension of the
ectoplasm over the upper hemisphere mainly; (c) the exposure of
the endoplasm at a small spot in the free pole and over a large
area at the attached pole; (d) the contrast between the structure
of the endoplasm in the free and attached hemispheres. (2) The
existence of a sharply differentiated ectoplasm, of three kinds of
endoplasm, and of the various substances of the germinal vesicle,
gives a degree of orginal diversity that is certainly surprising.
Our problem is to trace these substances in development, to
note the appearance of other substances, and from the behavior
of all in the normal and experimentally modified development, to
attempt to discover their respective roles in the formation of an
embryo. We should not attempt to prejudge the matter in ad-
vance by naming certain substances yolk or food-matters and
others formative substances. If these are true categories, it
should be possible to demonstrate it.
The following account will show that these substances are moved
by internal forces to definite locations in the embryo and become
parts of definite systems of organs. It will also be shown that the
localization, and part of the differentiation, is independent of the
process of cell division, though closely dependent upon interaction
with nuclear derivatives.
b. The Period of Maturation and Fertilization
I. Polarization..—As long as the germinal vesicle is intact
the arrangement of the various suleeees in the egg does not
conform in all respects to the position of the Ciera “alba onic
areas. [herupture of the germinal vesicle initiates a series of move-
ments of the substances by means of which they attain to their
1The substances of the egg are polarized before this process, but have a radically different
arrangement; a more exact term for the process would, therefore, be re-polarization, which is
objectionable in other respects.
168 Frank R. Lillie
definitive positions during the prophases of the first maturation
division. ‘This rearrangement of the substances may be called
the process of polarization, inasmuch as it takes place with refer-
ence to the polar axis of the egg. It produces a topographical
arrangement of the substances that corresponds in many essential
respects to the future embryonic areas. ‘Thus the history of the
maturation process is an important chapter in the development.
Fertilization, which occurs during this period, willbe considered only
incidentally, as I have nothing substantially new to add to Mead’s
fine account.
If the eggs of Chetopterus be taken and allowed to stand in sea-
water, the germinal vesicle breaks down, whether the eggs be
fertilized or not; the first maturation spindle forms and moves to
the predelineated animal pole; at the same time the various sub-
stances become polarized. “Thus polarization is independent of
fertilization. Unless the eggs be fertilized or stimulated in some
other definite fashion, they remain in the metaphase of the first
maturation division indefinitely (Mead ’g8), (Lillie ’o2). If the
eggs be so fertilized or stimulated, the polar bodies are formed and
the processes of development follow continuously.
The movements of the three classes of substances, ectoplasm,
endoplasm, and the substances of the germinal vesicle will be
described separately; they have reached practically their definitive
positions by the time that the first maturation spindle becomes
fixed at the periphery.
Ectoplasm.—Before the breaking of the germinal vesicle the
ectoplasm covers the upper two-thirds of the egg; as soon as the
germinal vesicle breaks down, it flows toward the vegetative pole,
and, even before the first maturation spindle has become fixed
at the animal pole, it has completely overflowed it (Figs. 2 and 4),
thus covering up the exposed endoplasm. The membrane accom-
panies the ectoplasm and so covers the entire egg. This move-
ment of the ectoplasm is clearly visible in the living egg as a series
of waves of the transparent external pellicle; but, owing to the
difhculty of orienting the living egg in this stage, I have not been
able to follow the course of the waves satisfactorily. “The sec-
tions, however, that enable one to trace the overflow of the vege-
Elementary Phenomena of Embryonic Development 169
tative pole, leave no doubt that they are associated with this
process.
At the end of the polarization period the ectoplasm around the
center of the vegetative hemisphere is as thick as anywhere else.
eo
9,9, 6
"@%
Cx
ss
eat
isi)
S59
Ss
Fig. 2. Axial section through a primary ovocyte, killed about ten minutes after coming into
sea-water, and three minutes after fertilization. The ectoplasm has already flowed to the
vegetative pole, leaving an exposed area of endoplasm at the animal pole. Part of the a endoplasm
has likewise flowed to the vegetative pole. The germinal vesicle has broken down and the maturation
spindle isin process of formation between the two primary asters. The residual substance of the ger-
minal vesicle is clearly seen. The chromosomes do not fall in the plane of the section. Letters same
as in Fig. 1.
The original opening in the ectoplasm at the animal pole has
become enlarged, and the outer end of the maturation spindle is
fixed here (Figs. 2-5).
Endoplasm.—Yhe endoplasm consists of three distinct parts
as already described, (a) the uppermost part laden with large
ar
170 Frank R. Lillie
spherules; (b) the non-spherular part overlying the germinal
vesicle; (c) the vacuolated part containing small spherules situated
below the germinal vesicle. In the process of polarization the
endoplasmic substances, a and }, reverse their relations both
in regard to one another and also with respect to the germinal
vesicle. Substance a flows around substance b and the ger-
minal vesicle toward the vegetative pole where it intermingles
Fig.3. A later stage of the same series, as in Fig. 2, killed twelve minutes after fertilization. It
shows the entry of the first maturation spindle into the residual substance of the germinal vesicle.
Orientation of section uncertain. Letters same as in Bice ie
with c, so that it is difficult for a time to distinguish them apart,
except that the two kinds of spherules may be recognized inter-
mingled (Figs. 2, 4 and 5). Substance 6 on the other hand
maintains pretty nearly its original position, and the substance of
the germinal vesicle passes up through it to the animal pole so as
Elementary Phenomena of Elementary Development 171
to lie above it. The definitive positions of these substances
is shown in Fig. 5. An intermediate stage is shown in Figs.
2 and 4.
Though endoplasmic substances a and c intermingle, they
do not lose their identity. This is shown by their subsequent
behavior; if, for instance, the eggs are allowed to remain unfer-
‘ r) See R
C) e ® 4. 2
at efe prlcc®
Pet sarcocees!
ed 90? =
Fig. 4. Axial section of a primary ovocyte of the same series, as in Fig. 2, killed twelve
minutes after fertilization. The maturation spindle 1s completely surrounded by the residual
substance of the germinal vesicle, and the latter is directed toward the animal pole where the ecto-
plasm is wanting. The nucleolus has become smaller and some of the chromosomes appear to
have arisen from it (compare Fig. 1). Letters same as in Fig. 1. s, spermatozoa.
tilized they will be found to have completely separated again in
the course of about two hours (see Fig. 39, p. 208, representing
a section of an egg that has stood two hours and seven minutes
in sea-water). [hese substances also separate out in normally
segmenting eggs and in unsegmented eggs that are under-
going differentiation.
172 Frank R. Lillie
The spermatozo6n normally enters the egg through the exposed
endoplasm of the vegetative pole, which usually appears slightly
collapsed at this time (Fig. 4) and less vacuolated, as though
there had been loss of fluid. “That the spermatozoon may enter
°°
Cy
°
Fig. 5. Axial section of a secondary ovocyte thirty-two minutes after fertilization. E. Ecto-
plasm; e.a., endoplasm a; e.b., endoplasm b; e.c., endoplasm c; r.s., residual substance of the
germinal vesicle; s.a., sperm aster.
through the ectoplasm is shown by the fact that the eggs fertilize
as readily after polarization as before.
Germinal Vesicle—In the case of the germinal vesicle we
have changes not only of position, but also in the character of
the substances concerned:
Elementary Phenomena of Embryonic Development 173
Fic. 10
Figs. 6 to 10. See text for detailed description. Behavior of substances of the germinal vesicle
as seen in a single living egg. Polar view. Fig. 6.11 A.M. Rupture of the germinal vesicle. Out-
flow of cortical substance of the germinal vesicle. Fig. 7. 11.04 A. M. Fig. 8. 11.07 A. M. Fig. 9.
11.15 A.M. Fig. 10. 11.20. M. a, Aster or polar view of spindle; E, ectoplasm; e, endoplasm; r.s.,
residual substance of the germinal vesicle.
174 Frank R. Lillie
As already described, one distinguishes readily in the living
germinal vesicle a clearer cortical zone and a central more
granular mass (Fig. 6). Soon after the eggs are in the sea-water
the membrane weakens at a number of points at once and
streams of the clear cortical substance can be seen to radiate
out in all directions into the surrounding protoplasm (Fig. 6);
these then form little islands of clear substance surrounding the
germinal vesicle, and around two of these islands radiations
begin to appear, and a spindle is formed between the two asters
thus arising and the latter then approach one another until they
are quite near together. In a polar view one can then see the
granular substance of the germinal vesicle fold around the spin-
dle (which contains the chromosomes as sections show) and
finally form a complete envelope around it and the inner aster.
Figs. 6 to 10, drawn from the living egg, illustrate this process;
the entire mass is moving to the animal pole during the process.
At first the residual substance of the germinal vesicle lies at one
side of the spindle (Fig. 7, 11.04 A. M.) and it is surrounded by a
clear zone proceeding from the original cortical layer of the ger-
minal vesicle; the residual substance of the germinal vesicle is
horseshoe shaped, and the spindle occupies the opening. Fig.
8 is a sketch of the same egg three minutes later (11.07); the limbs
of the horseshoe are bending toward one another so as to enclose
the spindle. Eight minutes later (Fig. 9, 11.15) the limbs of the
horseshoe have almost met around the spindle. At 11.20 (Fig. ro)
the limbs have fused completely and a slight indentation and
corresponding projection of the endoplasm mark the place of
union. [he polar view of the forming spindle is always the same.
If one has a side view instead of a polar view (Figs. 11 to 16),
some aspects of the process can be seen better; thus the fact that the
inner end of the spindle is completely surrounded by the granular
substance comes more clearly to view. Figs. 11-15 show
the spindle entering the residual substance. “They are drawings
of a single living egg taken at about two-minute intervals (com-
pare the section shown in Fig. 3).
‘Thus the germinal vesicle is transported practically intact to the
animal pole; during the process a considerable quantity of fluid
Elementary Phenomena of Embryonic Development 175
Fic. 15 ; Fic. 16
Figs. 11 to 16. For detailed description, see text. Behavior of substances of the germinal vesicle,
side view. Figs. 11 to 15. Five views of the same living egg drawn with the camera, showing
the origin of the maturation spindle and behavior of the cortical layer and residual substance of the
germinal vesicle. Fig. 11. 9.46 4. M. Fig. 12. 9.48 A.M. Fig. 13. 9.51 A. M. Fig. 14. 9-53 A: Me
Fig. 15. 9.54a.M. Fig. 16. A later stage drawn from another living egg. It will be observed that the
spindle arises between two separate primary asters and gradually sinks into the residual substance of
the germinal vesicle, which finally forms a mantle completely surrounding it with the exception of the
external end. The clear cortical layer of the germinal vesicle retains its original relation. Compare
also Figs. 6 to 10. a, Aster; E, ectoplasm; e, endoplasm; g.v, germinal vesicle; m.s., maturation
spindle; r.s., residual substance of the germinal vesicle.
176 Frank R. Lillie
has diffused throughout the cytoplasm, but this is the only loss.
The end-condition is similar in some respects to the “secondary
germinal vesicle”’ of Cyclops according to Hacker’s description (’02).
The sections enable one to follow many details that are obscure
in the living egg and to trace chemical transformations, obvious
as changes in staining reaction in the residual substance of the
germinal vesicle.
In the first place it is shown by the sections that the polarization
of the spherules begins immediately after the rupture of the ger-
minal vesicle. In the second place the chromosomes begin to
separate from the surface of the nucleolus as soon as the wall of
the germinal vesicle is ruptured, and the nucleolus (in conse-
quence!) appears shrunken and vacuolated (Fig. 4). In the third
place the staining reaction of the microsomes of the germinal
vesicle begins to change from acid to basic, first at the periphery
and later toward the center. [hus with a thionin and orange
stain one finds eggs in which the peripheral microsomes of the
germinal vesicle stain blue, and the central ones still stain orange;
with hematoxylin and orange the peripheral microsomes at this
stage take the haematoxylin and the central ones the orange. ‘This
cannot be satisfactorily shown in a figure without the use of color.
This change in staining reaction rapidly embraces all the micro-
somes of the germinal vesicle, and at the same time the whole
mass shrinks considerably owing to the diffusion of the original
droplets of the germinal vesicle.
During this period of diffusion of the fluid substance of the
germinal vesicle and the ensuing polarization of the ectoplasm
and endoplasm, the protoplasm as a whole possesses a much
higher degree of fluidity than before. The evidence for this,
apart from the observed diffusion of the fluid of the germinal
vesicle, is found in the change of contour of the sectioned eggs;
before the rupture of the germinal vesicle, and again after the
polarization is complete, the contours are perfectly regular in
practically every egg. But during the period of polarization the
contours are usually quite irregular, although the method of prepa-
ration is exactly the same. Moreover, the demarcation of the
various endoplasmic substances is no longer clear (Figs. 2 and 4).
'
Elementary Phenomena of Embryonic Development 177
The strongest evidence for greater fluidity at this time is found
in the fact that the ectoplasmic spherules are much more numerous
and smaller than they were previously or than they are subse-
quently (compare Fig. 1 with 2 and 4). Evidently there is a
reversible process of coagulation concerned, the spherules break-
ing into smaller particles as the fluidity increases, and setting or
coagulating again by a process of fusion.
Mead (’98) has given a very careful account of the origin of the
asters of the maturation spindle in Chztopterus. According to
his account a large number of asters arise around the ruptured
germinal vesicle; two of these, which he calls primary asters,
become large, and form the asters and create the spindle of the
maturation divisions. My observations confirm those of Mead in
almost every particular; though I have not found numerous asters
inthe sections. Indeed in my preparations Mead’s secondary asters
appear simply as fluid droplets from the germinal vesicle with very
slight indication of radiations. But as, according to my observa-
tions on the living egg, the primary asters likewise arise around
fluid droplets from the germinal vesicle, it seems quite possible that
Mead’s interpretation, that there is no essential difference between
primary and secondary asters, is correct. The primary asters
certainly arise separately; the observations on the living egg and on
sections are perfectly conclusive in this respect. They may at first
be near together or very far apart, but they soon exert an influence
on one another and a spindle arises between them (Figs. rand 12).
Figs. 1, 2, 4 and 5 illustrate sufhciently well for our present
purpose the history and composition of the chromosomes. ‘They
show at the same time the microscopic structure of the residual
substance of the germinal vesicle as it appears in sections, and
confirm the observations of its migration to the animal pole along
with the spindle. Thus Fig. 3 shows the spindle entering the
residual substance; Fig. 4 a slightly later stage, and Fig. 5 the
positions, structure and relations of the spindle and residual sub-
stance after they have attained the pole. “The microsomes are
distinguishable from ordinary cytomicrosomes only by their
slightly larger size. Before the germinal vesicle breaks down the
residual substance is represented by the coarse reticulum of the
178 Frank R. Lillie
germinal vesicle studded with innumerable microsomes that take
the acid stain. Whenthe germinal vesicle breaks down the retic-
ulum shrinks together with the partial escape of the contained
fluid and the staining reaction of the microsomes changes to basic
like the cytoplasmic microsomes; all the fluid, however, is not
squeezed out and it retains fluid droplets of considerable size that
give it a permanent reticular structure. The meshes are thick
and the microsomes are larger than the cytomicrosomes. It can
now be traced by virtue of these structural peculiarities.
The nucleolus is carried along, with the chromosomes that are
attached to it, by the residual Sb eee, and it 1s therefore found
at one side of the equatorial plate of the first maturation spindle
(Fig. 4), where it has been figured by Mead. It gradually dis-
appears and is lost from view.
The topography of the polarized ovocyte is shown in Fig. 5. It
will be seen by comparison with Fig. 1 what a complete rearrange-
ment of the substances of the unpolarized ovocyte has taken place;
however, no new substances have been formed, though the sub-
stances of the germinal vesicle have undergone considerable changes.
2. The Later Processes of Maturation and Fertilization.—The
formation of the polar globules and the presence of the sperm
nucleus in the egg do not involve any important changes in topog-
raphy of the substances, though the retreat of the egg nucleus to
the center of the egg after the formation of the second polar body
involves a more cone localization of the residual substance of
the germinal vesicle. Thus the topography of the odsperm is
essentially the same as that of the polarized ovocyte.
The problems of the nature of the maturation divisions, behavior
of the sperm and egg nuclei and origin of the cleavage centrosomes
do not fall within the scope of this paper. So far as my observa-
tions go they confirm in most respects the account of A. D. Mead
(98). I expect to take up the behavior of the chromatin in a
subsequent paper. It should be noted here, however, as bearing
on the later portions of the present paper that the number of
chromosomes in the maturation spindle is constantly nine,
arranged usually in the form of a circle of eight with one central
chromosome; the same number was found by Mead.
Elementary Phenomena of Embryonic Development 179
c. Action of Centrifugal Force in the Unsegmented Egg"
If the eggs of Chztopterus he taken and allowed to stand in
sea-water without fertilization the germinal vesicle breaks down,
the substances become polarized as already described, and the
first maturation spindle 1 is formed and moves to the animal pole,
where it remains in metaphase indefinitely (unless the egg be fer-
tilized or stimulated in some definite fashion). This coud Hone 1S
reached in from fifteen to thirty minutes, depending on tempera-
ture and some unknown factors.
If, at any later time before their death, the eggs be submitted to
a fairly strong centrifugal action (1500 to 2000 revolutions in one
minute) and then examined, it will be found that the endoplasm is
arranged in three distinct layers, viz: a small gray cap, a clear band
and a yellow hemisphere (Photographs A-H, and Figs. 17-22).
The gray cap and clear band together take up about one hemi-
sphere. ‘The ectoplasmic layer is not visibly affected (see photo-
graphs and Fig. 24) and forms, as before, a continuous layer with
an aperture at the animal pole. The maturation spindle is
usually found fixed at the periphery immediately after centri-
fuging, so that it is obvious that it has not been moved by the
centrifugal force.
iliese ageregations of substances bear no definite relation to
the polarity of the egg, for the maturation spindle may be found
either in the gray cap, which is then ring-shaped (Photograph H
and Fig. 19), or in the center of the yellow hemisphere, or in any
intermediate position. It is, however, most frequently found in
the hemisphere containing the gray cap.
If now the eggs be ‘antiga the polar bodies are formed at the
original animal pole, and thus may appear in the gray cap, or at the
center of the yellow hemisphere, or in any intermediate position;
though in this case, again, they are found more frequently in the
hemisphere containing the gray cap than in the yellow hemisphere
(see various conditions illustrated in Photographs A—H and in
Figs. 17-22).
1 The use of the centrifuge for the purpose of studying the composition of the protoplasm was sug-
gested to me by Dr. E. P. Lyon, who had already obtained results, about to be published, on the
ova of sea-urchins by this method.
180 Frank R. Lillie
Figs. 17 to 23. Showing the effects of centrifuging upon the living unsegmented egg. Compare
Photographs A-I. E, Ectoplasm; e, yellow endoplasm; c.b., clear band; g.c., gray cap; g.v.
germinal vesicle; , nucleolus; p./. polar lobe.
Fig. 17. Eggs in sea-water at 8.55 a.m. Centrifuged at 10.45 and fertilized. Drawn with camera
at 12 o’clock noon. The gray cap lies in the vegetative hemisphere.
Fig 18. Eggs in sea-water at 1.55 p. M. Centrifuged 2.30 p. Mm. Fertilized 2.45 ep. mM. Drawn
3-40 P.M. The gray cap extends into the polar lobe. Beginning of the first cleavage.
Fig. 19. Eggs in sea-water 8.50 a.m. Centrifuged 9.40 a.m. Fertilized 9.45 a. mM. Drawn 9.55
A.M. The gray cap surrounds the maturation spindle and is thus ring-shaped.
Fig. 20. Eggs in sea-water 1.55 p. M. Fertilized 2.03 p. mM. Centrifuged 2.50 p. M. Drawn 3.00
p.M. ‘The gray cap has passed entirely into the larger cell CD.
Fig. 21. Eggs in sea-water 8.50 a. m. Centrifuged 9.40 a. M. Fertilized 9.45 a. mM. Drawn
10.20 A. M. The gray cap lies opposite to the animal pole.
Fig. 22. Drawn 10.29 a.m. History otherwise the same as No. 21. Shows intermediate position
of gray cap.
Fig. 23. In sea-water 9.08 a.m. Centrifuged at same time before rupture of the germinal vesicle.
No stratification. Note the position of the nucleolus within the germinal vesicle.
Elementary Phenomena of Embryonic Development ISI
Fic. 18
Fie. 21 Fig. 23 Fic. 22
}
182 Frank R. Lillie
The polar lobe appears at the usual time preceding the first
cleavage, and always lies opposite to the polar globules, whatever
be the position of the yellow substance (Photograph G and Figs.
17, 18 and 20). ‘Thus, if the gray cap lies at the animal pole, the
yellow substance extends into the base of the polar lobe, as nar-
mally; or, inthe opposite case, the polar bodies lying atthe center
of the yellow hemisphere (Fig. 21), the gray cap may extend into
the base of the polar lobe (Fig. 18). The structure of the periph-
eral part of the polar lobe is the same in either case, and does not
differ from the normal.
It is therefore clear that the yellow and the gray substances are
extremes as regards their specific gravity; they move freely through
the ground substance of the Saauaylaicm. and are thus distri-
buted with reference to the direction of the centrifugal force, and so
may occupy any position with reference to the polarity of the egg.
However, polarity is the force that governs the position of these
substances in the normal development, causing aggregation of the
yellow substance in the vegetative hemisphere and of the gray in
the animal hemisphere. It is thus a stronger force than gravita-
tion, for these substances aggregate in the same polar sense
whatever be the position of the egg, but not so strong as the centrif-
ugal force employed. It is now clear why the gray cap is thrown
to the animal hemisphere more frequently than to the vegetative,
for the force of polarity has already caused a partial aggregation
of the substances before the centrifugal force is applied, and the
eggs, therefore, tend to rotate with the heavier pole in a distal
direction; but in a large percentage of the eggs, various causes
combine to overcome this, the principal one being no doubt the
crowding of the eggs in the bottom of the tube.
The process of ine, vage takes place with reference to the polarity
of the egg (Photograph I and Figs. 18 and 20) and its funda-
mental form 1s not ajfected by the chance disposition of the endo-
plasmic substances though minor abnormalities result from certain
jorms of distribution. anes the first two cleavages are always
meridional whatever the position of the yellow cbse and the
gray cap, and the third cleavage is equatorial and spiral in the
usual sense.
Elementary Phenomena of Embryonic Development 183
The appearance of stratification given by centrifugal action is
extremely striking, and it is so similiar to the banded condition
described for echinoderms by Boveri (o1a and ’o1b) and for Den-
talium by Wilson (05), that one cannot fail to be impressed by
the resemblance. Lyon has found that similar stratification may
be produced in the eggs of echinoderms, annelids and tunicates by
centrifugal action. So there can be but little doubt that it isafunda-
mental feature of egg-organization in the ova of bilateral animals.
It is a condition normally governed by the polarity of the egg,
and does not become obvious under normal conditions in the egg
of Chztopterus simply because the separation is not complete and
precise, and thus the strata shade into one another, the animal
pole being relatively light and grayish in hue and the vegetative
relatively dense and yellow.
By the aid of the centrifuge one can also demonstrate that the
substance of the gray cap is the residual substance of the germinal
vesicle. Ifthe eggs are centrifuged as usual immediately after they
come into sea-water, the germinal vesicle being still intact, one
does not get a gray cap or any banded appearance, but instead the
germinal vesicle is thrown to the surface with such force that it may
produce a protuberance of the cortical layer (Fig. 23), and the re-
mainder of the endoplasm appears yellow and not strongly polar-
ized by the centrifugal force; the cortex remains as before. The -
nucleolus always occupies the inner end of the germinal vesicle,
showing that it is undoubtedly heavier than the remainder of the con-
tents of the vesicle. Within the germinal vesicle there is no strati-
fication, the granular substance preserving its earlier distributed
arrangement. ‘These eggs then mature on standing, and the ma-
turation spindle moves to the periphery of the egg; in doing so.it may
take the longest course through the egg and become fixed at the
pole opposite to that occupied by the germinal vesicle, or it may
move to any other position. Thus it becomes practically certain
that the polar force governs the migration and leads it to the
animal pole. ‘The only other assumption would be that the spindle
determined polarity at whatever point on the surface it happened
to be fixed, and then it would be perfectly incomprehensible why
it sometimes migrates through the entire diameter of the egg.
184 Frank R. Lillie
If the eggs are allowed to stand eight to fifteen minutes in
sea-water after being taken from the female, so that in some the
germinal vesicle is still intact, and in others broken down, the
latter always show the stratification more or less pronounced after
centrifuging, and the former never show it, but instead the ger-
minal vesicle is always peripheral and contains a granular mass
similar to the substance of the gray cap.
Thus neither the gray cap nor the clear band are formed by
centrifugal force unless the germinal vesicle is broken down, and
the conclusion is inevitable that the entire gray cap is derived from
the germinal vesicle. The yellow substance does not aggregate
so closely before as after the germinal vesicle has broken down.
Hence it is probable that the appearance of the clear band is due
very largely to concentration of the yellow spherules at one pole.
It would seem that the endoplasm has become less viscid as a
result of the diffusion of substance from the germinal vesicle, so
as to permit closer aggregation of the yellow granules. The
structure of the gray cap completes the identification for it is the
same as the residual substance of the germinal vesicle (Fig.
24; compare figures of normal maturation).
The maturation spindle is usually found fixed at the periphery
immediately after centrifuging, whatever the direction of the
centrifugal force may have been; when, however, it is torn loose,
as sometimes happens, it is found in the clear band. ‘Thus it is
intermediate between the gray cap and yellow endoplasm in
specific gravity. It is clear, therefore, that when it is situated in
the region of the gray cap or yellow endoplasm, the centrifugal
force must cause it to exert traction on the cortex of the ovum.
That this is actually the case is shown by the form of the section
of many eggs killed immediately after centrifuging, in which there
is a very pronounced depression at the animal pole where the
maturation spindle is attached (Fig. 24).
These sections are very interesting in two other respects: (a)
When the spindle is torn loose from the periphery, it moves as a
whole and carries the chromosomes with it—chromosomes and
spindle are never found separated by the centrifugal force. This
bears witness to the extreme viscosity of the spindle area, for it
Elementary Phenomena oy Embryonic Development 185
can hardly be possible that chromosomes and spindle are of exactly
the same specific gravity: moreover the spindle preserves its form
intact, and is not bent or broken in any manner so far as | have
observed, though it must be subjected to considerable strain.
(b) If the eggs be fixed (killed) immediately after centrifuging, and
sectioned, stained and mounted in the usual manner, it is found
that the asters are almost entirely or entirely wanting, though
sections of the same eggs prepared without centrifuging show
well-developed asters at both poles of the spindle. The cyto-
plasm surrounding the spindle is dense and filled with microsomes
more numerous and smaller than usual.
A
O00 GS
AA Zp SO
Fig. 24 Section of an egg killed in picro-sulphuric acid immediately after centrifuging. The clear
band c.b. is seen to be densely microsomal. The ectoplasm retains its original arrangement. The
yellow endoplasm and the residual substance of the germinal vesicle are at opposite sides of the
egg. The maturation spindle retains its original position. A, Animal pole; c.b., clear band; E,
ectoplasm; e, endoplasm; m.s., maturation spindle; r.s., residual substance of the germinal vesicle;
V, vegetative pole.
The residual substance of the germinal vesicle has been stripped
off the spindle and has given place to more finely microsomal pro-
toplasm. ‘The sphere and centrosome, however, remain with the
spindle (Fig. 24). If the eggs are permitted to stand after centri-
186 Frank R. Lillie
fuging the radiations appear; but their character is different from
those of the normal egg, owing to the absence of the residual sub-
stance of the germinal vesicle around the spindle.
The most obvious interpretation of the clear band is that it
represents the pure ground substance cleared of spherules by
centrifugal force. This was, indeed, the interpretation that I gave
it when working on the living material. ‘The results obtained by
staining, in ornare lin, entire eggs killed in picro- -sulphuric or
picro-acetic acid, shows that this interpretation is only partially
correct; for in these eggs the original clear band appears intense
violet in color, and was not, for this reason, at first recognized;
in such preparations the gray cap is stained relativ ely lightly and
is seen to be highly vacuolated. ‘The yellow mass is unstained
and dense. Sections of such eggs (Fig. 24) show that the clear
band contains most of the snilplasnic microsomes (which, as
we have seen before, have strong affinity for basic dyes), embed-
ded in ground substance. Thus the centrifugal force has separated
the microsomes and spherules of the endoplasm, the former then
appearing as the clear band and the latter as the yellow mass. This
appears to me to furnish important evidence for the specific
nature of the microsomes as argued throughout this paper.
The result is very clear-cut and striking. The microsomes are
thus seen to differ from the spherules in specific gravity as well
as in the other respects already noted. Moreover, when the
spindle is torn loose from the periphery, it is invariably found with
the contained chromosomes in the clear band, thus showing that
the microsomes have the same specific gravity as the chromosomes.
This observation should be correlated with others described in this
paper, showing origin of microsomes from chromosomes.
The definiteness and fixity of polarity is one of the most striking
results brought out by centrifuging the eggs. The direction of
polarity is not altered by any arrangement given to the endoplas-
mic substances, and it asserts its power over the maturation
spindle under all the various circumstances of the experiments;
similarly it determines the location of the polar lobe under all
conditions, and, under normal conditions, regulates the distribu-
tion of the ectoplasm and the endoplasm.
Elementary Phenomena of Embryonic Development 187
The following statements may be made-concerning the polarity
of the egg of Chetopterus:
1. Polarity may be traced from the stage of the youngest
ovocyte continuously.
2. Inthe ovocyte the free end of the egg is the animal pole and
the attached end the vegetative pole.
3. It is not determined whether the odgonia are polarized or
whether the polarity arises in the ovocyte owing to the relations
of free and attached poles, or from some other external cause.
My own view is that the polarity is inherent and is a property of
the nucleus.
4. In the full-grown ovocytes and o6tid the polarity is un-
altered by any change of distribution of the granules of the endo-
plasm; it must, therefore, be a property of the residual protoplasm.
5. Polarity, however, determines the normal distribution of
the granules in the egg, and is thus a force stronger than
gravitation, because the distribution of the spherules is normally
fixed with reference to polarity, but may bear any relation to the
direction of gravitation.
6. Acentrifugal force may overcome the polarity and distribute
the spherules correspondingly.
The effects of polarity in the subsequent development will be
onsidered in Part IV.
2. Literature and Discussion
a. The Axis of the Ovarian Ovocyte and its Relation to Polarity
It appears to be the rule among annelids and molluscs that the
attached pole of the egg in the ovary becomes the vegetative pole
of the odsperm. It is true that the number of observations bear-
ing on this point is relatively small (Stauffacher ’93, Lillie ’g5,
Conklin ’o2 and ’03).
On the other hand Boveri's observations (ota and ’otb) make
it probable that in Strongylocentrotus the conditions are reversed.
There is no theoretical objection to such a contrast. On the con-
trary, as Conklin has argued in his very suggestive paper on
188 Frank R. Lillie
“Inverse Symmetry”? (’03), reversal of polarity would furnish a
very simple and complete explanation of the condition of inverse
symmetry. The considerations that Conklin brings forward in
this paper, together with the actual observations in He literature,
combine to anila: reasonably certain the hypothesis, that in all
animals the axis of the epithelial ovarian ova becomes the primary
axis of the segmenting ovum. It seems reasonably certain also
that the poles of this axis may be interchangeable, owing probably
to inverse polarization of the formative stuffs of the ovocyte.
b. The Ectoplasmic Layer
I have been surprised to find, since my observations on Chzetop-
terus were completed, what a large number of definite observa-
tions on the existence of an ectoplasmic layer in the eggs of various
animals were to be found in the literature. These have not been
recently collated and their importance 1s, therefore, not fully
realized by many embryologists. When brought together these
observations bring into one category the “polar rings,” “yolk-
lobe” and certain aha phenomena described by various AMOS:
I therefore give rather full citations.
In Clevane (Whitman ’78) the “polar rings”’ evidently corre-
spond to the two divisions of the ectoplasm of Chztopterus
(see Part IV, 1, a, of the present paper). These rings appear after
the formation of both polar globules and are situated near the
upper and lower poles, respectively. Each consists of a “trans-
parent fluid substance,” radiating lines of which at first extend on
the surface from each toward the equator of the egg; in my opinion
these indicate the origin of the rings from an ectoplasmic layer,
as in Rhynchelmis, though Whitman expresses no opinion con-
cerning the origin of this substance. The upper ring contracts
around the animal pole, but retains its central opening, which is
usually eccentric, thus agreeing exactly with Cheetopterus (see
Photograph ED ailre substance of the lower ring aggregates at
the lower pole in the form of a disc. Subseqmantr the substances
of both rings plunge deep into the egg, and the upper ring sub-
stance 1s PoAneeniried to the left posterior macromere, D, as in
39
Elementary Phenomena of Embryonic Development 189
Rhynchelmis; what becomes of the lower ring substance is uncer-
tain.
The conditions in the egg of Rhynchelmis (Vejdovsky *88—’92)
are most like those in Chetopterus. The condition before the
rupture of the germinal vesicle 1s thus described by Vejdovsky
(p. 32): “Unter der dusserst feinen Dottermembran, die man
kunstlich von der Eisubstanz tberhaupt nicht abheben kann,
erstreckt sich eine 0.003 mm. hohe Schicht des peripherischen
Plasma, welches sich in Pikrocarmin intensiver farbt und deshalb
ohne grosse Schwierigkeit wahrzunehmen ist (Holzschnitt Fig.
1, A). Sie besteht aus einer hyalinen Grundsubstanz, in welches
ausserst feine, aber doch deutliche, in Pikrocarmin intensiv sich
tarbende Plasmakornchen, eingebettet sind. Bei starken Ver-
grosserungen ist es nicht schwierig sicher zu stellen, dass diese
K6rnchen regelmassig schichtenweise und concentrisch in der
Grundsubstanz angeordnet sind, in einigen Fallen scheint es aber,
dass sie hier unregelmassig zerstreut sind. Diese Kornchen sind
denjenigen gleichzustellen, die man viel deutlicher an den Fasern
des Cytoplasmanetzes wahrnimmt. Im Leben ist diese Proto-
plasmaschicht braun.”’ According to this account it would ap-
pear that the ectoplasmic layer covers the entire ovum before
the rupture of the germinal vesicle.
After the formation of the polar globules the ectoplasmic layer
ruptures near the equator of the egg and flows to the two poles.
Cases were observed where the aco ates took place entirely
at the animal pole. The defect at the animal pole produced by
the maturation spindles appears to be covered up in some cases,
though it is always indicated for some time by a thinner spot.
Some eggs have no ectoplasmic layer, and these do not develop
beyond the stage of the first cleavage spindle. ‘The two ectoplas-
mic discs are transmitted entirely to the larger cell of the two-cell
stage. In the four-celled stage they are confined to the large
posterior macromere, and during this stage they sink into the
interior and surround the sphere containing the nucleus.
The behavior of the upper accumulation of ectoplasm is thus
strikingly different from the condition in Cheetopterus (see IV,
1,5). But the transmission of the lower accumulation to the large
190 Frank R. Lillie
posterior macromere agrees with Chetopterus. Enough has been
said to show what a remarkable similarity exists; the differences
will no doubt be explained at some future time.
In both Clepsine and Rhynchelmis the area of the egg between
the upper and lower accumulations of the ectoplasm is many times
larger than in Chetopterus.. This is correlated with the relatively
very small size of the entoderm cells in Chetopterus. Differences
in the functions of the ectoderm may also be a factor; thus all the
ectodermal cells in Chzetopterus are ciliated in contrast to Clepsine
and Rhynchelmis where cilia are lacking. In Chetopterus the
presence of ectoplasm seems to be essential fn the formation of cilia.
Wilson (’04, a) describes an ectoplasmic layer in the egg of
Dentalium; he finds thatitiscontinuous with the ‘‘lower protoplas-
mic area’ "(substance of the polar lobe)and with the upper’ ‘ proto-
plasmic disc.” “As the egg, still unfertilized, lies in sea-water,
the ectoplasm in the region of the upper disc slowly increases in
amount, and in some cases this region shows a faintly radiating
appearance around its periphery as if clear hyaloplasm were
flowing into it from the surrounding region.” It is evident that
Wilson’s “upper disc” corresponds to my “ectoplasmic defect”
at the animal pole; the author calls “attention to the fact that the
original disc is composed of very dense homogeneous protoplasm
that differs markedly in character from the alveolar protoplasm
of the ectoplasmic thickening that afterward extends over the
whole upper surface of the egg.” The resemblances between the
ectoplasmic layers in Dentalium and Chetopterus will come out
more clearly as the description proceeds. At present we may note
as points in common (1) the “alveolar character”? (Wilson) or
presence of specific spherules. (2) Connection with the polar
lobe and upper hemisphere. (3) Severance between the ecto-
plasm of the polar lobe and upper hemisphere later. (4) Exist-
‘ence of an ectoplasmic defect (upper protoplasmic disc of Wilson)
at the animal pole. Vejdovsky’s observations on Rhynchelmis
agree in all these respects, and Whitman’s on Clepsine 1 in most.
Mead (’98) finds in the egg of Cheetopterus ° ‘immediately inside
the outer pellicle a narrow Ore containing a single row of yolk-
granules regularly arranged.” This applies to the ovocyte of the
as
Elementary Phenomena of Embryonic Development 1g!
second order, and, at that stage, I also usually find the spherules
arranged in a single layer (Fig. 15); it will be observed that Mead
calls them “yolk-granules,” not having recognized their true
nature. Wheeler ('97) figures an ectoplasmic layer in the egg of
Myzostoma; it is characterized by the presence of granules larger
than microsomes, and like them staining very deeply in iron-alum
hematoxylin. “Vheir number and distribution are quite variable,
and they may even be entirely absent in some batches of eggs.”’
Wheeler believes that they are chromatin granules derived from
the disintegrating nuclei of nurse-cells. It 1s interesting to notice
in his figures that the ectoplasmic layer is absent at the outer end
of the maturation spindles, as in Chetopterus, Dentalium, Clep-
sine and Rhynchelmis. Conklin finds (’05) in the eggs of the
ascidian Cynthia a “peripheral layer of deeply-staining proto-
plasm in which the test cells were formerly embedded and which
contains no yolk, but numerous refractive spherules much smaller
than those of the yolk.” ‘The layer contains yellow pigment
“which seems to be associated with these small refractive spher-
ules.” As Conklin points out, Sobotta has observed and de-
scribed a similar layer in the egg of Amphioxus. Conklin has
traced its fate in Cynthia with the greatest exactness, and has
found that it forms a yellow crescent on the posterior side of the
egg, and that its substance enters into the composition of the
mesoblast cells.
The existence of so similar a layer in such widely separated
animals and its great morphogenic importance constitute strong
reasons for believing that it 1s likely to be found in all the principal
phyla. As will be shown in another place the assumption of its
existence in certain forms, in which it has not been described,
helps to explain other phenomena actually observed; for instance,
the presence of the polar lobe in many mollusca, and the absence
of flagella in abnormally differentiated eggs of Amphitrite (Scott)
(seep. 238),
c. Residual Substance of the Germinal Vesicle
There are many observations in the literature bearing on: the
existence of a large quantity of residual matter derived from the
192 Frank R. Lillte
germinal vesicle after the formation of the first maturation spindle,
though in most cases the observations are fragmentary and no
definite interpretation is given. Figures by Wheeler (’99), Mead
(98), Coe (99), Wilson ('o4a), Vejdovsky (°88—’92) and others
show a large amount of substance of the germinal vesicle remain-
ing after the formation of the first maturation spindle; but none of
these authors has traced its behavior in later stages. According
to Hacker (02) the entire substance of the germinal vesicle in
Cyclops passes to the animal pole where it forms his “secondary
germinal vesicle.” ‘This is like the condition in Chetopterus;
but Hacker fixed his attention on the chromosomes and did not
follow the residual substance farther. Without going into the details
of the various accounts it 1s clearly shown in the literature that, as
a rule, only a small proportion of the substance of the germinal
vesicle enters into the formation of the first maturation spindle.
Conklin’s observations (’05) are the most complete ones on the
subject of the residual substance of the germinal vesicle. He
finds, in Cynthia, that “as soon as the nuclear membrane has
dissolved the chromosomes, nucleolus and a granular mass from
which the spindle fibers are formed gather together into the center
of this area of nuclear protoplasm.” As the spindle forms the
entire area moves to the surface of the egg, and the clear proto-
plasm spreads out “into a cap or peripheral layer (Ciona) or may
form merely a somewhat flattened disc (Cynthia).’”’ After
fertilization the clear substance of the germinal vesicle flows to
the vegetative pole, “leaving the first maturation spindle sur-
rounded by only a small amount of protoplasm.” Here it receives
the sperm-nucleus and aster, and subsequently moves to the
posterior side of the egg and up to the equator; “finally, after the
meeting of the germ-nuclei near the posterior pole of the ege, these
nuclei and the clear protoplasm surrounding them move inward
to the center of the egg.” ‘At the close of the first cleavage the
nuclei and clear protoplasm move into the upper hemisphere, and
thereafter, throughout development, this hemisphere contains
most of the clear protoplasm and gives rise to the ectoderm.’ As
will be seen from the subsequent description, the behavior of the
residual substance in Chatopterus shows many points of similarity.
Elementary Phenomena of Embryonic Development 193
It seems probable, therefore, that the residual substance of the
germinal vesicle represents a specific formative stuff of essentially
the same character 1n different phyla.
d. Polarization
The phenomenon that | have termed polarization of the forma-
tive stuffs likewise appears to be a universal one. Here again
Conklin’s observations on Cynthia are the most complete; he has
described definite flowing movements of the ectoplasm, residual
substance of the germinal vesicle and of the endoplasm, beginning
with the rupture of the germinal vesicle, by virtue of which the
topography of the ovocyte is radically changed. ‘There is first a
movement of the ectoplasm and the residual substance of the
germinal vesicle to the lower pole of the egg, which corresponds
to the polarization described in this section. This is followed by
a bilateral arrangement which corresponds to what I describe in
Part IV, 1, a, as bilateral polarization. The rearrangement
of substances in the eggs of Ctenophores described by Fischel
((03), and in Echinids described by Boveri (ora and ’orb)
appears to correspond to the bilateral polarization in Chetopterus
(Part IV, 1, a), for it does not immediately follow the rupture of
the germinal vesicle but is a result of fertilization. Some of the
observations of Whitman, Vejdovsky and Wilson, already con-
sidered, belong here. Many authors have described or figured a
polar segregation of the yolk in the lower hemisphere following
the rupture of the germinal vesicle which is undoubtedly only a
part of the polarization processes taking place at this time.
Wilson, Yatsu and Zeleny have found that there is a progressive
limitation of potencies of parts of the egg of nemertines beginning
with the rupture of the germinal vesicle. Assuming that the vari-
ous formative stuffs have limited potencies this would be the
natural consequence of such a process of polarization as I have
described for Chatopterus, because the new topography is more
precise than the original one (compare also Conklin ’05).
194 Frank R. Lillie
IV. CLEAVAGE AND DIFFERENTIATION |
The problem now becomes to determine the distribution of the
various substances in the early development, to follow the forma-
tion of others and to investigate the relations of all to the various
forms of differentiation. “[o determine the normal distribution
of these substances I have studied the cell-lineage up to the time
of the formation of the mesoblast cell, and with the aid of sections
have determined the distribution of the various substances to the
different cells. “The use of intra-vitam staining has likewise con-
tributed to the study of the distribution of substances and to
the identification of new ones. For the purpose of ascer-
taining the relations of the substances to the various forms of
differentiation, | have made a renewed study of the differentiation
of unsegmented eggs, which I described in an earlier paper
(Lillie ’02). By the action of centrifugal force an abnormal
distribution of the endoplasmic substances has been produced,
and the effect of this on the form of the cleavage has also been
studied to a certain extent.
A statement of the more general conclusions may make the point
of view of the entire description clear. In the first place the
observations on cell-lineage show that the process of cleavage
does not produce an essentially different distribution of substances,
and that the parts of the trochophore are clearly mapped out in the
segmented ovum of sixty-four cells. The topography of the
unsegmented egg 1s, therefore, essentially similar to that of the
trochophore. In the second place it will be shown that some of
the substances (spherules) have in all probability a specific mor-
phogenic value, while others appear to have simply a nutritive
value, though a clear separation of the substances (spherules) into
these two categories has not been possible. The morphogenic
significance of ‘the substances is shown especially well by the
observations on the differentiation of unsegmented eggs, in which
clear regional homologies with the trochophore are found includ-
ing ectodermal, entodermal and mesodermal substances. In the
third place it will be shown that the activation of these morpho-
genic substances is accompanied by and probably dependent upon
interaction with nuclear derivatives.
Elementary Phenomena of Embryonic Development 195
t. Formative Stuffs in the Early Normal Development
Mead has given.a good account of the cell-lineage up to sixty-
four cells, and I have been able to confirm his results in prac-
tically all particulars. His description, however, was written
from a different point of view, and, though complete and accurate
as to the form of the cleavage, does not deal at all with the dis-
tribution of substances, which indeed he did not distinguish. It
is necessary, therefore, to describe some parts in considerable
detail, while for others I can rely on Mead’s excellent account.
a. The First Cleavage
The period of the first cleavage is meant to include the entire
time from the, dissolution of the membranes of the germ-nuclei
to the two-celled stage. It 1s characterized by the appearance of
the polar lobe involving division of the ectoplasm into two parts,
by the appearance of bilateral symmetry, and by formation of new
substances.
1. Polar Lobe and Division of Ectoplasm.—At about the meta-
phase of the first cleavage the ovum begins to elongate in a polar
direction and a slight constriction appears in an equatorial plane
considerably below the equator (Photograph Gand Fig. 26, p. 205);
thus the ovum becomes decidedly pear-shaped, the stem of the
pear being at the vegetative pole. In the region of the constriction
the ectoplasm is divided so that there is a gap between the part
situated below and that above it (Figs. 25 and 26); this gap is
permanent, and a vegetative polar group of the ectoplasmic
spherules is thus finally separated from the remainder. ‘The con-
striction separating this group deepens as the first cleavage
furrow begins to appear at the animal pole (Fig. 25); this furrow
lies to one side of the polar globules and on the same side of the
ege it passes into the constriction, so that the division becomes
decidedly unequal and the polar group of ectoplasmic spherules
(Fig. 25, p./.), passes entirely into the larger cell (CD). ‘The polar
furrow deepens during the later phases of the first cleavage and
thus produces a pedunculated lobe, the polar lobe, which contains
all the polar ectoplasmic spherules (see Photograph I) and also
196 Frank R. Lillie
some endoplasmic spherules in its base. At the height of forma-
ton of the polar lobe the ectoplasmic waves become extremely
pronounced all over it. During the completion of the first cleav-
age the constriction around the polar lobe gradually disappears
and the substance of the polar lobe forms that part of the larger
cell next the cleavage furrow, where the group of ectoplasmic
spherules keeps a superficial position.
Fig. 25. Longitudinal section, first cleavage, late anaphase. Posterior end to the left, anterior
to the right. The ectoplasm of the polar lobe has been separated from the remainder. c, chroma-
tin masses cut off from the chromosomes; c.v., chromosomal vesicles of the daughter nuclei. E.d.,
Ectoplasmic defect; p./., polar lobe; r.s., residual substance of the germinal vesicle.
- Mead has given a good description of the form changes of the
first cleavage, and has described the polar lobe, which he calls the
yolk-lobe, following the older terminology. “The term polar lobe,
introduced by Wilson, seems preferable to the name yolk-lobe,
and | therefore employ it.
The polar lobe is fundamentally an ectoplasmic formation.
The proof of this is experimental. We have seen that the endo-
plasmic substances may be given any arrangement with reference
Ele PIILE, ntary Phe r10Mena of Embr yonic Dex elopme nt 197
to polarity by the centrifuge; but that the ectoplasm is unaffected
and that the polar ¢ slcniles appear at the original animal pole even
though this be the center of aggregation of the large endoplasmic
Ppherules. Now the polar (epee invariably forms opposite to the
polar bodies, so that in the extreme case, reversal of position of the
endoplasm, the gray cap extends into the base of the polar lobe;
or the protoplasm of the clear band may extend into its base in
case of intermediate position of the endoplasmic substances
(Photographs G and I and Figs. 17, 18 and 19). Thus the posi-
tion of the polar lobe is independent of the position of the endo-
plasmic substances, and is determined by the polarity of the egg
and the distribution, with reference to the polar axis, of the ecto-
plasm. Inasmuch as the animal pole is devoid of ectoplasm the ecto-
plasmic substance of the polar lobe may be termed briefly the
polar ectoplasm.
2. Bilateral Symmetry.—The second striking feature of the
first cleavage is the appearance of bilateral symmetry, which
comes out “swags ble and permanently during the formation
of the first cleavage spindle. Prior to this time I ae been unable
to detect any positive evidence of bilateral symmetry; thus 1
comes to expression in a few minutes. Like polarity itself the
bilateral symmetry is independent of the distribution of the endo-
plasmic substances, and, in my opinion, can only be conceived as
due to a force like that of polarity. The evidence for this follows:
The first cleavage spindle forms approximately in the center of
the egg, but a little above the equatorial plane. Before this time the
egg shows a perfect radial symmetry so far as the distribution of
substances is concerned. Ina horizontal section the germ-nuclei
lie in the center of a mass of non-spherular protoplasm, which 1s
surrounded by a ring of endoplasm of even thickness, and this
again by an ectoplasmic ring of even thickness. As the spindle
forms, however, the endoplasmic ring becomes broader at one end
and narrower at the opposite end, as though the non-spherular
protoplasm containing the spindle had moved toward one side
of the egg; this side is the anterior, and the opposite side the pos-
terior face of the egg, and the axis of the spindle lies in the plane of
symmetry thus indicated, and therefore in the longitudinal axis
198 Frank R. Lillie
of the embryo. ‘Thus in the early anaphase the first cleavage
spindle lies eccentrically in a mass of non-spherular protoplasm,
and the center of the endoplasmic spherules has been shifted
toward the posterior end (Figs. 25 and 26).
At the same time the centrosphere at the posterior end becomes
much larger than that at the anterior end (Fig. 26); this is
very marked at the end of the prophase, and is, indeed, the
first clear indication of the posterior end.
‘Treadwell ('97) has shown that, even when the first cleavage is
equal in annelids, it bears the same relation to the axis of
the embryo. But it should be noted that, when the first
cleavage is unequal, as in Chetopterus, we have demonstrative
evidence that the bilaterality thus established involves more
than the mere determination of an axis; it involves also
certain embryonic proportions of prospective significance. The
cell CD is not only posterior in position, but it is larger and
different to a certain extent 1n its composition, and behaves
radically differently from the cell, 4B; in fact, the first cleavage
predelineates the proportions and properties of the anterior and
posterior ends of the embryo.
The gradual determination of the plane of bilateral symmetry
furnishes a fascinating problem. It is difficult or impossible to
say which of the phenomena observed to be involved are primary
and which secondary; it seems probable that the primary deter-
mining factor has altogether eluded observation, and that the
orientation of the spindle, the enlargement of the posterior
centrosphere, and the shifting of the endoplasm and of the
spindle are all consequences of some more remote cause, VIZ:
a second process of polarization’ analogous to that following
the breaking of the germinal vesicle, but with reference to a new
axis placed at right angles to the polar axis—the sagittal axis of
the embryo.
'T have called the shifting of substances that defines the bilateral symmetry a second process of
polarization because, so far as I can see, there is no immediate explanation of the phenomenon.
Certainly the unequal cleavage (and the determination of bilateral symmetry that goes with it) is
not due to any chance distribution of the substances of the egg; for on no theory of chances can
one explain the uniformly normal relation of composition and of mass between the cells 4B and CD.
Elementary Phenomena of Embryonic Development 199
In centrifuged eggs the first cleavage is invariably normal with
relation to the sole = , and usually unequal whatever the positions
of the endoplasmic substances (see Photograph I, and Figs. 18
and 19). Thus the cell 4B may receive most, or in other cases
practically none, of the endoplasmic spherules. ‘There is certainly
a tendency in the former case toward equality in size of the two
cells, but the tendency toward inequality is usually markedly the
stronger of the two. Thus, just as the polarity of the egg is inde-
pendent of the distribution of the endoplasmic substances, so also
is the bilaterality. And conversely just as polarity determines
the polar distribution of these substances so does the force of
bilateral polarization acting at right angles to polarity normally
determine their bilateral arrangement.
This conclusion is in full agreement with my earlier opinion on
the organization of the egg of Unio (or) where I distinguished
polarity and bilaterality as fundamental primitive features of the
egg organization. In his fine study of the organization and cell-
lineage of the Ascidian egg, Conklin (’04) likewise states his
opinion that the median plane and the posterior pole are deter-
mined by the “structure of the egg”’ and not by external incidents
such as the path of the spermatozo6n. I have chosen the terms
polarity and bilateral polarization as preferable because they
express the opinion, which, I believe Conklin also had in mind,
that they represent conditions antecedent to the distribution of
substances in a polar or bilateral sense, and hence more funda-
mental. :
We know polarity and bilateral polarization only as conditions
or forces that determine definite distributions of certain substances.
Beyond this they can be defined only in a negative manner; thus
they are not due to any visible aggregation of unlike substances;
they are not determined by the path of the spermatozoén, nor by
chance location of germ-nuclei. They might be conceived to be
electrical or magnetic phenomena, but all the evidence of the
influence of electricity and magnetism on development runs
counter to this idea. But however obscure the nature of such
forces may be they are clearly demonstrated to exist (see General
Discussion, * "Principle of Unity”).
200 Frank R. Lillie
The first division is a differential one with reference to the sub-
stance of the polar lobe, and to the amounts of other substances.
Thus CD receives the entire area surrounding the polar globules
that is devoid of ectoplasm (Fig. 29), it also receives all of the
substance of the polar lobe and the larger amount of the endo-
plasmic spherules. On the 4B side the ectoplasm and the endo-
plasmic spherules come up to the first cleavage furrow at the
animal pole; whereas on the C'D side they stop some distance from
it (Fig. 29). These points are of importance with reference to the
subsequent development.
3. Residual Substance of the Germinal Vesicle and Nuclear
Derivatives.—There remain yet two features of the first cleavage to
consider, viz: the fate of the residual substance of the ger-
minal vesicle andthe formation and distribution of new atelier
derivatives.
The residual substance of the germinal vesicle forms a mantle
around the first cleavage spindle (Fig. 25, r.s.) and is thus divided
approximately equally between 4B and CD. After the first
cleavage is complete it is difficult to distinguish it by the usual
means. But by means of the centrifuge it can readily be shown
that it is still a distinct substance, and that certain of its properties,
at least are still the same. If the two-celled stage be centrifuged a
segregation 1s produced in the same manner as in the unsegmented
naleaed egg; that is, a stratification of three substances appears
in each cell—a gray cap, a clear band and a yellow mass. ‘These
may be placed in a polar direction or in any other direction. If
the centrifugal force acts at right angles to the axis of the egg, the
eray suberinte goes to the ereett in ae upper cell, and lies against
the cleavage wall in the lower cell; while the yellow endoplasm
lies against the cleavage wall in the upper cell, and against the
most distal surface of the lower cell (Fig. 27). Other variations
need not be described. It will be seen that it is possible in this
way to demonstrate the continued separate existence of the resi-
dual substance of the germinal vesicle.
Similarly one can demonstrate it in the four-celled and eight-
celled stages; Fig. 28 illustrates this for the eight-celled stage.
Later cleavage stages were not centrifuged but I have other evi-
Elementary Phenomena of Embryonic Development 201
dence of its independent persistence to a later stage. In centri-
fuged eggs in which it passes originally entirely into one cell it is
possible to follow it, on account of its massed condition, up to
late cleavage stages in which its position is just internal to the nu-
cleus. Finally, if the results of staining intra-vitam with neutral
red are to be trusted, we havea method that enables one to follow
it step by step with perfect accuracy into the trochophore.
Fig. 27. To show the effects of centrifuging in the two-celled stage. Drawn from the living mate-
rial. c.b., clear band; e, endoplasm; g.c., gray cap.
Fig. 28. To show the effects of centrifuging in the eight-celled stage. Drawn from the living
material. Explanation of letters same as Fig. 27.
That the nuclei of cleavage stages set free certain substances at
each prophase is a familiar idea, that has been best set forth by
Conklin. It has, however, been impossible to follow these sub-
stances far by cytological methods, so that their fate is largely a
matter of conjecture. In the egg of Chetopterus such substances,
“oxy-chromatin,”’ are similarly set free, and, in addition to these,
there is liberated at each mitosis a group of large granules that can
be followed through at least one cell-generation in each case.
These are the bodies that Mead described in the first cleavage as
nucleoli.
Their history in the first cleavage is briefly as follows: “They
arise in connection with the chromosomes from the segmentation
nucleus and separate from them in the prophase of the first divi-
sion. So that the equatorial plate consists of a certain number
202 Frank R. Lillie
of chromosomes with intermingled granules. The latter are
hardly larger at this time than the ends of the chromosomes, and
they stain in iron-hematoxylin even more intensely than the chro-
mosomes themselves. Only the chromosomes divide and in the
Fig. 29. Outline of longitudinal axial section of the completed two-celled stage. To
show the transmission of chromatin masses cut off from the chromosomes exclusively to the
larger cell. The fine dotted outline shows the boundary of the spherular endoplasm. £, Ectoplasm;
E.d., ectoplasmic defect;c, chromatin masses cut off from the chromosomes; N, nucleus; /.E.,
polar ectoplasm.
anaphase the granules lie midway between the daughter chromo-
somes among the interzonal fibrils (Fig. 25, c). During the ana-
phase they become fewer and larger, no doubt by fusion with each
other. In the telophase the cleavage plane passes anteriorly to
them, thus leaving them invariably in the larger cell CD. So far
as I could see, not one of them is left in the smaller cell. Fig. 29
showing the mid-body of the first cleavage is a characteristic view
Elementary Phenomena of Embryonic Development 203
of this stage, and shows with what certainty these granules may be
traced into the larger cell. “The origin of the bodies from the
chromosomes and their differential distribution to one cell are
suggestive of an important role in differentiation.
Thus a new group of granules arises during the first cleavage
from the chromatin. A similar process is repeated in later cleav-
ages, but whether or not it occurs to the same extent in all the cells
I am unable to say. These granules attain the size of spherules
by fusion; they then mingle with the a group of spherules of the
endoplasm, and can no longer be distinguished. The repetition
of this process up to sixty-four cells would produce a group of
spherules staining in haematoxylin as numerous, probably, as the
original a group of the endoplasm. It is possible, therefore, that
spherules of the a group are continually disappearing and being
replaced. Certain appearances in spherules of the a group during
the cleavage favor this view; many are found that appear eroded or
wasted to a crescent, a condition that I have not observed prior to
the beginning of cleavage. It is impossible, therefore, to say in
the late cleavage, how many of the spherules of the a habitus are
original members of this group, and how many have been derived
from the chromosomes during cleavage.
The observations are instructive as bearing on the question of
differentiation of nuclei. The assumption that chromosomes
may divide differentially has not the slightest foundation in the
observed facts of karyokinesis, excepting in the maturation
divisions. On the other hand the hypothesis that cytoplasmic
differentiation is dependent on nuclear differentiation appears to
me a necessary corollary of our growing knowledge of the charac-
ters of the chromosome complex. The above observations on
differential distribution of nuclear derivatives shows a method by
which not only may nuclear determination be realized, but also by
which differentiation of nuclei may be obtained. If, for instance,
this process of chromatin diminution takes place in certain cells
and not in others, differentiation of the nuclei might result; when,
on the other hand, the nuclear derivatives receive a differential
distribution in daughter cells of different prospective tendencies,
the problem of the determination of these cells seems simplified.
204 Frank R. Lillie
Thus nuclear determination and differentiation may exist even
though every chromosomal fission be integral or equational.
I have described the first cleavage in considerable detail, because
it includes part of the process of segregation of substances, par-
ticularly the bilateral polarization, and also illustrates the origin
of new substances during the cleavage period. The account of the
subsequent cleavages may now be divided into two parts. In the
first will be given a very brief account of the form of the normal
cleavage (cell-lineage) and the fate of the cells. In the second
part the distribution of the substances in these cells will be traced.
b. The Form of Cleavage
The cleavage follows the usual annelid type (Figs. 30-37), 7. ¢.;
the ectoderm is derived from three quartets of micromeres formed
by alternate dexiotropic and leotropic equatorial cleavages of the
four macromeres, 4 (left anterior), B (right anterior), C (right
posterior) and D (left posterior). The mesoblast comes from the
cell 4D; the entoderm from the cells 34, 3B, 3C,and 4D. The
somatic plate is derived from the cell 2D or X.
Certain special features of the cleavage deserve particular
attention. Inthe four-celled stage D is much the largest of the cells,
C comes next, 4 and B are the smallest (Figs. 30 and 31). ‘The
differences between the cells 4, B, and C are, however, relatively
slight. In the second cleavage the polar lobe is indicated by a
protuberance of the vegetative pole of the cell CD toward the left
side; its material passes entirely into the cell D. Again in the
third cleavage there is usually a protuberance at the vegetative
pole of D (Fig. 32) indicating the location of the substance of the
polar lobe, which may, therefore, be followed readily into the cell
1D. The polar globules are attached a little posterior to the first
cleavage furrow on the line of the furrow between C and D, a
position that is retained throughout the cleavage (Figs. 31 and 35).
The ectoplasmic defect is transmitted to the cell rd (Figs, 30, 33,
36 and 38).
The third cleavage is dexiotropic and approximately equal,
though the ectomeres ra, rb, rc, and 1d are slightly smaller than
the “macromeres” r4, 1B, rC and 1D. ‘Thus in the upper
Elementary Phenomena of Embryonic Development 205
Fie. 35 Fic. 36 Fic. 37
Figs, 26, 30 to 37. To illustrate the form of the normal cleavage in Chetopterus. Drawn from
preparations.
Fig. 26. Anaphase of first cleavage.
Fig. 30. Four-celled stage from in front. Note that the ectoplasmic defect is confined to the
quadrant D.
Fig 31. Four-celled stage from the animal pole.
Fig. 32. Four-celled stage from behind. Early anaphase of third cleavage showing polar lobe
in the D quadrant.
Fig. 33. Formation of the first generation of micromeres, of approximately the same size as the
macromeres.
Fig. 34. Eight-celled stage from anterior end.
Fig. 35. Eight-celled stage from animal pole.
Fig. 36. Division of eight to sixteen cells to show large size of 2d.
Fig. 37. Approximately sixty-four-celled stage seen from behind. Outline copied from Mead
(97). The small circles in the cells represent the ectoplasmic spherules (diagrammatic).
In all of the figures on this plate except the last, the ectoplasm is indicated by the dotted contour
line. The relation of this line to the animal and vegetative poles should be noted.
206 Frank R. Lillie
quadrant the cell rd is much larger than its mates, similarly in the
lower quadrant 7D is the largest cell (Figs. 33 and 34).
In the fourth cleavage (eight cells to sixteen cells) the cell 1D
divides unequally so that its upper product 2d is larger than the
lower, the ““macromere” 2D (Fig. 36). The division of 1/4, 1B,
and rC are approximately equal; thus 2d is by far the largest cell
of the second quartet. In the sixteen cell-stage the cells rd and 2d
are much the largest cells in the egg. The orientation of the egg
Fig. 38. A sagittal section through a stage of about sixty-four cells. The small upper cells are the
apical cells. The ectoplasmic defect will be noted in the posterior apical cell to the observer’s right.
E, Ectoplasm; e.c., endoplasm c; En., endoderm cells; E.d., ectoplasmic defect; M., mesoblast cell.
X derivatives of first somatoblast.
and the identification of the cells is thus a relatively simple matter.
Fig. 37 (outline of cells after Mead, 97) illustrates the arrange-
ment and prospective significance of the cells of the sixty-four cell-
stage. Only the superficial part of each cell is indicated and this
Elementary Phenomena of Embryonic Development 207
is not always a good measure of relative size. ‘The distribution of
the ectoplasmic spherules is indicated by the small circles in the
cells with approximate accuracy.
c. Distribution of Substances in the Cells
No cell is pure in regard to the formative stuffs it contains, but
each cell, up to a late stage at least receives both ectoplasm and
endoplasm, with the exception of the entoderm cells which appear
to receive no ectoplasm (Fig. 38). The arrangement of the sub-
stances is the same in all ectodermal cells: (1) Externally a layer
of ectoplasmic spherules; (2) the nucleus in a mass of micro-
somal cytoplasm; (3) just internal to the nucleus a mass of non-
spherular substance; (4) within this a group of endoplasmic
spherules staining black in iron haematoxylin (endoplasm a), and
(5) next to the segmentation cavity a group of large spherules
staining in orange G formed by segregation and fusion of the
smaller endoplasmic spherules (endoplasm c) already described.
The arrangement is similar in the mesoderm cell (though the polar
ectoplasm was not distinguished in the section figured), and, with
the exception of the absence of the ectoplasm, in the entoderm cells
also.
This arrangement of substances is invariable; each cell is
polarized; the axis of polarization of each is a radius of the egg,
and the central ends are homologous. Thus each cell exhibits a
stratification of substances similar to that of the entire egg.
There is, however, one difference in the arrangement of the
substances in the single cells and in the entire ovum. In the latter
the c endoplasm lies above the a endoplasm, whereas in the former
the c endoplasm is most distal. It must be remembered that
there is a polarization of the whole segmented egg as well as of the
individual cells; the c and a endoplasmic substances retain the
same relative position in the entire segmented egg as in the unseg-
mented. It is as though a cavity had been formed in the center
of the c endoplasm of the unsegmented egg (Fig. 39) and nuclei
placed around the periphery just within the ectoplasm, and cleav-
age planes cut through between nuclei to the cavity. ‘Thus the
208 Frank R. Lillie
polarity of the cells is directly derived from the entire ovum.
Nevertheless it is immanent in each cell, for, however much the
arrangement of the cells may be disturbed, the arrangement of
substances in the individual cells remains the same.
We have seen that the polar ectoplasm passes entirely into the
D quadrant. Between the two and the four-celled stage it forms a
polar lobe that lies at first against the 4 quadrant, but shifts to the
B quadrant as the cells come to rest. When the spindles are formed
for the third division a very small polar lobe appears in D (Figs.
Fig. 39. Axial section of an unfertilized egg that had stood in the sea-water two hours
and seven minutes before killing. The a and cspherules of the endoplasm have segregated out
(e.a., and e.c.; compare Fig. 5). A clear layer has arisen between the ectoplasm (£) and the
a endoplasm; the outer centrosome of the maturation spindle has divided once, and the inner one
several times.
32 and 40). Sections taken at this time show that part of the
polar ectoplasm plunges into the interior of the egg (Fig. 40, p.£.).
This is very conspicuous in the four-celled stage containing the
spindles for the next division. A tongue of endoplasm then
extends across, severing the portion lying internally from the
portion that remains superficial in position (Fig. 40). It is possi-
Elementary Phenomena of Embryonic Development 209
ble that the upflow of the polar ectoplasm may carry some of it
above the line of the next cleavage furrow, so that a certain amount
may go into rd. An upflow of endoplasmic spherules goes on at
the same time in all the cells both along the cleavage planes and
also next the surface.
go009
Paste
: ry : Sinks
Q
rey ttt
e@@ {ee
Fig. 40. Sagittal section through the cells B and D of the four-celled stage. The section passes
a little to one side of the middle line; in the median section the spindles of the third cleavage were
formed in metaphase, and the ectoplasmic defect was confined to D. The small polar lobe is con-
fined to the D quadrant and contains all of the polar ectoplasm, p.E. Part of the latter is passing
into the interior of the cell along the plane between B and D. Most of the striated spherules in the
interior of the cells, however, belong to the c endoplasm, and it is impossible to distinguish exactly
the boundary line between them and the polar ectoplasm.
The substance of the polar lobe (polar ectoplasm) is thus far
from being pure in its distribution, for a small portion of it may
pass into the cell zd, and the remainder is divided in two parts of
which the central part is probably transmitted to the first somato-
blast (2d or X), and the superficial part, to the second somatoblast
(4d or M). I am not, however, perfectly certain in regard to
these points, though it is probable from the position of the
material; both Wilson (’o4a) and Crampton (’96 ), moreover,
210 Frank R. Lillie
have shown experimentally that substance of the mesoblast is
contained in the polar lobe.
These observations agree closely with Wilson’s interesting
experimental results on Dentalium; among other things he found
that removal of the first polar lobe involved absence of the apical
organ which normally arises in the D quadrant; the fact that some
of the polar endoplasm probably enters into the composition of
the cell rd might explain this curious result. Wilson, moreover,
observed firecthy that substance of the polar lobe entered into the
formation of both first and second somatoblasts.
It has been a difficult task to determine the fate of the resi-
dual substance of the germinal vesicle in the normal cell-lineage
by direct observation. In the section on staining intra-vitam it
will be shown that it is possible to trace it with a high degree of
accuracy by this method. I will, therefore, add pee wrale some
observations made under experimental conditions that confirm
the observations, made with vital staining, that this substance is
distributed mainly to the first generation of micromeres: Seeing
that cleavage takes place in centrifuged eggs always with reference
to the original polarity and not to the induced stratification of
substances, it often happens that the residual substance of the
germinal vesicle, now in the form of the gray cap (Figs. 17 and
22 and Photographs), is located in the lower hemisphere.
In such cases it can be seen to stream quite rapidly toward the
animal pole during the process of cleavage, so that by the eight-
celled stage it 1s usually found in the upper quartet. If, however,
by any chance the third cleavage plane has come in so as to isolate
it Wholly or in part in the lower quartet it always comes to occupy
the uppermost corner of the cells in which it is found (Figs. 41 and
42). his phenomenon may be observed with the greatest ease,
because this substance in its massed condition contrasts strongly
with the other cell-constituents.
There can, therefore, be no doubt of a strong inherent tendency
for this substance to aggregate in the upper hemisphere. Under
normal conditions it is originally located in this part of the egg,
so that there is every reason to believe, on the basis of these
observations alone, that normally it 1s distributed mainly to the
Elementary Phenomena of Embryonic Development FEMA
cells of the first quartet of ectomeres and their descendants. The
reader is referred to the sectionon vital staining for more detailed
evidence concerning its ultimate fate.
The distribution of the ectoplasmic spherules, excluding the
polar group, corresponds exactly to the distribution of cilia (see
Fig. 37). There can, therefore, be no reasonable doubt that they
are specially concerned in some way with the production of cilia;
this conclusion is practically demonstrated by the observations on
the differentiation of unsegmented eggs. I would expect, there-
fore, to find in true prototrochal larve of annelids, that the dis-
tribution of the homologous substance would be confined to the
prototroch, and would thus form a narrower band than in the
S)
Fic. 41 Fic. 42
Figs. 41 and 42. Two eggs centrifuged before cleavage, now in the eight-celled stage. The stippled
areas represent the substance of the gray cap, which tends toward the highest point (i.e., nearest to
the animal pole) in the cells to which it is confined. Drawn from the living eggs.
uniformly ciliated larvae of Chztopterus. The upper division of
the ectoplasm in Rhynchelmis has an entirely different fate accord-
ing to Vejdovsky: like the lower accumulation (my polar ecto-
plasm) it is restricted to the D quadrant, where it lies near the
animal pole; in the four-celled stage both polar accumulations flow
into the interior of the egg and surround the nucleus and _peri-
plast; subsequently the material appears to be transmitted mainly
to the “.mesomeres”’ (first and second somatoblasts).
It would appear, therefore, that the upper division of the
ectoplasm has fundamentally different functions in Cheetopterus
and Rhynchelmis. It is possible, however, that it 1s really com-
212 Frank R. Lillie
posed of two substances in both forms, and that, in Cheetopterus,
I have overlooked the part corresponding to the main mass in
Rhynchelmis, and that Vejdovsky has overlooked the part in
Rhynchelmis corresponding to the main mass in Chetopterus.
Seeing that the Rhynchelmis embryo is without locomotor cilta,
it Sa otaled be natural for the substance forming the superficial
layer of the ectoderm cells of Chzetopterus and agetu nied with the
formation of cilia to disappear in its phylogenic history. It
is difficult to believe that substances having so similar an original
disposition and structure as the upper and lower divisions of the
ectoplasm in Chztopterus and Rhynchelmis are entirely different
in their morphogenic functions (compare Vejdovsky’s Fig. 30,
Plate IV, with my Fig. 25).
Undoubtedly it would be possible by a detailed cytological study
of each cell in each stage of the cleavage to follow more accurately
the fate of the substances with which we have been concerned.
A beginning has been made on this problem, but it has proved so
difficult, that it seemed desirable to present the facts already
determined, rather than delay their publication, and greatly
. increase the extent of this already too long publication.
The main results concerning the distribution of substances in
the normal development are:
(1) The ectoplasmic defect at the animal pole is the place of
formation of the apical organ with its flagella.
(2) The polar ectoplasm enters into the cells rd, 2d and 4d.
(3) The remainder of the ectoplasm forms the superficial layer
of the ectoderm cells.
(4) The entoderm cells receive no ectoplasm.
(5) All cells receive some of each kind of endoplasm.
(6) New substances arise from the nuclei and are differentially
distributed in some cases at least.
(7) The residual substance of the germinal vesicle is distributed
mainly to the first quartet of micromeres.
d. Intra Vitam Staining
The capacity of living protoplasm to take up stains has been
investigated by a large number of workers. Alfred Fischel (’99)
Elementary Phenomena of Embryonic De velopment 213
was the first to apply the method to embryological research; his
idea was to attempt in this way to differentiate distinct elements of
the ovum and if successful to follow them in the embryonic devel-
opment. “Hinsichtlich des letzteren Punktes galt es mir, im
besonderem, als erwiinschtes Ziel, vielleicht ermitteln zu konnen,
dass wahrend der Furchung eine Verteilung bestimmter Elemente
des Eies auf ganz bestimmte Zellen, also gewissermassen eine
Teilauslese der Plasmaarten der Eizelle statthat.” Fischel
worked with several stains on the ova of a number of different
animals, but obtained the best results with neutral red in the
development of Echinus microtuberculatus. He found that
stained granules appeared in a zone around the spindles and that
they had such avidity for the stain, that they would completely
decolorize weak solutions and become intensely red. During the
resting stage of the nuclei the granules scattered throughout the
cell. ‘They were not distributed differentially in the development,
but each cell contained them.
Garbowski (’o4) is the only other author, so far as I know,
who has applied the method to embryological work; his problem
was to unite a stained portion of one egg with an unstained portion
of another and to follow the subsequent development of the grafted
parts. His results, however, do not bear directly on the problem
under consideration. My own purpose was precisely the same as
Fischel’s, namely, to find if it were possible to stain constituents of
the egg protoplasm intra vitam and, if so, to follow their history in
the subsequent differentiation with special reference to differential
distribution of such substances. Both results were obtained; cer-
tain granules take the stain very intensely and are distributed in
avery precise fashion to a particular region of the embryo. Although
several stains were used, I shall describe the results from only
one, neutral red, which I found, like Fischel and Garbowski,
incomparably the best for the purpose.
To obtain the results that I describe one must not use a strong
solution, for then the stain becomes diffuse and the eggs do not
develop far in it. The best results were obtained from eggs
placed immediately after fertilization in a solution of 75 parts sea-
water plus two and one-half parts of a saturated solution of neutral
214 Frank R. Lillie
red in sea-water. ‘This solution is a very faint rose color, and the
eggs develop in it perfectly normally for more then twenty-four
hours. If there are many eggs in the culture they may entirely
bleach the solution, all the stain being absorbed by certain gran-
Fic. 43 Fic 44
Fic. 45 Fic. 46
Figs. 43 to 46. Drawings of living eggs reared in neutral red solution.
Fig. 43. Polar view, stage of first maturation spindle, which is represented by the central circle.
The parallel lines represent the spherular endoplasm. Eggs fertilized at 10.18 a. M.; put in seventy-
five parts sea-water plus two and one-half parts of a saturated solution of neutral red in sea-water at 10.20
A. M.; drawing from the living egg at 10.43 a. M. The dots around the maturation spindle represent
the red-staining granules.
Fig. 44. Later stage of another egg seen from the side; the red-staining granules lie above the
germ-nuclei.
Fig. 45. Stage of first cleavage spindle seen from the animal pole. The two small circles represent
the polar globules. The red granules surround the spindle.
Fig. 46. Two-celled stage. Resting nuclei. Red granules scattered in the upper hemisphere.
=
Elementary Phenomena of Embryonic Development Dales
ules that become intensely red; such granules retain the stain
many hours or permanently, even if the eggs are transferred to pure
sea-water. If the stain be made about twice this strength, the
eggs develop abnormally after three or four hours; moreover, a
diffuse pink color appears in the endoplasm also, but disappears
from it very quickly after transfer to pure sea-water, though it
remains in the granules that stain in more dilute solutions. Thus
the stain is specific for certain granules, and the afhnity of these
granules for the stain 1s very intense.
If, then, the stain be of the proper strength one obtains invari-
ably the following results when the eggs are put in immediately
after fertilization and are allowed to remain. Up to the time of
the formation of the first maturation spindle there is practically
no stain in the egg, though by careful examination with an oil
immersion lens one can see certain minute, scattered, bright red
granules in the ectoplasm. In some cases there appears around the
first maturation spindle a complete mantle of bright red granules
larger than the ordinary cytomicrosomes and smaller than spher-
ules (Fig. 43). [hese agree in position with the residual sub-
stance of the germinal vesicle. In other cases these granules do
not stain until the second maturation spindle is formed or even
later.
When the germ-nuclei are formed, these granules form a band
above them and later surround them (Fig. 44). When the first
cleavage spindle is formed, they form a mantle surrounding it
completely (Fig. 45) and are thus divided between the two cells.
As the nuclei of the two-celled stage come to rest, the red granules
spread out in the upper hemisphere 0 each beneath the ectoplas-
mic spherules, 7. ¢., at the periphery of the non-spherular substance
(Fig. 46).
As the nuclei elongate for the second division the red granules
again accumulate around them and form a mantle to the spindles,
and are thus divided between the four cells, in which, as the nuclei
come to rest, they again spread out in the upper hemisphere
(Fig. 47).
In the preparation for the third cleavage (four cells to eight
cells) the red granules are again attracted to the nuclei and spread
256 Frank R. Lillie
out along the periphery of the spindles. But in this case the
larger proportion is aggregated around the upper aster, and a
relatively small number only around the lower (Fig. 48). Thus
the first generation of ectomeres receives by far the larger propor-
_ tion of the red granules, and this produces a most striking orienta-.
tion of the egg, the upper quartet being a bright red, and the lower
only faintly tinged, except in the upper left hand corner of each
macromere, where the red granules congregate after the cleavage ts
completed, and thus joretell their distribution to the second quartet
of ectomeres (Fig. 49). The cell D furnishes the most striking
illustration of this on account of its large size.
I am not sure that all of the red granules remaining in the
macromeres are distributed to the cells of the second quartet;
but certainly the larger number are, so that very few of the original
red granules remain for the third quartet.
In the later cleavage the general result is so clear as to be easily
visible with the low powers; the upper half of the segmented egg
has an intense red stain, the portion derived from the second
quartet very little, and below this there is almost none at all.
This difference continues to increase in intensity during the
formation and growth of the trochophore. At about twenty hours
the exumbrella of the trochophore (Fig. 51) is brilliant, the red
stain is distributed in great blotches that define a broad band
interrupted dorsally and leaving only a small area at the apical
pole with smaller red spots, in the center of which is the apical tuft
of cilia. The subumbrella in comparison to the exumbrella is
very lightly stained but the better stained specimens show a few
red granules in the ectoderm that appear to define the somato-
blastic plate; I am not, however, sure of the distribution of these
granules in the subumbrella, which vary greatly in different
cultures.
The bright red stain is practically confined to the ectoderm; the
entoderm and mesoderm have at most a faint rose tinge. As the
trochophore becomes older, the broad prototrochal band becomes
relatively narrower, owing no doubt largely to the expansion of
the head vesicle in front of it.
If now the cells of the prototroch containing the large masses
.
j
i
Elementary Phenomena of Embryonic Development O58 |
of red are examined with the oil immersion lens, it is found that
the red mass 1s situated internal to the nucleus. Five substances
may in fact be recognized in each cell: (1) the layer of ectoplasm
with its characteristic spherules which are slightly stained;
(2 and 3) the nuclear area containing a large vacuole; (4) the
bright red mass, analyzable under the 2 mm. lens into a dense
group of large red spherules; (5) an aggregation of yellow endo-
plasmic spherules (Fig. 52).
Now in a late cleavage stage it will be found that the arrange-
ment of substances is the same with this exception, that the red is
in the form of relatively small granules, and the vacuole is not yet
formed (Fig. 50). From which it follows, either that the red
granules have grown to the size of spherules, or that agglutination
and fusion have taken place among them. ‘The origin of spherules
from smaller granules is thus demonstrated.
The question now arises, what is the origin of these red-staining
granules that have so precise a distribution in the development.
One thinks at once of the residual substance of the germinal
vesicle with which the aggregation of red granules has the follow-
ing points in common: (1) the original distribution around the
maturation and the first cleavage spindles; (2) the position in late
cleavage just central to the nucleus; (3) the size of the granules,
intermediate at first between cytomicrosomes and spherules; (4)
the distribution to the upper hemisphere.
One is, however, met at once by the difhculty that the amount
of these granules in the later development exceeds considerably
the original amount of the residual substance of the germinal
vesicle. ‘This difficulty may, however, be met by assuming that
similar granules arise from other nuclei than the germinal vesicle;
there is very definite evidence for this view detailed in the account
of the differentiation of unsegmented eggs.
But a more serious difficulty arises; if these red granules are
indeed the residual substance of the germinal vesicle, one should
find that the gray cap formed from this substance in centrifuged
eggs takes the red stain. Now, this is not the case; and to save
the hypothesis one can only assume that the denseness of the
ageregation induced by the centrifugal force is unfavorable to the
218 Frank R. Lillie
Figs. 47 to 52. Drawings of living eggs reared in neutral red solution.
Fig. 47. Four-celled stage. Resting nuclei. Red granules scattered in upper hemisphere.
Fig. 48. Third cleavage-spindles formed. Majority of red granules accumulated around the
upper half of the spindles.
Fig. 49. Eight-celled stage. Resting nuclei. Same egg as Fig. 48, twelve minutes later. Details
drawn only in D quadrant. In 7rd the red granules are uniformly distributed; in 7D they occupy the
upper left hand corner, or region of the cell 2d.
Fig. 50. Single cell of about the sixty-four-celled stage. The red granules are now larger and lie
just internal to the nucleus.
Fig. 51. Trochophore of twenty-four hours, reared in neutral red solution, the black areas
show the distribution of the stain. The largest areas in three irregular rows are inthe prototroch. As
the larva rotated, the dorsal interruption of the prototroch came repeatedly into view.
Fig. 52. A single prototrochal cell drawn with oil immersion lens in life. The red granules are
now very much larger than before. E, Ectoplasm; e, endoplasm; N, nucleus; V7, vacuole.
hdeediinn dl teens hotel
219
Elementary Phenomena of Embryonic Development
220 Frank R. Lillie
penetration of the stain. What one does observe is that similar
scattered red granules appear on the surface of the clear band,
and that these become more abundant in the upper part, and
spread over the entire animal hemisphere, whatever be the position
of the gray and yellow masses, as in the normal development.
Now the clear band always lies against the gray cap, and, as this
gradually dissolves and spreads out, it seems probable that the
red eranules appearing on the clear band are derived from the
gray cap. It would thus appear that the residual substance of the
germinal vesicle does not consist entirely of erythrophilous gran-
ules; but it is probable that the original red granules are derived
from this substance.
This is at any rate the conclusion to which I have come. It
should be possible to observe this without any uncertainty in the
living egg, if the hypothesis is correct; but when I began to realize
the significance of the question the season had passed; and I have
to rely on the observations made before I realized that the gray
cap was the residual substance of the germinal vesicle.
I expect to return to this question another season; but I have
little doubt of the correctness of this identification of the red
granules distributed to the exumbrella with part of the residual
substance of the germinal vesicle and other nuclear derivatives.
The resemblance in position, size of granules, and general be-
havior is too precise to leave much room for doubt.
2. Differentiation Without Cleavage
In 1902 I published a paper on Differentiation Without Cleav-
age in the Ege of the Annelid Cheetopterus pergamentaceous, in
which I showed that under certain conditions, eggs of Cheetop-
terus might become ciliated, and exhibit other phenomena of
differentiation without undergoing any process of cleavage.
Similar results have been obtained for other annelid ova by
Treadwell (02) and Scott (03 and ’06). One striking fact noted
in Chetopterus was that such ciliated unsegmented ova some-
times exhibited what appeared to be regional homology with the
trochophore. But the structure of the ciliated ova was very
Elementary Phenomena of Embryonic Development 221
variable and such homology could not usually be demonstrated.
My general conclusion at that time was that cell division is not
necessary for embryonal differentiation of certain kinds; and that
the usually disordered and variable character of the forms develop-
ing from unsegmented ova indicates that the essential role of
cleavage in normal development is the localization of processes
of different kinds.
However, the conclusion in no way explained how differentia-
tion of specific cell-organs could arise in an unsegmented mass of
protoplasm with only one nucleus. ‘The most obvious inference
was that the unsegmented ovum already contained substances of
specific morphogenic value, that played their roles whether dis-
tributed in cells or not. ‘This idea has been more or less definitely
in mind since the original experiments, and I, therefore, took up
the subject in Cheetopterus again in the summers of 1904 and 1905.
I found that a variety of methods might be employed to bring
about differentiation without cleavage:
(1) By exposing the unfertilized eggs for about an hour to the
action of potassium chloride in sea-water. (For details of method
see Lillie *o2.)
(2) By treating fertilized eggs with the same solution but in
higher concentration (Lillie ’02).
(3) If ova are fertilized after standing two to eight hours in sea-
water a variable proportion develop without undergoing seg-
mentation, the proportion being greater when the ova have
remained longer without fertilization. Or such eggs might seg-
ment at first and the blastomeres fuse together, the subsequent
development being without cleavage. ‘The fertilization was often
polyspermic in such cases.
On July 28, 1904, a batch of eggs from one female was divided
into three parts, which were fertilized after standing three hours,
four hours and five hours, respectively, in sea-water. None of
these segmented to any considerable extent. “The next morning
the four-hour lot was swarming with ciliated specimens, all oe
which had developed without cee On July 30 some eggs
were divided into two lots one of which, A, was fertilized after
standing in sea-water one and one-half hours and the other B,
-
222 Frank R. Lillie
after two and three-quarter hours. The cleavage of lot A was
irregular from the start but only about 40 per cent developed
without cleavage. In lot B, on the other hand, over go per cent
failed to segment or the cells fused later on, and most of these eggs
differentiated without cleavage. (For details of these experiments
see the section on multinucleated, unsegmented eggs.)
(4) Fertilized eggs placed at a temperature of 10-14° C. for
about twelve hours remain unsegmented and differentiate when
restored to the room temperature, usually without segmentation.
In Experiment 8, 1904, two lots of eggs, A and B, were fertilized
at 4.52 and 5 P. M., respectively; each was divided into two lots,
A1and A2, Brand Bz. §8.A1 and 8.B2 were placed at a tem-
perature of about 14° C. about the time of the formation of the
first polar body; 8.A2 and 8.B1 were placed at the same time
at a temperature of about 16° C. When 8A.1 and 8.B2 were
examined the next morning at about g A. M. they were unsegment-
ed and they showed almost as sharp a segregation of the residual
substance of the germinal vesicle as could be produced with the
centrifuge. The preparations show that the maturation spindle
remained unchanged in many cases and degenerated in others.
In 8.A2 and 8.B1 there were two classes of eggs, viz: young
normal larve and eggs like 8.A1; clearly the temperature in this
case was near the lower margin of the temperature range for
cleavage. A large proportion of the unsegmented eggs then un-
derwent differentiation without cleavage at a very rapid rate.
(5) I have some evidence that an*abnormally high tempera-
ture may produce the same effect (only one experiment not quite
conclusive, as differentiation was not followed to a period of forma-
tion of cilia).
Careful examination of this mode of development, however,
produced, showed, what I had entirely missed before, that the
differentiation proceeds by the segregation and differentiation of
the substances described in the preceding parts of this paper, but
which I had not recognized at the time of my first study.
In describing the phenomena of differentiation without cleavage
I shall leave out of account a good many accompanying phenom-
ena which are not directly related to the problem in hand and which,
Elementary Phenomena of Embryonic Development 228
moreover, were sufhciently noticed in the earlier account (Lillie
’02), such as the ameboid movements occurring at various stages
of the process, and the more or less abnormal cleavage of a variable
proportion of eggs in each experiment.
The unsegmented eggs that undergo differentiation may be
divided into two classes according as they are uninucleated or
multinucleated; the former constitute the type in the KCI cultures
and are relatively rare in the fertilized cultures; the latter may owe
the multinucleated condition either to polyspermy or to an original
cleavage which is subsequently lost by fusion of the blastomeres.
The multinucleated unsegmented eggs differentiate nearly as
rapidly as normal ova (cilia formed in six or seven hours) whereas
the uninucleated ova differentiate more slowly (cilia formed in
eight to nine hours).
a. Uninucleated Ova
The conditions are simpler in the uninucleated ova in some
respects and the records more complete; they may, therefore, be
considered first. The process of differentiation depends on segre-
gation of the cytoplasmic materials already described, growth of
the nucleus to an enormous size, interaction of nuclear derivatives
and cytoplasm, and a final rearrangement and differentiation of
substances. Among the many variable phenomena, these are
constant, and may, therefore, be considered as primary.
1. Early Segregation of Formative Stujfs.— The orginal segre-
gation of material, whether the eggs be fertilized or not, 1s like the
normal up to the period preceding the first cleavage; the polar
globules may or may not be formed (Lillie ’02); the polar lobe may
appear and is then retracted; but cleavage does not take place.
Then the egg-nucleus together with the nonspherular protoplasm
begins to sink in toward the center of the egg. At the same time
endoplasmic spherules slowly arise toward the animal pole, as in
the normal development, between the nonspherular protoplasm
and the ectoplasm (Fig. 53). The ectoplasm also begins to flow
towards the animal pole andis soon aggregated mainly inthe upper
hemisphere (Figs. 54 and 55); the polar ectoplasm remains at the
vegetative pole (Fig. 55). ‘The original opening in the ectoplasm
224. Frank R. Lillie
at the animal pole may persist and the spherular endoplasm
reach the surface here (Figs. 53, 55, 56). he layer of spherular
enodplasm is usually considerably thinner on one side, show.
ing that there is a bilateral polarization of the substances like
the normal. The nucleus has enlarged considerably. This
occurs about one and one-half hours from the beginning of the
experiment.
Fig. 53. Section of differentiating uninucleated unsegmented egg. E, Ectoplasm; E.d., ecto-
plasmic defect; e., spherule bearing endoplasm; N, nucleus; p.E., polar ectoplasm. Eggs put in
ninety-three parts sea-water plus seven parts 2 M KCl at g a. ., transferred to sea-water
at 10 A. M. Preserved 10.39 A. M., one hour and thirty-nine minutes from the beginning of the
experiment. The dotted outline represents the boundary of the spherule-bearing endoplasm; the
broken line, the ectoplasm.
The ectoplasm now flows more rapidly towards the animal
pole and thus forms a relatively narrow polar ring;in some cases
the opening disappears and the ectoplasm becomes heaped up in
a mass (Fig. 59); as thereis relatively little endoplasm in this
part of the egg there appear three strata (Figs. 54, 55 and 56).
This occurs about two to four hours from the beginning of
the experiment. Thus in Experiment 3.1, 1904, I put some
unfertilized eggs into ninety-five parts of sea-water plus five
parts of 5/2 M KCl at 10.47 a. m., and the eggs were trans-
Elementary Phenomena of Embryonic Development 225
Fie. 54 Fic. 55
Fic. 56 Fic. 57
Figs. 54 to 58. Stages in the differentiation of uninucleated unsegmented eggs drawn from living
material. E, Ectoplasm; e, endoplasm; E.d., ectoplasmic defect; p.E., polar ectoplasm; r.s. re-
mainder of gray cap (residual substance of germinal vesicle).
Fig. 54. Eggs put in ninety-five parts sea-water plus five parts of a two and one-half M KCI solu-
tion at 3.15 p.M. ‘Transferred to sea-water at 5 P.M. Drawn at 5.15 p. M., two hours from the begin-
ning of the experiment.
Fig. 55. Eggs put in ninety-five parts sea-water plus five parts of a two and one-half M KCI solu-
tion at 10.47 a. M. Transferred to sea-water at 11.52 A.M. Drawn about 2.45 p. M., four hours from
the beginning of the experiment.
Fig. 56. Same history as Fig. 55.
Fig. 57. Eggs put in eighty-five parts sea-water plus fifteen parts of a two and one-half M KCl
solution at 11.10 a. M. Transferred to sea-water at 12 o’clock, noon. Drawn 4.12 p. M., five hours
from the beginning of the experiment.
226 Frank R. Lillie
ferred to normal sea-water at 11.52 A. M., and the conditions
shown in Figs. 55 and 56 were found at about 2.30 and
2.45 P. M., respectively. It will be noticed that there is quite
a pronounced constriction near the equator between the
unlike substances; this often becomes so deep as to produce the
impression of a cleavage. The substance of the polar lobe is indi-
cated in both cases, and in one (Fig. 55) an ameboid prominence
is formed at the animal pole, owing to extrusion of endoplasm
through the ectoplasmic defect occurring there.
In about half an hour to an hour more a very striking change
takes place in the majority of the eggs (uninucleated, unseg-
mented). A mass of yellow spherules that have continued to rise
toward the animal pole pushes out through the defect in the
ectoplasm and forms a protuberance at
the animal pole (Figs. 57, 58, and Photo-
graph L). ‘Thus the ectoplasm becomes
a broad band encircling the ovum
between the polar masses of yellow
endoplasm. (For section of such a
stage, see Fig. 60.) The nucleus tas
_now become as large as the original
germinal vesicle and it lies in the non-
spherular protoplasm beneath the ecto-
plasmic band. In some cases the
ectoplasmic defect at the animal pole
Fig. 58. Eggs taken from female :
at g A. M., centrifuged at 10.45 A. M. becomes covered up (Fig. 59), and then:
Fertilzed 10.50 4m. Drawn about “¢hisg banded form does mot acculinesmen
3-15 Pp. M., four hours and _ thirty-five
cases the ectoplasm simply becomes
more massed.
There are some reasons for thinking that the polar masses of
endoplasm are of different composition, thus it will be noticed
that the original lower mass (Photograph L) affects the photo-
graphic plate less strongly than the upper mass, though to the eye
they appeared alike in color.
The mere separation is also evidence for some difference in
composition, though it is to be admitted that it does not constitute
decisive proof. A third reason is found in the presence in the
minutes after fertilization.
|
/
|
i
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Elementary Phenomena of Embryonic Development 227,
lower mass of a small reticular area usually clearly marked at this
stage (Fig. 60) and always absent in the upper mass. ‘These facts
are mentioned only as indicating that the original (or early
induced) diversity of substances in the endoplasm may be greater
than one would suppose. ‘The opposed masses in this case corre-
spond. roughly to the endoplasm of the first generation of ecto-
meres (upper mass) and of the other cells (lower mass). The
distinction is not between endoplasmic substances a and c, for
these occur in each mass.
2. Concerning the Growth of the Nucleus—Nuclear growth is
usually accompanied by nuclear division involving the formation
and division of a definite number of chromosomes at periodic
intervals. Now in the eggs that we are describing the nucleus
does not divide, but it grows and undergoes periodic changes that
correspond to the periodic changes of dividing nuclei. ‘hus
in any preparation the nuclei of some eggs are composed mainly
of nonstaining substances, with only a few scattered stained gran-
ules similar to the “nucleoli” cast off at each normal division
(Figs. 59 and 60), and other nuclei in ova on the same slide, and
thus of precisely the same age and mode of preparation, are dense
masses of intertwined chromosomes (Fig. 61). “These conditions
undoubtedly represent successive phases of nuclear activity, and
demonstrate a periodic succession of chromatic and non-chro-
matic stages. Inasmuch as the number of chromosomes in the
older and larger nuclei is much greater than in the younger and
smaller, it cannot be doubted that there is a periodic formation
and division of chromosomes as in the normal cleavage.
The egg shown in Fig. 61 was killed three hours and seventeen
minutes after the beginning of the experiment. After making due
allowance for the slower rate of development it would correspond
in age to about a normal sixty-four celled stage, which would
contain, if unfertilized, g x 64 = 576 chromosomes. A calcu-
lation of the chromosomes inthis uninucleated egg gives a roughly
corresponding number; the nucleus ran through four sections and
there were considerably over 100 chromosomes in each. (All in
the one section figured could not be shown in the figure.) The
number corresponds so closely to the theoretical requirements,
228 Frank R. Lillie
Fig. 59. History same as 53, preserved two hours
that it has the value of an
actual demonstration of the
view expressed in the last
sentence of the preceding
paragraph.
The arrangement of the
chromosomes in Fig. 61 is
suggestive; they are not uni-
formly distributed, but are
arranged in four groups.
The number of groups in
the entire nucleus was at
least double as many. The
only explanation of such a
and nineteen minutes from beginning of experiment. grouping that occurs to me
The ectoplasm aggregated in animal hemisphere; ecto- is contained in the hypoth-
plasmic defect covered up; a and c endoplasms sepa-
rating out.
of which there were nine,
tend to remain together.
‘There is no other evidence
for this than I have given.
In any event the steady
increase in number 1s a
strong support for the in-
dividuality hypothesis.
That the nucleus does
not divide under such cir-
cumstances may be attrib-
uted to the absence of cen-
trosomes and _ spheres,
which are usually lacking
in the uninucleated unseg-
mented eggs in the early
esis that all descendants of
each original chromosome,
Fig. 60. History same as 53, preserved four hours and
eighteen minutes from beginning of experiment. Longi-
tudinal section of three-banded form; original animal
pole probably to left. Nucleus in achromatic condition.
Vacuolated area to right characteristic of eggs in this
condition.
stages. The reason for this defect is not very clear, especially in
the case of those eggs where the stimulus to development is fur-
nished by normal fertilization. But, even when the stimulus is
ae
Elementary Phenomena of Embryonic Development 229
supplied by potassium chloride, certain eggs form asters and spin-
dles, so that the potentiality of these structures must be sup-
posed to reside in the egg proto- Se
plasm. The series of preparations Be x
upon which I rely for most of ‘a i
the data concerning the nuclei is, eRe, \
from a potassium chloride series, yee \
inwhich the polar bodies were not | ee \
formed;! it is thus possible that, | Sue |
in this case, there is a connection \ AUP
between the omission of the pro- )
cess of formation of the polar \ /
bodies and the absence of asters. a J
Such an explanation might also tee, Lees
apply ue the uninucleated, vee Fig. 61. History same as 53, preserved three
Memtcdeiec. round ine fertilized joics and seventeen midutes from beginning of
series; these constitute a relatively experiment. See text for description.
small proportion of all the eggs in such a culture, and in a
normal lot of eggs some fail to fertilize, and do not form
polar bodies; if we supposed that some extract of the sperm
stimulated differentiation in such eggs we would get the above
result.
3. Interaction of Nucleus and Cytoplasm.—After the nucleus
has reached about the size of the germinal vesicle a strong mutual
attraction between the nucleus and the ectoplasm begins to be
apparent, when the nucleus is in the chromatic condition. This
is shown in one of two ways: Either the ectoplasm is drawn into
the interior of the egg where it forms a mass in contact with the
nucleus, or the chromatic part of the nucleusis drawn out to the
ectoplasm. It is interesting to note that the former corresponds
to the normal procedure in Clepsine (Whitman ’78) and Rhynchel-
1Tf the polar bodies are not formed, the looped or elongated form of chromosome does not appear
in the uninucleated, unsegmented ova until between one and two hours from the beginning of the
experiment. Previous to this time one finds only irregular forms similar to the chromosomes of
the maturation spindles. Thus the chromosomes apparently carry out the maturation divisions
within the egg but without forming daughter nuclei, before the form of chromosome characteristic
of the cleavage divisions appears.
230 Frank R. Lillie
mis (Vejdovsky ’88—’92).
| When the ectoplasm has
fil ll na Md lM ee : gathered in the interior
HA AAT oS it collects on one side on
the large nucleus, and a
very important change
takes place. A la rege
quantity of the chromatin
ageregates on the margin
of the ectoplasm, and,
while the remainder of
nucleus returns to the
achromatic condition,
this portion diffuses in
Fig. 62. Eggs put in ninety-five parts sea-water plus five the form of search Patt
parts 23 M KCL at 11.10 a. M., five hours and ten minutes cles (microsomes) between
from beginning of experiment. For description see text. the ectoplasmic spherules,
which are thus completely
2
— me
eee?
at
oe
<68
oi
085
— 28
0%9 88
8,
ts
66 8,
eee?
A —
=
Z
‘e
eo 0 _@
e
Ve
/ fo
interpenetrated by a chromatic
network. Eggs in which the
ectoplasm has been attracted
‘into the interior apparently do
not develop farther.
When the ectoplasm remains
external the chromatic part
of the nucleus is drawn to it.
Fig. 62 illustrates a case in
which there were two accumu-
lations of the ectoplasm on
opposite sides of the ege; the 8:
attraction acting on the nucleus ‘
has drawn the latter out into Fig. 63. History same as 62. For description see
a broad band extending from ee
one mass of ectoplasm to the other. The chromatic portion of the
nucleus is next the ectoplasm on each side, leaving an achromatic
nuclear bandinthe center. The chromatin is broken up into small
particles which are scattered between the ectoplasmic spherules.
Elementary Phenomena of Embryonic Development 231
A single section, such for instance as is shown in Fig. 63, shows
the outline of the nucleus still intact; the chromatin in strands
with large varicosities is seen stretching off toward the main mass
of the ectoplasm, and the farther we trace it from the center of the
nucleus the more the chromatin is broken up; radiating lines of
granules can be seen extending from larger masses of chromatin in
between the ectoplasmic spherules, so that all intermediate stages
between masses of chromatin and scattered microsomes may be
seen. Many similar sections occur on the same slides. Such
granules are very much less abundant between the ectoplasmic
spherules in stages preceding this.
The living material furnishes the most conclusive proof of the
migration of the chromatin halo of the nucleus. _ If the living eggs
be examined under pressure at this time, one sees a gray halo
around each large nucleus (Photograph J and Fig. 58); a little
later this may be seen to stretch out toward a surface aggregation
of ectoplasm and to flow into it losing its identity in the larger
mass (Photograph kK).
The intermingling of a large part of the nucleus, distinguishable
in the living condition as a gray halo and in sections as a periph-
eral chromatin layer, is one of the most striking and suggestive
phenomena concerned in the differentiation of the uninucleated
unsegmented eggs. Concerning the facts there cannot be a par-
ticle of doubt. The process can easily be observed in the living
condition, and even photographed, as Photographs J and K
prove. he preparations are even more conclusive, if possible,
for one can study the details more leisurely. Nor is there any
doubt, in my opinion, that the chromatin particles become micro-
somes. ‘The gradation from larger chromatin masses to un-
doubted microsomes is perfect, and proceeds in an orderly direc-
tion from the nucleus outward (Figs. 62 and 63).
The interpretation of this phenomenon must proceed from the
assumption that it is the result of a process occurring in the normal
development, probably in each generation of cells, but which has
been inhibited by the abnormal nuclear conditions, until it breaks
loose by sheer force. Its striking character is due to the summa-
tion of a number of relatively inconspicuous processes of the nor-
222 Frank R. Lillie
mal development. It, therefore, furnishes to some extent a meas-
ure of the amount of chromatolysis within the same period of the
normal development.
The result necessitates a more careful inquiry into the normal
conditions; at present it does not seem advisable to attempt a
detailed comparison. I may say, however, that the process ap-
pears to me to represent more than the formation of the chromatic
granules that lie between the groups of daughter chromosomes
in the normal anaphase (see description, p 202,and Fig.25);because
I believe that such granules escape into the cytoplasm periodically
in the uninucleated unsegmented eggs, and do not accumulate
within the nucleus. ‘The matter is still under investigation.
Finally it should be noted that such distribution of chromatin is
not limited to the ectoplasm, but occurs probably in the endoplasm
also in later stages.
4. Later Distribution of Formative Stuffs —That the ectoplasm
undergoes a radical change by this intermixture of chromatin is
proved by two striking phenomena: (1) The mass of chromatin-
charged ectoplasm immediately overflows the remainder of the
substance of the egg, usually, but not always, covering it com-
pletely; (2) the staining reaction of the ectoplasmic spherules
changes radically; whereas previously they could not be stained
with iron haematoxylin without overstaining the rest of the egg,
they now take the hematoxylin strongly. These two phenomena
will be considered separately.
Overflow (unicellular gastrulation). I observed this process
first in the summer of 1904. The most complete records are in
Experiments 5 and 1 of that season, from which I take the follow-
ing account: Experiment 5 was started at II.I0 A. M. July 28,
1904. Division 1 of this experiment was treated as follows:
The eggs were put into a mixture of ninety-five parts sea-water
plus five parts 23 M KCl at 11.10 a.m. Part a was transferred
to sea-water at 12.05 P. M. (fifty-five minutes). The designation
of this experiment is thus 5.1.a. At 3.20 P. M. many eggs had reached
the elongated condition shown in Figs. 57 and 58 with two terminal
masses of yellow endoplasm, and a broad equatorial band of ecto-
plasm through which the large nucleus could clearly be seen. At
Elementary Phenomena of Embryonic Development 233
5 p. M. the ectoplasmic band in many eggs had ruptured on one
side transforming it into a saddle-shaped mass, and the two masses
of endoplasm met and fused on the opposite side of the egg (Fig.
64). The nuclear area is situated beneath the ectoplasmic
accumulation.
It is undoubtedly at this time that the chromatic matter of the
nucleus intermingles with the ectoplasm, though | did not observe
this in 1904 for the simple reason that the eggs were not examined
under pressure. But in 1905 this phenomenon was observed as
already described.
Fic. 65 Fic. 64 Fic. 66
Figs. 64 to 70. Later differentiation of uninucleated unsegmented eggs from life.
Fig. 64. Form produced by rupture of ectoplasmic band on one side. Eggs put in ninety-five
parts of sea-water plus five parts 2 M KCl at 11.10 a. mM. Transferred to sea-water at 12.05 P. M.;
drawn at 5 p. M., five hours from beginning of experiment.
Fig. 65. Slightly later stage than 64.
Fig. 66. Same egg as sixty-five drawn three minutes later; the ectoplasm has overflowed a large
part of the endoplasm.
All stages of the rupture of the ectoplasmic band were observed;
the notes read “all stages may be seen; the granular band (ecto-
plasm) first becomes broader on one side, then the yolk masses
(endoplasm) approach and fuse and the band ruptures. Thus
the yolk has the characteristic shape shown in the figure.”
Then the ectoplasm accumulates in a mass (Fig. 65) and
234 Frank R. Lillwe
immediately begins to spread rapidly over the surface of the egg
(Figs. 66 and 67) until there is a complete surface layer of ecto- |
plasm. The notes read “This overflow of the gray matter (ecto-
plasm) can easily be watched as it takes place rapidly, and all |
stages may be seen at once on a single slide.” “Two drawings of
the same egg (Figs. 65 and 66) accompanying Experiment I, 1904,
show that a considerable part of the process of overflow may
take place in three minutes. An uncovered spot, which prob-
ably corresponds to the position of the polar ectoplasm, may persist
for some time, and the egg thus affords a striking picture, as of
epibolic gastrulation ina unicellular mass of protoplasm (Fig. 67).
Wf),
gnu
Fic. 67 Fic. 68
Fig 67. Another egg; slightly later stage. Unicellular “‘gastrula.”
Er Fig. 68. Ciliated uninucleated unsegmented egg drawn about twenty-three hours from beginning
of experiment. The vacuoles are about in the position of the prototroch of the larva.
In many cases the process of overflow is incomplete, or does not
involve the yolk mass at all, thus leading to conditions described
beyond. ‘There is usually more or less endoplasm included in the
overflowing mass; in some cases a very considerable quantity, so
that the layer established by the overflow may be quite thick.
This is a parallel condition to that found in the ectoderm cells
normally, in which, as described on p. 207, only the layer external
to the nucleus is composed of ectoplasm, and the remainder of the
cell of endoplasm.
Change of Staining Reaction. Sections made for the purpose
of studying the process of overgrowth show that it does not begin
until the ectoplasmic spherules have been so modified by the
Elementary Phenomena of Embr yonic Dew elo pment 235
intermixture of chromatin that their staining reaction to iron
hematoxylin has changed considerably. Although this approxi-
mates their stain to that of some of the endoplasmic spherules,
there is not the least difhculty in distinguishing them. ‘The ecto-
plasmic spherules are smaller, or at least more uniform in size,
less spherical, and are massed differently; the substance in which
they are embedded likewise appears different from that of the
endoplasm, so that the total effect of the ectoplasm even when its
spherules stain black is entirely different from that of endoplasm.
It appears as if the ectoplasmic spherules become completely
impregnated with chromatin, just before the process of over-
growth takes place; the change in staining reaction is difficult to
explain otherwise. [he formation of cilia follows very soon after
the process of overgrowth; thus it cannot be doubted that the
impregnation of the wenplasine spherules with chromatin stimu-
lates their latent potencies.
Sections stained in thionin and orange G show that the dispersion
of the chromatin is by no means confined to the ectoplasm; but
that the endoplasm as well is thoroughly interpenetrated with
particles of chromatin, which are beyond all question microsomes.
The same thing may also be seen in the iron hematoxylin prepara-
tions, but it is not shown in so striking a manner as by the thionin
and orange stain. The bright orange spherules contrast beauti-
fully with the brilliant blue microsomes clustered around them
and in all the interspaces. With thionin and orange staining the
ectoplasmic spherules continue to take the orange stain even after
the intermixture with chromatin but they take the blue also to a
certain extent, so that their general effect is olivaceous.
We have seen that in some cases the nucleus draws the ectoplasm
into the interior of the egg, so that it is completely surrounded by
the endoplasm. It is probable that this is the reason why many
eggs fail to reach the stage of differentiation of cilia. [hus
Experiment 9, 1901, very few of the eggs became ciliated; but the
preparations show that, in practically all, the nucleus grew and
the ectoplasm segregated in the usual fashion. But the sections also
show that the internal migration of the ectoplasm is very char-
acteristic of the series.
236 Frank R. Lillie
ie Formation of Cilia and Other Cell Constituents.—The
formation of cilia follows hard on the heels of the ectoplasmic
overflow. Some uninucleated eggs begin to revolve eight or nine
hours after the beginning of the experiment, and others not until
much later. “The majority, in the case of the potassium series,
never reach this stage at all, probably, as suggested before, owing
to failure in the process of overflow.
Typically the entire ectoplasmic surface becomes ciliated, but
the cultures show great variety of conditions owing to the following
circumstances: (1) A good many eggs break into two or more
U
\
\
yy be I
Sys
Fic. 69 Fic. 72
Fig. 69. From the same culture as 68; about same age.
Fig. 70. From same culture as 68; about same age. The endoplasm of the egg has been con-
stricted off.
parts owing to excessive development of constrictions between
segregated substances (compare Fig. 69). Thus are produced
some non-nucleated parts, some parts nucleated and without,ecto-
plasm, other parts nucleated and with relatively little endoplasm.
(2) Fusions are very common (see Lillie ’02). (3) A larger or
smaller part of the endoplasm may remain exposed (compare
Figs. 68 and 69).
It is, therefore, not surprising that good examples of clear
regional homology with the entire trochophore are rare. Some
are, however, found. (Ihe regional homology may be very
precise in multinucleated unsegmented eggs; see next section.)
The best example of it in uninucleated eggs is shown in Fig. 1 of
my Ig02 paper. (See also Figs, 68, 69 and 70 of the present
Elementary Phenomena of Embryonic Development 227
paper.) The reason for the existence of a regional homology is
so clear that discussion is hardly necessary. Fig. 70 shows a
ciliated part corresponding only to the upper halves of Figs. 68
and 69 and resembling only the exumbrella region of the trocho-
phore.
In general the following statements may be made concerning
the differentiation of the ca amc eeed eggs. (1) Organs are never
formed, but only such structural dsmene as may occur in single
cells of the trochophore. (2) Organs may, however, be simulated
by the aggregation of the Bice cnc matter of an organ, for
instance in the case of the yellow endo-
plasm, which simulates the gut of the
trochophore, or the row of large vacu-
oles situated near the upper margin
of the yellow endoplasm (e. g., Fig.
69) which simulates the row of vacu-
oles of the prototroch. (3) The struc-
tural elements appear in the same
order of time as in the trochophore.
(4) The distribution of structural ele-
ments tends to resemble that of the Fig. 71. Ciliated unsegmented egg
trochophore. (5) he yellow Prides about twenty-eight hours old. Most of
the endoplasm has been consumed.
plasm (yolk ?) is used up, apparently fae
for the maintenance of metabolism,
in the ciliated unsegmented eggs precisely as in the larva (see
Fig. 71). (6) The apical flagellum is never formed.
Inasmuch as a number of conditions were figured and described
in my earlier paper (Lillie ’02) and because multiplication of
examples would be unprofttable, I shall content myself with
illustrating in a very few cases the general statements just made.
The first and second statements really require no further
elaboration; Figs. 68-71 sufficiently illustrate them.
The order of appearance of the structural elements is:
(a) cilia; (b) intermediate vacuoles; (c) ectoplasmic vacuoles. |
was much interested in attempting to discover the mode of origin
of the cilia. From the fact that their distribution, both in the nor-
mal larva and unsegmented eggs, is the same as that of the ectoplas-
Ze
Z
Se
238 Frank R. Lillie
mic spherules, I was at first inclined to believe that each spherule
was the germ of a cilium, and expected, therefore, to find the same
number of cilia and spherules. The number of cilia seems, how-
ever, tO exceed the number of spherules, and they do not arise
directly from the spherules but jrom the microsomes between or on
the surface of the spherules. This can be seen very clearly by
focusing on the surface of an egg that 1s acquiring cilia with a
2 mm. oil immersion lens. But there is a very intimate relation,
and one can convince himself after the rate has slackened that the
spherules vibrate in unison with the beat of the cilia.
The intermediate vacuoles appear between the ectoplasm and
the endoplasm soon after the formation of cilia. They are prac-
tically confined to the original ectoplasmic hemisphere, and are
specially well dev eloped nelly © just above the margin of the endo-
plasm, thus in a position corresponding to the prototroch. ‘They
appear to correspond to similar large vacuoles situated internal
to the nucleus in the cells of the exumbrella and particularly of
the prototroch in the larva. But, as they are confined by cell-
walls, they tend to fuse and thus to produce a fluid-filled space
separating ectoplasm and endoplasm (see figures).
The ectoplasmic vacuoles are much smaller and more pedeunie en:
and do not appear until about twenty-four hours from the begin-
ning of the experiment (Fig. 71). They are usually massed in a
particular region, corresponding to their distribution in the tro-
chophore where they are very much more abundant in the exum-
brella. In the ciliated ova of this age, the yellow endoplasm is
much reduced in amount, and the eggs as a rule do not live much
longer.
(6). It is a curious fact, observed also by Scott in his observa-
tions on Amphitrite, that the apical flagella are never found in
these ciliated eggs. ‘This indicates a combination of factors in
the normal development that is seldom or never realized under
the conditions of the experiments. The explanation is probably
as follows: The apical flagella arise at the animal pole, and in
Chetopterus one can frequently see one polar body at least at
their base. This is, however, the place where the endoplasm
comes to the surface; the apical flagella are therefore endoplasmic
Elementary Phenomena of Embryonic Development 239
in their origin. In the overflow of the ectoplasm in the unseg-
mented eggs this exposure of the endoplasm is seldom, or never,
preserved. Thus the formation of the apical flagella would be
prevented.
The study of the cytological phenomena in sections has been
carried only as far as the overflow of the ectoplasm (about eight
hours). Up to this time it is possible to follow the history of the
original substances of the egg consecutively. I have not studied
the later cytological details; the changes that take place in the
structure of the cytoplasm are very profound and complex.
b. Multinucleated Unsegmented Eggs
These are characteristic of cultures of fertilized eggs so treated
as to inhibit the process of cleavage or to induce fusion of blasto-
meres. [here is a strong tendency towards polyspermy, if eggs are
allowed to stand for several hours in sea-water before being fer-
tilized. Such eggs may break up at the first division into a con-
siderable number of blastomeres, and, in many such cases, the
blastomeres soon fuse together, and cleavage planes do not
again appear. The protoplasm of the egg of Chetopterus is
extremely susceptible to external conditions, and one of the most
common evidences of this is the fusion of blastomeres.
In Experiment 7.B., 1904, for instance, the eggs were taken from
the female at 9 A. M., and were allowed to stand in sea-water until
11.42, when they were fertilized; a series of eggs was preserved and
from them entire mounts and sections were prepared. In the
entire mounts of eggs preserved twelve minutes after the addition
of the sperm it can readily be seen that there are from three to
twelve or more sperm nuclei in each egg. These were also seen
in the living eggs under pressure. [he polar bodies were formed
normally in about twenty-five minutes. Figs. 72 to 75 show the
form changes of a single egg, from 12.32 to 12.47 P. M. At first
this egg became so lobulated that it seemed as though it were divid-
ing into about ten cells at once; then these lobes gradually dis-
appeared and the eggs returned to their spherical condition.
At 12.54 the great majority of the eggs had returned to the spher-
240 Frank R. Lillie
ical condition; about 5 per cent appeared like normal two-celled
stages; these were probably monospermic.
In Experiment 7.A. some of the same eggs were fertilized at
10.35 A. M., having remained in the sea-water one hour and thirty-
five minutes before fertilization. About 60.per cent of these
segmented fairly normally, the blastomeres fused together in the
remainder “Intermediate conditions were rare or absent.”
(Quoted from notes.)
‘Akal
Vo
Fie. 72 Fie. 73
Mii | | jie
|
Wana DD
i
|
Fic. 74 Fic. 75
Figs. 72 to 75. Four views of the same polyspermic egg. The eggs were put in sea-water at 9
A. M. and allowed to stand until 11.42 a. M. when they were fertilized. Fig. 72 was drawn at 12.32
p. M.; Fig. 73 at 12.37; Fig. 74 at 12.44; Fig. 75 at 12.47. At 12.50 the egg was perfectly spherical
again and showed no sign of cleavage.
I expect to make a special study of the polyspermy, for which
I have the preparations. Soin this place I shall consider only
a few points. In the polyspermic eggs multipolar spindles arise,
and a very uneven distribution of the chromatin results. ‘This
is followed by the formation of nuclei of very unequal size, and
in the following divisions the mitoses are irregular. ‘Thus there
=
Se, = ae
es
Elementary Phenomena of Embryonic Development 241
is an enormous increase of the chromatin, and-soon the most of
the nuclei cease to be separate in the resting condition, and form
a vast reticulum situated between the ectoplasm and endoplasm
in the upper half or two-thirds of the egg (Fig. 76). A few
small nuclei may remain separate. In the ensuing periods of
division the individual karyokinetic figures cannot be observed,
and there is a veritable riot of centrosomes, chromosomes, spindle
fibers and astral radiations.
Fig. 76. Optical section of whole mount; multinucleated unsegmented egg, preserved five and
one-half hours after fertilization. The nuclei formed an extensive reticulum; the apparently separate
nuclei of the figure are the optical sections of the broad strands of the nuclear reticulum.
During this period the ectoplasm becomes very thick at the
animal pole, and the endoplasm forms a ball that is attached to
the surface at the vegetative pole by the substance of the polar
lobe (polar ectoplasm) (Figs. 77 and 78).
Thus it will be seen that the presence of numerous nuclei or of
an extended nuclear reticulum, situated between ectoplasm and endo-
plasm, results in a different arrangement of the ectoplasm and endo-
242 Frank R. Lillie
plasm from that found in the uninucleated ova where the nucleus 1s
central in position. In the multinucleated ova the polar distribution
of substances of the ovum does not differ essentially from the normal.
It appears to follow that the curious modes of aggregation of the
ectoplasm and the endoplasm in the uninucleated ova are due, in the
first place, to the lack of the restraining influence of numerous nuclet,
and, 1n the second place, to the fact that, when nuclear influence 1s
established by the enlargement of the nucleus, it proceeds from a
single center in place of many.
yy tit
9009900006
Fic. 77
Fig. 77. Multinucleated unsegmented egg drawn from life four hours and ten minutes after fer-
tilization. The eggshad been allowed to stand in sea-water one hour and fifty minutes before fertilizing.
Fig. 78. Multinucleated unsegmented egg drawn from life eight hours after fertilization. From
a culture in which the eggs had been centrifuged one and one-half hours after fertilization.
The maintenance of the normal distribution of ectoplasm and
endoplasm in the unsegmented multinucleated ova results in a
very perfect homology of the ciliated unsegmented ova with the
trochophore. A few examples may illustrate this. Fig. 77 is
from a living egg, six hours old. The polar globules show the
position of the animal pole; the ectoplasm, endoplasm and nuclei
(appearing almost like vacuoles in the living egg) have the posi-
tions described for Fig. 76. At the pole of the egg nearly opposite
to the polar globules is seen a mass of spherular protoplasm, that
corresponds exactly in position and appearance to the mesoblast
cells in the normal embryos, which can very readily be seen in the
living condition. ‘Thus we can readily homologize ectoplasm,
Elementar yy Phenomena of Embr yonic Der elopme nt 24.3
endoplasm and mesoblast substance with those of the normal
larva. Moreover, they have the same positions with relation to
one another and to the polarity of the egg as in the normal embryo.
The main difference is that the absence of cell-walls permits all
of the endoplasm to ageregate in a single mass; whereas in the
normal larva it is divided between the cells of the ectoderm, ento-
derm and mesoderm, though occupying in each kind of cell a
position next the segmentation cavity.
In some cases the substance of the polar lobe is partly separated
from the yolk-mass proper (Fig. 78). ‘This affords a fine demon-
stration of the existence of such a substance at the lower end of
the yolk-mass. The presence of this substance is perfectly
characteristic. It is very adhesive, as union by it of two or three
ova is very common.
An interesting addition to the information concerning such un-
segmented Eated eggs 1s given by staining intra vitam with
Reueral red. It is then found that there is an Aecumulices of red
granules in the upper hemisphere that correspond precisely in
position and staining reaction with those of the trochophore
(see Part IV, d). The only difference is, that in the unseg-
mented egg they are more massed, a condition naturally resulting
from the absence of cell-walls.
For completeness of demonstration of the principle of germinal
localization these differentiated, and yet wholly unsegmented, ova
leave little to be desired.
c. Literature and Discussion
Differentiation of entirely unsegmented eggs has been observed
in two other genera of polycheeta, in Podaee by Treadwell (’02)
and in Amphitrite by Scott (03 and ’o6). Bullot (’04) failed to
observe it in Ophelia, though he looked for it, and he therefore
cast some doubt upon its occurrence. I would only point out
that the occurrence of segmented ciliated embryos in his cultures
by no means demonstrates the absence of the phenomenon of
differentiation without segmentation; for Treadwell, Scott and |
have all observed the occurrence of ciliated ova both segmented
and unsegmented in the same culture-dish. Bullot’s figures show
244 Frank R. Lille
that, in the unfertilized cultures of Ophelia eggs, segmentation
was seldom or never, even approximately, normal.
It is clear from the descriptions of these three authors that the
occurrence of a certain amount of segmentation 1s more common
under the conditions of the experiment in the forms that they
described than it is in Chzetopterus, which seems to be peculiarly
favorable for the observation of differentiation without any
cleavage.
To this list of observations may be added, in all probability,
those of Bastian (’05) on a peculiar form of differentiation in
Rotifer eggs. Indeed, unless one is willing to adopt Bastian’s
explanation that particular species of ciliates may arise by direct
metamorphosis of rotifer eggs, no other explanation is possible
than that he has observed cases of differentiation without cleavage.
His peculiar interpretation so colors the description of the observa-
tions, that it is difficult to utilizethem. But it is probable from the
figures that in some cases the eggs form cilia without any cleavage,
and that, in other cases, the eggs segment or break into several
smaller parts, each of which then differentiates farther without
cleavage.
Scott summarizes his results on the differentiation of unfertilized
eggs of Amphitrite as follows: “ Under the conditions of the experi-
ments certain forms of development occur with or without cleav-
age and with or without the formation of polar bodies in the unfer-
tilized egg of Amphitrite. Such development takes the form of
nuckear divisions, the early differentiation of a layer of ectoplasm,
the growth of cilia, the appearance of vacuoles that are found in
the fertilized egg of the same age, the development of a brownish
pigment, the ameboid movements of the cytoplasm that are con-
nected with cleavage, the ameboid movements at a later stage of
development that appear entirely independent of cleavage, and
the change in shape of the egg, which in most cases at least is
connected with incomplete, arrested or abortive division of cyto-
plasm. The apical tuft of cilia which is characteristic of trocho-
phores from fertilized eggs is always absent.”
Scott justly emphasizes in his theoretical considerations “the
int-mate relation that exists between cytoplasmic and nuclear
Elementary Phenomena of Embryonic Development 24.5
differentiation; the correlation in development between these two
factors is very complete where a normal organism results. Inas-
much as the cessation of development is a cumulative process,
that is the abnormalities appear in successive transformations of
the asters and nucleus, we must look upon the cessation of develop-
ment as due to incomplete reactions between nucleus and cyto-
plasm.”
Treadwell (’02) observed that ciliated embryos might arise in
Podarke without cleavage, and that the differentiation might be
carried very far in such cases. One case that he describes 1s par-
ticularly interesting (Fig. 12 of Treadwell); two eggs are fused to-
gether and are entirely unsegmented. ‘Around one portion of
the fused mass is a ring of cilia, occupying very much the position,
with respect to the cell, of the prototroch in its relation to the
trochophore. Not only are the cilia present, but around the em-
bryo, underneath the ciliated band, is an area free from the ordi-
nary pigment of the rest of the cell, but containing granules of
a faint yellow color, agreeing in this respect Sein with the
prototroch band of the signin egnaglnone In this embryo there
is, then, not merely a differentiation—without cleavage—of cilia,
but of the characteristic protoplasm accompanying these cilia.
Or, in other words, we have here in the unsegmented embryo,
not merely a differentiation of cilia, but a differentiation of proto-
troch cilta.”
The possibility of a considerable amount of embryonal differen-
tiation without either nuclear or cytoplasmic division may be
considered established. ‘This in itself is a fact of considerable
importance, for it disposes effectually of all theories of develop-
ment that make the process of cell-division the primary factor of
embryonal differentiation, whether in the form of Weismann’s
qualitative nuclear division, or of Hertwig’s cellular interaction
theory. Further, the phenomenon establishes firmly, as I pointed
out in 1g0T, the view that the role of cell-division in development
is primarily a process of localization. This view is a fundamental
part of the doctrine of formative stuffs and is held as such by
Wilson, Conklin and others. It may of course be variously
elaborated, according to whether it is held, for instance, that cell-
246 Frank R. Lillte
walls are impermeable partitions, as Conklin believes (O05, p2102),
or may permit passage of materials through (e. g., by perforation)
as Vejdovsky appears to have observed. ‘The meaning that is
given to the term “‘cell-division’”’ would be important in elabora-
ting the theory; for myself I had meant to include in it only the
processes of division of the chromosomes, separation of daughter
nuclei and the origin of the cell-wall between the daughter nuclei;
movements of the substances within the cell, for instance, would
be excluded. It seems to me, however, that the elaboration of a
theory concerning the role of cell-division in development has
to meet with considerable difficulties. I would agree, in general
with Conklin, that the localization pattern is something more
fundamental and constant than the cleavage pattern; yet the
fixity of the latter in all ova of the determinate type of cleavage
is a marvelous thing, and cannot be fully explained by any mosaic
principle (see General Discussion, p. 254).
V. GENERAL DISCUSSION
Embryological and cytological study has advanced beyond the
stage of opinion represented by current theories of development.
It is generally agreed that the complex phenomena cannot be re-
duced to the operation of any single factor, and many possible
factors have already been more or less thoroughly discussed, but
there is no agreement as to the relative importance of the various
accepted factors, nor as to their relations to one another in the
various embryonic processes.
I. Relation of Nucleus and Cytoplasm
There can be no doubt that the fundamental morphological
composition of the protoplasm of the egg of Chatopterus corre-
sponds to the terms of a granule theory and not to those of a filar,
reticular or emulsion theory. That a threadlike, reticular or
alveolar formation may arise in such protoplasm is unquestionable,
but such conditions are secondary ones, due to particular modes of
ageregation of the elementary constituents. It is not necessary in
adopting a granule theory to go as far as deVries and Altmann
Elementary Phenomena of Embr yonic Development 24.7
and assume that the granules (pangens of deVries) are the only
living elements in ‘ne cell, like the bacteria in a zooglcea. The
microsomes appear to be the primary living elements of the cyto-
plasm; I would not venture to assert that thes are the only living
elements.!
Similarly there can be little doubt that the larger granules, the
spherules, are products of the microsomes. ‘The origin of some
of these from aggregations or growth of microsome-like bodies
could be slainise eed by intra vitam staining; moreover, the
spherules follow the microsomes in time of origin in the ovogenesis.
Again there can be no doubt that a very large proportion, at
least, of the microsomes are of nuclear origin. A great many
occur in the residual substance of the germinal vesicle, and, in the
history of the uninucleated unsegmented eggs, swarms of micro-
somes can be seen to proceed from disintegrating chromosomes
just prior to the period of differentiation.
Thus my conclusions correspond to certain of the terms of
deVries’ theory of intracellular pangenesis (1889), provided that
we identify the microsomes with deVries’ pangens. ‘This theory
involves the following assumptions:
1 That the entire living protoplasm is composed of pangens, which consti-
tute the only living element in it. The pangens are invisibly small.
Now the microsomes are not invisibly small, and I regard them
only as the primary living elements; not as the sole living
elements.
2. There are as many different kinds of pangens as there are independently
variable characters, or independent “factors” composing the complex of the char-
acters of the species.
The only cytological evidences that the microsomes are of
different kinds are (a) that they produce different kinds of spher-
ules, and (>) that they proceed from nine different sources in
Chzetopterus, viz: the nine chromosomes of the egg-nucleus.
3. All the various kinds of pangens of a species occur in the nucleus, and
those existing in the cytoplasm come from the nucleus.
1 Indeed a large part of the discussion as to what elements of the cell are living and what are not
living seems to me to be purely academic, and likely to remain so, until we possess much more
satisfactory knowledge of the mechanics of the vital processes.
248 Frank R. Lillie
This agrees with my opinion concerning the microsomes of
Cheetopterus.
4.'. Uhe cytoplasm contains essentially only those kinds of pangens that enter
into activity init. Thus in each variety of cell the immerse majority of the
pangens remain inactive in the nucleus, and only those leave the nucleus and
enter into activity in the cytoplasm chat represent the specific cell characters to
be expressed.
On the cytological side there is no evidence to correspond to this
idea. On the contrary all the chromosomes appear to be active
in each cell.
5. Pangens multiply both in the nucleus and also in the cytoplasm.
This is certainly the case as regards microsomes in the nucleus,
and possibly also in the cytoplasm.
The theory of intra-cellular pangenesis has anticipated cer-
tain observations that may be made concerning the elementary
phenomena of development. But it is defective in two important
respects: (I) it assumes a degree of original diversity, and certain
modes of behavior of the pangens (such as the inactivity of the
vast majority in each species of cell) that find no justification in
our cytological knowledge, but only in alleged theoretical neces-
sit es; (2) it provides no explanation of an essential part of embry-
onic development, viz: the spatial arrangement of organs and
their sequence in time, in short, the unity of the organism by which
alone is the possibility of self-sustenance guaranteed. Weis-
mann’s theory of germ-plasm proceeds yet farther in unwarranted
assumptions as to he original complexity of the germ-plasm, but
includes an explanation or the spatial arrangement of organs and
their sequence in time, by providing for these factors in the
architecture of the germ-plasm.
2 The Original Diversity of Organization
All theories postulate a certain original diversity or complexity
of organization as the starting point in embryonic development.
Now we must inquire what we mean by original? For some the
fertilized ovum constitutes the starting point; but it is clear, on
consideration, that it is relatively far removed from the actual
Elementary Phenomena of Embryonic Development 249
origin. Only that can be original which is continuous through the
series of generations, viz: the germ-plasm in Weismann’s termin-
ology, which we identify with the chromosome group. The
entire history of the ovogenesis, as Wilson has repeatedly pointed
out, forms part of the embryonic development. ‘The original
diversity of organization is, therefore, contained in the specific
chromosome group, which observation has shown to be transmitted
from generation to generation.
The next question is as to the degree of original diversity within
this chromosome complex. I would maintain that the estima-
tion of this should rest primarily on the observable facts and not
on a mental projection to the lower plane of germ-plasm of the
complexity of the higher plane of the adult organization. ‘The
visible diversity of the chromosome complex is usually only quan-
titative, that is, it consists of a definite number of parts, the in-
dividual chromosomes. In some cases qualitative differences are
also observable, for the individual chromosomes may differ in size
and behavior (Montgomery ’o1, Sutton ’02, Wilson ’05); Boveri
(02) has shown also for the echinids, by some ingenious experi-
ments that, though all the chromosomes are alike morphologi-
cally, the individual chromosomes are probably non-equivalent
physiologically. There is, therefore, good reason for believing
that there are at least as many different kinds of original substances
in the germ-plasm as there are chromosomes. Considerations as
to valency of chromosomes may, perhaps in the future, tend to
equate differences in the number of chromosomes in different
species; recent observations of McClung (’05) have furnished
some arguments along this line. To maintain that there are no
more original germinal substances than there are actual unit
chromosomes may, perhaps, be too extreme a position; but it
seems to me sounder by far and likely to prove more fruitful as
a working hypothesis than the assumption that the germ-plasm
is a microcosm of “determinants” of all the characters of the
species.
I do not believe that any considerations as to the potencies of
the germ-plasm are valid as arguments for the amount of the
original diversity, because, if the validity of such arguments be
250 Frank R. Lillie
recognized, there remains no standard but the arbitrary judgment
of the individual. Apart from the a prior: difficulty of accounting
for the phenomena of heredity there is no reason for assuming the
existence of a large number of original germinal qualities. But,
seeing that any species 1s as distinct from other species in thestage
of germ-plasm as in the adult condition, the original germinal
qualities, whatever they may be, must bear the stamp of the species.
At present we have no accurate means of estimating the degree
or nature of the differences between the chromosomes, but I believe
that certain statements may be made about them that follow
logically from our present knowledge. In the first place, the
differences cannot correspond to the differences between organs
or regions, either of the embryo or adult, because the doctrine of
the individuality of the chromosomes teaches that each cell receives
a descendent of each chromosome. ‘The whole economy of nature
forbids us to believe that each cell possesses arm, leg, brain, liver,
lung, etc., chromosomes, of which only one* class enters into
activity in any given tissue, the remainder lying idle. The fact of
the uniform distribution of all chromosomes to all tissues proves
conclusively, either that all chromosomes are alike, or that each
represents some character of the entire organism. As we have
accepted the view that they are originally unlike, we must adopt the
second alternative.
The only observations that we have connecting a particular
chromosome with a particular set of characters are those of
McClung (’02) and Wilson (’05), according to which the accessory
or idiochromosome is a sex-determinant. Now sex is preémi-
nently a character of the entire organism. My hypothesis is, that
each chromosome represents some such character or group of
characters. It is difficult to imagine what such characters may
be; we need a new morphology for their enumeration, and it is
to be hoped that this will come from the breeders’ experiments;
for the only clue that we have to the relation between chromosome-
characters and species-characters, consists in the parallelism
between the reduction-phenomena in the germ-cells and the
Mendelian ratios in inheritance. We might hope, therefore, to
get at the nature of chromosome-characters by an enumeration
Elementary Phenomena of Embryonic Development 251
of the various kinds of characters that are inherited in Mendelian
proportions. To attempt this in detail would be too great a task,
but they include such characters as color (e. g., inheritance of
albinism, or green and yellow endosperm of peas, etc.), stature,
pubescence, etc., that are not special characters of particular
organs but of the whole organism.
We must be careful, however, to avoid a pitfall here in assuming
that there is any resemblance between species-characters and
chromosome-characters. [here is at most only correspondence
due to genetic connection; and any imaginable degree of knowl-
edge of the unit species-characters would not furnsh a_ particle
of information as to the nature of the original unit germinal
characters.
3. Properties of the Whole (Principle of Unity)
If the first step in any theory of embryonic development must
be certain postulates concerning the original diversity of organiza-
tion, the second step must be an explanation of the physiologi-
cal unity of the organism in all stages of its development; for the
two main facts concerning any organism are that its parts are
diverse, and that it is, nevertheless, a physiological unit. The
traditional view, held by many embryologists at the present day,
is that the physiological unity arises in the course of embryonic
development by the secondary adaptation of originally inde-
pendent parts to one another. But this explanation has, in my
opinion, become untenable, and must be replaced by the view
that there are certain properties of the whole, constituting a prin-
ciple of unity of organization, that are part of the original inherit-
ance, and thus continuous through the cycles of the generations, and
do not arise anew in each (compare Lillie, ’or, p2 275) Weis=
mann places this principle of unity of organization in the archi-
tecture of the germ-plasm, but, as [ cannot accept his view of vast
complexity of the germ-plasm, neither can I accept this princi-_
ple in the sense of Weismann. My own views agree most nearly
with those expressed by Whitman in his paper on the Inadequacy
of the Cellular Theory of Development (’93). In this paper
252 Frank R. Lillie
Whitman uses the term organization to express what I have termed
above, properties of the whole or principle of unity.
If any radical conclusion from the immense amount of investiga-
tion of the elementary phenomena of development be justified,
this is: that the cells are subordinate to the organism, which pro-
duces them, and makes them large or small, of a slow or rapid rate
of division, causes them to divide, now in this direction, now in
that, and in all respects so disposes them that the latent being
comes to full expression. We see this in the adaptiveness of the
process of cleavage of the ovum (Lillie ’95, ’99; Conklin ’96-’97;
Meisenhemier ’99), in the regeneration of a starving planarian
constantly suffering a diminution in the number of its cells while
its structure 1s increasing in complexity (Lillie ’0o, Schultz ’o4),
in “regulation,” and in all cases of morphallaxis (Morgan ’oo),
whether in a protozoan or a metazoan. ‘The organism is primary,
not secondary; it is an individual, not by virtue of the cooperation
of countless lesser individualities, but an individual that produces
these lesser individualities on which its full expression depends:
The persistence of organization is a primary law of embryonic
development.
I believe that this conclusion is strongly reinforced by my
observations on differentiation without cleavage; for here we see
the various substances of the ovum marshalled in order, disposed
in a bilateral arrangement and fashioned in the form of a larva;
and we see the cilia and other cell-constituents arise in the appro- .
priate locations—and all this without the need of even a single
nuclear division. ‘The question arises whether these phenomena
could not be explained by assuming appropriate attractions and
repulsions between the elements of the different classes of sub-
stances, the spherules and microsomes. Although such attrac-
tions and repulsions undoubtedly exist, and although they appear
to me to constitute an important elementary morphogenic factor,
yet I find the assumption inadequate to explain the orderly
arrangement of the processes collectively. It seemed at first that
the polarity of the ovum might be explained by assuming that
the arrangement of endoplasmic substances was produced by mutual
attractions and repulsions; but it was found that no alteration of
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Elementary Phenomena of Embryonic Development 253
the arrangement of the endoplasmic substances modified the
direction of polarity. Similarly the bilateral polarization of the
first cleavage spindle and many aspects of the later cleavages
appear to be independent of any chance arrangement of endo-
plasmic spherules.
Thus there is an apparent inversion in the sequence of embry-
onic phenomena, by virtue of which those characters that we
would expect to appear late, such as the general form and pro-
portions of the embryo, manifest themselves first, and thus lend
to the subsequent phenomena an adaptive aspect; as though that
which was to be explained preceded the phenomena that could
alone account for it. Jt 1s obvious, however, that the adaptiveness
of development does not constitute an explanation, but 1s, on the
contrary, itself one of the chief phenomena to be explained.
The principle of unity transcends all forms of visible diversity
hitherto observed; it is a property of the whole distinct from the
discernible properties of the parts. Undoubtedly it is capable of
further analysis, and it must ultimately be derived from particular
relations and properties of material particles. In embryonic
development it reveals itself first by axial polarization, second by
bilateral polarization and determination of the localization pattern,
third by adaptation in cleavage, etc. Analysis of some of these
phenomena may some day give a clue to this most mysterious of
embryological phenomena.
Morgan (04) has attempted an “Analysis of the Phenomena
of Organic ‘Polarity,’”’ based on the phenomena of regeneration.
He concludes that “by means of three assumptions—of toti-
potence, of heterotropy and of organization-power—we can
explain the main features in the result. Each assumption is,
moreover, a direct deduction from an experiment or observation.”’
In a footnote he adds, that “the same explanation applies to the
development of the egg.”’ By “‘heterotropy”’ he means that “the
material,” though totipotent “‘is somewhat different at every level,
and that this difference corresponds in kind to the character of
the body at each level.” The “organization power,” as I un-
derstand it, is the same as the “centripetal influence’? which,
“acting from the surface inward, determines the organization of
254 Frank R. Litlie
the new parts. ‘The action of this centripetal influence is on the
new parts as a whole, and determines the relative location of
each organ.” Later on he suggests that “for want of a better
term, we may provisionally call the property of living material to
assume a specific form, the property of formative organization.”
It is clear that Morgan here offers an analysis of much more
than is generally included under the term polarity; indeed he
offers an analysis of regeneration and embryonic development
asawhole. It is interesting to note that he defines the factor of
“formative organization’’ so as to be similar to what Whitman
calls organization simply, and what I have called influence of
the whole. It would be essentially the same idea, if it included
heterotropy also, and the latter appears to me to be subordi-
nate to the former principle.
4. The Mosaic Theory of Development
The mosaic theory of development of Roux, excepting that
inessential part concerning qualitative nuclear analysis, has been
strongly supported by the facts of cell-lineage and the recent
work of Boveri (ota), Fischel (03), Wilson (04a and b),
Conklin (05a and b), Zeleny (05), Yatsu (04) and others.
Wilson, Conklin and Fischel especially have shown conclusively
that in mollusca, ascidians and ctenophores, the mosaic character
of development is based on the principle of germinal localization
originally enunciated by His. The work of these authors is too
recent and well known to need review here; I only wish to say that
I fully accept the results, and am prepared to abide by the theo-
retical necessities resulting from the assumption that the cleavage
mosaic in Chetopterus is as definite a mosaic of potencies as it is
in Patella, where each cell of the cleavage-mosaic up to the thirty-
two celled stage, or later, differentiates after isolation in sub-
stantially the same manner as when forming part of the whole.
But Wilson (o4b) thinks that this ool is inconsistent with
the conclusion stated by me in 1got, “that the entire organism in
every stage of its development exercises a formative influence on
all of its parts,” although he does not doubt “that this position,
Elementary Phenomena of Embryonic Development 255
with proper qualifications, is well grounded.” He then goes on
to say that “it is clear that the primary localization of formative
stuffs in the unsegmented egg is essentially an act of the “organism
as a whole;”’ and even though a complete preformation and pre-
localization of specific stuffs for every cell and tissue were assumed
—and I believe with Boveri and Fischel that such an assumption is
not necessary or even probable—we should not escape the neces-
sity for assuming such action of the whole.” But in the same
paragraph, while assenting to Whitman’s saying that “organiza-
tion precedes cell-formation and regulates it,” he takes issue with
him on the ground “that the cytoplasmic aggregation or “organ-
ization” is a progressive or epigenetic process.”
As I understand Whitman, “organization”? is what I have
called “action of the organism as a whole;”’ Wilson has either
understood the matter differently, or has forgotten this, when he
identifies “cytoplasmic segregation” and ‘organization.’ Organ-
ization, or action of the organism as a whole, 1s something
that precedes and regulates cytoplasmic localization as much as
it does cell-formation. If this fact were clearly kept in mind I
think that Wilson would find less difference between Whitman’s
views (and mine) and his own, than he suspects; for the same
mistaken identification of organization with cytoplasmic localiza-
tion reappears in the succeeding remarks.
But I do not mean to assert that my views are identical with
those of Wilson even when this allowance is made, for he seems
to believe that the action of the organism as a whole ceases when
once the localization pattern is determined, and that thereafter
It is a question of self-differentiation of independently developing
parts with a certain amount of correlative interaction’ of cells.
1Tt seems necessary to make a special statement of the opinion that I hold concerning the distinc-
tion between “action of the organism as a whole’’ and the principle of correlative differentiation. By
the formerI mean, to use the words of my paper on the organization of the egg of Unio, “‘that the
entire organism in every stage of development exercises a formative influence on all of its parts.””
The principle of correlative differentiation, as I understand it, involves all actions of the intraorganic
environment, as I expressed it in my “Experimental Studies on the Development of the Organ in
the Embryo of the Fowl” (1903), that is, “that the rate, degree or mode of differentation of any
embryonic rudiment is dependent on some part or parts of the same organism external to itself.” It
will be seen that, as thus conceived, there is an important difference between the two principles, and
that the principle of correlative differentiation would not include the principle of action of the
organism as a whole without a considera>dle extension of its usual meaning, which seems to me
undesirable and likely to be confusing.
256 Frank R. Lillie
It appears to me on the contrary, that the action of the organism
as a whole 1s a continuous process; that physiological unity exists
in every stage, and not merely sporadically, no matter to what extent
the mosaic principle may apply. We have to deal with diversity
in unity, and unity in the midst of diversity as the two funda-
mental properties of organisms. ‘The phenomena of regenera-
tion and regulation are incomprehensible on the basis of a
pure mosaic theory of development. Wilson does not fail
to recognize this principle here and there, as in cases of
regulation, but it seems to me that he has not given it sufficient
weight, and has not kept clearly in mind the distinction
between this principle and. the derivative condition of germinal
localization.
I.would like also to give what seems to me the explanation of
Wilson’s conclusion that “cytoplasmic segregation is a progressive
or epigenetic process,” based on the results of Boveri, Yatsu,
Zeleny and himself. In general it may be said that these authors
have found that there is a progressive limitation in the potencies
of parts of the egg from the time of the rupture of the germinal
vesicle to the eight-celled stage or beyond. Now in Chetopterus
there is a visible segregation of substances already present, de-
scribed in Part III, 2, beginning with the breaking of the germinal
vesicle, and continuing to the third cleavage at least, by means of
which a new germinal topography is produced. Assuming that
these substances have limited potencies, as has been demon-
strated for similar substances in other eggs, the redistribution of
them would inevitably bring about such a result as these authors
have described, because the new topography is much more precise
than the original one. The rearrangement is an “epigenet-c
process,’ if you please; but only the topography is new, not the
substances. ‘There is a great deal of evidence in the literature
that a similar redistribution is characteristic of the maturation
period in most phyla. On the other hand I believe that a true
epigenetic process begins with the first cleavage in the production
of new substances from the nuclei, and undoubtedly plays a part
in the progressive limitation of potency of the blastomeres; but,
I believe, only a small part at first. (Lo avoid misunderstanding,
Elementary Phenomena of Embryonic Development 257
I would state that it is my view that the specific formative stuffs
of the later development arise after the process of cleavage has
begun.)
Differences in time of origin of formative stuffs no doubt exist
in different kinds of eggs, baie I do not believe that the differences
are so extreme as some would appear to think. Determinateness
of cleavage may be a measure of the extent and precision of their
localization prior to cleavage, but I think it must be a very inexact
measure. I fully agree with Whitman that “cell-orientation may
enable us to infer organization, but to regard it as a measure of
organization is a serious error;’’ however, one must keep in mind
that “organization” precedes and controls “localization,” and
not confuse the two terms. When this is done I can also agree
with Wilson that “‘a highly differentiated initial cleavage pattern
is, therefore, ipso facto evidence” (in some degree), “of a high
degree of initial cytoplasmic /ocalization”’ (last italics mine).
5. Concerning Formative Stuffs
Much has been written of late concerning formative stuffs.’
(Morphoplasmic stuffs of Wilson, organ-forming substances of
various authors.) Unfortunately their actual physical character-
istics have been but little examined; indeed, in most cases the so-
called organ-forming substances are really germinal areas prob-
ably including a variety of substances, and distinguishable only
by their localization and by the useful but superficial character
of color. As I have attempted to show, the specific character of
the substances in Chztopterus is given by the spherules. It is
certainly reasonable to expect that formative stuffs in other ova
may be differentiated by microchemical methods. Previous
observations on this point have been more or less incidental, so
that generalization would not be profitable.
According to the theory of formative stuffs developed in the
preceding Pages, a series of stages characterizes each kind before
it reaches its definitive croleeien condition. First, its origin,
1These “formative stuffs’? do not conform to the older conceptions of Sachs, who postulated cir-
culating fluids of formative function. They are supposed to be varieties of protoplasm that probably
do not circulate exte sively from cell to cell.
258 Frank R. Lillie
in the form of microsomes, from the nucleus; second, the forma-
tion of granules of a different order from the microsomes, the
spherules; third, the addition of new nuclear derivatives. (The
third stage is necessary for there is no final stage of differentiation
in non- maeleed parts; in the uninucleated ceeomeared eggs the
third stage 1s clearly marked morphologically by the diffusion of
chromatin particles among the spherules.) Fourth, definitive
histogenesis (the morphogenic reaction).
The theory of formative stuffs doesaway with any ‘“‘determinant”’
hypothesis. “Characters” are not due to “unfolding” of the
“potencies”? of “determinants”? but are results of morphogenic
reactions between two or more formative stuffs. “The “character”
need no more be preformed in the reagents (formative stuffs) in
the case of a morphogenic than in the case of a chemical reaction.
But I do not mean to imply that the morphogenic reaction is a
simple chemical reaction, nor that, after it has taken place, the
character need appear at once in its definitive form. ‘The mor-
phogenic reaction is probably often of the nature of a response to a
stimulus, a phenomenon of irritability, and the definitive “char-
acter’’ resulting may come to expression slowly.
6. Nuclear Specification
The logical consequences of the preceding conclusions cannot
be avoided: it 1s clear that the nuclei cannot produce the same
kinds of formative stuffs in successive stages of ontogenesis, unless
we assume that the organism as a whole manufactures different
things out of the same substances at different times and in different
places. ‘This appears to be so improbable an assumption that
there is no escape from the conclusion that the nuclet undergo pro-
gressive differentiation; in other words, that each successive onto-
genetic stage is preceded by a corresponding nuclear phase.
The soealble modes of nuclear specification are: (1) that different
chromosomes represent successive ontogenetic phases, and that,
therefore, in any given phase the majority are inactive. The
objections to this view appear to me to be fatal; for, in the first
place, there is not a sufhcient number of chromosomes to satisfy
Elementary Phenomena of Embryonic Development 259
the conditions of the hypothesis; and in the second place, there are
grave objections to the hypothesis of inactive chromosomes in any
stage of development. ‘This is, however, substant ally the view
that Wilson presents (’05, p. 292). (2) The second possibility
would be that all chromosomes are active in the various stages of
ontogenesis, but that only a part of each is active at any given
stage. In other words, that each chromosome contains the deter-
minants of all stages, as Weismann supposes, and that these enter
into activity successively. This would, however, presuppose an
enormous amount of original diversity, an idea that we have
already specifically rejected, besides being open to the general
objection that the major part of each nucleus would necessarily be
supposed to be inactive. (3) Both the foregoing are essen-
tially preformation hypotheses. ‘The third possibility is that the
postulated progressive development of nuclei is essentially an
epigenetic process.
It appears to me that all the well-established physiological and
embryological data point toward this conclusion. Yet, from
Nageli down, nearly every writer on the subject of heredity postu-
lates a degree of original preformation in the idioplasm_ or
nucleus, corresponding to the amount of morphogenic activity
supposed to be exercised by it in all stages of the life history. Ac-
cording to Nageli “every perceptible character is represented in the
idioplasm by a rudiment” (Abstammungslehre, 1884, p. 23). Ac-
cording to deVries it is necessary to assume original specific pangens
for every heritable property (Intracellular Pangenesis, 1889). Weis-
mann postulates a determinant for every independently varying
part. The embryologists have usually not entered into this ques-
tion; but Wilson has gone so far as to state (05a) “that the germ
consists of two elements, one of which undergoes a development
that is essentially epigenetic, while the other represents an original
controlling and determining element. ‘The first is represented by
the protoplasm of the egg. ‘The second is the nucleus, which, as
I have attempted to show, must apparently be conceived as a kind
of microcosm or original preformation, consisting of elements
which correspond, each for each, to particular parts or characters
of the future organism.”
260 Frank R. Lillie
According to these writers, therefore, all the characters that are
ever to be impressed by the nucleus on the cytoplasm are repre-
sented by original preformations in the nucleus. Such a conclu-
sion appears to me to be practically a negation of the evidences of
our senses. If such a degree of original diversity is really pre-
formed in the chromosome-complex, it 1s inconceivable that it
should not reveal itself to some one of our senses by variety of
behavior or reaction. Moreover, there is not room in the known
laws of chemical combination for such diversity of substances
within the chromatin of a species as these preformation hypotheses
require. It seems to me that all a priori considerations should
be ruled out of court, unless we are willing to transform
biology into a branch of metaphysics dealing with potencies and
latencies.
If nuclear specification is to be considered an epigenetic process,
the causes thereof may be conceived either to be in the environ-
ment of the nucleus, viz: the body of the cell, or to lie within the
nucleus itself. In the former case we should have to assume that
the locations of nuclei in different parts of the original germinal
topography may act as stimuli on the nuclei to differentiate them
in various directions. ‘The original cause of cytoplasmic diver-
sity has been traced back to the nucleus, but I do not think that it
is necessarily illogical to reverse the order and assume that the
cytoplasmic diversity may be a cause of new nuclear diversity.
On the contrary it is a widespread biological phenomenon that
secretions of the organism react on the organism itself in various
ways.
On the other hand I cannot avoid the conclusion that the pro-
gressive development of nuclei is to a great extent a process of self-
differentiation. ‘here are certain unavoidable corollaries of the
argument bearing on the question. If descendants of each orig-
inal chromosome are transmitted to each cell of the organism, and
if each chromosome, therefore, represents some character of the
entire organism, as we concluded before, it must follow that each
has a series of forms of expression suitable to the successive onto-
genetic stages. Thus, if chromosome x, for instance, expresses
itself by pigmentation in the adult condition, it must have had
Elementary Phenomena of Embryonic Development 261
some different form of expression in stages prior to the appearance
of pigment. There is, in other words, a correspondence between
the mode of expression of a chromosome, and the ontogenetic
stage reached by the entire organism. There must, therefore,
be a progressive evolution of the chromosomes of the same general
character in all cells; but this need not exclude local specialization
of the nucle also.
The differentiation of any particular cell would therefore be the
result of an interaction between a specific formative stuff or stuffs,
inherited from previous generations of cells, and a new
material derived from the nucleus. Variation in either factor
would give a different result. ‘Thus, for instance, all cells of the
embryonic epidermis might be supposed to contain similar stufts
originally; their special lines of differentiation would then be
dependent on the nature of the final nuclear stuff. If, now, we
suppose that this may vary with the different external conditions,
the line of differentiation would vary with the latter..
To take a specific instance, we may explain Lewis’ (’04) inter-
esting discovery that a lens may arise in tadpoles from any part
of the embryonic epidermis that is brought into suitable relations
to the optic vesicle, by the hypothesis that the stimulus of the
optic vesicle causes a different kind of nuclear secretion in the
epidermal cells acted on than in others. In this case the postu-
lated nuclear differentiation would result from the environment.
If such a case may be considered typical, we might perhaps
generalize by saying that the progressive evolution of chromosomes
common to all cells is a process of self-differentiation, but that
local specifications may result from action of the environment.
According to this conception, therefore, there is an orthogenic
and epigenetic progressive development or evolution of the somatic
nuclei during the development of the individual, that is common to
them all, and in addition local specifications characteristic of parti-
cular regions and organs. ‘The latter are subordinate modifications
within the limits set by the ontogenic stage reached in the evolu-
tion of the somatic nuclei of the individual. “There would also be
involved in the general conception a theory of continuity of the
germ-plasm, similar to Weismann’s, viz: that the nuclei of the
ZOD Frank R. Lillie
“Keimbahn” undergo neither evolution nor specification, except
such evolution as may be phylogenic in its character.
Without developing the idea any farther in this place I think it
will be seen that the view is not inconsistent with the biogenetic
law, and that it may be made part of a larger theory of phylogenic
development. It seems to me that some such ideas as these
result logically from our present cytological knowledge and, indeed
they have arisen in my mind in the attempt to interpret current
cytological conceptions. ‘They are presented in no dogmatic
spirit, but in the hope of stimulating discussion.
7: Summary of Discussion
The main points of the discussion may be summarized thus:
(1) The chromosome group of the species contains the total
sum of the material transmitted from one generation to another.
(2) [he microsomes arise from the chromosomes and constitute
the primary cytoplasmic element. They produce the various
formative stuffs.
(3) The original diversity, by which I mean the actual degree
of heterogeneity of the chromosome group, is probably relatively
slight.
(4) There is an original principle of unity, action of the organ-
ism as a whole, which expresses itself by axial and bilateral
polarization (thus determining the segregation pattern) by adapta-
tion in cleavage, and probably in various other ways, and which
is continuous from generation to generation. Only its mode of
expression changes and this in accordance with the stage of develop-
ment of the organism. ‘The unity of the organism does not arise
by the secondary process of division of labor.
(5) Apart from the postulated original diversity and the action
of the organism as a whole, the entire development is epigenetic.
(6) Each chromosome probably represents in each stage some
property of the entire organism.
(7) Each ontogenic stage is preceded by a corresponding nuclear
phase; in other words, nuclear evolution is the primary factor in
the determination of embryonic stages.
ae
Elementar 1 Phenomena O Embr YONIC Develo brnent 26
y ; 3
(8) Nuclei probably also undergo local specification as a result
of varying intraorganic environment, and possibly also through
action of the organism as a whole.
(9) By virtue of the two modes of nuclear evolution and specif-
cation, different kinds of formative stuffs arise at successive phases
of the ontogenesis and in different parts of the embryo.
(10) The final histogenesis of any cell depends upon interaction
of the formative stuffs already present in the cytoplasm with the
last formative stuff derived from the nucleus.
(11) The nuclei of the “ Keimbahn” undergo neither evolution
nor specification except such as may be of a phylogenic character.
264 Frank R. Lillie
LITERATURE
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266 Frank R. Lillie
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Elementary Phenomena of Embryonic Development 267
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268 | Frank R. Lillie
DESCRIPTION OF PHOTOGRAPHS.
The author is greatly indebted to Misses Catharine Foot and E. C. Strobell for the photographs
which were taken and printed by them.
All the photographs are from living eggs. The magnification in all is 237 diameters.
All the eggs shown were centrifuged, about 3000 revolutions in one minute, and (with the excep-
tion of A and H) then fertilized. 4 to I inclusive, show the early stages of such eggs up to the first
cleavage, one and one-quarter hours. J, K, L and M were taken from six and one-quarter to seven
hours from the time of fertilization.
The first group illustrates the stratification of the egg substances produced by the centrifuge and
its relation to polarity, the axis of the egg standing vertical in each case (except in Photograph A, where
the axis is not known). The large, dense area seen in each photograph is the massed spherules of the
endoplasm, the smaller is the ‘‘gray cap”’ or residual substance of the germinal vesicle. The clear
band is seen between them. The photographs show the ectoplasmic layer very distinctly, especially,
by contrast, just external to the endoplasmic mass.
Except for the outlines of the polar bodies in B, D, F, G and I, the photographs have been repro-
duced without retouching.
A. Living egg, unfertilized, shortly after centrifuging. The polar area cannot be seen, hence
the relation of the strata to the polarity is not known.
Band C. Two views of one egg with two polar bodies. Centrifuged 8.45 a. mM. Fertilized
9-17 A. M. Photographed forty-eight minutes after fertilization. C is a high focus to show the gray
cap, B a lower focus to show the polar bodies. The plane of stratification is nearly at right angles to
the axis of the egg.
D. Centrifuged 8.45 a. mM. Fertilized 9.17 A. M. Photographed 10.13. M. There are two polar
bodies. The plane of stratification is inclined about go° to the axis of the egg.
E. History same as D. Photographed 10.20 a. mM. There are four polar bodies, a condition
observed only once. Plane of stratification inclined about 45° to the axis of the egg.
F. History similar to D. There are two polar bodies. The polar lobe is beginning to form.
Plane of stratification inclined about 120° to the axis of the egg.
G. Same egg as shown in F. Photographed five minutes later.
H. Centrifuged 9.08 a. m. Unfertilized. Photographed 10.45 a. M. The gray cap surrounds
the maturation spindle and is therefore ring-shaped. During the time that has elapsed since centri-
fuging the gray cap has spread out considerably. ,
I. History similar to D. Two-celled stage nearly complete. Polar lobe is nearly at its height.
Its substance contrasts strongly with the endoplasm. Most of the gray cap is in the smaller cell and
the larger is filled almost entirely with endoplasm.
J. Centrifuged 9.08 a.m. Fertilized immediately. Photographed 3.30 p. M., six hours and twenty
minutes after fertilization. This is a uninucleated unsegmented egg showing the large clear nucleus
and the chromatin halo surrounding it; distorted and flattened by pressure.
K. History same as J. Photographed 4.10 p. M., seven hours after fertilization. The chromatin
halo has risen to the surface where it is mingling with the ectoplasm. Egg distorted by pressure.
L. History same as J. Photographed 3.52 Pp. M., six hours and forty minutes after fertilization.
Egg is flattened by pressure. Three-banded condition. The shadow of the ectoplasmic band may
be seen on the surface of the nucleus.
M. History same as J. Photographed 3.45 p. m., six hours and thirty-five minutes after fertiliza-
tion. Three eggs are shown in one field of the microscope, the one to the left is segmented (abnormal)
the central one is multinucleated unsegmented; the egg to the right is uninucleated unsegmented.
Shown for comparison of the three main conditions found in such cultures.
ELEMENTARY PHENOMENA OF EMBRYONIC DEVELOPMENT IN CHAETOPTERUS
FRANK R. LItLieé
Journat or Experimentar Zoorocy, Vou. U1,
REGENERATION OF GRAFTED PIECES OF
PLANARIANS
BY
LILIAN V. MORGAN
Witnh SEVENTEEN FiGurREsS
Experiments in grafting pieces of Planarians were undertaken in
order to discover whether regeneration of short pieces grafted on
to longer pieces would be influenced by the polarization of the
longer piece. It has been found, in Hydra by Peebles! and by
ate and possibly in Tubularia by Peebles, that long pieces
have such an influence on short pieces.
After repeated attempts in various ways to graft two pieces,
it was found that in some species of flatworms, a fair proportion
of pieces would grow together if held in place between wet pieces
of paper. Thick tissue paper was used, tough enough to stand
the necessary manipulation, and thin enough to be almost instantly
soaked in water, and when wet to cling closely to the pieces of
worm. A small piece of wet paper is first laid on a flat surface of
parafhn (hardened onthe under side of a small dish). As it 1s very
important that the pieces to be grafted should be brought together
immediately after they are cut, a second piece of wet paper 1s
held in readiness, and the whole worms are placed with a camel’s
hair brush on the first paper. The worms are then cut at
the level required, and the pieces are turned in the desired
relation to one another, and quickly covered by the second piece
of paper. If the pieces have not stayed in exactly the right posi-
tion, they can be shifted to a certain extent by pushing with a dull
1Peedles, F., 00. Experiments in Regeneration and in Grafting of Hydrozoa. Archiv f.
Entwickelungsmech., Bd. x.
King, H. D., ’or. Observations and Experiments on Regeneration in Hydra viridis. Archiv f.
Entwickelungsmech., Bd. xiii.
JourNat or Exprrmentar ZodxoGy, VoL. 11, No. 2.
270 Lilian V. Morgan
knife on the upper piece of paper. For a moment they will remain
in place, but in order to keep them quiet for the length of time
necessary for them to grow together, they are held at the sides
by the edges of pieces of cover-slip pressed down on the outside
paper close to the worms. ‘lwo or three fine needles stuck
through the paper into the parafhn at the ends of the worms keep
them from moving apart. The small dishes are placed in a
moist chamber—a large tightly-covered dish with a little water
on the bottom—and kept in the dark. It is all important that the
erafting pieces should be neither too wet nor too dry. They
should be transferred to the paraftin plate with very little water,
and the surplus drops should be drained off the pieces of paper
after they are soaked. If the worms are too wet they do not
stick, and if they are too dry they disintegrate. At the time that
the worms are cut, the tips of both tails are cut off in order that
the pieces shall be as quiet as possible. The grafting pieces are
left in position from eighteen to twenty-four hours. At the end
of that time, after the cover-slips and needles have been removed,
a little water is added to float the upper paper, which is then very
carefully taken off. Since it is dificult to handle very small
pieces, the worms were cut anteriorly at the desired levels, and
grafted. Only after the pieces had grown together was the one
which was to be the short piece in regeneration cut off near the
union to make it the required length.
The method was first worked out with Planaria maculata,
collected at Van Courtland, N. Y., and at Cold Spring Harbor,
L. I. ‘They are apparently the same species but the two forms
have some constant and marked differences. Only ten grafts of
these worms were successful. [he main part of the work was
done with Phagocata gracilis from a fresh water pond at Fal-
mouth, Massachusetts.
A. REVERSED GRAFIS PERFECTLY UNITED
The anterior cut surface of a short piece was grafted to the
anterior cut surface of a longer piece in order to see whether a
reversed head or a normal tail would regenerate at the exposed
posterior cut surface of the short piece.
ee
Regeneration of Grafted Pieces of Planarians 271
©
The worms were cut at three different levels: (1) At an anterior
level a short distance behind the eyes near the region of the head;
(2) at a middle level through the pharyngeal chamber; (3) at a
posterior level behind the pharyngeal chamber through the region
of the tail. All possible combinations (except two) of these levels
were made. In each case, the combinations are designated by the
initials of the regions through which the cuts were made, viz:
head, middle, and tail regions; the capital letter stands for the
longer piece, the small letter for the shorter piece. “Thus, “ Mh”’
indicates that the combination is made up of two worms, one cut
through the pharyngeal chamber in the middle region of the
worm, the other cut behind the eyes through the head region;
the two pieces were joined by their anterior cut-surfaces and
when grown together, the second worm was cut off posteriorly,
leaving only a short piece (h) grafted to a comparatively much
longer piece (M) of the other worm. The results of the experi-
ments are noted in the following table:
TABLE I
Regeneration of short pieces of Phagocata gracilis grafted in a reverse direction on to longer pieces
THe SHort ComMpoNENT PRODUCED AT ITS
Free Posterior SURFACE OTHER Diep
1 1 ee REsuLTS |
i | |
Head Tail | Closed Knob
= ee: ie |
Hh 8 I 5 6 4
Hm == 10 2) 2 =
(Ht) -— -- — _— =
Mh 2 — — — =
Mm Z 2 10 I =
Mt = 3 8 I I
(Hh) -- -- — _— =
Tm 4 = — —_ =
Ae 2 I = | =
18 17 33 10 5
Hh — 5 —_— I =
(bh long) | |
DyD. Lilian V. Morgan
The last column of the table may be left out of account; it
simply records the cases which died after being grafted, before
they showed any results one way or the other. The next to the
last column will be ignored for the present. The first three
columns give the cases where the graft was good, the cut surfaces
were squarely attached, and a certain proportion of the short
pieces regenerated.
It is clear that the exposed cut surface of the short piece is a
posterior surface, from which would normally regenerate a tail,
if the piece were not attached to the other worm. It has been
shown in Planaria maculata! that a head is formed at both the
anterior and posterior surfaces of very small free pieces, but this
I believe has not been found to be the case in Phagocata gracilis;
even exceedingly small free pieces of this worm regenerate a head
at the anterior end, but invariably a tail at the posterior end.
If then from the free posterior surface of a short piece a head
develops, the probability is that the short piece 1s in some way
influenced by the longer piece.
The table shows that out of sixty-eight cases, thirty-three (nearly
half) closed in, regeneration was prevented, and the combination
remained headless. Combinations of this sort are always sluggish
as compared with normal worms or with worms with regenerated
heads, normal in direction or reversed; they have lived, however,
for weeks without showing signs of deterioration. Of the
thirty-hve which regenerated, about half (eighteen) produced
heads, and the other half (seventeen) produced tails. This pro-
portion of heads and tails occurred for the various combinations
of the three regions of the worms all taken together, but if each
kind of combination is considered by itself the proportions are
very different.
Hh. Short pieces from the head region, reversed and grafted
to the head region, regenerated in nine cases out of fourteen, and
of these nine cases eight produced heads (see Fig. 14, 15), and
only one a tail. Varying the size of the shorter piece was tried.
In five cases, the shorter piece (h) was left about twice as long
‘Morgan, T. H., ’04. The Control of Heteromorphosis in Planaria maculata. Archiv f. Ent-
wickelungsmech., Bd. vii.
Regeneration of Grajted Pieces of Planartans 272
as the short pieces in all the cases recorded in the first line of the
table, but yet much shorter than the long piece (H). In all these
cases, a tail and not a head regenerated.
Hm. Ten out of twelve pieces from the middle region, reversed
and grafted to the head region, regenerated, but all ten without one
exception produced tails.
Mh. ‘Two short, reversed pieces from the head region grafted
to the middle region both regenerated and produced heads.
Mm. Middle region on middle region regenerated in only four
cases out of fourteen, and of these, half (two) produced heads,
and half (two) tails. :
Mt. No heads were produced and only three out of eleven
pieces regenerated and produced tails.
Tm. Four cases regenerated, and all produced heads.
Tt. Regeneration in only three out of eleven cases; two of the
three produced heads, and one a tail.
Of the ten grafts of Planaria, one (an Mh) made a head, prob-
ably from the posterior surface of h, but the history was not closely
enough followed, to be certain of the origin of the head; another
graft (Hh), made a tail at first, but when the new tail had been cut
off, near the line of union with the longer piece, a head developed.
It appears from the data for Phagocata that the reversed head
region readily produces a head; it occurred in all the cases, but
one, when it was grafted to a head region (if the piece h was
very short), and in both cases that were tried on the middle region,
On account of this uniformity, it did not seem necessary to try
ith.
The results from the reversed middle region are much more
variable. In the first place, a much larger proportion of these
grafts, than of grafts of the head region, did not regenerate at all.
Of those that did regenerate, all ten grafted onto the head region
produced tails, half of those grafted onto the middle region pro-
duced heads and half tails, but all four on the tail region pro-
duced heads.
The reversed tail region in far greater proportion than the head
or the middle region closed and did not regenerate, but the experi-
ments show that even the tail region, if it does regenerate, may
274. Lilian a Morgan
produce a reversed head; for although of six regenerated pieces
(three grafted to the middle, and three to the tail) four produced
tails, yet two of those grafted to the tail produced heads.
In these experiments, there is no regularity as to the kind of
regeneration of the reversed grafted pieces; but the results show
that using each of the three regions of Phagocata gracilis as stock,
with some of the other regions as graft, and each as graft, with
some of the others as stock, heads may be produced. But some
of the possible combinations of the different regions developed
only tails. “The head region as graft on different stocks, and the
tail region as stock with different grafts produced the largest pro-
portion of heads in these experiments. It is possible. that when
tails are produced, the smaller piece, although very short com-
pared with the longer, may be long enough to be beyond a limit
within which a piece may regenerate a reversed head. On looking
over the notes to seewhether there were data exact enough to throw
any light on the point I found in three cases, where the pieces were
measured or their length noted and heads were produced that the
small piece was shorter than in two cases where the pieces were
measured and produced tails. But another piece (which was
longer to begin with) closed in, and although it was afterward
cut twice until it Was very short, it even fen produced a tail.
The closing-in, in itself, could not have affected the kind of
regeneration, for other pieces which closed-in, and were after-
ward cut, produced heads; these were not actually measured.
B. IRREGULAR CASES
Having considered the regeneration of a head or a tail at the
exposed posterior surface of squarely erafted and perfectly united
pieces, the other sorts of regeneration will be examined. ‘The
results recorded in the fouten column of the first table are ex-
panded in the first part of a second table.
‘There are two main classes of examples:
I. First, when the original cut surfaces did not graft perfectly
across the cut ends, surface to surface, but one, or the other, or
each of the cut ends. was not covered completely by the other
Regeneration of Grajted Pieces of Planarians 275
TABLE If
Phagocata gracilis (Column 4 of Table 1)
I. Heap rrom Exposep Epcr | Il. Heap at ANGLE or Grarr |
oF ANTERIOR SURFACE OF From |
|
_— ‘UNCERTAIN
Long piece| Short Both Long piece Short | Both
Hh — -- —_ No. 94 (k) | No. 3 (k) | No. 1 (k) =
(one eye) | No. 19 (k)
No. 10 (k)| No. 95 (k)
H long h = = = = — No. 43 (t) =
(four eyes)
Hm No. 18 (t) |
No. 89 (k)| — = = a = oh
Mm =a No. go (k) _ — = ae a
Mt > = = — —_— — No. 86
Planaria Maculata
feels Mens ee a — = _ e —_ a a eee
Hh | No. 2 (k) | a No. 7 (t) — — | = No. 4
| No. 3 (k) (two heads) |
Hen = = — No. 8 (t) = = ey
Ht No. 13 (k) — No. 11 (t) -- — | No. 12 (t) —
| (two heads) (three eyes) |
1The behavior of the exposed posterior surface of the short component is indicated in the table by
the small letters ‘‘t”? and “‘k;” (t), when a tail was formed; (k), when the piece closed-in, and formed a
knob, which did not regenerate in the posterior direction.
surface, but was left in part free. From such a free surface, a
head regenerated, and whatever its origin, whether from the short
or from the long piece, it gradually assumed (in the examples
whose history was followed) the size and position of a head of the
large piece.
In Planaria maculata No. 13 Ht (Fig. 24, B,) the small grafted
piece was from the beginning but a mere fragment at one edge of
276 Lilian V. Morgan
Fig. 1. Phagocata gracilis, combination Hh. A, One day after grafting. B, Eleven days after
grafting. C and D, Diagrams of the anterior nervous systems of combinations Hh, fourteen days after
grafting, drawn from several sections with the camera lucida. The worm of Diagram C had regenerated
a reversed head. £, Diagram of the anterior nervous system of a combination Hh, fourteen days after
grafting, where h had closed in. a, Line between the large and small components of the grafted com-
bination. 5, Line between old and new tissue.
Fig. 2. Planaria maculata No. 13 Ht. 4, After regeneration of the large component had begun.
B, Eight days later than 4.
Fig. 3. Phagocata gracillis No. 89 Hm. 4, One day after grafting. B, Twelve days after
grafting. a, Exposed anterior surface of the long component H. 56, Exposed posterior surface of the
short component m. », Line to which m had been absorbed forty-one days after grafting.
Fig. 4. Phagocata gracilis No. 18 Hm. 4, Five days after grafting. B, Ten days after grafting.
C, Forty-three days after grafting. x, Outline of the new head sixteen days after grafting.
Fig. 5. Phagocata gracilis No. go Mm. a, Exposed anterior surface of m.
Fig. 6. Planaria maculata No. 2 Hh.
Fig.7. Planaria maculata No.7 Hh. C, About sixty-eight days later than 4 and B.
Fig. 8. Phagocata gracilis No. 94 Hh. A, Twenty-two days after grafting. B, Diagram of the
nervous system of A.
Regeneration of Grafted Pieces of Planarians
278 Lilian V. Morgan
/ 5
the anterior cut surface of the large piece, and the large piece
regenerated freely; the small piece was gradually absorbed, and
the combination appeared seventy-one days after grafting like a
normal worm.
Phagocata gracilis, No. 89 Hm (Fig. 34, B,) was amore inter-
esting case, because the union took place in such a way that part
of the anterior cut surface of the long piece was not covered by the
small piece and was drawn over to one side. Regeneration took
place from the exposed portion, and the new tissue grew much
longer on the short side than on the long. A head was thus
formed in the normal position of the head of the large piece.
The small piece closed in at its exposed posterior surface, and was
at first large enough to take part in sticking to the bottom of the
dish, but was gradually absorbed, and forty-one days after
grafting formed merely a small excrescence on the side of the
large worm. It had the same appearance fifty-six days after
erafting.
Phagocata No. 18 Hm (Fig. 44, B, C,) was interesting because
of morphallaxis which took place involving both pieces. The
small piece when grafted covered about half the anterior surface
of the large piece, and was at first pushed before the large piece
as it crawled. A narrow head regenerated at the exposed half of
thesurfaceof thelarge piece. “The small piece gradually changed its
position and turned back at the side of the large piece, developed a
tail at its free posterior surface, and crawled with the large piece
and in the same direction. [he head, in the meantime, became
not only as broad as the large piece, from which it originated, but
as broad as both components together. “The worm died before
it became certain whether the smaller tail was going to be absorbed
although it seemed to have diminished.
Phagocata No. go Mm (Fig. 5) was a caseof head growing
from the partly exposed anterior surface of the smaller piece.
The head assumed the position of the head of the larger piece,
and the closed-in posterior part of the small piece formed a slight
protrusion from the side of the large worm.
Planarians Nos. 2 and 3 produced heads from part of the
exposed anterior surface of the large piece. ‘Their origin was nor
Regeneration of Grafted Pieces of Planarians 270)
followed closely. In one (Fig. 6), there seemed to be two heads
from the large piece, the small piece being stuck to the middle of
the cut surface between the heads.
In Planaria No. 7 (Fig. 74, B, C,) the grafted pieces stuck
merely by their edges, leaving most of the anterior surfaces of
both pieces exposed. [wo vigorous worms regenerated, with
complete heads and tails, and crawled or attempted to crawl in
opposite directions, attached only by the corners of their heads.
The eyes at first were very irregular and never became perfectly
normal, but were, except one of them, multiple eye-spots in two
clusters in each worm. ‘The worms lived for weeks, one of them
being sometimes dragged along, while the other, which had a
better hold, crawled normally. Usually they crawled side by
side, the head of one being doubled up in the angle between the
two worms (as seen in Fig. 7/4, B).
Planaria No. 11, Ht, was a two-headed combination, one head
and one tail were smaller than the other and seemed to be under-
going absorption. The origin of the regenerated parts was
uncertain.
II. The second class of irregulat grafts comprises somewhat
different phenomena from the first.
In Phagocata No. 94, Hh (Fig. 84), the new head probably
came from the large piece, and it may have originated from an
exposed edge (as in class I, just considered), but in No. ro Hh,
and No. 3 Hh, the head seems to have originated from the small
piece, not from a free surface, but in another way. The union of
the two pieces in both cases seemed to be perfect, but the line of
union, instead of being straight across the large piece (Figs. 9, 10),
was at an acute angle with its long axis. No. 1o died before the
head was complete, but a head (Fig. g) seemed clearly to be
developing from the small piece, starting with new tissue that
regenerated in the angle between the old tissue of the small piece
and the line of union with the large piece, 1. ¢., the head developed
from one side of the anterior surface of the small piece, though in
contact with the large piece. [he head grew in the direction
which was nearly anterior in relation to the large piece, and the
posterior surface of the small piece closed in, and became a knob
280 Lilian V. Morgan
Fig. 9. Phagocata gracilis No. 10 Hh. A, One day after grafting. B, Ten days after grafting.
C, Eighteen days after grafting. x, Line where the posterior end of h was cut off a second time.
Fig. 10. Phagocata gracilis No. 3 Hh. 4, One day after grafting.. B, Later stage. C, About
twenty days after grafting. D, Eighty-three days after grafting. E, Diagram of the anterior nervous
system of D.
Fig.11. Planaria maculata No.8 Hm. 4, Five days after grafting. B, Seven days after grafting.
Fig. 12. Phagocata gracilis No.1 Hh. 4, One day after grafting. B, Fifteen days after grafting.
C, Twenty-nine days after grafting.
Fig. 13. Phagocata gracilis No. 19 Hh. A, Four days after grafting. B, Twelve days after
grafting. C, Forty-seven days after grafting.
Fig. 14. Phagocata gracilis No. 95 Hh. A, Fourteen days after grafting. B, Diagram of the
anterior nervous system of I 4.
Fig. 15. Phagocata gracilis No. 43 H long h.
Fig. 16. Planaria maculata No. 12 Ht.
Fig. 17. Phagocata gracilis, dorso-ventral Hh. A, One day after grafting. C, Twenty-nine days
after grafting. B, Another specimen, six days after grafting. D, E, F, Diagrams of different positions
of a dorso-ventral graft after regeneration. J, II, Worms. 4, B, Heads; solid lines represent ventral
surfaces; dotted lines, dorsai surfaces.
Regeneration of Grafted Pieces of Planarians 281
IIB
MAKE!
KID) A /
282 Lilian V. Morgan
at the side of the worm. No. 3 (Fig. 10) was somewhat different.
The small piece was wedge-shaped, narrowest at the anterior end
of the slanting line of union with the large piece. From this
narrow part regenerated most of the new tissue from which the
head developed; the broad side of the piece lengthened a little
at first as though to make a tail, but later closed in and was being
absorbed. ‘he head differed from the reversed heads of Table I,
in that it did not develop in the middle of the exposed posterior
surface, but at the thin edge of one side. At one period a single
eye was seen, and also a second pigment spot, which later dis-
appeared, and only one eye remained in the narrow abnormally
shaped head.
Planaria No. 8 Hm (Fig. 114, B) developed a head at the for-
ward angle of a slanting graft, but its origin was not studied.
The small piece made a tail.
In the examples of a single head (next column of Table II)
made up in part from both components of the compound worm,
the head (where the history was followed) originated from an
edge, perhaps slightly exposed of the large piece.
In Phagocata No. 1 Hh (Fig. 12) the point of origin of the head
was the anterior edge of a slanting graft. In No. 19 Hh (Fig. 13)
new tissue developed from the large piece, especially at the sides
of the straight graft. But only one edge grew anteriorly, and the
other side of the graft was thrown back. In both No. 19 and
No. 1, new tissue from the small piece also regenerated at the line
of the graft, and took part in the formation of the head, and one
eye came from each component. ‘The posterior edge of the small
piece closed. In No. 19, the division between the large and the
small piece was always marked by a notch at the forward end of
the head; the compound head was always somewhat lateral
In position.
The first steps in the regeneration of No. 95 Hh (Fig. 14) were
not followed; there resulted from a grafted pair, one worm having
an eye from each component, and with a knob from the small
piece at the side. A combination of H long h (No. 43, Fig. 15)
produced a worm with one head, but with four eyes, and with
two tails.
Regeneration of Grajted Pieces of Planartans 283
Planaria No. 12 Ht (Fig. 16) produced one head with three
eyes, and two tails, and a pharynx developed in the smaller
regenerated tail.
Like the perfectly attached unions, the irregular combinations
also usually resulted in one way or another in the formation of com-
plete worms derived from parts of two worms. If the point of
attachment of the two components is very limited, two nearly
complete worms may be regenerated, as Planaria No. 7 (Fig. 7),
but usually, even though regeneration of two worms begins, the
smaller duplicate parts are later absorbed. If part of an anterior
cut surface remains exposed after grafting, the point of regenera-
tion is thereby determined, and the new. head grows at that point;
it may be from the larger component, Pl. No. 13 (Fig. 2), Ph.
No. 89 (Fig. 3), Ph. No. 18 (Fig. 4), or it may be from the smaller
Ph. No. go (Fig. 5):
If a cut anterior surface is not exposed, new tissue may still
grow at the line of the graft, and, one edge growing forward, form
a head, at first lateral, then more and more anterior in relation to
the larger component. Such a head may be derived from one
component, No. 94 (Fig. 8), No. 3 (Fig. 10), No. 10 (Fig. 9g), or
from both, No. 1 (Fig. 12), No. 19 (Fig. 13), No. 95 (Fig. 14).
In all these cases, whether the head be derived from a free sur-
face, or from the growing edge of the line of the graft, the posterior
part of the smaller component (whether it closes in or whether it
regenerates a tail) is almost always gradually absorbed, leaving
one complete worm whose body and tail are derived from the
larger component, and whose head is derived from the larger com-
ponent or from both, or even from the smaller only. In the last
case, No. go (Fig. 5), No. 3 (Fig. 10), No. ro (Fig. 9), the tail 1s
absorbed of that same component from which is derived the head
of the final combination. In the combination worm, however
the head may arise, the larger body persists as the body of the
worm. Part of a second head may also regenerate, but it 1s
usually abnormal and in a crowded position [No. 43 (Fig. 15),
PieNo. 12 (Fig. 16)| and perhaps degenerates. One of two eyes
degenerated in No. 3 (Fig. 10). A siege case of morphallaxis i in
the formation of the final unit is seen in No. 18 (Fig. 4), where the
284 Lilian V. Morgan
head (derived from the large piece) at first occupied but half the
width of the large piece, the small piece regenerated a tail, which
grew back to a posterior position before it was absorbed, and the
fewd widened until it extended across the width of both compo-
nents. Allthe observed cases of a head being formed at one edge
of the line of graft without a free surface are of thecombination Hh.
C. DORSO-VENTRAL GRAFTS
Another set of experiments was carried out to see what sort of
regeneration would take place if two worms were so attached by
their anterior cut surfaces that the dorsal surface of one worm was
continuous with the ventral surface of the other, 7. ¢., one Worm was
turned over on its back before grafting together the anterior ends.
After many unsuccessful attempts to get the pieces to stick
together, six grafted pairs were obtained from pieces cut anteriorly
in the head region (dorso-ventral Hh), and four combinations of
tail regions (dorso-ventral Tt). In one Hh, part of an anterior
surface remained exposed, and two normal worms regenerated
and pulled apart. After about a week, all the other five Hh com-
binations without exception, had regenerated in one and the same
way. From both flat surfaces at the line of union of the two pieces
a head regenerated; at the same time the small piece had regener-
ated a tail at the free posterior cut surface (a normal product), and
the result of such a combination is that a larger worm crawls
about carrying attached to it, posterior to the eyes, another smaller
worm at first shorter than itself. In other words, two worms
appear to be attached to each other by the “napes” of their necks,
the larger one carrying the smaller one curled up above it (Fig. 17
4, B,C). As the smaller one grows, it also tries to attach itself
and sometimes the combination is so twisted that both worms
crawl at once. The two cases of dorso-ventral graft obtained in
Planaria maculata regenerated in the same way. ‘The dorso-
ventral grafts of Phagocata of the combination It behaved like
those of abe Eoaeion Hh in two cases where they could readily
be observed; in the other two, two heads showed, but the pieces
were very small and not carefully made out.
Regeneration of Grafted Pieces of Planarians 285
The preceding description of the regeneration of dorso-ventral
combinations does not fully represent the state of the case; for,
when the worms are of different colors and the grafted combina-
tion is more carefully examined, it is seen that the head of the
crawling worm has its dorsal surface pigmented like the dorsal
surface of the worm carried above it. In other words, the head is
a composite of ventral material regenerated from one worm, and
dorsal material regenerated from the other worm, and both heads
belong in part to both worms. ‘The relations of the heads to the
old parts are shown in a diagram (Fig. 17D) of a side view of the
combination; the dorsal surfaces are represented by dotted lines,
the ventral by continuous lines. If worm J is loosened from its
crawling surface, the heads may be thrown in opposite directions
(Fig. 17£), and now the worms appear to be attached by their
throats, and worm / appears to possess head B instead of head 4.
If allowed to return to the position of least resistance, head 4 will
again be thrown forward from the larger worm /, and the ventral
surfaces of worm J and head 4 will be continuous in the crawling
position on the bottom of the dish. Diagram F, representing a
position midway between D and £, willshow most clearly how the
composite heads have grown from the upper and lower edges of
the graft.
In dorso-ventral grafts, there 1s no chance for the nerves of the
two worms to unite, and this may be a factor in the production
of two heads at the graft, and hence of no regeneration of a head
at the free posterior cut surface of the small piece. From this
point of view, the results of the experiments fall directly in line
with the results of the plain grafts of Tables I and II.
The relations of the nervous system have not been worked out
in all cases, but many combinations were sectioned and showed
results of interest. As far as the nerves have been studied, the
general rule holds that whenever a head is developed, its nerves
are connected with the longitudinal nerve-trunks of one or both
components of the graft, and if there is a single head involving the
smaller component, the longitudinal nerves of the small compo-
nent are connected with those of the larger component; the single
head may be a reversed head on the small piece, or a laterally
286 Lilian V. Morgan
compound head, the derivation of the head being judged by the
position of the eyes. If a reversed head 1s developed at the pos-
terior surface of the small piece, it is found in section that the
longitudinal nerves of small and large components meet squarely
end to end and thus the nerves are perfectly continuous, and in
the new head, sometimes (Fig. tC’) apparently normal “brain”
and intercerebral commissure are developed; sometimes the
commissure is very small. If a tail is formed again, the longi-
tudinal nerves of the two pieces unite end to end, but there is no
brain or commissure. In one specimen, the longitudinal nerves
in the new tail are connected at intervals by smaller nerves as
they are throughout a normal worm.
In a case of what was supposed to be reversed regeneration
(Fig. 1D), the anterior nerves are in the small component,
and with a large connecting nerve are shunted off to one side
connected with only one longitudinal nerve of the large compo-
nent; the left nerve of the small component is continuous with the
right nerve of the large component; the connecting nerve leaves
the longitudinal nerve at the level where the normal commissure
should connect the two longitudinal nerves of the large piece;
but exactly through this region passes the line of the na and
the normal commissure 1s “ha slightly developed. “This worm
was thought to be among those which showed externally perfect
head regeneration, but it may have been confused in embedding
with closed-in pieces since the eyes have not been found in the
sections. [he chief interest lies in the one-sided connection of
the nerves of the larger and smaller components.
In grafts in which the small piece closed in, and in which no
regeneration occurred, there was found no brainor commissure. In
the examples that were sectioned, the longitudinal nerves ended
abruptly, sometimes with a few nerve branches in place of the
commissure, and in one case, a third large nerve in the knob that
remained of the small component (Fig. 1£) was connected by
a few nerves with one of the longitudinal nerves.
The union of the longitudinal nerves of the two components
seems to take place at the time that the pieces grow together; the
nerve-trunks are continuous in surface view when the grafted
Regeneration of Grajted Pieces of Planarians 237
pieces are first released from the papers (eighteen to twenty-four
hours after being placed together), and in sections of a graft then
killed, the nerves are found united. ‘The line of union of the two
pieces of worm can sometimes be seen in sections of worms killed
some time after the pieces grew together because of the accumu-
lation at the line of union of parenchyme nuclei.
In Phagocata No. 94 (Fig. 85) the nerve of the closed-in knob
of the small piece has formed a closed ring with branches. ‘The
ring is united with the longitudinal nerve of the side of the large
component on which the knob 1s attached.
In No. 95 (Fig. 146), where one eye has developed in each com-
ponent of the graft, the longitudinal nerves of the large com-
ponent are connected by a commissure, the eye of the ee com-
ponent is innervated by nerves from the brain connected with one
longitudinal nerve; from the commissure a branch passes to the
nerve ring in the knob derived from the small component, and
anteriorly from this ring a large branch nerve is developed from
which the eye, derived from the small component, is innervated.
Again this branch and the brain in the other side of the large
component are connected by nerves but slightly developed in the
normal position of a commissure.
In No. 3 (Fig. 10o£), where there is but one eye which 1s derived
from the small component of the graft, the longitudinal nerves
of the large component are connected by a commissure and are
also connected anteriorly by the nerve of the smaller component
of the graft, forming with the commissure a complete ring. “Two
anterior nerves, which also are connected together arise, one from
the longitudinal nerve of the large component, the other from the
nerve ring of the small component, and from this a nerve extends
to the one eye.
The fact that regeneration at line of graft a slanting occurred in
cases of the combination Hh only has not been explained. It
might be supposed that the material of the head region most
easily forms a head, the heads being here produced even See aN the
usual outlet for regeneration, 1. e., a free surface, is blocked by the
graft. In line with this view would be the case of No. 3, w here the
reversed head grew, not in the middle of the piece, but at the thin
288 Lilian V. Morgan
edge of the piece, that is the point where the tissue was nearest
to the normal place for head-regeneration. ‘The question as to
whether (as in Tubularia)'a head regenerates sooner or more
readily at different levels from behind forward, has not been
studied. It is shown by the results given in Table I, that
regeneration of a head even in a reverse direction may take place
at any level of the worm, and the condition of the nervous system
in two of the class of cases under discussion suggests that a nerve
ending freely is a factor in regeneration. In neither of the
worms sectioned (Figs. $8 and 14) do the longitudinal nerves of
the two components meet squarely as in the worms with reversed
heads (Fig. 1). In the cases where the line of graft slants, the
free anterior end of a longitudinal nerve trunk may then, as in
dorso-ventral grafts, be a factor in determining the position of
the new head, although there is no exposed cut surface of either
worm.
It would be of interest to know the condition of the nerves in a
case like Planaria No. 2 as compared with Phagocata No. 19,
and Phagocata No. 58. In Planaria No. 2 (Fig. 6), it seems that
two heads have regenerated from the anterior surface of the
large component of the graft, one head each side of the small
closed piece. In Phagocata No. 19 (Fig. 13), new tissue grew
from the large component at both sides of the line of graft, but a
head developed on one side only, and was later composed in part
of the smaller component of the graft. In Phagocata No. 58, the
small piece closed in, the anterior surface of the larger component
regenerated on both sides of the small piece, and the new tissue
completely surrounded it, but a head never developed. The
worm was watched for ninety-six days.
Reviewing the results as far as worked out, it appears that in
all cases of graft, the eye and its nerve are developed from the
same component of the grafted worm. With two worms cut off
anteriorly there are four exposed anterior nerve ends. When
these, by the conditions of the graft, cannot unite, two perfect
‘Morgan, T. H., and Stevens, N. M.’o4. Experiments on Polarity in Tubularia. Journ. of Exp.
Zool., vol. i.
Regeneration of Grafted Pieces of Planarians 289
heads are formed (dorso-ventral grafts and a very one-sided
eraft like Planaria No. 7, Fig. 7). When they can unite end to
Baa (pertect plain gratts), es do so, and regeneration of the
anterior ends of the nerves is excluded; if a head is then developed
it is not the anterior, but the posterior, exposed ends of the nerves
that come to innervate the eyes, and may even form normal brain
and commissure, or may regenerate in a way less like a normal
anterior nervous system. ils irregular orafts, various ways of
combining the nerves of the sell component with those of the
large lead) to different arrangements of the nerves of the two sides.
When the small component ¢loses in, its nerves may end freely
posteriorly, as in square grafts (Fig. 1£), or may form a ring
attached in various ways to the nerves of the large component. In
No. 94 (Fig. 8) the ringas shunted in with one longitudinal nerve
of the large component. In No. 3 (Fig. 10) it is joined with the
commissure and with one longitudinal nerve and innervates the
one eye which is derived from the small component. In No. 95
(Fig. 14) the ring is connected by a large nerve to the commissure,
sends a large branch to the eye derived from the small component,
and the branch is joined by a few nerves with the anterior nerve
of the other side of the large component.
The relations between the kind of regeneration (as seen in
surface views) and the condition of the nervous system is what
would be expected, though apparently perfect regeneration of a
head and eyes may be accompanied (as in Leptoplana littoralis)*
by imperfect regeneration of cerebral commissure and brain.
Some points in regard to the closing-in of the exposed posterior
surface of the small component may be further examined. Clos-
ing-in of the posterior end of the small component never occurred
where there was regeneration of two heads in the anterior tissue
of the components, either complete heads [dorso-ventral grafts
(Fig. 17) and Tabie II, column 3, Planaria No. 7 (Fig. 7) and
No. 11], or partially complete [Table II, Phagocata No. 43
(Fig. 15), Planaria No. 12 (Fig. 16)]. It always occurred (except
1Morgan, L. V. Incomplete Anterior Regeneration in the Absence of the Brain in Leptoplana
littoralis. Biol. Bull. vol. ix.
290 Lilian Ve Morgan
in two cases), Table II, Phagocata No. 18, (Fig. 4) and Planaria
No. 8 (Fig. 11), where a single head originated in some way from
the anterior end of one or both components (Figs. 2, 3, 5, 8, 9,
10, 12, 13, 14). It often occurred in plain square grafts (Table I),
but never when the shorter component was not very short (Table I,
H long h). Closing-in of the anterior surface of various kinds
of Planarians, when the worms are simply cut across without
grafting, 1s a very common phenomenon; it has often been
observed and is frequently noted in the literature on regenera-
tion in different species and genera of Planarians.' Closing-in
of posterior cut surfaces of ungrafted worms, so far as I know,
has not been observed.
It might then appear that when the posterior surface of the small
component closes in (Table I, column 3;- Table I, columns 1, 2,
4, 5 and 6, except Phagocata No. 18 and the compound-headed
worms Phagocata No. 43 and Planaria No. 12), it acts like an
anterior cut surface of an ungrafted worm. If this were always
true, it would follow that when the anterior surface of the small
component takes part in the formation of the head, and at the
same time, the posterior surface closes in, both surfaces of the
small component act like anterior surfaces of a cut worm. On
looking closely at the facts, it will be seen that the closing-in of
the posterior surface of the small component never occurred
when all of the anterior surface of the small component took part
in the formation of the head (dorso-ventral grafts and column 3
of Table II); in those cases a tail was formed from the short com-
ponent posteriorly. “The cases where head-formation occurred
anteriorly, and at the same time closing-in occurred posteriorly
in the small component are cases of slanting grafts and of all other
grafts where part only of the anterior surface of the short com-
ponent contributed to the new head (Table II, columns 2, 5 and 6,
‘Morgan, T. H., ’98. Experimental Studies of the Regeneration of Planaria maculata. Archiv
f. Entwickelungsmech. d. Organismen, Bd. viii. Lillie, F. R.,’o1. Notes on Regeneration and Regu-
lation in Planarians. Am. Journ. of Physiol, vol. vi. Schultz, E.,’02. Aus dem Gebiete der Regenera-
tion. IJ. Ueber die Regeneration bei Turbellarien. Zeitschr. f. wiss. Zool., Bd. Ixxii. Morgan, T. H.,
04. Notes on Regeneration. Biol. Bull., vol. vi. Child, C. M.,’o5. Studies on Regulation. IV, V
and VI, Journ. of Exp. Zodl., vol. i. Morgan, L. V., loc. cit. :
Regeneration of Grafted Pieces of Planarians 291
the origin of the parts was not followed in Phagocata No. 43 and
Planaria No. 12). The condition of the nervous system in slant-
ing grafts indicates that there is a difference between the closing-in
of these posterior surfaces and the closing-in of the posterior sur-
faces of reversed square grafts without regeneration at the line
of graft.
The longitudinal nerve trunks of the short component in the
slanting grafts (Figs. 8, 10 and 14) did not join end to end with
those of the long component, but formed a closed ring joined in
some other way with the longitudinal nerves of the long compo-
nent. In contrast to this the posterior ends of the longitudinal
nerves of closed-in pieces of square grafts without regeneration at
the line of graft ended freely. It may then be true that the closing-
in of reversed square grafts is like the closing-in of the anterior cut
ends of ungrafted worms, while the behavior of the nerves may be
a factor in the closing-in of the posterior ends of the small pieces
in slanting grafts where part of the anterior surface contributes
to the new head. ‘The hypothesis that the closing-in of reversed
square grafts without regeneration is a phenomenon of an anterior
surface or of one acting like an anterior surface leaves unexplained
the case of a graft where the short piece at first closed in, but after
it was twice cut off produced a tail; as the shortness of the piece
seems to be a factor in the production of reversed heads, it is
unexplained why the piece when longer acted like an anterior
surface, when shorter made a tail.
SUMMARY
1. Short pieces of Phagocata gracilis, cut from different parts
of the worm, and grafted by the anterior surface to the anterior
surface of long pieces from different parts of another worm, some-
times produce heads, sometimes produce tails and sometimes
close in at the exposed posterior surface.
2. The numbers of the combinations cut from different regions
of the worms, which either failed to regenerate, or produced heads,
or produced tails, are fully summarized in Table I, page 271, and
on page 272. The number of grafts that was studied of any one
292 Lilian V. Morgan
combination was comparatively small, and with large numbers
the proportions in the results might be different.
Longer pieces from the head region of a worm, reversed and
grafted on the head region of another worm, regenerated tails
at the exposed posterior surface of the short component.
3. When (because of imperfect grafting or for some other
cause) regeneration occurs at some point other than the exposed
posterior surface of the small component, various results follow.
Examples of Phagocata and Planaria are fully summarized in
Table II, page 274, and on page 275.
4. Ifthe conditions of the graft are such that part of an anterior
surface 1s exposed (even if only a very small fraction of the whole
surface) a head is regenerated from the exposed part of that sur-
face. A head may regenerate from each component of the
graft (Fig. 7), or only from the large component (Figs. 2, 3, 4,
6), or from the short component (Fig. 5), according to which
surfaces are partly exposed.
5. A single head formed in this way (no matter from which
component or in what position it originates) gradually grows to
the size and position of a head of the larger component, the
smaller duplicate parts of the compound worm are absorbed,
and one complete compound worm results.
6. Ina few cases where both long and short components were
cut through the head regionsofthe worms, a head regenerated at one
edge of the line of graft, although there was apparently no exposed
(anterior) surface (Figs. 8-14).
7. In the last cases (paragraph 6) except one (Fig. 13, and
one where the original line of graft is not recorded, Fig. 8) the line
of graft was not straight across the worms, but at an acute angle
with the long axis, and the head regenerated at the forward angle
of the graft. The heads were derived from one component
(Figs. 8 and 10), or from both (Figs. 12-14).
8. Where the nervous system of these grafts was studied, it
was found that the longitudinal nerves of the two components did
not squarely unite, and probably anterior ends of the longitudinal
nerves remained free.
g. These heads like those of paragraph 4 acquired by degrees
Regeneration of Grafted Pieces of Planarians 293
the size and approximate position of a head of the large com-
ponent, and the small knobs or tails at the exposed end of the
small components showed (where their history was followed) that
they were being absorbed.
10. [wo compound worms with extra eyes were observed
(Figs. 15, 16) but their history was not recorded.
11. Another kind of combination, “dorso-ventral” grafts,
tried with one combination of Planaria and with two combinations
of different regions of Phagocata, produced uniform results
(except in one case of imperfect graft). In the grafted combina-
tion, the ventral surface of one worm was continuous with the
dorsal surface of the other. All the examples produced double
worms, the two heads being at the line of graft, on opposite sides
of the combination, and each head being derived in part from each
component of the graft (Fig. 17).
12. [he heads in these cases regenerated at the normal place
for head regeneration, but not from a free cut surface. ‘The cut
anterior ends of the longitudinal nerve trunks must, however,
have ended freely.
13. he exposed posterior surface of the small component in
each case of dorso-ventral graft regenerated a tail, and never
closed in, and the small component became in a short time a more
or less nearly full-sized worm, attached to the other worm.
14. In single-headed compound worms where the nervous
system was studied, it was found that the longitudinal nerve
trunks of the two components are connected in one way or another
and the anterior nervous system and the innervation of the eyes
are derived from the two components according to which part of
the new head arise from each.
15. In square grafts, the anterior ends of the longitudinal
nerves of the two components unite end to end. ‘The posterior
ends of the longitudinal nerves of the short component may form
commissure and brain, if a reversed head regenerates, but they
may end freely if the posterior end of the short component closes
in.
16. In slanting grafts, which regenerate a head at the line of
graft, the longitudinal nerves of the short component form a ring
204 Lilian V. Morgan
connected in one way or another with the longitudinal nerves of
the large component.
ii mike nervous system of double worms, like that of ne 7s
and of dorso-ventral grafts (Fig. 17) has not been studied, but the
conditions of the graft are evidently such that the longitudinal
nerve trunks of the two components could not unite, and in accord-
ance with this is the fact that double worms (not one compound
worm) are formed.
New York, January, 1906.
Contributions from the Zodlogical Laboratory, Syracuse University.
Pee PERIMENTS ON HE BEHAVIOR OF TUBICOLOUS
ANNELIDS
BY
CHAS W. HAR GMa
With Turee Ficures
During the past summer it came in my way to collect
numerous colonies of the serpulid annelid, Hydroides dianthus,
which is very abundant in the waters about Woods Hole. Find-
ing them to be well adapted to aquarium life [ kept colonies under
observations upon my laboratory table during almost the entire
summer, and made such experiments and observations on their
reactions and various forms of behavior as suggested themselves
from time to time.
In connection with the observations upon Hydroides, at the
suggestion of Dr. |. P. Moore, I also included species of Pota-
milla and Sabella, though they were very much less numerous
than the former species. I am under obligations to Dr. Moore
for identifying the several species.
As is well known, many of these annelids are remarkably
sensitive to the slightest disturbances of various sorts, such as
vibrations, the intervention of shadows, etc. In connection with
interesting observations concerning the habits of various tubico-
lous worms Dalyell,’ remarked concerning Amphitrite bombyx
that “it is impatient of light,’’ withdrawing into its tube in-
stantly upon the interception of the light.
1The Powers of the Creator Revealed. London, 1853. Quoted from Andrews’ four Mor ph.,
vol. v, p. 287.
Tue JourNnat or ExperiMENTAL ZOOLOGY, VOL. 111, No. 2.
296 Chas. W. Hargitt
Claparede,' has also called attention to a similar feature in
Branchiomma_ kollikeri, stating that “it is very sensitive to
changes in the amount of illumination, for a slight movement of
the hand at a distance of a meter from the aquarium, causes all
the animals to withdraw into their tubes as soon as the shadow
falls upon them. Yet Sabellas, having no eyes, remained im-
mobile and unaffected.”
It will be seen from some of the following observations that the
reference to Sabella is more or less incorrect, since our species of
Sabella, at least, are quite well provided with eyes, and are also
subject to the same stimuli as are others, differing only in degree.
Fig. 1. Several individual tubes of Hydroides dianthus, showing general aspects when
growing freely upon shells or similar substratum. (Somewhat less than natural size.)
Similar observations have been made also by Darwin? and
others upon earthworms, the significance of which is probably
of the same general character as the former. Still later ob-
servations upon species of Tubicolide have been made by Andrews,
Loeb, Nagel, and others, which will be considered in detail in a
later connection.
1Annelides Chetopodes du Golfes de Naples. 1868. Quoted from Andrews Jour Morph., vol. v,p. 287.
?The Formation of Vegetable Mould through the Actions of Worms. 1881.
Behavior of Tubtcolous Annelids 297
My observations extended to the following named species:
Hy droides dianthus, Potamilla oculifera, and § Sabella microph-
See lane. chiefly the first. “hese were available in considerable
numbers, and collected from various shells about the docks of the
United States Fish Commission, from shells, various bivalves,
Venus, Pecten, etc., from rocks dredged from depths varying from
two or three fathoms to fifteen to twenty in Vineyard Sound and
Buzzards Bay. The other species were obtained in part from
among colonies of Cynthia collected from the docks, and in part
among colonies of Hydroides. ‘Their numbers were smaller
than those of Hydroides and the observations correspondingly
Fig. 2. Colony of Hydroides dianthus growing in complex mass from flat rock base. The
various aspects of the mouth of the tubes may be easily distinguished, showing vertical, lateral
and downward relations referred toin the paper. (Somewhat less than natural size.)
less extended. As will be observed in a later connection the
limitations of experiments on species of Potamilla and Sabella
were due in part to their comparative indifference to the various
tests applied.
EXPERIMENTS ON HYDROIDES DIANTHUS
The general character of these annelids is so well known that no
particular account is necessary. The photographs of several typi-
298 Chas. W. Hargitt
cal conditions will show quite enough to make clear the habitat and
modes of growth. Colonies growing upon shells are seldom large,
while those growing upon rocks are frequently quite large, often
including from thirty to fifty, or even more, distinct specimens, each
inhabiting its own tube, but forming inextricable masses vari-
ously intertwined, and among which are usually various other
annelids, corals, hydroids, etc., the whole comprising a most 1n-
teresting ecological community, as well as a most beautiful display
of richly varied form and color, rarely surpassed among the almost
infinite variety of marine life. It may be noted in passing that
most of this richness and variety of coloration is to be found 1n the
Fig. 3. Colony of Protula intestinum, from Bay of Naples. The serpentine aspects of
the tubes referred to inthe paper are easily recognized. The coiled tubes to be seen upon the
central tube of the colony is particularly interesting as clearly indicating the indifference of the
creature to the influence of gravity. (Somewhat less than the natural size.)
annelids themselves, a fact which has been long known and
commented upon, but little understood. ‘This feature will be
further considered in connection with the several accounts in
which it may be involved.
Behavior of T ubtcolous Annelids 299
Though I had often observed the general sensitiveness of these
creatures to sudden intervention of shadows of various sorts, my
attention was particularly attracted to the matter in the present
instance by the observation that shadows of even the slightest
degree, such as those produced by a strip of white paper, or even
a glass rod, seemed quite as effective a stimulus as those produced
by the hand or other opaque screen. And this was the more
noticeable in that the specimens were before a north window, and
thus in diffused light.
Having determined upon a series of observations and ex-
periments, the colonies were arranged in several aquaria, certain
of which were placed upon a table before a window facing south,
the others upon a table facing northwest, the latter also in such a
position that a sixteen candle-power incandescent lamp was
available for certain experiments at night.
First Series
The first series of experiments was made _ to determine exactly
the character of the stimulus to which the reactions of the worms
were due; that is, whether they were the result of differences of
the intensity of light, or whether to the suddenness of the stimulus,
or the apparently negative effects of shadow stimulus. Loeb (’93)
had pointed out, in a simple experiment made by opening and
closing the shutter of a window before which specimens of
Hydroides uncinata were living in an aquarium, that “only the
decrease in the intensity of light acts as a stimulus upon the
animals.’’
This experiment I repeated many times and under variously
modified conditions. The general fact that only the decrease of
light, or in other words the shadow, is effective was abundantly
confirmed. Drawing the shade before the window, interposing the
hand or a sheet of paper, always sufhced to insure the prompt re-
traction of the animals. Now by continuing the presence of the
shadow, the worms extended themselves quite as before. When
thus extended the curtain, or other intervening object, may be
removed, allowing the increase of light to its former intensity.
300 Chas. W. Har gitt
This was variously done, in the case of the curtain, vertically; in
the case of the hand, screen or cardboard, etc., by either lateral,
downward, or vertical withdrawal; but in every case the added
increment of light produced no effect whatsoever upon the
behavior of the animals.
The experiment was further varied by employing artificial
light, an incandescent electric light of sixteen candle-power, hung
just over the table upon which the aquarium was placed. Allow-
ing the specimens to become fully extended, which was quite as
common by night as by day , in darkness as in light, the light was
suddenly Heche) directly i in their faces, so to speak, but in no case
was there evidence of any specific reaction. On the other hand, the
sudden extinction of the light almost always gave the same re-
sponse as the shadow, as could be seen by immediately turning
on the light again.
Again the experiment was made in bright sunlight. Allowing
the specimens to fully expand under an appropriate screen they
were then subjected to the sudden increment of full sunlight but
without the slightest reaction. “This was varied by covering the
aquarium with a dark box; leaving them in total darkness until
fully expanded, then quickly removing the box and allowing the
full sunlight to fall directly upon them gave the same negative
result as in the former.
There can be no doubt, therefore, that the reaction is not due
to simply a difference of light-intensity alone. For whether in
diffused or direct sunlight, whether in natural or artificial light,
the response is to the shadow, sudden diminution of light, a
purely negative condition. But it may well be doubted whether
this can be properly designated as simply negative phototropism
or heliotropism. ‘The phenomena are much too complex to be
explained by any single factor. Details on this point will be taken
up in a later connection, however.
The experiments were varied by interposing the screens at a
distance of from a very few inches, perhaps two or three, to five,
ten, fifteen, twenty, etc., up to forty or fifty, but without materially
affecting the results. In the diffused light before a north window
the shadows from the greater distances were not always equally
Behavior of T ubtcolous Annelids 301
effective, but before a south window when the light was bright,
even though diffused, the results were as certain and effective as
at the shorter distances. No attempt was made to determine the
limits beyond which the shadows might become too indefinite to
act as a stimulus.
Furthermore, the experiments were still varied by varying the
time or rate of the shadow movements. It was found that
ordinarily little difference could be detected as to the reactions
under a swift and a slow motion as ordinarily made by the move-
ment of the hand. Butif pains were taken to insure a very rapid
movement, as by propelling an object before the aquarium by
mechanical means, such as a spring, it was possible to pass the
shadow of a small object before the specimens without producing
sufficient effect to act as a stimulus. Likewise it was possible to
interpose a screen by such very slow degrees as to avoid any direct
reaction on the part of the specimens.
Another experiment was tried to test the effects of a constantly
recurring, or rhythmic shadow. ‘This was effected by arranging
a pendulum so adjustable as to secure a rhythm of from about a
quarter of a second to a full second. With the full second move-
ment there was more or less constant reaction with each passing
shadow. With the half second movement it was found that, after
the first few beats, a considerable portion of the worms failed to
respond at all; and with the quarter second beats almost all the
colonies became indifferent to the presence of the passing
shadows.
These results are extremely interesting as indicating the possible
relations of the reactions to protective or adaptive ends. May it
not be possible that in these rhythmic shadows we have a simula-
tion of the more or less rhythmic shadows resulting from the
ripples or wave action, which are phenomena of more or less
constancy and of course affecting in much the same way specimens
living in the shallower waters !
In connection with this matter of rhythmic shadows it was
observed that where experiments were repeated with any con-
siderable frequency specimens sooner or later became somewhat
irresponsive, often failing entirely to react to any of the usual tests.
302 Chas. W. Hlargitt
This I am inclined to regard as the result of fatigue. But it may
naturally be asked if this might not be equally well explained as
the result of a condition similar to that induced by the rhythm
just considered ?
That it is a matter of fatigue, or a closely related phenomenon,
rather than the latter, seems to me strongly indicated by the fact
that it results from any form of shadow stimulus which may be
applied, such as a vertical or lateral; slow or rapid; made by the
hand, by a small rod or broad screen; in short, by any or all of
the various tests applied successively or in any order whatsoever.
These tests applied singly and at irregular intervals rarely failed
to produce the appropriate reaction. It was only when repeated at
such rapidly recurring intervals as to leave insufficient time for
readjustment that the animals gradually became irregular and
indefinite in their behavior. At almost every point the results
simulated so intimately the fatigue phenomena of higher organisms
as to leave a very strong impression of its similarity, or even
identity, differing, if at all, chiefly in degree rather than in
character.
Second Series
The second series of experiments was directed to a determination
of the exact localized areas involved in the various reactions. The
first step toward a solution was the observation that when the
crown of gill flaments was directed away from the source of light,
as would be the case when the opening of the tube, and conse-
quently the direction of the head was turned from the light, it
frequently happened that the worm failed to respond to the usual
shadow stimulus. ‘This was variously repeated and with cumula-
tive evidence to the effect that the outer, or under surface of the
gills was distinctly less sensitive to these stimuli than the inner
surface. This is only what might naturally be expected, since
it is the inner surface which in expansion sustains direct relations
to sources of contact or approach from the surrounding medium,
while on the other hand the outer surface in the unfolding and
expansion of the plumose filaments would be least exposed.
Behavior of Tubicolous Annelids 303
Furthermore, it was found by close observation that the terminal
portions of the filaments were likewise more sensitive than the
basal portions, which 1s again what might naturally be anticipated.
If one watched carefully the behavior of a specimen following a
retraction of the gills it would be seen that the following protru-
sion Was a very cautious process, so to speak. ‘The tips of
the gills would be gradually extended, barely protruding beyond
the margin of the tube, and in this position the creature would
wait for a time, the filaments in the meantime actively vibrating,
as if searching for any source of danger. Next they would be
protruded somewhat farther, with another pause, and finally the
entire crown would be protruded and gradually expanded. ‘The
same process Was frequently observed, but in reverse order, in a
contraction following a very slight stimulus.
Following an enrol vigorous stimulus, as a very dense
shadow or a mechanical disturbance, the worms would frequently
remain for some time deeply withdrawn, and in again expanding
would do so with unusual caution.
This was most strikingly demonstrated in the following ex-
periment of further attempting to locate the sensitive areas by a
process of graduated excision of the gill filaments. ‘This was
attempted by a quick snip with scissors as the specimens were
quietly expanded near the surface of the water. The operation
was found, however, to be an exceedingly difhcult one, owing to
the lightning-like rapidity of the contraction of the worm at the
slightest disturbance. I succeeded in several cases in clipping off
about one fourth of the terminal portion of the filaments, and
found in every case that there followed an appreciable loss in the
acuteness of the sensory reaction, though as will be seen in the
later discussion of “mixed stimuli,’ it is not beyond doubt that
this apparent loss was due to the interposition just here of another
stimulus, that resulting from the mutilation, which for the time
being superseded that of the light. This is further suggested in
the fact that immediatelly following such an excision the creature
seemed to go through various maneuvers, rubbing the filaments
over each other in the most curious fashion, protruding and with-
drawing them in very unusual ways. And it was further ob-
304 Chas. We Hargitt
served that within perhaps a dozen hours the worm behaved in
quite a normal fashion, and seemed to have regained much of its
former sensitiveness, though its responses were less definite or
exact than before.
Attempts to excise further portions of the same gills upon a
following day showed the operation to be a much more difficult
matter than in the first instance. ‘This was not only due to the
same extremely rapid reaction mentioned before, but also to the
fact that the creature had apparently acquired a degree of caution
in protruding far beyond the tube, and had likewise apparently
become much more easily disturbed than before. ‘This became
even more marked upon a third or fourth attempt. It was as if
it had acquired an experience which served much as in higher
animals, and will have a peculiar significance in some of the later
discussions.
I finally succeeded in excising entirely the gills down to the base
of the palps in the case of several specimens by substantially the
same process, except that it was necessary, in order to secure this
result to place the scissors about a quarter of an inch below the ori-
fice of the tube, and then to cut off that much of the tube, and in
the process “catch’’ the crown of the retreating worm. Ina few
other cases I forced the worms out of the tubes by thrusting a
bristle into the smaller portion of the tube, posterior to the worm,
and then gradually irritating it till it emerged entirely, or so far as
to allow careful excision of the entire gill area, after which the
worm was allowed to readjust itself in the tube.
This last expedient was found, however, to give less satisfactory
results than the former, as a rule, since it often happened that by
the process of forcing the worm from the tube there was great
hability of injuring it sufficiently to seriously interfere with the
subsequent experiments, or to so modify the reactions as to give
inconclusive results.
As a result of these several experiments it was clearly demon-
strated that the sensory centers are within the gill areas, and chiefly
the more distal portions, though with the basal third of the gills
intact the creature still retains more or less of sensory activity.
But with the entire gill area removed close to the palps, the sensory
Behavior of T ubtcolous Annelids 305
power is wholly lost for the time being. It must be stated inci-
dentally that the capacity of regeneration is well developed in
these worms, and new tissue begins to make its appearance within
about two days, and with this regeneration there is recovered in a
more than proportional measure, the sensory function. The
entire gill is regenerated in about two weeks, or in some cases
perhaps less.
It will be evident, I think, from these several experiments, that
the sensory area is quite definitely circumscribed in this species,
and it may also be stated in this connection, that it is equally so in
the other species included within the present account. But it
remains to consider the further question, whether there be any
special sensory organ, answering the structural or functional
purposes of an eye? In reply it may be said that such organs,
the so-called eyes, have long been known in many of these worms,
and that they have been generally regarded as having a visual
function. ‘The citation from Claparéde in an earlier section of
this paper is one of many which might be given in support of the
view.
On the other hand it is likewise equally well known that there
are not lacking many species, not only among worms, but ccelenter-
ates, molluscs, etc., which are totally devoid of anything of the sort
and are nevertheless, quite as sensitive as species having an abun-
dant supply of these “eyes.’’ Such is the case, for example, with
species of Hydroides. So far as known the gills are wholly devoid
of anything like the “‘eyes’’ of Potamilla or Sabella; and yet the
latter are, so far as my observations go, much less sensitive than
the former.
Andrews several years ago made a critical study of the eyes of
annelids, comparing them with similar organs of molluscs. Com-
menting on points of the comparisons, he remarks, ““In_ both
cases the animals respond very quickly to slight sudden changes
in the intensity of illumination, bivalves seeking safety by retreat
within the hard shell, the annelid Saiceertae into firm tubes.’
But he continues, “The great number and position of these
organs suggests doubts as to their usefulness as eyes, the same
that have been made to the like organs of Arca”’ (op. Citi. 2379)
306 Chas FLargitt
This author found no structure which was in any degree com-
parable to the “eyes’’ in the other species investigated, including
Potamilla and Sabella. Yet he found that “‘the seat of sensation
is also in the branchiz; when these are cut off more and more, the
animal still reacts till nothing but the bases of the branchial stems
remain.’’ ‘This last point differs slightly from my own obser-
vations as just given above, though it would seem to be rather
a difference of degree than of kind.
It may be mentioned in passing that Dr. A. L. Treadwell, who
has carefully studied the histology of the branchiz of Hydroides,
assured me that he was not able to distinguish anything compar-
able with the eye-like organs found in other species of annelids.
It seems fairly certain, therefore, that we have in these annelids
a condition of highly sensory activity, more or less definitely
localized, yet without any specialized organs, as sensory centers,
or media of photic coérdination. Whether it shall be found upon
further investigation that there exists in these annelids certain
specialized cells, similar to those found by Langdon! in the earth-
worm, which may be regarded as having a similar function must
be left for the future to determine, though the probability of such
sensory cells may be confidently anticipated.
Third Sertes
A third series of experiments was conducted in relation to the
effects of colored light upon the reactions of these worms.
The general influences of various parts of the spectrum on
organic processes are too well known to call for special details. It
will be sufficient to refer to the observations of Lubbock,? Graber,’
and Engelmann,‘ as examples of many who have given attention
to the matter. Others will be cited in connection with particular
phases to be considered later.
My experiments with colored light were made with dark ruby
glass such as 1s commonly used in photographic dark rooms, and
‘Langdon, F. E.: Jour. Morph., vol. xi, 1895, p. 193, etc.
*Lubbock: Ants, Bees and Wasps, 1882, p. 186, et seq.
8Graber, V.: Grundlinien des Helligkeits- u. Farbensinnes der Tiere, 1884.
‘Engelmann, Th. W.: Ueber Licht- u. Farbenperception niederster Organismen, Arch f. d. gesam.
Phyisol., Bd. xxix, 1882.
Behavior of Tubtcolous Annelids 397
with deep blue cobalt glass. No attempt was made to determine
whether the glass was approximately monochromatic. However,
for the purposes involved, it answered fairly well.
The first experiment consisted in the very simple process of
interposing a plate of colored glass between the aquarium and
window, with no attempt to protect other portions of the aquarium
from the diffused light of the room. As might have been antici-
pated, under the circumstances no definite results were distinguish-
able.
The next experiment was made by enclosing the entire aquarium
under a dark box, one end of which was fitted with a colored
glass. [he specimens were left here for about a half hour before
observations were attempted, and then no differences could be
distinguished. After another half hour further tests were made,
but with no appreciable differences as compared with those under
natural light. In this instance the red glass was used. ‘The
experiment was varied by interposing blue glass, and again the
aquarium was left for about the same time, and no differences
being apparent, the apparatus was left for another hour before
testing, and again with negative results. Finally it was left over
the entire night and tested about 8 a. M. the next day, but again
with negative results. As will be seen by the later experiments
the apparent failures were due to the fact that immediate atten-
tion was not given to the matter of tests and observations.
At 8.15 the aquarium was placed under the red glass
and testing immediately, it was observed that there were no
responses to any of the former tests, even perfectly opaque screens
like a thick pasteboard, gave only negative results. hat 1s,
reactions were wholly inhibited. After an hour, further tests
showed that specimens were quite recovered and reacted to any
of the usual tests quite promptly and energetically. Repeating
the tests at intervals during another hour they gave the same
positive reaction which had been shown under normal conditions.
Removing the red screen entirely, in order to repeat the tests under
blue glass, it was found that the entire colony was strangely
irresponsive; none of the usual tests making any impression what-
ever upon them. Repeating the tests in every way possible failed
308 Chas. W. Hargitt
to secure any reaction for at least two minutes, when it was noted
that the specimens were apparently recovering froma sort of
dazed condition, and within five minutes all were reacting quite as
promptly as under normal conditions.
The specimens were left for about forty minutes under natural
conditions and were then placed under the red light again and
carefully observed, and with the same results as in the last
experiment except that it was found that after the first effects of
inhibition due to the red light, they were found to gradually
recover sensitiveness in from five toten minutes. Testing as before
at intervals of from five to ten minutes during half an hour they
reacted quite as under normal conditions. [hen again the red
screen was removed and the animals carefully watched and tested
as before with the same results, namely, that immediately follow-
ing the removal of the screen they were found to be quite indifferent
to any of the usual tests but that in from two to five minutes they
had quite recovered and reacted as under normal conditions. By
actual count the reactions were as follows: At the end of one half
minute none; end of one minute one specimen; end of one and one-
half minutes four specimens; end of two minutes ten specimens;
end of two and one-half minutes all but five of the colony of
twenty-five specimens. These did not fully recover normal
activity until some six minutes had elapsed.
At two o’clock they were placed under the blue screen and care-
fully watched as before. ‘There was at first the same inhibition
of reaction as in the former case. At the end of two minutes the
first indication of recovery of sensory activity was noticed. Re-
peated at intervals of one minute it was found that in four minutes
apparently the entire colony became normally responsive. ‘Tests
made at intervals of about five minutes for nearly half an hour
seemed to show that there was a higher average of response than
under the red light. At 2.25 the screen was carefully removed
and tests showed specimens to be acutely sensitive, respond-
ing to the slightest shadow.
The experiment was repeated, after adding a new and fresh
colony to the aquarium, and with almost identical results.
Behavior of Tubicolous Annelids 309
Again at four o'clock the same experiment was repeated, and
withthe same results, the creatures losing for about five minutes any
sensory discrimination, but quickly recovering it with apparently
greater acuteness than under natural light. “This was likewise the
case at the conclusion of the experiment at six o’clock, when the
screen was removed, every specimen reacting with great prompt-
ness and vigor.
‘These experiments were repeated again and again, under vary-
ing conditions of light, temperature, etc., but with so large a
measure of constancy as to preclude the possibility of the opera-
tion of merely accidental or incidental causes.
As has been noted in an earlier section, the coloration of these
creatures is very variable, ranging from orange-red on through
yellow, dull brown, blue and purple of all leis of pomiacen
to almost pure in some cases. In connection an the experiments
on colored light occurred the query, whether there might be any
possible relations between the various colors of the worms and the
exposure to varying intensities of light > While their arrangement
in the colonies gave no apparent support to an afhrmative probabil-
ity, still it seemed well to carefully test the individual reactions of
the more conspicuously colored specimens. ‘This was accordingly
done in repeated instances but with entirely negative results.
Whether in natural light or under the influences of artificial and
colored light; whether among a group of bright orange-colored spe-
cimens, or in deep purple colored specimens, not the slightest
individual difference could be distinguished. And the same was
equally true whether the experiments were made with colonies just
taken from the natural habitat or those longin the aquarium. — It
may be assumed therefore that these rather remarkable colors have
little or no adaptive relations to light, any more than to selective
protection. It seems most remarkable that in a single colony of
these worms numbering perhaps thirty or more specimens, and
probably arising from a single brood or generation, all this range
of color variation should be found, and that without any apparent
significance in relation to their habits or life history, yet such seems
to be the case here as in many other equally well known instances
among this and other phyla or classes.
310 Chas. W. Hargitt
In this connection may be mentioned an illustration of obser-
vations cited by the writer in an earlier paper,! namely, the decline
of color brilliance under the artificial conditions of the aquarium.
In Hydroides this was quite marked, as was also the case with
Protula. The colors in which this was first noticeable were the
reds, orange and yellow, though it was not lacking in the bluish
tints. These changes were less evident in Potamilla and Sabella,
the colors of which are less striking.
It might be inferred from what has gone before that these color
changes would have little effect upon the sensory responses of the
specimens, and such was the case. While as already shown, any
evident decline in vigor was likely to involve a corresponding
decline in sensory qualities, but there was nothing to indicate that
there was any necessary relation between these phenomena and
that of decline of color.
EXPERIMENTS ON OTHER ANNELIDS
Reference has been made repeatedly to Potamilla and Sabella,
but no special account has been given of definite experiments made
upon them. As already stated, the numbers of these specimens
obtained were comparatively few, and the experiments much less
extensive than upon Hydroides. But one species of each was
found, namely, Potamilla oculifera, and Sabella microphthalmia.
In size and habit they are very similar. ‘Their tubes are not
calcareous and rigid as with Hydroides, they are much smaller
in size, with a habitat among sponges, ascidians, etc. [heir general
behavior is similar to that of Hydroides, but very much less striking.
The experiments made upon them were the same as have been
already described for the other species. In various experiments
with light of varying intensities—to electric and colored light, to
touch, etc., they gave no reaction which was not much more clear
and convincing in the former. ‘To colored light their behavior was
almost uniformly negative, as was also the case with that of the
electric lamp. Furthermore, their reaction time was much more
sluggish and uncertain than in the case of Hydroides. Their
1Science, vol. xix, 132, 1904.
Behavior of Tubicolous Annelids 311
reaction to shadow stimuli, while similar to that of Hydroides, was
as before very much less striking. It was frequently the case that
only after some two or three more or less dense shadows had been
cast over them that a reaction followed, and then comparatively
slow, and so to speak, deliberate-like. ‘Tactile stimuli, and exci-
sion of portions of the gills induced, asin Hydroides, apparent
caution and more promptness of reaction.
Concerning the behavior of Protula intestimum a brief reference
has already been made in another section. It may be worth while
to cite a few additional observations which serve to confirm and
accentuate those of the other species. Protula is a large tubicolous
annelid, quite common in the Bay of Naples which he Hydroides
secretes dense calcareous tubes curiously coiled in serpentine as-
pects, asshownin Fig. 3. “Uhesetubesare often 180 mm. ormorein
length and from 5 to 8 mm. in diameter at the opening. ‘The color
is senele of a more or less uniformly bright orange red, and a col-
ony of these creatures fully expanded “ise a most brilliant
picture.
Their reactions to the various stimuli already mentioned are
very similar to those of Hydroides, though somewhat more
erratic. hus it frequently happens that while at one time re-
sponses to shadows are very prompt and decisive, at another time
they may be apparently quite indifferent. Perhaps on the follow-
ing day this may all be changed and the colony acutely sensitive
to the slightest variation in the intensity of light. Furthermore,
they seem less adapted to continuous aquarium conditions than
do species of Hydroides, a lowering of vital tone being more or
less evident after about a fortnight. ‘This is also associated with a
decline in the color tone of a more marked degree than 1s the case
with the former species. And with these changes the reactions
to various stimuli, especially light, become very uncertain or
even entirely lacking.
GENERAL DISCUSSION
The foregoing experiments and observations bring before us
certain clearly defined facts which call for further consideration
and explanation. ‘This may be facilitated, perhaps, by a com-
212 Chas. W. Hargitt
parison with similar facts from other sources, some of which have
already been referred to. Patten,’ whose somewhat extended
observations and study of the eyes of molluscs 1s well known, has
offered suggestive comments concerning their reactions to varying
intensity of light. For example, he has observed that in Arca,
whose sensory organs are very numerous and highly complex, the
reaction is much less marked than in species of Avicula in which
the slightest shadows, such as that of a pencil, causes instant
response. He makes a similar comparison between Cardium and
Pecten, the former of which is extremely sensitive to varying light
intensity but only slightly so in reference to mechanical disturb-
ances like a jar of the aquarium or the action of waves; while the
latter is highly sensitive to mechanical stimuli but much less so to
shadows, though in this case again the sensory organs are both
numerous and complex.
Hence he concludes that “ We are led to suspect the presence of
some other factor which must, when known, account for the
apparent agreement in functional powers between two organs so
widely different in structure.’’
Concerning Pecten, Patten also observed the phenomenon of
fatigue, to eee reference has been made previously, finding that
‘specimens experimented upon frequently become erratic in
responses, “so that finally, even quite deep and sudden shadows
may produce only restless or uneasy movements, or perhaps no
effects at all’’ (ibid., p. 614).
Darwin’s observations concerning the reactions of earthworms
to light are too well known to call for more than passing notice, ex-
cept to point out that this critical observer expressed no hesitation
in ascribing to these creatures definite light perceptions, nor did he
hesitate to suggest the cooperation aan coordination of other
factors in nemo about the varying results obtained in his
experiments. For example, he observed that, under the conditions
of feeding, mating, etc., or where they were otherwise preoccupied,
they either failed entirely to react to the light stimulus, or did so in
very different degrees of promptness or directness. He even
IMitt. Zod]. Sta. Neapel, Bd. vi, p. 608, et seq.
Behavior of T ubtcolous Annelids 3 13
suggests that at times their behavior indicated changes of nervous
states, “as if their attention were aroused or as if surprise were
felt.’”
Under the later development of the theory of tropisms, and its
extension to the phenomena of animal behavior, its dominance
has relegated the earlier views to the limbo of discarded anthro-
pomorphisms so called. Without essaying any review of the pros
and cons of this problem, it may be said that already a reac-
tion has taken place and frankness compels a reconsideration
of some of these discarded and discredited views. Such a review
has already been made by Jennings’ so far as it relates to the lower
organisms, and his conclusions must, it seems to me, be equally
true for many if not most higher animals as well.
Among those who have given especial attention to the behavior
of annelids, the most important are Loeb,’ and Nagel,‘ and to a
less extent Radl.°
As is well known, Loeb maintains without hesitation that the
behavior of these annelids in their relations to light is governed
by the same laws as is that of plants. Indeed, concerning the
general orientation of these creatures he contends that they are
governed by two fundamental influences, namely, gravity and
light, and in support of this view describes various experiments,
and gives illustrations as to various details.
Concerning the influences of gravity as a factor in the case of
the species i am unable to find any adequate confirmation of
Loeb’s views. An examination of the several photographs pre-
sented herewith will, it seems to me, show that neither in the case
of single individuals nor in that of colonies is there any such uni-
formity as to position or relation as would indicate the predominant
influence of any single factor or force. Where single specimens
are found growing either upon a shell, or upon a solid and fixed
1The Formation of Vegetable Mould. London, 1881.
2Contributions to the Study of the Behavior of Lower Organisms. Washington, 1904.
3Der Heliotropismus der Tiere und seine Ueberlinstimmung mit dem Heliotropismus der Pflanzen.
Wiirzburg, 1890.
‘Der Lichtsinn augenloser Tiere. Jena, 1896.
5Untersuchungen tiber den Phototropismus der Tiere. Leipzig, 1903.
314 Chas. W. Hargitt
support, as a rock, the general position is more or less prone, with
a varying serpentine aspect, as shown in Fig. 1. In certain cases
the mouth of the tube would be found in one direction, and in
others quite otherwise. Sometimes the head projected upward,
often downward. This is better shown in some measure in the
case of colonies. While here the general tendency of the tubes
is vertical in growth, many are found diverging laterally, and in
not a few cases directly downward, as may be seen in Fig. 2.
In this figure the open mouths of several tubes may be observed
near the top. This colony is attached to a flat stone, whose center
of gravity would serve to maintain a constant position, hence the
variously coiled tubes are evidence of a correspondingly shifting
behavior on the part of the worms during their growth. This 1s
perhaps still better seen in Fig. 3, a colony of Protula, several
of which I studied at Naples. While here as in the former
figure, the general aspect of the tubes 1s toward the vertical, still
their lower serpentine coils show the varying conditions of
orientation at different periods of growth. But if further evidence
be needed it is found in the tube coiled about the larger central one
the mouth of which opens almost directly downward. But
furthermore, colonies kept in the aquaria for nearly two months,
during which time the tubes had grown almost half an inch, showed
not the slightest evidence of any response to gravity. They did,
however, show unmistakable evidence of adjustment to conditions
favorable for respiration, and for the capture of food; and these
I regard as of far more importance in determining orientation
than either gravity or light.
Zeleny,’ who has reared the larva of these serpulids in connec-
tion with his investigations upon their regeneration and regula-
tion, has incidentally recorded brief, but very interesting observa-
tions concerning the behavior of the young worms in relation to
“gravity, light and food, but was able to find no general rule,
although some groups seem to be arranged with respect either to
maximum food-obtaining ability, or with respect to a lateral
stimulus of unknown character.’’
Biol. Bull., vol. viii, p. 309.
Behavior of Tubicolous Annelids 315
While not sufficiently extended to warrant definite conclusions,
these observations go to corroborate my own as to the impor-
tance of adjustments in reference to respiration and obtaining
food.
Concerning the reactions to light Loeb! holds very decided
views, and has taken Nagel sharply to task concerning a sugges-
tion that the behavior of such creatures as responded to the
stimulus of shadows might be due to something akin to a sense of
apprehension as to the approach of an enemy or other threatening
danger. In opposition to this he undertakes to show the necessity
for the existence of some organ of perception similar to a cerebrum
in order to enable the creature to possess any instinct, or sense of
self-preservation, such as Nagel’s suggestion implies. Loeb
suggests, on the other hand, that the effect of light may tend to
induce a muscular expansion or stretching so that the worm
emerges from the tube. If now, a sudden diminution of light
follows, asin the case of a shadow, an opposite muscular reaction
is Set up, and consequently the worm rapidly withdraws into the
tube, and the sudden retraction of the worm is only the expres-
sion of the rapid extension of the contraction wave of the worm’s
musculature.
Of the relations of light to many of the phenomena of life there
is not the slightest doubt. “That in many cases these relations may
be so intimate as to warrant designating them by such terms as
phototropism, heliotropism, etc. But that there 1s any such
correlations involved in the aspects of behavior under review as to
warrant regarding them in the light of cause and effect seems not
only doubtful, but wholly inadequate. As Radl has remarked,
“Diese Erklarung Loebes ist vielleicht ebenso einfach wie
unrichtig’’ (op. cit., p. 78).
In the present case there are many difficulties in the way of the
rigid application of any theory of tropisms. In the first place
the widely differing degrees of responsiveness among closely
related species of slightly differing habitat is difficult to explain by
this means. Again, the varying behavior under slightly differing
1Archiv f. d. gesam. Physiol., Bd. Ixvi, 1897, p. 461.
316 Chas. W. Hargitt
conditions, as for instance, where specimens have been rendered
wary under attempts to excise the gills, or where a given specimen
has been placed for a time so near the surface of the water that
it has been compelled to extend itself in an unusual manner to
bathe its gills at all, under which circumstances it often fails
entirely to react to the ordinary stimulus. In this case we have
apparently what Jennings has designated as “ physiological states’’
namely, conditions in which the internal changes involved in
respiration perhaps, have served to render the animal indifferent
for the time to stimuli which under normal conditions are very
constant and effective in their action, or perhaps as, suggested
above, complex or mixed stimult.
-That light of itself is not a determining factor in impelling the
worms to emerge from the tubes is evident in that this attitude
occurs 1n darkness as well as in light, by night as well as by
day.
Furthermore, it must be recalled in this connection that the
particular stimulus involved in these observations, as previously
pointed out, is not light at all directly, but the lack of light, or
the shadow. ‘The response is, therefore, induced by a negative
stimulus, if such an apparent paradox be tolerable in relation to
phenomena of behavior. Of course, it is not overlooked that
Loeb has designated these and similar reactions as due to “nega-
tive heliotropism.’’ At the same time it is not clear that in the
present case we are dealing with phenomena at all comparable
with those associated with negative heliotropism as ordinarily
understood. For as already observed, the phenomena are not
in themselves negative. They are not dependent upon any given
degree of light, or rather darkness, but to the suddenness of the
change.
Rawitz' long ago called attention to exactly this peculiarity in
describing the reactions of species of acalephs. Referring to the
suggestion of Drost concerning the effect of shadows as stimuli,
he says “Ein Reiz kann immer nur von etwas positivem, also in
diesem Falle vom Lichte, ausgetibt werden, niemals aber von
'Der Mantelrand der Acalephen. Jenaische Zeit. f. Naturwiss, Bd. xxii, xxiv, xxvii.
Behavior of T ubicolous Annelids 3 17
einer Negation. Und Schatten ist eine Negation, die des Lichtes
namlich,”’ etc.
But it will be asked, if we are not to regard these reactions as
due to some form of light stimulus, whether it be positive or
negative, nor perhaps even to photokinesis, how then shall they
be interpreted? For myself they seem much better explained as
activities concerned with protective adaptation than by any merely
mechanical or mechanico-chemical adjustments. If the tubes
in which these creatures have their homes have been acquired as
protective adaptations, and if it be granted that they, in common
with other annelids, have some sensory capacities, including
light perceptions, then it would not seem a far call to interpret
the phenomena in question as sensory reflexes which have become
instinctive through multiplied generations of struggle with
various predatory enemies, whose approach cast fateful “shadows
before.”’
It only remains to consider, briefly, the reactions involving the
influence of colored light. Ina general way the principal features
concerned under this head have long been known. Experiments,
have shown that violet rays have a definitely higher phototropic
quality than have those of lower refrangibility, such as orange
and red.
Nagel* has called attention to the same facts in relation to
molluscs, though he enters into few details, and frankly admits
the desirability of additional observations. He believes that all
the colors of the spectrum, except red, have a measure of stimulus
for such organisms as show, like his mussels, sensory reactions
to the presence of light.
Loeb? has also made similar observations, and concludes that
“since the heliotropic phenomena appear only weakly or not at
all behind dark red glass, while they occur just as in diffuse day-
light behind dark-blue glass, the few red rays which penetrate
the dark-blue glass cannot be responsible for the heliotropic
phenomena which take place so energetically behind this screen
but can be due only to the activity of the more refrangible rays.’’
1Der Lichtsinn augenloser Tiere, Jena, 1896, p. 53-
*Physiological Studies, vol. i, p. 18.
318 Chas. W. Hargitt
A comparison of these views with the facts cited in connection
with my observations will show that in several particulars there
are obvious differences. In the first place, my observations show
that, though there is an inhibitory quality in the dark-red rays
when first brought into relation with the worms, it is only
temporary. Within some ten minutes, or after time has allowed
the specimens to become adjusted in their sensory activities to
the new conditions, they become almost if not quite as responsive
to the shadows asunder normal light. And, moreover, the same
thing happens under the catches of the dark-blue rays. Here
hare 4 is a brief period of inhibition, as in the case of the red rays
though the adjustment is more rapid. | Usually within five minutes
fhe animals have become accustomed to the new light and behave
quite as under normal light. I have pointed out in the earlier
connection that it was not certain that the red glass with which
my experiments was made was monochromatic, bit since it was
sufficiently so to render the light passing through it nonactinic,
it may be assumed that it was sufhiciently so to prevent the
passage of the essentially blue or violet rays, at any rate in sufh-
cient measure to act as a stimulus. It seems not improbable that
the apparent discrepancy concerning the effects of the red rays
may be due to the fact that a longer time is required in adjustment
to these rays than to the blue.
In the next place, and here I have seen no similar records, the
most striking difference appears in the fact, previously mentioned,
that following the removal of the red screen there was a most
remarkable inhibition of all sensory responses under the effects
of normal light. For several minutes, not more than five, the
animals behaved in the most striking manner, much as if asleep,
and recovered sensory activity in much the same way as if awaken-
ing. Nothing of this sort was apparent when suddenly brought
from under the influences of the blue rays. Here if anything
their sensory activities seemed even more than normally alert.
However, while this behavior seems more or less peculiar and
singular, I am inclined to believe its explanation may be found
along the same line of influence which has already been cited,
namely, the inhibitory effects of the colored light when first applied.
Behavior of Tubicolous Annelids 319
Under both the blue and red there was a period of inhibition
for some time varying in the two cases, as pointed out above, but
most effective in the case of the red. Recovery of sensory activity
under these changed conditions involved, of course, some physio-
logical change in the sensory apparatus in response to the
peered conditions. Now in the sudden emergence from the
low refrangibility of the red rays which had involved the preceding
adjustment there was involved likewise another physiological
change but in the reverse order.
SUMMARY
1. Under the varying degrees of light intensity furnished by the
sixteen candle-power incandescent lamp, the diffuse light from
north and south windows, and direct sunlight, the results of all
experiments involving increased intensity of light were uniformly
negative. On the other hand, experiments involving a sudden
decrease of light intensity gave results as uniformly positive.
However, the behavior does not seem to be essentially comparable
with that usually designated as negative heliotropism.
2. Experiments continued without interruption for some time
gave rise to behavior analogous to that of fatigue.
3. Various experiments involving the direction of light contact,
the excision of branchia, etc., showed that the sensory areas are
located in the branchial filaments, chiefly the inner and terminal
portions.
4. When the animals are brought under the influence of red
and blue light sensory activities are for a time inhibited. ‘This
is more marked under the red than the blue. On the other hand,
when brought suddenly from the colored light into normal white
light there is apparently an intensified sensory acuteness due to
the blue light, while the effects of the red seem to have been just
the opposite, namely, to positively inhibit sensory response for a
period of from two to five minutes.
5. Species of Potamilla and Sabella behave in essentially the
same manner as do those of Hydroides, though with less acuteness,
promptness and certainty. This is somewhat remarkable since
320 Chas. W. Hargitt
bd
these species are abundantly supplied with “eyes,’’ which are
entirely lacking in Hydroides.
6. Species of Protula likewise behave in essentially the same
manner as do the others, though apparently somewhat more
erratic and uncertain in their reactions.
7. [he experiments tend to discredit the theory of tropisms,
since no single factor, such as light or gravity, furnishes an
adequate explanation.
8. The experiments strongly suggest the presence in the gill
filaments of these creatures of sensory cells and nerve endings
through which are coordinated by means of nervous centers the
various aspects of behavior toward protective or physiological
ends.
INHERITANCE OF DICHROMATISM IN LINA AND
GAs FROMUDEA
ISABEL McCRACKEN
In a series of experiments with the dichromatic species of beetle,
Lina lapponica, as described in a previous paper,’ it was deter-
mined by breeding through a series of four generations that the
behavior in heredity of the alternate characters of the species
spotted-brown” and “black” follows, in general, Mendelian
behavior.
This was evidenced by the fact that the broods from a first
cross between spotted individuals and black individuals are either
wholly spotted or the broods are made up of individuals, some of
which (and the majority) are spotted, and some are black. In
other words, it appeared that “spotted-brown”’ was either com-
pletely or partially dominant, depending upon unknown causes.
That black is a recessive character was evidenced by the fact
that it frequently did not appear in a first cross between S and B,
in which case it was extracted in the next generation and thereafter
bred true.
The proportions between alternate characters from hybrids in
Lina showed no parallelism with typical Mendelian proportions,
however, and called for further experimentation.
oe
EXPERIMENTS WITH LINA
I have during the past season (1905), sought to determine the exact
proportion of dominant to recessive in successive generations bred
each generation from hybrid dominant parents. ‘The individuals
used as parents in each generation were spotted individuals, from
broods composed of both spotted and black individuals, that had
1 Inheritance of Dichromatism in Lina lapponica, Journal of Experimental Zodlogy, vol. ii,
pp. 119-136.
Journat or Experimentat Zoétocy, Vor. 11, No. 2.
222 Isabel McCracken
proven their hybrid character by producing black as well as spotted
offspring.
Diagram I shows character of matings and color character of off-
spring throughout four generations from hybrid parents. _S stand-
ing alone signifies a brood in which S was completely dominant,
S B standing side by side signifies a brood consisting of both
spotted and black individuals, the spotted being present in larger
numbers (or partially dominant).
Sirs Saxis
[sexs] eel
|
S and S B Smandesmb
eo SPP aA0
S andS B Seb
| : |
Sand’ SB SeB
| |
|
Sand SB
Diagram 1. Showing pedigree through four generations from hybrid S.
Inthe past year’s experiments, as in the previous ones, the hybrid S
parents of the first generation produced two sorts of broods, broods
in which S was completely dominant and broods in which S was
partially dominant. In 1904, by breeding consecutively from
completely dominant S broods, pure S broods were obtained in
the “third generation. Breeding in 1905 consecutively from
hybrid S parents chosen from broods in which S dominated
partially, in each generation both ‘sorts of broods were obtained,
that is, completely dominant S broods and partially dominant S
broods. In the partially dominant S broods the proportion of
dominant to recessive gradually increased through the fifth genera-
tion when hibernation began.
It may be noted here that last year (1905) there occurred in
broods both from dominant and recessive parents, an occasional
wholly melanic individual, thorax as well as wing covers being
totally black. Such individuals were utilized for a study of
“sport-variation’”’ in heredity.
Inheritance of Dichromatism 323
Table I gives a summary of the data in each generation from
S x S parents, including total number of broods, number of broads
in which S lease completely, number of broods in which S$
dominated partially (designated as “ mixed broods’’), total number
of melanic or sport individuals, total number of individuals and
proportion of S to B in partially dominant 5 broods.
TABLE I
Total | Propor-
No. Com- Total Total Total tion of
- ra pletely No. of No. of No: of, | S28
ate Domi- Mixed Melanic | Individ- | in Mixed
eee nant S Broods. Sports. uals. Broods.
Broods
Pirsh GER eTAhlOM! ecto e--1= 118 13 105 10 | 3442 | Bas BU
Second generation ......... 45 27 18 I * 1050 | Heit Bal
Third generation .......... a2 121 21 I AZO Senet
Fourth generation.......... 19 16 3 ° 549 |. 26nia
The increasing proportion of spotted individuals produced in
successive generations shows a progressive dominance of the S
character. That B might eventually be excluded from the partial
dominant S line seems highly probable, but conclusive evidence
cannot be obtained on this point until success attends hibernation.
It seems, however, that we may consider S as dominant in a
greater sense than that denoted by Mendelian terminology. It 1s
progressively dominant in a way not accounted for by Mendelian
law.
EXPERIMENTS WITH GASTROIDEA
The relation of dominant to recessive in alternative characters
of the kind studied is more clearly brought out in the following
series of experiments with Gastroidea dissimilis.
Gastroidea dissimilis is a dichromatic species belonging to the
same family of beetles as Lina lapponica, the Chrysomelidz.
When an adult of this species first issues from its pupal case,
the wing covers are softand yellow. During the process of harden-
324 Isabel McCracken
ing all individuals become at first a smoky brown and later black.
From this black condition the individual color-development pro-
ceeds gradually in one of two directions. In one series of indi-
viduals there is a gradual deepening of the pigment to a shiny
permanent deep blue-black condition. In the other series there is a
passing from the black condition to a shiny permanent bright
green condition. ‘The color is fixed in each series within two or
three hours of issuing.
We find, therefore, in Gastroidea, in its color-development, the
same condition found in Lina, that is, a primary condition through
which all individuals pass (and in which one series of individuals
remains), and a secondary condition into which one series of
individuals only passes.
The two colors, black and green, are therefore represented in the
species, no intermediates having been observed during the course
of the experiment involving the handling of many thousands of
individuals (about 26,000). The beetle is small, about 5 mm.
long, feeds on dock or rhubarb and breeds from late in February
until early in September, producing normally five or six genera-
tions ina year. Under laboratory conditions the breeding season
was prolonged through December and seven generations were
reared from a lot collected March 16, 1905.
The experiment began with two hundred adults, one hundred
black, one hundred green. ‘These had been mating out of doors,
blacks and greens promiscuously, and ovipositing had begun.
It was comparatively certain that there were no pure bred indi-
viduals in the collection, since presumably crossmating had been
going on ever since dichromatism had been established in the
species.
The first eggs were obtained March 16. These hatched March
24 and issued as adults April 23. At this season, therefore, mature
adults were obtained in less than forty days from the egg.
Females oviposited five or six days after maturing, each female
ovipositing ten to fifteen egg masses during the following few
weeks with from twenty to thirty eggs in a mass, frequently run-
ning as high as fifty eggs in the mass. With this material as a
nucleus, | sought to determine whether there was a behavior of
Inheritance of Dichromatism 325
the color-alternatives in heredity corresponding to that of Lina
lapponica. My plan was to breed successively from completely
dominant hybrids, if such were found to be present, to breed suc-
cessively from partially dominant hybrids, and, finally, from
recessives that had from one to several pair of dominant ancestors.
In the following tables results in total are tabulated for seven
generations of laboratory reared lots.
Table II gives the data of the first generation reared from out-
door collected adults. Individuals used as parents for these data
were confined in the laboratory with mates of similar color. Since
it is altogether possible that some or all of the females had mated
with individuals of alternate color before laboratory isolation, the
results indicated give little more than known parentage for second
generation data.
TABLE II
First Generation
Coleen Namber ss |) Number | Number | Total No.
Character of of of of Proportion of B : G in
of | Green Black | Mixed Indi- Mixed Broods.
Matings. Broods. Broods. | Broods. viduals.
GXG 33 ° | 6% 580 | 1 B in each of six G broods.
Beeb al fo) | 2 39 1207 ii), 6) 1
*One B individual in each six broods.
The single black individuals occurring in otherwise green broods
of G x G are possibly due to earlier matings of the female parents.
The data from the Bx B matings seemed to point to B as the
dominant type, the actual results being presumably somewhat
modified by uncontrolled matings previous to collection.
In succeeding generations the same method of mating was
followed as with Lina lapponica in 1904, that is, females of one
brood were confined in a breeding-jar with males of another and
vice Versa.
326 Isabel McCracken
For second generation data five categories of matings were
established as follows:
A-G X G, each parent from broods of BX B parentage that had produced mixed broods.
B-G X G, each parent from broods of G X G parentage that had produced broods of green only.
C-B X B, each parent from a mixed brood of B parentage.
D-B X B, each parent from broods of BX B parentage that had produced broods of black only.
E-G X B or B & G, the G parent from a pure G brood, the B parent from a completely dominant
B brood.
Table III gives a summary of the data of these matings.
TABLE III
Second Generation
|
Total
Mating bs | | No. | No. | No. of “ Proportion of
Cate- reac | Parents: Green | Black | Mixed |” ® B: Gin
gory. | aay Broods. | Broods. | Broods ee Mixed Broods.
ae a | as = ee
A 133 << 133 GX G (mm!) 16 fo) 337
B GXG GXG A | fo) 98
(e BX B BX B(m) cy | 5 25 740 eee 4
D BX B BX B (cd?) ° | I fo) 22 But 1 brood reared.
GX Gl|eo>cBg) | |
: BX Bi or \ | fo) I 15 384 3.6 21
Bo X GQ J | |
m! in parenthesis means that the individuals mated were from mixed broods.
cd? in parenthesis means that the individuals mated were from broods in which B was completely
dominant.
This data shows that the recessive greens (G) breed true under
either condition A or condition B. The dominant blacks (B)
produce, under condition C, either broods in which B dominates
completely or mixed broods in which B dominates partially.
Since but a single brood was reared under condition D, the data
are insufhcient. B x G produces either completely dominant B
broods or mixed broods.
For third generation data five categories of matings were estab-
lished as indicated by Diagrams 2, 3, 4, 5 and 6.
Inheritance of Dichromatism
Diagram 2. Mating category A.
First matings............ BxB Bx B BxB BxB
ea [ee ene [ara
| | |
irstspenisces.scse.co ce B-BG B-BG B-BG B-BG
| | oar ed)
| |
Second! gene..:---...-.-- G G
|
hinds pene -oaneesee
Diagram 3. Mating category B.
Birshamatings..) see. BxB Bx B BxB Bix B
[ena oe eae lea
| | | |
IBIS fea Clee eeen an seeeerce= B-BG B-BG B-BG B-B-G
[ | | |
| |
Second! pene sssc-----c-- B-BG B-BG
| |
Diagram 4. Mating category C.
Birst)matings: --:---.-.- BxB Bx B Bx B Bx B
Partie es |: = lie! fies
| | | |
Birst renss.-< 50652-35055 B-BG B-BG B-BG B-BG
eae acs 2-2 | |
| |
Second) gents. ---..-.- B-BG B-BG
| |
B-BG
Diagram 5. Mating category D.
Mirstymatingss.----.... Biss /B Bx B BEXEB, Bx B
al ed ee) S|
| | | |
Binsthgensen-: acccessss<s B-BG B-BG B-BG B-BG
| | | |
| |
Second penl....---.. ---- B-BG cae
B-BG
a2 7
328 Isabel McCracken
Diagram 6. Mating category E.
First matings............ Bx B GxG Bx B GxG
(Sal (ae Kae Pease
| | | |
BKStROUs.-voeresensoes B G B G
| S| | a
| |
Second! gente--.-.---== B B
[Mi . Rae Et Seal
|
‘Mewtistel) Fee ecccbocccenoe BG
Inspection of these diagrams shows that the original black
parents (B x B of the first matings, except in Diagram 6) may be
considered hybrids inasmuch as they produce both black and green
individuals.
Table IV gives a summary of the results of third generation
matings.
TABLE IV
Third Generation
Ma- | | | Total Proportion of
ting Great- Grand No. | No. No. | No.of | B:A in
Cate-| gtand- | scents. Parents. Green | Black | Mixed | [pdj- Mixed
gory. | Parents. | Broods. | Broods. | Broods. | yiduals.| — Broods.
aos = fe
A BX B |GXG GXG | 29 ° ° 733
Be |) BoB | BSB Ga) Goce | 27 ° rk 653
C BEX Be | BBG) BBG) fo) 8 19 688 27 Ons
D Bx BBX By Gad) Bee BiEd)) ° 18 3 518 Pie, BT
BX B \ 420 7, B11
E W \
GxG{ BEX G BX B (cd) eae 15
*One B individual in a brood of nineteen, otherwise G individuals.
Comparing categories A and B, we find, with the single excep-
tion of one individual, that broods of green individuals only are
produced by G x G matings, whether there are one (Category B)
or two (Category A) generations of green parentage.
Comparing Categories C and D, we find that under each of
these conditions, that is, whether the B parents are chosen from
broods in which B dominates completely, or from mixed broods,
Inheritance of Dichromatism 329
two kinds of broods result, broods in which “‘black”’ dominates
completely, and mixed broods. ‘The comparative number of
broods in which “black”? dominates completely is, however, much
greater in Category D than in Category C, and the comparative
number of black individuals in mixed broods is much greater in
Category D than in Category C. ‘This shows a decided difference
in the influence of the black character in these two categories.
In the fourth generation, ten categories of matings were estab-
lished as indicated in Table V. This table gives a summary of
the results of these matings.
TABLE V
Fourth Generation
SES | G-great- Great No. | No. !No. of} Total Ear
ting = Grand- i f ; . B:G in
grand- grand- Parents. G’n | Black | M’d |No. of E
eat parents. parents. Re Broods | Broods|Broods Indi’d. MGs
gory | roods.
A BOG Ben Ga Gi(m)) iG XG: GxXG righ, Wp ey late 457
B BX B |BX B@)|GXG(@m)|«Exe 17 ro) ° 404
Cc |BXB |BX Bi) |BX B@)|GxG(m)| 6 ° ° 69 |
Be By) |
D Gx Gf BXG BSB Gai) GX G(m) ll, So ° 322, |
E |BXB |BX B(cd)| BX B(m)|GxG(m)| +4 jae 73
F BSB BX B(m) BX B(m) | Bx B aS Aisey || eG an
G | BX 8B |BX Bd)|BX Bcd px B a i og ) 199
| BaxeBi| | |
H eExc{i2s% BX B(cd)|B x B(m) Se) 3 for |) as) Or
Beer) | |
I excels ~ © BX B(m) |B yx B(m) OF Ny a2 aaltanes ZO STAs pcal
1 | BX B\||BxX B(cd)| BX Bled) 5 z 3 , ‘ aM
FEyeGie Gs NGS :f'
Comparing Category D of Table IV with Category G of Table
V we find, as in Lina lapponica, that by selecting individuals in
which there is complete dominance, the recessive character is
eventually eliminated.. In other words, the fourth generation
from hybrid parents, along the line of complete dominance, breeds
true to the dominant character, no recessives appearing in the
offspring.
330 Isabel McCracken
Comparing Categories A, B, C, D and E in Table V we find
G x G breeding true in the fourth generation whether the imme-
diate parents only are green or there has been a lineage of green
for one, two or three generations.
In the fifth generation, seven categories of matings were estab-
lished as indicated in Table VI. ‘This table embodies a summary
of these matings.
TABLE VI
Fifth Generation
| | |
a \eecs| ce Gréat- | Wes fvoul Neste cles eee
ting Grand- lpsadtet BE bate __ | No. of|B : G in
Cate- Fee grand: Brand. parents. | hare Indi- | Mixed
Oa parents. | parents. parents. | Bd’s Bd’s|/B ’s| wide Bea
| | |
at eats | ey
A | BX BY) GX G GXG GXG GG) 325) onl ones29)
B |BXB|BXB GXG GXG GX G]}24| o| o| 679 |
CB eB eBe@eB BX B GXG GG |) 9s) 10) || onl) waz0n|
Ds | BAcB) | BX B BxXB BX B DAA eileen cll <c75
D |/BXB|BXB BX B BX B GXG|14/| 0} ©| 370)
ED | BGBa Bx 8 a) | BS Bi Ga) Bi Bim) PBs 1B) 708935 8 | 1336 eG su
F |BXB/ BX B (cd)| BX B (cd)| BX B (cd)| BX B 1s | 0 | 397
BX B(cd)| | BX B(cd)) | BX B(cd)
Gabe B GxG , GXG ee ey Ole?) 6 | He Gee
1The letter G in this connection indicates ‘‘ great.”
Data in Table VI shows G XG continuing to breed true in
Categories A, B, C and D, that is, whether there has been a line
of green parentage for four generations or the immediate parents
only are green. Comparing Category F of ‘Table V with Category
E of Table VI we find an increasing proportion of broods in which
black is wholly dominant and an increasing proportion of black
individuals in the broods in which black is but partially dom-
inant. In Category F, Table VI (compare Category G, Table
V) black in the completely dominant line continues to breed
true.
In the sixth generation six categories of matings were estab-
lished as indicated in Table VII. This table embodies a summary
of the results of these matings.
338
Inheritance of Dichromatism
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ar am ea : ‘ SX |\(p) ax a | (p>) ax a| (p) ax a] \(P) axa) lax a
1:91 61+ 9 8 ° qed @maxa! @Maxa| ™axa| @™axad| axa a
1: gz | 6vt 9 5 ° aX @ (P)a Xa! (P)axXal (P)AXa]| (PAXA| AX a a
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TIA ATavL
332 Isabel McCracken
The data in Categories A and B show single black individuals
in each of three otherwise green broods out of a total of forty-four
broods, and a total of 1317 individuals of G x G parents. Unless
this can be attributed to a mishap due to accident in the handling
of breeding jars (a possibility where hundreds of breeding-jars are
being daily cleaned and cared for) this is a return of latent B in
normally recessive G. It will be noticed that such an exception
to the general behavior in G x G matings was also recorded in
Table IV (third generation).
Comparing data in Category F, Table VI, with data in Category
D, ‘Table VII, we find a discrepancy in the behavior of what was
apparently pure B, that is likewise unaccounted for unless by
accident or reversion. Table VII, Category D, records the
presence of a recessive G (possibly a /atent G in Castle’s termi-
nology)! in each of six broods from pure B parentage.
Otherwise the behavior of G and B in the sixth generation is
consistent with their behavior in preceding generations in similar
categories.
In the seventh generation, nine categories of matings were
established, as indicated in Table VIII.
*Castle, W. E., 1905: Heredity of Coat-colorin Guinea pigs and Rabbits. Carnegie Inst. Pub., No.23.
333
Inheritance of Dichromatism
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334 Isabel McCracken
Categories A, B, C and E show consistent behavior of recessives.
Category F shows the possibility of complete domimation of B
even from a line of partially dominant ancestry.
The proportions in Categories D and G remain consistent with
proportions in corresponding categories of previous tables.
‘Table [IX embodies a comparison of broods of seven generations
reared successively from B x B in which the parents were partial
dominants, that is, black individuals from mixed broods con-
taining a larger number of black than green individuals. This
table slows the proportion of B:G (of dominant to recessive)
in successive generations, and the rate of increase from generation
to generation of the dominant B.
This data shows a progressive dominance of B, which either
reduces G to a latent in the seventh generation or wholly eliminates
it.
TABLE IX
2 Prop’n
Total eile | Total No. | Total No. of B:G
Nolce Navara! Broods B ECOL B -
Broods. | Individs. Conese) ae) Mixed
o, Dominant. |Dominant. BREGATS
First generation (Table IT)............. 41 1207 2 39 T2391
Second generation (Table IG) ....... 30 740 5 25 it guid Bu
Third generation (TableIVC) ........ 27 868 8 19 2a ONL
Fourth generation (Table VF) ........ 13 250 8 Hog) 5H
Fifth generation (Table VIE) ......... 43 1336 35 8 8.531
Sixth generation (Table VITE) ........ 14 419 8 6 16:1
Seventh generation (Table VIII F)!..... 10 333 10 ° All B
‘Not absolutely, but practically, comparable with categories of other generations with which it is
compared.
COMPARISON OF RESULTS IN LINA AND GASTROIDEA
The fact that in one species (Gastroidea) black is the dominant
character, while in the other species (Lina) black is the recessive
Inheritance of Dichromatism 235
character, shows that there is nothing in the character as such
that makes for its actual behavior.
That green is the final color in the color-development of the
individual in Gastroidea has already been pointed out. In the
paper previously referred to, it was pointed out that black is the
final color in the color-development of individuals in Lina lap-
ponica. We have, therefore, a similarity of behavior in the estab-
lishment of the definitive color in the two-color series in each
species. [hat is, we have in each species a primary color-con-
dition through which all individuals pass and in which one series
remain (“ black”. in Gastroidea, “spotted” in Lina). We have
in each species a secondary color-condition into which some of
the individuals pass and there remain (“green”’ in Gastroidea and
“black” in Lina). It has been shown that it is the final or second-
ary color-condition that becomes the recessive character in each
species.
If it could be aionth that the primary or first condition into which
each species enters represents the older or ancestral condition, we
would then have in Lina and Gastroidea a condition found by
Castle in pigmented versus albino and long versus short-coated
guinea pigs; that 1s, a dominance of the ancestral or older character.
SUMMARY OF RESULTS
1. In Gastroidea dissimilis “black” is dominant, “green” is
recessive. In Lina lapponica “black” is recessive and “ spotted-
brown” is dominant, though not typically so. In each species
the dominant color is that appearing first in the color-development
of the maturing adult, and the recessive color is that appearing
last in the color development.
2. In each species the recessive character breeds true at once
(with the possibility of the recurrence of the latent dominant).
The dominant character breeds true in the third or fourth genera-
tion from a hybrid through the completely dominant line (with
the possibility of a recurrence of the latent recessive).
3. In each species there is progressive or accumulative domi-
nance from generation to generation through the partially domi-
nant line.
336 Isabel McCracken
In conclusion it is evident that under the conditions of the breed-
ing experiments with Lina lapponica and Gastroidea dissimilis,
the heredity of the alternate characters in dichromatic species
differs materially from typical Mendelian heredity of alternate
characters. In the latter, the relation between dominant and
recessive characters is a perfectly stable one, assuming the
definite numerical proportion of 3:1. Inthe former, there is
apparently an actual prepotency of the dominant character that in
the long run effectually eliminates or reduces the recessive char-
acter to a latent one.
Entomological Laboratory, Stanford University,
December, 1905
Contribution from the Biological Laboratory of Clark University
LOCOMOTION OF AMCEBA AND ALLIED FORMS
BY
ORIS P: DELLINGER
Witn Two Pirates AND TWENTY-NINE FIGURES IN THE TEXT
Few subjects are more fundamental or have been more
assiduously studied than the movements of the Amceba. ‘The
earlier writers maintained that the explanation of these move-
ments was to be found in the contractility of protoplasm. M.
Schultz, Briicke, DeBary, Kthne, Haeckel and others held this
view. They did not fully analyze their theories, however,
although Briicke did postulate an internal contractile frame-
work to account for the streaming of protoplasm.
Early in the discussion, objections were raised to the contractile
theory. Wallich (’63) called attention to the fact that the
currents of protoplasm in Amcebe did not begin at the posterior
end or in the interior but that they commenced at the point of
advance and extended backward. Biitschli, Hofmeister, Nageli
and others made similar observations and on that account re-
linquished the contraction theory. All these observations were
made by looking down on the Amceba from above, while if it is
viewed from the side, the difficulties can be readily explained.
BERTHOLD’S THEORY
Many other theories followed, none of which need be mentioned
until Berthold’s. In the year 1886, he advanced the opinion that
the protoplasm of Amcebe behaves much the same as a drop of
fluid which is spreading on a solid surface. If a drop of inorganic
fluid is caused to adhere more strongly at one side than the other,
it will roll toward the more adherent side. He believed this
movement to be exactly what we have in Amcebe. Jennings
('04, p. 208) says: “By a proper arrangement of the conditions
Tue Journar or Expertmentat Zoéxocy, Vor. 11, No. 3.
338 Oris P. Dellinger
almost every detail of amceboid locomotion may be clearly imitated”’
(by a drop of fluid). Berthold further compares the movements
to a drop of water fleeing from a rod wet with ether; but, as
Butschli points out, the character of the movements here is entirely
different from those of a drop adhering to a solid surface.
Butschhi, in discussing Berthold’s view, says (p. 190): “I consider
it as Incorrect to suppose that Amcebz really adhere to the solid
substratum. I do not entirely dispute the fact that local adhesions
at the hinder end, or occasionally also in the pseudopodia during
their retraction, may come under observation. On_ the other
hand, I consider it certain that an extensive adhesion is absent.’’
Berthold was obliged to offer some other explanation to account
for the protrusion of a free pseudopod, as in that case there is no
B
L ENTS roe
Fig. 1. Diagram of the currents in a drop of clove oil in which the surface tension is lessened
on one side. The drop elongates and moves in the direction of a. (After Jennings.)
Fig. 2. Diagram of the currents in a progressing A. limax. A, top view; B, side view. (After
Rhumbler from Jennings.) :
surface to which to adhere. He explained it by a contraction
theory. We shall see later that neither form, currents, nor
adhesion at the point of advance, required by Berthold’s theory
are present.
SURFACE TENSION THEORY OF BUTSCHLI AND RHUMBLER
Butschli (’92) and Rhumbler (’98) followed with the surface
tension theory. According to these two authors, and also many
others, the Ameceba is a drop of complex fluid which moves about
as a result of local changes in surface tension. ‘The currents in
such a drop are forward in the central axis and backward along
the surface (Fig. 1). Fig. 2 will show the same currents in the
Locomotion of Am@ebe and Allied Forms 339
2)
Ameeba, according to Rhumbler. It is evident that this theory
would explain the putting out of free pseudopods, but, as Butschli
points out, it would not account for the very fine pseudopods of
actinospherium-like forms. Unfortunately for their theory the
necessary currents are not present. Even Butschli calls attention
to the fact that he could not always observe them. Neither is
the Ameeba the shape of a drop of fluid acting under changing
surface tension and, what is more to the point, there are definite
points of attachment. As Jennings points out, this theory would
not explain many phenomena of the free pseudopods.
MOVEMENTS OF AMCBA AS DESCRIBED BY JENNINGS
Great credit is due Jennings (’04) for the careful experiments
and observations by which he throws doubt on the surface tension
theory of Rhumbler and Butschli, and brings forward his con-
tractiontheory. Forthis reasona careful review of his work will be
taken up before going on to my own observations and experiments.
Jennings bases his view on the following:
(1) Experiments and observations to determine what move-
ments take place.
(2) Observations as to the shape of advancing Amcebe.
(3) Belief that the anterior edge is closely applied to the sub-
stratum.
(4) Observations which seem to indicate contractility of the
ectosarc.
His experiment consisted in mixing soot with water in which
Amoeba verrucosa was moving. Particles of the soot became
attached to the surface of the Amceba and by watching these, he
could distinguish what currents were present. He sums up his
results as follows:
‘In an advancing Amceba substance flows forward on the upper surface, rolls
over at the anterior edge, coming in contact with the substratum, then remains
quiet until the body of the Ameeba has passed over it. It then moves upward at
the posterior end, and forward again on the upper surface, continuing in rotation
as long as the Ameeba continues to progress. The motion of the upper surface
is congruent with that of the endosarc, the two forming a single stream’’ (Fig. 2):
340 Oris P. Dellinger
According to his description, Jennings derives the shape of an
advancing Amceba from two observations. These are as follows :
“Tn an Ameeba that was creeping on the lower surface of a cover glass, I was
able to define with some accuracy the parts that were attached and those that
were not. A small flagellate was moving briskly about between the Amceba and
the cover glass but its excursions were limited by a visible line-running parallel
with the anterior edge of the Ameeba and extending at the sides back to about one-
third the animal’s length from the rear. The zone between this and the margin
was pressed close to the glass, and was evidently attached to it. The more
pointed posterior end was held quite away from the glass, leaving a broad passage-
way through which the flagellate finally escaped.
“The results of this observation were confirmed by another. An Ameeba
verrucosa in full career was suddenly turned on one lateral edge by a strong current
from a rotifer, and its upper edge coming in contact with the cover glass, it
remained in that position some time without change of form. It could be seen that
the under surface was concave, the edges very thin and flat, while the posterior
portion was thick and arched’’ (p. 145).
Fig. 3. Diagram of the movements of a particle attached to the surface of A. verrucosa in
side view. As the Amceba moves forward from position 7, the particle moves forward to the positions
shown in 2, 3, 4,5 and6, (After Jennings.)
Fig. 4. Jennings’ conception of the shape of an advancing A. verrucosa, seen from the side. * The
anterior edge is thin and applied closely to the substratum while the posterior end is raised high in
the water. The Ameba is attached only by the anterior third to the substratum. (After Jennings.)
Fic. 5. Shape of advancing Amebe in general. (After Jennings.)
7
a
Locomotion of Amebe and Allied Forms 341
From these observations Jennings concludes that the form of
the advancing Amcebz verrucosa is that given in Fig. 4, and of
Ameoebe in general, that of Fig. 5, in which we see the thin
anterior edge applied closely to the substratum while the thick
posterior end 1s raised high above it. On the same page he gives
a diagram of an advancing anterior edge and on the preceding
page, the following observation and conclusion:
“A certain feature of the advance of the anterior edge seems of much significance.
Each wave seems to arise just behind the previous anterior boundary line and over-
laps it, leaving it buried. ‘This line often remains visible for a short time after the
new wave has been formed. ‘The new wave rolls over the preceding one in such
a way that its original upper surface becomes applied to the substratum. ‘This
is demonstrated by the rolling under of small objects on the upper surface of the
advancing wave. A diagram of the movement at the anterior edge is given in Fig.
45. [he movements can be imitated roughly by making a cylinder of cloth, laying
it flat on a plane surface, and pulling forward the anterior edge in a series of waves.
The entire cylinder then rolls forward just as the Amceba does.
“The essential features of the movement seem to be; (1) the advance of the wave
from the upper surface at the anterior edge; (2) the pull exercised by the wave on the
the remainder of the upper surface of the body, bringing it forward. Most of
the other phenomena follow as consequences of these two. ‘The flowing forward
of the granules of the endosarc seems to demand no special explanation, since a
fluid containing granules within a rolling sac must necessarily flow forward as
the sac rolls. By the movement forward of the anterior end a space is left free;
by the rolling forward of the posterior end the fluid is piled up and pressed upon
and must flow forward into the empty space in front. Possibly there may be
other causes at work in producing the endosarcal currents, but such currents
would be produced without other cause in a sac moving as Amceba does.’’
From the above it will be seen that Jennings’ conception of an
Ameeba is an elastic sac filled with a fluid. If we give the sac
contractility, which from observations (p.147) he has a perfect
right to do, we have the Amceba from which he derives his
theory of the movements.
Further discussion of his paper will follow my own observations
and experiments, but | may add that, although I can confirm
some of his observations, I think, had he studied good side
views of his specimens, he would have reached far different
conclusions.
4
342 Oris P. Dellinger
AMEB AND DIFFLUGIA STUDIED FROM ABOVE AND FROM THE SIDE
In connection with a comparative study on the cilium, it was
necessary for me, as many students of contractile protoplasm have
done, to begin with the pseudopod of the Amceba. Before I had °
proceded far, it became evident that I must repeat Jennings’ (04)
observations; the more so because a study of prepared slides of
the pseudopod led me to doubt his conclusions as to the structure
of Amcebe.
I did not confine myself to Amoebz but gave considerable
attention to Difflugia, Actinosphaerium and Euglena. Difflugia
is practically an Ameeba in a shell, and has pseudopods that move
in a particularly definite way. Apparently little attention has
been paid to this form. Jennings (’04) and Penard (’go) refer
to it, the latter calling attention to the activity of its pseudo-
pods.
It was necessary to answer the following questions before I
could proceed:
1. How does an Amceba form a pseudopod f
2. What evidence have we for a contractile substance in Amceba
and how is this substance distributed ?
3. How does an Ameeba attach itself?
4. What determines its form while in motion ?
5. What causes Amcebe to advance in a certain direction?
My first observations were made in the usual way by watching
the forms from above. I used Difflugia acuminata, D. spiralis,
Ameeba proteus, A. limax, A. verrucosa, A. radiosa and several
small forms of Amoeba which I did not determine. Amoeba
proteus was especially well suited to my purpose, as it was large
enough to be easily seen with the unaided eye.
Difflugia
Diff_lugia acuminata extends a slender pseudopod to its full
length free in the water. ‘That it is free is evident from its
motions. ‘The pseudopod then becomes attached at its tip (Fig. 6)
and contracts, drawing the mouth of the shell up to the point of
attachment. A new pseudopod appears pari passu, generally
Locomotion of Amebe and Allied Forms 343
near the base of the first (Fig. 6), and increases in length as the
first is contracted. ‘The new pseudopod, which is fully extended
when the mouth of the shell reaches the point of attachment,
swings into the line of advance and becomes attached. The
cycle is then repeated. Figs. 6 and 8 show the position of the
Fig. 6. Diagram of Difflugia acuminata showing changes from one pseudopod to another.
a, a, a,a,ashow points of attachment; b,b,b, b,b, the new pseudopod at different stages of its formation.
(The diagram was made with camera lucida by drawing the paper at right angles to the line of
advance.)
mouth and pseudopod at different stages in the change from one
pseudopod to another. Fig. 7 shows four successive positions of
the shell at the time when a pseudopod becomes attached, and also
the lines of advance of the shell and of the tips of new pseudopods
as they form and are carried forward. That the pseudopods
while free take no part in drawing the shell forward is evident
from the figure.
Often Diffugia acuminata apparently moves with a single
pseudopod, in which case the new pseudopod is extended directly
34.4 Oris P. Dellinger
over the tip of the old. I obtained evidence to prove this from a
specimen in which the second pseudopod appeared at the side
instead of over the end of the first (Fig. 9). It is evident that the
}
Fig. 7. Diagram showing position of mouth at time the pseudopod was attached and the lines of
advance of mouth and of forming pseudopods. 7,2, 3 and 4 show positions of mouth when attachments
a,b,c and d are made. Dotted line indicates advance of the mouth and the solid lines follow the tips of
the forming pseudopods. (Camera lucida.)
Fig. 8. Diagram of D. acuminata as it advances. At position 7,a is attached; at 2,} is attached
and c is appearing; at 4, the mouth of the shell is well up to the point of attachment of 6 andc¢ is long;
at5, cis attached; at 6,c has contracted and d is appearing; at 7, d has become attached. (Camera
lucida.)
Fig. 9. D. acuminata with new pseudopod appearing near the tip instead of near the base.
Fig. 10. Diagram of currents in advancing D. acuminata. The substance of the contracting
pseudopod flows over into the new pseudopod.
last two methods of locomotion are but variations of the first.
‘The movements were sometimes complicated by combining these
methods.
The particles in the contracting pseudopod flowed around into
the new pseudopod (Fig. 10), giving the exact picture of the flow
Locomotion of Amebe and Allied Forms 345
of particles in retracting and protruding pseudopods of Amcebe.
Another point of resemblance was the shrunken and wrinkled
appearance of the pseudopod as it contracted.
Difflugia spiralis carries its shell when in motion much as a
snail does, and thus the mouth and base of the pseudopods are
not visible unless viewed from the side. Yet watching it from
above one sees the end of a pseudopod suddenly appear, wave
about for a time, and then become attached. ‘The shell then
moves over it, at the same time another pseudopod appears and
the above is repeated.
Amaebe—Absence of Rolling Movement in all Except Ameba
V errucosa
My first experiment with the Amceba was the one Jennings per-
formed with lampblack. I tried at first Amoeba proteus, A. limax
and some smaller Amcebe which I did not determine. In these
forms I never obtained his results, although I repeated the
experiment many times. ‘The attached particles would merely
oscillate back and forth a little but never revolved as he describes
(Fig. 11). The position of the particles on the Amcebe made no
difference. I watched particles attached to the anterior end, to
the posterior end, and to the middle, but never found any revo-
lution.
I next experimented with Amceba verrucosa. ‘The particles
became attached much more readily in this form and I found the
currents exactly as Jennings describes them. Diatoms, granules
and particles of all kinds passed to the anterior end, over the edge,
then stopped until the Amceba passed over them, when they were
picked up and the above repeated (Fig. 3).
‘There may be rotation in other species but | am convinced that
it is not common. Evidence for this will appear later in the
paper.
I next gave my attention to the direction of the currents of
protoplasm. By watching the granules it is easy to tell just
what the currents are. In general the granules flow forward
rapidly in the middle and spread out in all directions at the
346 Oris P. Dellinger
anterior end. Some of the granules at the border stop while those
in the center flow on. ‘These may remain quiet for some time
and then enter the current, or some may not enter the current until
the body of the Amceba is almost past, when they enter at the
posterior end. ‘The large granules of the endosarc oscillate back
ao
Fig. 11. A, diagram showing position of the particles of lampblack on an advancing Ameba at
different stages of the advance. B, the same Amceba twenty minutes later. During all that time
they had oscillated back and forth but had kept nearly the same position. (Camera lucida drawing.)
Fig.12. Diagram showing movements of a large particle in the endosarc during different stages
of the advance. (Camera lucida drawing.)
Fig. 13. Diagram of A. proteus moving in a clear field.
Fig. 14. Diagram of A. proteus moving in the presence of small algz on which it is feeding.
and forth but keep the same relative position (Fig. 12). The
currents at the anterior end are like those in a pseudopod formed
free in the water. ‘The granules do not flow forward in straight
lines but zigzag along as if flowing azound obstacles. In
Locomotion of Amebe and Allied Forms 347
Amoeba verrucosa there are definite streams and channels
which anastomose, and the flow reminds one of the flow of
corpuscles in a frog’s web. Diatoms flow forward with the other
granules if lengthwise with the currents. If lying across the
channels they appear tobe caught and move with the Ameeba itself.
In Ameeba verrucosa there is also a peculiar jerking of the
particles at the posterior end at regular intervals, as if at this point
there were regular flows forward. I was unable to explain this
while studying them from above but a side view revealed the
cause.
The anterior end of Amceba verrucosa 1s very regular i in contour
and advances evenly in all direct‘ons. Here again it is an ex-
ception, for in all other species studied the anterior end advances
by a series of lunges, whichare never directly forward but alternate
from side to side of the direct line of advance. When there are
many pseudopods put out in front, as is often the case, the one
through which the Amceba advances alternates from side to side.
This phenomenon probably accounts for the absence of rolling
of the ectosarc in these forms.
The various shapes of advancing Amoebe find an explanation
in the reactions of the animals to food. If Amcebz are traveling
in a clear field or moving from one mass of debris to another, they
move by long loops (PI. II, Figs. 7, 8, 9) or maintain a long, slender
form and are attached at cael points (Fig. 13 and PI. ie Higs1a).
When moving and feeding on algz they reach out many pseudo-
pods and then present a palmate form (Fig. 14, and PI. I, Fig.13).
Other observations made were:
The shape and wrinkled appearance of the posterior end
indicate that the substance is contracting in this region.
2. The particles in Amcebz that are rolled along retain their
relative position and do not flow as in an advancing Amceba.
This observation is easily made by rolling an Amceba about with
a rod. ‘This gives good evidence for two things: First, that the
Ameeba is not an elastic sac filled with a fluid, which if rolled
along would produce the endosarcal currents (Jennings, quoted
above). The flow of the particles in the endosare must be due to
something else. Second, that the endosarc has a definite struc-
348 Oris PR Dellinger
ture that holds the particles firmly, however much the Amceba
is rolled about.
There is no question as to the methods of movement in Difflu-
gia. A slender pseudopod reaches out, attaches at the tip, and
contracts, drawing the shell forward. A new pseudopod is
formed by an aurlsos of the substance from the contracting
pseudopod. The new pseudopod 1 is full length when the Hens
of the shell reaches the point of attachment of the old and swings
in to the line of advance and attaches. ‘The above cycle is then
repeated. .
The similarity of the two forms suggests that Amoeba move
by the same general method. [he movements of an unidentified
Ameeba fies some support for this view. ‘This form extends
a slender pseudopod free in the water, attaches it by the tip and
draws the rest of the body up to this point. It then establishes
a point of attachment close behind the first, and freeing this, the
above is repeated. My observations before obtaining a side view
indicated that Amoeba proteus moves in the same way.
STUDY OF AMC@B AND DIFFLUGIA FROM THE SIDE
The discussions of Amcebz for the past fifty years call to mind
the story of the two knights and the shield. Ifa single observa-
tion of an Amceba accidently turned on its side by a passing
rotifer has given ground for a theory of the shape, attachment, and
movements of Amcebze, it becomes clearly necessary to devise
some means by which Amcebe and similar forms can be studied
in side view. Such a view will give points of attachment and
support, and upon these depends an understanding of the strains
and contractions which produce the movements both for advance
and for the formation of pseudopods. So far as I can find, no
mention of the study of Ameeba in this way is to be found in the
literature.
‘The apparatus devised is very simple. One edge of an ordinary
slide is ground squareand polished. Long cover slips are cemented
to this with the edges extending beyond the polished surface
so as to form a narrow trough (Fig. 15). With the microscope
Locomotion of Amebe and Allied Forms 349
brought to a horizontal position, specimens pipetted into the
trough move along the edge of the slide and are easily observed in
side view.'
Difflugia
Difflugia spiralis moves exactly like D. acuminata (Fig. 16);
that is, it extends a pseudopod free in the water, attaches it near
the tip (z, Fig. 16) and draws the shell forward. As the shell is
drawn forward a new pseudopod appears (2, Fig. 16), grows in
length as the first is shortened (3, Fig. 16), is full length when the
mouth of the shell reaches the point of attachment, and is then
Polished Edge
wy
WE.
Fa
‘7 6 Cover Glasses
Fig. 15. Diagram of apparatus designed to study Protozoa in side view.
swung into the line of advance and attached. The attachment
of the pseudopod in both cases is near the tip and is definite.
When Difflugia moves with one apparent pseudopod, as
described above, the new pseudopod is put out as a direct
continuation of the old. It is always short and the points of
attachment are closer together than when moving with two
pseudopods (Fig. 17).
Difflugia spiralis often creeps on a vertical surface. At such
times the points of attachment can be easily demonstrated by
jarring the table. The unattached parts vibrate while those
attached move with the glass. This same form often creeps on
the ceiling (Fig. 18), and then the movements are the same as
when on the floor.
1The cover slips were cemented with varnish and baked in an oven while closely pressed to the slide.
Only a trace of cement was used and great care exercised to prevent any of it reaching the polished
surface.
350 Oris P. Dellinger
Amabe
Advancing Amoeba verrucosa is attached at the anterior and
posterior ends and is free everywhere else (Fig. 19). The ante-
rior end is extended along the substratum but is apparently free
Fig. 16. Diagram of D. spiralis changing from one pseudopod to another. At position 7, the first
pseudopod is attached; at 2, a new pseudopod is appearing; at 3, the mouth of the shell is well up to
the point of attachment of the first and the new pseudopod is reaching over toattach; at 4, the new
pseudopod is attached.
Fig. 17. D. spiralis moving with one pseudopod.
Fig. 18. D. spiralis creeping on the ceiling.
Fig. 19. Side view of advancing Ameeba verrucosa showing the various shapes it assumes at different
stages of advance. At position 7, a new attachment has just been formed at the posterior end and it
is quite thick. At position 3, the Amceba is just ready to form a new attachment at the posterior
end. Jt is seen that the anterior end is not thin, and although put out along the substratum is not
attached except at definite points.
from it. Itis thick and blunt (Fig. 19, and PI. I, Figs. 1, 2, 3 and
4). There is a sudden flow of the substance forward when the
attachment at the posterior end is released and the Ameeba in this
region becomes much thicker (z, Fig. 19, and Plate I, Fig. 1).
Locomotion of Amebe and Allied Forms 351
This accounts for the jerking motion noted when observed from
above. ‘The flow of the substance forward is due to the pull from
the attachment at the anterior end and contraction of the pos-
terior end. As the substance flows forward the posterior end
becomes thinner andis drawn up to the point of attachment (2, 3,
Fig. 19). A new attachment is then formed at the anterior end
and the one at the posterior end is released. ‘The strain due to
the pull from in front causes the sudden flow forward. ‘The old
attachment at the anterior end becomes in part the new attachment
at the posterior end, but when the sudden flow forward occurs, the
attached surface is extended back much farther.
It is difficult to understand how Jennings arrived at the con-
clusion that the anterior end is thin. On the other hand the ob-
servations from which he derives the shape, and on which he bases
his view as to the thickness of the posterior end and point of attach-
ment, are easily explained. If the Amceba 1s seen in the stage of
its movement shown in Fig. 19, the posterior end is thick but
the anterior end is not thin, neither is it the only point of attach-
ment. The escape of the flagellate, which he describes might be
explained by the change of attachment at the posterior end, or by
the Ameeba being jarred loose (PI. I, Fig. 4).
Admitting the shape as he gives it, it does not seem to me that
he explains the endosarcal currents. It is true they would occur
in a sac rolling on the floor, but unfortunately for his explanation
the sac he observed was hanging from the ceiling, in which case
the particles should fall down toward the posterior end instead of
flowing forward as he finds them. If Fig. 4 is inverted we have
the Amceba as he saw it.
All other forms of Amoebe advance much more like Difflugia.
They extend the anterior end free inthe water and attach it at or
near the tip and then contract. At the same time the posterior
end is contracting and the substance thus pushed and pulled
forward goes to form the new anterior end. ‘This continues as
long asthe Amoeba advances (Figs. 20, 21, 22 and 23, also Plt Ts
Figs. 6, 7 and 8). Often the anterior end is pushed along the
substratum but no attachments form except at definite points.
In other cases the anterior end is lifted free and then curves
259 Oris P. Dellinger
2)2)
down to the substratum and attaches, forming a long loop (PI. II,
Figs. 1 to 10). ‘The posterior end is then released and the sub-
stance flows over to the anteriorend. At the same time another
anterior end is extended. Amoeba proteus frequently uses this
method (Fig. 24) and a small undetermined form appears to move
a ai ere
A Bae
Zi
aA B A B GC Deemed
mu 2. 3 4 5 4
A A B B25.A B B B
Fig. 20. A. proteus, side view showing shape and points of attachment and the method generally
used by this form in advancing. At position r, the Ameeba is attached at a and } and forms a long
loop; at position 2, a new anterior end has been extended; the points of attachment at a and b are well
defined and the substance is drawn up into posts; at position 3, attachment a has been released and a
new attachment formed at ¢; a new anterior end has been pushed out at 4 and brought to the
substratum and attached. The Ameeba continues this as long as it advances.
Figs. 21 and 22. Side views of small undetermined Amoeba showing points of attachment.
Fig. 23. Side view of A. limax showing points of attachment.
Fig. 24. Side view of A. proteus showing how it moves by long loops.
Fig. 25. Side view of the small Ameeba referred to above, showing how it loops along in changing
from one point of attachment a, to another. At2,attachment b is just forming; at 4, attachment a
is released.
in this way altogether (Fig. 25). The points of attachment are
always well defined and never extensive (Fig. 20, and PI. I, Figs.
6, 7 and 8). Sometimes they are far apart, the Amoeba forming
a long loop, or they may be close together (Fig. 26).
Locomotion of Amebe and Allied Forms 353
The substance is often drawn up into well defined posts. The
attachments are formed quickly and at no time was there any
evidence that a viscid substance is present. An Amceba was
jarred loose from the slide by striking the table when the parts of
the body that had been in contact were easily seen. ‘The sur-
face at these points was perfectly flat and looked as if it had been
planed off.
The granules at the anterior end move outward in all directions
but never backward. At the posterior end the granules flow
eee
2G. Pa ah 28
1 2 3
a
Fig. 26. A. proteus, side view, showing points of attachment close together. The anterior end is
just reaching out to form a new attachment.
Fig. 28. Side view A. proteus creeping on the ceiling.
Fig. 29. A. proteus at rest showing points of contact. The Ameba does not seem to touch with an
extensive contact.
forward in a steady, even stream. ‘The currents are really the
same as seen from above, the upper and lower surface then be-
coming the lateral edges. Particles attached to the surface oscillate
but are never carried around the Ameeba.
An Ameeba proteus covered with carmine granules was advancing
in the usual way. ‘The particles moved along with the Amceba,
keeping the same relative position. “wo particles almost oppo-
site each other on the upper and lower surface of the advancing
anterior end kept the same distance from the tip as it advanced.
The one on top never rolled over and passed to the under side.
354 Oris P. Dellinger
Particles attached to the tip would sometimes pass to the upper
surface. At no time did particles move along the upper surface
to the anterior end and then pass underneath. On the other
hand, particles on the under surface flowed along with particles
on the upper surface. I am convinced that in species studied
there was no flowing of the upper surface over the anterior edge
to the under side. |
Ameoebe often creep on a vertical surface or, by inverting the
cell, on the ceiling, and at suchtimes the shape is the sameas when
creeping on the floor (Fig. 28). A large Ameeba proteus creeping
on the ceiling was jarred loose and tried to attach itself again.
It had partly succeeded when it was loosened at the new attach-
ment. In rounding up it then went through the stages shown in
Fig. 27. The entire process took about fifteen minutes. If the
l 9 3 4
Fig. 27. Side view of A. proteus that was jarred loose from the ceiling at all but two points of
attachment, which were near the anterior end. Shapes it assumed in rounding up. It hung in position
3 for ten minutes during which time all the large granules in the endosarc kept the same position.
Ameeba is a sac filled with a fluid in which the endosarcal particles
are suspended it seems that these particles would settle to the
bottom. ‘This was not the case. Since particles do not flow as they
would in a sac, we must assume a more definite structure in the
endosarc. Whatever structure is assumed must account for the
behavior of the endosarcal particles while the Amoeba is moving
and at rest. In other words, the endosare must be so constituted
as to allow the particles to flow at one time and to hold them
securely at another.
Locomotion of Amebe and Allied Forms 355
Other observations were:
1. Amoebz at rest do not touch with an extensive contact
(ig220).
2. Amoebe often change form without advancing. They do
this in the presence of Paramoecia and appear to be fishing for
them.
3. Amoebe when rolled about, or when starting to move, after
a rest, extend pseudopods in all directions. Diffugia when
turned on its back will do the same. In either case as soon as a
pseudopod becomes attached the animal moves off in the direc-
tion of the point attached.
CONCLUSIONS
What answer to the questions asked at the beginning of this
inquiry are to be found in the above observations f
1. Advancing Amcebz at no time approximate the shape of a
drop of fluid adhering more closely at one side than the other.
Neither do they behave like a drop acting under changing surface
tension. I think we are warranted in saying also that they are
not sacs of contractile ectosarce rolling about. Such an assump-
tion would not explain the phenomena in moving Amcebe as we
have found them from a side view.
2. The definiteness with which Difflugia extends a pseudopod,
swings it about, brings it into the line of advance and attaches tus
the manner in which the shell is drawn up to this point of attach-
ment; and the creeping about over a vertical surface and the
ceiling, indicate that in this form there 1s a contractile substance.
3. In advancing Amcebe the movements are essentially those
of Diffugia. Often they are exactly the same. The anterior
end is extended free in the water and attached. ‘There is then a
contraction of the substance back of this point and a flow of the
substance toward the anterior end results. The movements of
Amoebz are due to the presence of a contractile substance.
From observation we are warranted in assuming that such a
substance is present.
356 Oris P. Dellinger
4. The granules of the endosare do not- flow as if they were
suspended in a fluid in a contractile sac. On the contrary they
are held in definite positions under conditions that would indicate
that the endosarce has definite structure. A coarse reticulum
of contractile substance distributed through the endosare would
account for the phenomena as we have observed them.
I wish to acknowledge my indebtedness to Dr. C. F. Hodge,
under whose direction the research was made, for the many sug-
gestions and the encouragement given me. Also to Mrs. A.
Forrest Dellinger for help in preparing the drawings and plates.
Clark University, Worcester, Mass.
January 23, 1906.
Locomotion of Amebe and Allied Forms 357
LITERATURE CITED
BERTHOLD, G., ’86.—Studien ttber Protoplasmamechanik. 332 pp., 7 pl. Leipzig.
BrtckeE, E., ’62.—Die Elementarorganismen. Sitzb. d. K. Akad. Wien. Bd.
xliv, II, Abth.
Burscu1t, O., ’92.—Untersuchungen tiber mikroskopische Schaume und das Proto-
plasma. 234 pp., 23 fig., 6 pl. Leipzig.
HormetstER, W., ’65.—Ueber die Mechanik der Bewegungen des Protoplasmas.
Flora, pp. 7-12.
Jennincs, H. S.,’04.—Contributions to the Study of the Behavior of Lower Organ-
isms. pp. 130-230. Carnegie Institution, Washington.
Kutune, W., 64.—Unters. ber das Protoplasma u. die Contractilitat. Leipzig.
NAGELI, C.,’55.—Die Glitschbewegung, eine besondere Art der periodischen Bewe-
gung des Inhalts in Pflanzenzellen. Pflanzenphysiol. Unters.
Heft I. Pp. 49-53.
PENARD, Euc., ’90.—Etudes sur les Rhizopodes d’eau douce. Mem. de la Soc. de
Physique et d’Histoire Naturelle de Genéve. T. 31, pp. 1-230.
’o02.—Faune rhizopodique du bassinsdu Leman. pp. 714. Geneve.
Ruumster, L., ’98.—Physikalische Analyse von Lebenserscheinungen der Zelle.
Bewegung, Nahrungsaufnahme, Defakation, Vacuolen-Pulsation,
und Gehausebau bei lobosen Rhizopoden. Arch. f. Entw.-mech.
der Organismen, Bd, vii, pp. 130-350.
Scuuttze, M., ’63.—Das Protoplasma der Rhizopoden und der Pflanzenzellen.
Leipzig.
Wa tticu, G. C., °63.—On an Undescribed Indigenous Form of Amceba. Ann.
and Mag. of Nat. His. Bd. ii, pp. 287.
358 Oris P. Dellinger
DESCRIPTION OF PLATES
Plates I and II are photographs of living Amceba taken with B & L 3 objective and No. 1 eyepiece
All but Nos. 10 and 13 in Plate II are of the same Ameba.
Pirate I
Fig. 1. Amoeba verrucosa, side view. Just afterattachment is formed at the posterior end. The
Ameeba is thick at the posterior end and has a definite point of attachment in that region. The
anterior end is also attached and the lines observed from above are seen to run to the point of
attachment at the anterior end.
Figs. 2 and 4. A. verrucosa, side view. In Fig. 4 the Ameba is just ready to form a new attachment
at the posterior end. It is noticed that the anterior end is not thin but is thick.
Fig. 3. A. verrucosa side view. Was creeping on the ceiling and was jarred loose except at the
anterior end. The anterior end is rounded and is not thin.
Fig. 5. A. verrucosa, top view.
Fig. 6. . A. proteus, side view. The Ameeba is attached at a and b and the anterior end is being
advanced to form a new attachment. Note the definiteness of the points of attachment.
Fig. 7. A. proteus, side view. The Ameeba is attached at a and b. The substance at bis drawn
upinto a well-defined post. Theanteriorendis coming to the substratum to form a new attachment.
It moved from ¢ to d while the plate was being exposed. A new pseudopod is appearing at e.
Fig. 8. The samie Amceba a few seconds later. e has grown in length. Attachment at a is
releasing and another has been formed at f.
Prate II
Figs. 1 to 6. A. proteus, side view. Changing from one attachment to another. At position 7, the
anterior end has reached over to attach. At 2, the attachment at the posterior end is released.
At 3,4 and 5 the substance is flowing over to the anterior end. At 6, the substance is well over, and
the Ameeba is putting out a new pseudopod at the anterior end.
Figs. 7, 8 and 9. Ameeba proteus,side view. Moving by long loops. The Ameeba has just brought
the anterior end to the substratum but has not attached it. It slides along and is not attached until
the position in Fig.g is reached. The substance then flowed over to the anterior end as in Figs. 1 to 6.
Note the smallness of the pseudopods at the posterior end on which the Ameba is resting.
Fig. 10. A. proteus, top view. Showing form taken when moving in a clear field. The Amoeba ©
in this photograph is attached at the anterior and posterior ends and probably forms a loop.
Figs. 11 and 12. A.proteus, side view. Upon small pseudopods, which is often the case. A new
anterior end is being extended in 77 and is brought to the substratum and attached in 72.
Fig. 3. A. proteus, top view. Moving in the presence of alge, with numerous pseudopods at the
anterior end.
LOCOMOTION OF AMCBA AND ALLIED FORMS. Oris P. De.iincer. LAL ANATID, I
Tue JourNar or Expermmentar Zoorocy, Vor. 1, No. 3.
LOCOMOTION OF AMCEBZ AND ALLIED FORMS. Oris P. DeLLINGER. PLATE II
Tue JourNat oF ExprertmMentar Zoérocy, Vor. m1, No. 3.
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CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE
MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE,
E. L. MARK, Direcror.—No. 180. 7
LIGHT REACTIONS IN LOWER ORGANISMS
I. STENTOR CCERULEUS
BY
Ss. O. MAST
Professor of Biological Science at Hope College, Holland, Mich., U. S. A.
Wirth Six Ficures
RempLITCrOd UCHOLME Ee Reiter = cis eo. Ne o.ciake aa ole wad o tYs those ototoa ed oa ere Senet Sees 359
2. Material and apparatus ........ SO HOC Oe Io Ae BOO ric aan Gc ca OBA acoE 360
QMO) SERV ACLON SH peeeey eats choi clexcien= ons io) tiyouele Ves sieie ehsvaveiaPe arco aah eid scree ae noes RE RE 366
PA oimae VEG VITUS SO LEDIEOLS Meee se soit oie iy yie reveal yess ciel ON sie 6 eieie [oasis lo mice fea raie Seer ae 366
Ame loni ght ormaniin tensity. <ctacs see ate «ec reeeieiets eke aco tts s Secale aioe ete 366
bemuovlightipraded tan initensi Gyiscts (yese)=ive 2) iste shes see eios cae Sos erevelehave ol stove Ie cia alee pleats 371
BaP ACEACHCOnS COHLOLS faiy crevasse Sato, ior Se os REIS toe wera oer as mee Cacia om tere 377
Ore bhresholdetorlrchitss tum ilay sec syst ca hers see tei nePensioie ce cietese Sao cleree PeeO EN 381
ZN Vigiecneranimales sided! lumina ted ictmx\s)<screiers lave Ae are @ eich eve levon clare eicietsisareeaisters 381
IBITS UETHGEROG Sperqereyeun arererelalersc ore tele Mine «ae IE ero cis eet ee ee 382
Slacoiays (aetilaveyel Soh Bcc Gian ae ta SERRA en a A AMG mermaid Seas hc 383
intial hat oboe ata JEBe atte Done DOE H TOC eed ore SOB neAG ata sa0c 384
Due vathitheanimal:sranterior end iJlluminateds2-4--c- s01- een ee eieeeee earn 384
AMR S UI TLLTIVALS MRRP C TR eo eeeroFer vies ars: ti clove cs ciaisteiie'w Sualln eosiGidione dierelengs peers ais avavostanae sete ere 392
Fem DIDO pT ap i varrraraates foystop-farsvers ci wievasr Si cieoatatsiora Sg ays sjorcle.evele Gisra.slerelelere Meee oa ail eree elms 393
Os. ARENGIDY. 36 so. bow Sa adea Oo COCOA CORES FEO ances Sea Anan ON en cape anGunad ce doe 394
I. INTRODUCTION
The light reactions of Stentor cceruleus have been described
in detail by Jennings (’o4a, p. 29) in a paper, entitled “Re-
actions to Light in Ciliates and Flagellates.’’ His work shows
conclusively that such reactions do not take place in accordance
with the tropism theory as set forth by Loeb, Verworn, or Holt
and Lee, but that the animals orient by means of “motor
reactions,’ 2. e., by turning toward a structurally defined side
(aboral), when stimulated, and then proceeding on a new path
at an angle with the old one. My experimental results, while
THE JOURNAL OF ExPERIMENTAL Zo6 oGy, Vor. 111, No. 3.
360 S. O. Mast
differing in a few minor details from those of Jennings, lead to
the same general conclusions. Our methods, however, have been
quite different. Jennings laid particular stress on the detailed
movements of the individuals, while I directed most careful
attention to the regulation of the stimulus. Moreover, he dealt
with some phases of the subject more extensively than I did,
while I dealt with others more extensively than he did. My
experiments were, however, entirely completed and much of
this paper was written before the work of Jennings was publish-
ed. The facts that, working entirely independently of each
other on the same organism and with different methods, we
have obtained results which lead to the same general conclusion
and that this conclusion is not in agreement with the commonly
accepted tropism theories, have led me to prepare this paper for
publication even at the risk of repeating some matters already
published by others.
I wish, before continuing further, to express my appreciation of
many valuable suggestions by Dr. G. H. Parker, under whose
direction the work was pursued, and my indebtedness to Prof.
E. L. Mark, not only for granting exceptional facilities for
carrying on the work but also for his encouraging interest.
I am also under obligation to Dr. H. 8. Jennings for helpful
criticism.
2. MATERIAL AND APPARATUS
The animals used in the following experiments were obtained
by letting aquatic plants collected in a pond known to contain
Stentor, decay in battery jars nearly filled with water. Stentors
begin to multiply rapidly after Paramecium disappears or while
it is decreasing. If, however, the jars contain more than about
one-tenth as much vegetable matter as water, fermentation
takes place to such an extent that the Stentors are apparently
all killed, for while in such cultures there are usually numerous
Paramecia, Stentors never appear, or if they do, there are very
few of them. The cultures should be kept in diffused daylight
or total darkness at a temperature of about 22° C. If condi-
tions are favorable, Stentors usually appear about ten days after
Light Reactions in Lower Organisms 361
the cultures are put up, and then multiply very rapidly, frequently
becoming so numerous that the walls of the jars to which they
attach themselves, especially near the surface of the water,
become densely covered with them in the course of a few days;
but they soon decrease in numbers again and frequently dis-
appear altogether within a week. It was found that if a few
stalks of hay were added to cultures about to decrease in
numbers they could be kept in good condition much longer than
otherwise. Frequently it happens that after the Stentors seem
to have completely disappeared in a given culture they reappear
and multiply very rapidly. The disappearance and reappear-
ance of Stentors in a culture is due largely, as Peters ('02) has
shown, to chemical changes brought about by the decaying
vegetable matter. While working on the following experiments,
several cultures were constantly kept on hand. ‘This insured
an abundance of good material at all times.
Stentors, as is well known, are usually found most abundantly
on the shady side of the vessel in which they are kept. If
put into a dish containing water and placed in front of a window,
they swim about for a time seemingly at random, but soon orient
and then swim from the source of light, apparently as nearly
parallel with the light rays as possible. How and why do they
orient? Do they move parallel with the rays of light, and if so,
why? What is the minimum light intensity which causes orienta-
tion? Is this a constant?
Answering these questions involves quantitative as well as
qualitative work. In order to make quantitative work worthy
of the name, it is, as Jennings (04b, p. 507) has well stated, at
least necessary to know what we are trying to measure, 1. €., We
must understand the reactions of the organism with which we
are working in detail, and know the precise value of the stimulus
as it reaches the organism; and to know this we must understand
the physical and chemical properties of the stimulus. The
results and conclusions of the following experiments present a
striking illustration of the importance of the latter. Strasburger
(’78) found that certain swarm spores were positive in large
vessels and negative in hanging drops in the same light intensity.
362 S. O. Mast
Rothert (’03) obtained similar results, and both offered quite
extensive theoretical explanations for the cause of this; but
neither got at the truth of the matter because they did not
recognize the effect of the curvature of the surface of the hanging
drop on the light as it entered the water. Chmielevsky (’04)
proved conclusively by theoretical considerations and by means
of photographs that the intensity of a hanging drop of liquid is
frequently greatest in parts of the drop farthest from the source
of light, so that it is more than probable that the organisms in
hanging drops in the experiments of Strasburger and of Rothert
reacted to light precisely as they did in the vessels.
In the following experiments particular stress was laid on
the study and regulation of the light used as a stimulus. The
work was carried on in a large basement dark-room in which
the temperature varied but little. “This room was supplied with
gas, carbon filament incandescent lamps of various candle-power,
a carbon arc lamp, and six-glower and _ single-glower Nernst
lamps. Moreover, the room was so situated that sunlight, direct
and diffused, could also be made use of. The Nernst single-
glower lamp was found to be the most satisfactory source of
light for all experiments, both quantitative and qualitative, pro-
viding the intensity required was not great. Such a glower
consists of a single, small, straight rod composed of oxide of
zircon. ‘Lhe rods are about I mm. in diameter and vary from
Icm. to 2.5cm. in length, thus producing when heated a compact
source of light. ‘They are heated in air by an electric current
and need not be protected by a globe, so that much reflection
and refraction are avoided. ‘The glowers were generally not used
in connection with the regular Nernst lamp, but were mounted
in front of an opening in a box the inside of which was painted
dead black (Fig. 1, d, c), to prevent reflection from the back-
ground. The box used was nearly cubical in form, each side being
about 35 cm. long. The end containing the opening was made of a
heavy asbestos pad to avoid danger from fire, since the tempera-
ture of the glower becomes very high. In case one glower did
not -produce light of sufficient intensity, three were so grouped
that a cross-section would form a small equilateral triangle.
Light Reactions in Lower Organisms 363
When these were heated they appeared much like a single
glower somewhat enlarged. We have, then, in the Nernst
glower, arranged as described above, a light the direction of
the rays of which can be pretty accurately controlled, certainly
more nearly so than the direction of rays from carbon
incandescent or arc lamps. But the light intensity of the
Nernst glower, while it probably varies less than that of
the carbon arc, certainly varies more than that of the carbon
incandescent. It varies with the voltage, the age and composi-
tion of the glower, the temperature of the room and especially
with currents of air. Most of these factors were, however, under
control. The light intensity with known voltage and tempera-
ture was frequently measured; the voltage was recorded from
time to time during the progress of nearly all quantitative ex-
periments by means of a meter in the circuit; the temperature of
the room was nearly constant and drafts of air were avoided as
much as possible. But as a matter of fact the variation under
the most favorable conditions was so great that delicate quantita-
tive results must be accepted with caution. The variation
in light intensity, and its relation to the variation in voltage are
indicated in the Appendix (p. 394).
A modification of a piece of apparatus devised by Sabine and
Yerkes (Yerkes, ’03) was used extensively in the following experi-
ments and found very serviceable. By means of it a field of light
can be produced which either is uniform in intensity through-
out or gradually increases in intensity from one end to the
other. he rays producing the field are all practically perpen-
dicular to the plane of the field and there is little diffusion. The
defects in Oltmanns’’ method of producing light of graded
1Oltmanns (’92) produced in an aquarium light gradually increasing in intensity from one end t
the other, by placing a hollow prism filled with a mixture of India ink and glycerine-gelatine between
the source of light and the aquarium. The India-ink mixture of course absorbed only a little light
at the thin end of the prism, but gradually more toward the thickerend. Oltmanns and others assumed
that the light rays in the aquarium under such conditions were parallel with each other and perpen-
dicular to the side through which they entered. That this is not true, is clear from a theoretical as
well as from a practical standpoint. The India-ink mixture contains numerous solid particles of
carbon in suspension, which, together with particles in suspension in the water in the aquarium,
unquestionably diffuse the light more or less.
364 S. O. Mast
intensity, which have been pointed out by Strasburger (’78,
p- 588), Miss ‘Towle (’oo), and others, seem tobe largely if
not entirely avoided in this method. ‘The piece of apparatus
used in these experiments will be referred to as the light-grader.
Its construction will be readily understood from the accompany-
ing diagrams (Figs. 1, 2).
The walls of the apparatus are all light-proof and dead black
inside, so as to prevent reflection. ‘The outline of a cross-section
at any point is square. ‘he upper portion of the front wall of
the vertical part of the apparatus is hung on hinges forming a
door. From the bottom of this door is hung a loose vertical
curtain, which can be so opened that observations can be made
without admitting light. The glower is parallel with the minor
axis of the lens. It is mounted in front of a small opening
in a light-proof box, painted inside dead black, which thus
forms a non-reflecting background. The glower and stage
are at the conjugate focal points of the lens, and, therefore, at
equal distances (50 cm.) from it. ‘The plano-convex cylindrical
lens used is 25 cm. long, 10 cm. wide and has a radius of
curvature of 12.5 cm.
A cylindrical lens will not form a single definite image of an
object, but rather a series of images, since by means of it light
is focused only in reference to one plane. If, then, the object,
e. g., a Nernst glower, is placed at one of the conjugate focal
points so that the distance from the lens to the glower is equal to
that from the lens to the image, and the glower is so arranged that
it is pependicular to the axis of the lens, the image will not
consist of a narrow band of light as large as a glower, which would
be true if the segment of a sphere were used as the lens, but it
will consist of a comparatively large field of light, the length of
which is proportional to the functional length of the lens, while
the width is equal to the length of the glower, regardless of the
functional width of the lens (see Fig. 2). But since the amount
of light which passes through the lens is directly proportional
to the functional width of the lens and the width of the field is
constant, it is clear that the intensity of light in the field, if we
disregard the amount of light absorbed by the lens, must also be
Light Reactions in Lower Organisms 365
Fic. 1
Fig. 1. Avvertical section of the light-grader. The lens a, which is a segment of a cylinder, has
its longitudinal axis lying in the plane of the section; b, stage; c, Nernst glower; d, non-reflecting
background; e, mirror; f, light rays; g, opaque screens.
Fig. 2. Stereographic view of light, lens and image. a, Lens; b, field of light graded in intensity and
produced by the image of the glower; c, Nernst glower; d, opaque screen containing a triangular
opening and lying on the upper flat surface of the lens; f, light rays.
366 S. O. Mast
theoretically proportional to its functional width. Direct meas-
urements of the light intensity with different functional widths
of the lens proved this to be true within the limits of error. If,
then, the lens be covered with an opaque screen containing a tri-
angular opening, the base of which is parallel with the minor axis
of the lens as represented in Fig. 2, there will result a rectangular
field of light in which the intensity gradually diminishes from the
end produced by light which passes through the base of the trian-
gular opening to the opposite end, where theoretically it fades
into darkness. Practically, however, it was found to be impos-
sible to cut the apex of the triangular opening so as to prevent an
apparent line at the end of least intensity. Since the light
intensity of the field is proportional to the functional width of
the lens, it is evident that the rate of diminution in inten-
sity depends upon the ratio of the altitude of the triangular
opening to the length of its base, 7. e., decreasing the altitude
or increasing the base causes an increase in the rate of diminu-
tion and vice versa. ‘The facts that a field of light, either uni-
form in intensity or graded in intensity, can be produced by
merely changing the form of the opening in the screen over
the lens, and that the intensity can be changed by altering the
width of the opening, and readily calculated for any width if it
is known for a given width, make this a very desirable piece
of apparatus for quantitative as well as qualitative work.
3. OBSERVATIONS
A. Moving Stentors
a. In Light Uniform in Intensity
In studying the reactions of Stentors to light-rays_per-
pendicular to the plane of the field, animals were put on
the stage of the light-grader in a shallow aquarium! contain-
ing about 3 mm. of water taken from the culture jar and
‘The aquarium used was made by enclosing a space of desired size on a clear piece of plate glass
with a ridge of paraffin attached to the glass with balsam.
Light Reactions in Lower Organisms 367
carefully filtered. A field of light of the desired size and
intensity, produced by regulating the opening in the screen,
was thrown into the middle of the aquarium. The shallow-
ness of the water restricted the movements of the animals
almost entirely to the plane of the field and thus prevented
orientation. « If, under these conditions, a uniform field of light
about 16 candle-meters in intensity is thrown into the aquarium
and a considerable number of Stentors introduced and evenly
scattered, it will be noticed in the course of a few seconds that
nearly all of the Stentors have left the light area (a), as repre-
sented in Fig. 3. These animals, as stated above, can not orient
toward the source of light; how, then, do they get out of the
light area and how do they keep out?
Fig. 3. Distribution of Stentors as seen in the light-grader 30 seconds after a field of light 16
candle-meters in intensity throughout was thrown into the middle of the aquarium containing
water 2 mm.deep. a, Field of light, natural size; b, shaded area of aquarium. In this experiment the
animals could move only in directions perpendicular to the rays of light.
After being stirred up, as they are when transported from
the culture jar to the aquarium, they move rapidly, so that those
in the light area soon swim out of it, but without any noticeable
change in their course as they pass from light into darkness. The
animals in that case get out of the light area by mere random
movements. But when animals in the dark happen in their
random movements to come in contact with the light area, they
suddenly stop, swing their anterior end towards one side, and then
continue on a new path which forms a definite angle with the old
one. The new course may take them back into the dark area
at once, but if it does not, as frequently happens, they soon stop,
turn again towards one side and ‘begin again on a new path.
368 S. O. Mast
This reaction may be repeated several times before the animal
succeeds in getting out of the light area; indeed, Stentors were
frequently seen to repeat this reaction until they finally got
out of the light area at the side opposite the one at which they
entered. ‘hus we see that the animals are kept out of the
light area by means of what Jennings has called the motor
reaction. :
It is evident from the preceding description that the light
rays in this experiment are practically perpendicular to the
plane of the field, and that the movements of the animals are
almost entirely restricted to thisplane, so that the rays strike them
perpendicular to their longitudinal axis; therefore the direction
of the direct rays certainly can not affect their movements.
But it may be argued that there is sufficient light reflected
from other individuals and from particles of various other
substance in the water to affect the direction of movement of
Stentors and that they leave the light area because of this
reflected light, to which they orient. That this is not true is
clearly shown by the fact that Stentors are found swimming
at random in the shaded regions close to the edge of the light area,
as represented in Fig. 3. “The shaded area contains practi-
cally as much reflected light as the light area, so that if the re-
flected light affected the direction of movement at all, we should
expect it to affect that of the Stentors in the shaded area as well
as that of those in the light. But if this were true, we should
expect the an'mals in the shaded regions to be less numerous near
the edge of the light area than elsewhere; we should also expect
to find them oriented toward the light area, but this is not the
case. The animals are as numerous in this region as they are
farther from the light area and are here found swimming at
random. ‘That the motor reaction is not due to the temperature
is evident from the fact that Paramecium, which is more sensi-
tive to heat than Stentor (Jennings, ’04a, p. 31), does not respond
when it passes into the light area. Thus we must conclude that
the animals do not leave the light area by orienting to reflected
rays, but that they are carried out of this area by random move-
ments caused by the stimulation of the light, and that they are
Light Reactions 1n Lower Organisms 369
Oo _=
kept out by means of a motor reaction induced by an increase
in light intensity as they attempt to enter.
If the light is not very intense many of the animals, after hav-
ing given the motor reaction several times without succeeding
in getting out of the field, become quiet or continue on in a
straight course, no longer responding with the motor reaction.
Such animals have apparently become acclimated. Some ani-
mals give the motor reaction apparently as soon as their ante-
rior end touches the field of light, others do not respond until
they are entirely within the field of light, while still others
pass into the field three or four millimeters before reacting, and
if the light is not extremely intense, there are always some which
apparently are not affected by the light at all. “hese responses
show clearly that there is great variability in the sensitiveness
to light among animals from the same culture, and this is true
even under the most favorable circumstances. “The variability,
together with the fact that Stentors readily become acclimated
to light, makes quantitative work exceedingly difhcult.
The fact that the motor reaction is given by some animals as
soon as their anterior ends reach the light area, shows that stimu-
lation of this endis sufficient to induce a reaction, butit does
not show that the posterior end 1s not sensitive, nor does it show
that the posterior end is less sensitive than the anterior. This
question will be dealt with later.
In the experiment described above, Stentors were frequently
seen to enter the field of light so that their paths form rather
acute angles w:th the edge of the field. Animals thus entering
the field, in responding with the motor reaction, often turned
toward the center of the field, and in so doing were of course
carried farther into the light area; whereas, had they turned
toward the edge they would immediately have been carried
back into the dark area again. This indicates, as Jennings (‘04a,
pp. 33-35) very clearly showed by direct observation, that they
always turn toward a structurally defined side regardless of the
region stimulated. That this is really what takes place, can be
demonstrated as follows: Water to the depth of about one
centimeter is put into the aquarium in the light-grader so that
370 S. O. Mast
the Stentors can readily swim in all directions. If, now, by
means of a mirror held beneath the aquarium, a field of light
about 20 candle-meters in intensity is suddenly flashed on
animals swimming at random in the dark, nearly every animal,
no matter in which direction it is moving,—from the source of
light or toward it,—stops almost instantly, turns, and then starts
on a new path, 7.e., responds with the motor reaction. If the
animals are numerous, they may be seen literally to turn in every
direction at the same instant. If reaction takes place in accor-
dance with the tropism theory as defined by Loeb, Verworn, or
Holt and Lee, we should of course expect all animals not oriented
to turn from the source of light, but we should not expect those
which happen to be already oriented when the light is flashed
on them to turn at all. This, however, is not the case. As
stated above, practically all turn; some of those moving at right
angles to the rays turn toward the source of light and others
from it; and of those already oriented, some turn to the right,
others to the left. This is a most striking and convincing
experiment. It strongly supports the direct observations of
Jennings by which he demonstrated that Stentor always turns
toward a structurally defined side and shows clearly that there
is no apparent relation between the direction of the rays and the
direction of turning.
When Stentors which are oriented to a given light respond with
the motor reaction to an increase in intensity of the light, they
are for the time being thrown out of orientation, but by repeated
response to the more intense light they soon become oriented
again. If, however, the light intensity is increased very much
(125+ candle-meters), they may again respond after having be-
come oriented. As a matter of observation, under such condi-
tions the responses in many individuals are repeated in such
rapid succession that the animals appear to be turning about a
pivot at their posterior end.
Jennings (’o4a, pp. 49-50) writes: “When a large number of
Euglena are swimming toward the source of light, if the illumi-
nation is suddenly decreased in any way, they give the typical
motor reaction described in my previous paper as a response to
Light Reactions in Lower Organisms 371
other classes of stimuli (Jennings, ’00, p. 235). ‘That is, they
turn at once toward the dorsal side.’’ Euglena are positive,
so that when they are swimming toward the source of light
they are oriented; when, under such conditions, they respond
to a decrease in light intensity, they are, as was found to be true
in case of Stentor, thrown out of orientation. ‘These reactions
seem decidedly fatal to any theory which assumes that animals
remain oriented because when in this position similar surfaces
on opposite sides of the body, or locomotor appendages on such
surfaces, are equally stimulated.
We have thus far shown that when Stentors pass from a dark
into a light region they respond with the motor reaction. Will
they respond likewise when they pass from a region of one
intensity to that of another?
Two adjoining regions of different light intensity can readily
be produced by reflecting the field of light in the light-grader
so that the reflected field overlaps the field produced by direct
light. Where the two fields overlap, the light will of course be
more intense than elsewhere, although it is exceedingly difb-
cult to see any line of demarcation between these regions of
different light intensity; but when Stentors swimming in the
regions of lower intensity happen to strike this plane they respond
with the motor reaction much as if they had come in contact
with a glass wall. If, however, they strike the plane while
swimming from the more intensely illuminated region, they pass
on apparently unaffected. It is decidedly interesting as well
as instructive to watch the results when these animals reach
the practically invisible plane between the regions of different
light intensity; if moving in one direction, without any response
whatever; if moving in the opposite direction, with a marked
reaction.
b. In Light Graded in Intensity
Holt and Lee (01, pp. 471-475) found that if Stentors are put
into an elongated aquarium in which the light becomes gradually
more intense from one end to the other, they collect at the dark
37/2 Se OMViast
end of the aquarium. ‘These investigators produced such light
conditions by means of Oltmanns’ prism, as follows: “Parallel
rays of light fell horizontally at right angles to the long axis of a
narrow trough destined to receive the organisms. Between
the trough and the incoming light there intervened a prismatic
screen. Thin at one end, it there let almost all the light
through; but becoming gradually thicker it gradually diminished
the intensity of the rays till at the opposite end the screen was
opaque and intercepted all the light.” ‘If numbers of blue
Stentors are put into a trough that is moderately illuminated
under the conditions described above, the animals at first swim
away from the light until they encounter the farther wall of the
trough. ‘They then swim backward a little distance and start
off in a new direction, as Jennings has described for other species
of Infusoria, some toward the light end of the trough and some
toward the dark end. Soon they strike the wall again, and
again start off in one direction or the other, and this series of
movements is repeated many times. The preponderance of
movement is toward the dark end, and in time by far the
majority are found there swimming about.’’ Holt and Lee
explain the cause of the preponderance of movement toward
the dark end by applying Verworn’s hypothesis. “The animals
swimming from the side of the aquarium nearest the source of
light toward the opposite side are, they say, so deflected toward
the dark end, because the side of the animals facing the light end
of the aquarium is more strongly illuminated than the opposite
side. ‘his is due in part to the decrease in light intensity from
one end of the aquarium to the other, but more especially to
the fact that the side facing the more highly illuminated end of
the aquarium receives considerable light which is reflected from
the water and sides of the trough at the well lighted end. The
fact that an appreciable quantity of light is thus reflected is
sufficiently attested by Strasburger (’78) and Miss Towle (’00).
‘Thus it is clear that Holt and Lee in their explanation of the
cause of the “preponderance of movement toward the dark end”
make use largely of two factors, the swimming from the source
of light and the light reflection from the more highly illuminated
Light Reactions in Lower Organisms 373
end of the aquarium. Can these factors be eliminated and, if
so, will the animals still collect at the darker end of the aqua-
rium? If, with these factors eliminated, they still collect at the
darker end, it is clear that the hypothesis of Verworn, as applied
by Holt and Lee, will not explain the cause of such a collection.
A field consisting of rays practically perpendicular to the plane
of the field and graded in intensity can readily be produced in
the light-grader by placing a screen containing a triangular open-
ing over the lens, as described above (pp. 364-366). If, now, a
considerable number of Stentors are put on to the stage in the
light-grader in an aquarium containing thoroughly filtered water
about two millimeters in depth, and such a field of light is thrown
Fig. 4. Distribution of Stentors as seen in the light-grader one minute after a field of light graded
in intensity was thrown into the middle of the aquarium containing water 2 mm. deep. The shallow-
ness of the waterforces the animals to move at right angles to the light rays. a, Field of light; b, shaded
area; the light intensity atc is 16 candle-meters; at d, 4 candle-meters and ate, 1.6 candle-
meters. The intensity at f was so low that it could barely be distinguished from that in the
shaded area.
into the aquarium perpendicular to the bottom, it is found that
in a short time nearly all the animals have left the more in-
tensely illuminated regions of the field, while there are as many
in the region of lower intensity as there are in the shaded area.
This result is represented in Fig. 4.
If the light area is made as large as the aquarium, the animals
collect at the darker end. In this experiment there are a good
many animals in the light area when the light is first turned on
and each one will cause diffusion of light, 7. e., will be a secondary
source of light, reflecting rays parallel with the plane of the field.
374 Se Oo Masi
Those at the most highly illuminated end of the field will reflect
more light than those at the darker end. May not, then, the
collection of the Stentors at the darker end be due to orientation
to these reflected rays, as has been suggested by some who believe
ray-direction to be the primal cause of orientation? An argu-
ment has already been presented in this paper (p. 368) which
seems to indicate very clearly that the reflected light would not
under the above conditions affect the direction of movement
of the animals. But to test this more in detail and to learn
precisely how the animals react in collecting at the darker end
of the aquarium, a square field of light 2.5 cm. ona side was
thrown into the middle of an aquarium on the stage in the light-
erader. The field of light was 125+ candle-meters at one end
and o+ candle-meters at the other. The aquarium contained
water about two millimeters deep, taken from the culture jar
and carefully filtered. Special precautions were taken to pre-
vent diffusion of light by dust particles on the lens, on the water,
or on the mirror used to reflect the light after it had passed
through the aquarium into a dark chamber, where it was absorbed
(see Fig. 1, p. 365). One Stentor at a time was taken with a fine
pipette from a dish kept in the dark, then carefully dropped
into the center of the field, and its reactions studied. In this
way the effect of light reflected from numerous animals was of
course eliminated. The movements of the animals, practically
restricted to the plane of the field by the shallowness of the water
in the aquarium, could be distinctly seen with the naked eye, but
of course their structure could not be made out. As soon as an
animal was released in the center of the field it responded with
the motor reaction several times in rapid succession, moving
but a short distance between successive responses, until it appar-
ently became acclimated to the intensity of light to which it
was subjected. If at the end of this period it happened to be
headed so that in moving forward it did not pass from regions
of lower to regions of higher light intensity, it usually continued
making nearly a straight path until it got out of the light area.
If, however, it happened to be oriented so that its movements
carried itinto regions of higher light intensity, it continued only
Light Reactions in Lower Organisms 375
“SI
a short distance before again responding with the motor reaction,
and such response was repeated at short intervals, until the
animal happened to become so directed that when it moved
forward it no longer passed from regions of lower to regions of
higher intensity. It thus continued on a straight course out
of the field, there being no longer any stimulation to induce the
l /
eo
yy
yY ~
, x Se
Re “ | :
Ss : ~
~
™s
da
Fig. 5. Each square represents the field of light at its actual size. The rays of light strike the
plane of the field practically perpendicularly. The higher light intensity at the side 7 was 125+ candle-
meters, and that at the opposite side, d,o-+candle-meters. The lines within the square represent,
approximately, the paths of twelve Stentors taken at random from a dish in the dark and dropped
one at a time as nearly into the center of the field as possible. The angles in the paths indicate
points where the animals gave the motor reaction.
motor reaction. It will be seen from this description that
Stentors in light graded in intensity, moving perpendicular to
the direction of the rays, do not orient and then continue in a
definite direction. “They may continue moving in any direction
excepting in such as would carry them from regions of lower to
regions of higher light intensity. ‘This fact will become more evi-
dent by referring to the accompanying figures (Fig. 5), which were
376 S. O. Mast
made by tracing the paths of the animals on a sheet of paper as
they proceeded on their course. With a little practice it was
possible to watch the animals and draw at the same time, so
that while these tracings do not represent the paths in detail very
accurately, they at least represent the general course taken by
the animals.
It will be seen from these figures that only one of the twelve
animals left the light area at the most highly illuminated end,
and that this one continued responding with the motor reaction
at short intervals until it got out of the area. Evidently it did
not happen to become headed in such a direction that movement
after a reaction would no longer carry it into regions of higher
light intensity. It will also be seen that there is absolutely
no indication of orientation with reference to degrees of light
intensity, 7. ¢., the animals move continuously in any direction
except such as would carry them rapidly into regions of higher
light intensity. Subsequently 64 additional animals were put
into the graded field one at a time. Of these, four left the feld
at the end of highest light intensity, 26 at the end of lowest
intensity, and 34 at the two sides.
We have in the above experiments eliminated the two prin-
cipal factors (p. 372) upon which Holt and Lee based their
explanation of “preponderance of movement toward the dark
end,’ and have found that such movement still takes place.
Evidently, then, their explanation will not hold. Jennings
(o4a, p. 48), after stating this explanation and describing the
motor reaction of Stentor due to light stimulations, continues
with the following paragraph:
“There is evidently nothing in this account [referring to the
observations of Holt and Lee] which is inconsistent with the
method of light reaction which I have described. On the
contrary, the reason why the organisms finally swim toward the
dark end and gather there becomes much more evident when
the reaction method that I have described is taken into con-
sideration. Let us suppose that the Stentors, after striking
the back of the trough, turn in equal numbers toward D [dark
end of trough] and toward L [light end of trough]. In those
Light Reactions in Lower Organisms 277
swimming toward D the anterior end is directed away from the
source of strongest light (due to reflection from the lighted end
of the dish L), and the animals are passing into a region of less
intense light. ‘hereis thus nothing to cause the ‘motor reaction,’
with its accompanying change in the direction of movement.
In the Stentors swimming toward L, on the other hand, the
strongest light falls on fae anterior end, and the organisms are
passing into a region of more intense light. Either of these
factors taken separately may, as we have seen, cause the motor
reaction (the turning toward the right aboral side), thus changing
the directionin which the Stentors swim. ‘The animals which
start to swim toward L will therefore soon be turned, and only
when the direction of movement is toward D will there be no
cause for further change.”’
In the experiments described above, we have substantiated
Jennings’ ideas as set forth in this paragraph; we have shown
that the preponderance of movement toward the darker end is due
to the motor reaction induced by an increase in light intensity,
which consequently prevents continuous movement toward the
more highly illuminated end of the aquarium.
It should, however, be kept in mind that these experiments
were completed before Jennings’ paper was published, and there-
fore were in no way influenced by his explanation.
B. Attached Stentors
In cultures many of the Stentors are usually found attached
by the posterior end to solid objects of various kinds near the
surface of the water. The relative number of attached and free
individuals, however, depends upon the condition of the culture.
In a given culture under certain conditions nearly all the animals
may be free, whereas, in the same culture perhaps only a few
days later, nearly all may be attached. From casual observa-
tions, it was thought that the number of free animals depends
upon the rate of reproduction. In some cultures it was difficult
to study the reactions of free-swimming animals, because they
attached themselves again almost immediately after being de-
378 S. O. Mast
tached. On the other hand it was difficult in some cultures
to study reactions of fixed Stentors because the least disturbance
seemed to cause them to separate from their support.
Attached Stentors exhibit great variability in sensitiveness to
light. For while the reactions are marked in some cultures when
they are subjected toa sudden change—. g., either to an intensity
of 120 candle-meters or to rather weak diffused daylight—in others
only slight reactions are induced by sudden exposure either to
strong direct sunlight or to the light from a carbon are of 250
candle-power at a distance of 25 cm. (4000 candle-meters). In
testing quite a number of cultures at different times, from Decem-
ber to the following August, I did not find any in which there was
no response to sudden exposure to direct sunlight; but in many
cultures there were individuals that did not appear to be affected
at all, and, as stated above, there were some cultures 1n which the
response was not definite. It is, therefore, not surprising that
Jennings was unable to get any light reactions from attached Sten-
tors in the cultures that he studied. He says (’o4a, p. 32): “Such
individuals do not react at all to light. When light is thrown on
them they remain in the positions in which they are found at the
beginning, neither contracting nor in any way changing their posi-
tion. No matter whether the light is weak or strong, and with-
out regard to the direction from which it comes, fixed Stentors give
no reaction and show no orientation with reference to light. “The
contact reaction apparently inhibits the light reaction completely.’ ’
This, as we have seen, is true only under certain conditions,
but the interference of contact reactions probably has something
to do with the variation, which is much greater in attached than
in free swimming animals. Contact reactions probably also tend
to keep the animals in such a position that their longitudinal
axis is perpendicular to the surface to which they are attached,
for they are usually found in this position-even when attached to
vertical surfaces or to the upper surface of solids, and when attached
to such surfaces they must maintain their position against gravi-
tation.
The reactions in detail are as follows: If Stentors in favorable
Light Reactions 1n Lower Organisms 379
condition are put into a dimly lighted aquarium containing water
a few millimeters deep, they soon become attached to the bottom,
take a position such that their longitudinal axis is approximately
vertical and soon become quiet. If, now, the light is slightly in-
creased, they begin to swing about their point of attachment, pre-
sumably turning toward their aboral sides. If, however, the light
is suddenly increased considerably, they at once contract, but
in the course of a few moments they expand again and then
slowly swing as they do when the light intensity 1s only slightly
increased; after this they soon become quiet or break loose.
These reactions are entirely independent of the direction of the
light rays. ‘They are precisely the same in animals hanging from
the surface film or from a cover glass floating on it, whether
illuminated from above, below or from the side. ‘There is, how-
ever, some evidence that the same reaction is induced by light
of lower intensity when thrown on the anterior end than when
thrown on the posterior, indicating that the anterior end 1s more
sensitive than the posterior (p. 389.)
Attached Stentors illuminated from the side do not orient, as
might be expected at first thought. Experiments with reference
to this point were repeated many times with animals in various
conditions and with light varying in intensity from very strong
direct sunlight striking the animals at various angles, to that
which would barely induce a reaction; but in no case was there
any definite indication of orientation. Assuming the tropism
theories to be correct, we should, of course, expect the animals
to orient, 7.¢., to turn until symmetrical portions are equally
stimulated and then stop, and we should not expect any reaction
in animals already oriented when subjected to light stimulation,
unless the stimulations were intense enough to cause contraction.
But, as stated above, such 1s not the fact; when slightly stimulated,
the Stentors swing about their point of attachment regardless of
the direction of the rays, and if illuminated from the side they do’
not stop swinging when the anterior end becomes directed away
from the source of light. Evidently, then, the reactions of attached
Stentors are not in accordance with the tropism theories. Are they
in harmony with orientation by means of motor reactions?
380 S. O. Mast
The anterior end of Stentor, as will be shown later (p. 389),1s more
sensitive to light than any other portion of the surface of the body,
so that when it is turned from the source of light the stimulation
onthe animal as a whole is weaker than when it is turned in any
other direction. It is this which keeps free-swimming animals
oriented after they have once attained such a position by means
of the motor reaction (Jennings, ’o4a, p. 45). Why, then, do not
attached Stentors remain oriented, when, in swinging about their
point of attachment, they happen to reach a position in which
their anterior ends are directed from the source of light? We
have shown that free-swimming animals which are already
oriented respond with the motor reaction if the light intensity 1s
suddenly increased and thus are thrown out of orientation, and
that if animals hanging from a surface film are suddenly illumin-
ated from above they begin to swing about their points of attach-
ment; so that the mere fact that the anterior end is directed from
the source of light is not sufficient to prevent the motor reaction,
1. €., turning toward a structurally defined side. We have also
seen that attached Stentors are readily acclimated to light and,
as this process of acclimation proceeds, we should expect to find
a stage at which the turning of the anterior end from the source of
light would cause enough reduction in. stimulation to prevent fur-
ther swinging, 7.¢., to inhibit the motor reaction, and this is
probably true. We find attached Stentors when not stimulated
arranged so that the longitudinal axis is perpendicular to the
surface to which they are attached. With these animals accli-
mation has proceeded so far that there is no longer sufficient stimu-
lation when the anterior end is directed from the source of light
to induce the motor reaction. We should, therefore, expect them
to maintain a position perpendicular to the surface to which they
are attached. .
If the light intensity, then, is great enough to cause a sufhcient
-stimulation when the anterior end is turned from the source of
light, attached Stentors respond with the motor reaction which
prevents orientation; but if it is not intense enough to induce the
motor reaction under these conditions, the tendency to take a
position perpendicular to the surface on which they are attached
Light Reactions in Lower Organisms 381
again prevents orientation, so that the fact that attached Stentors
do not orient is precisely what we should expect if the animals
orient by means of motor reactions.
In closing this section of the work, let me state clearly that,
while I have found absolutely no evidence of tropic responses in
any of my experiments with Stentor, I do not wish to be under-
stood as intimating that difference in intensity on opposite sides
of these organisms, or even that the direction in which the light
rays pass through them, may not affect the direction of their
motion. I shall, however, state again, as I have stated several
times in the preceding pages, that the reactions of Stentors are not
in accord with the tropism theory as defined by either Loeb,
Verworn, or Holt and Lee.
C. Threshold for Light Stimuli
a. With the Animal’s Side Illuminated
Since the threshold for light stimuli varies greatly with different
individuals and in the same individual under different conditions,
it is evident that the mere determination of it without correlating
the physiological conditions of the animals with the individual
variation in the threshold can be of little value. We have, how-
ever, one problem that can be attacked without such correlation.
I have assumed in some of the preceding discussions, that the
anterior end of Stentor is more sensitive to light stimuli than
any other portion of the surface. Now, this assumption can be
tested if the threshold can be ascertained when animals under
any given conditions are illuminated on the side, and again when,
under the same conditions, they are illuminated on the anterior
end. ‘To obtain the threshold for animals with the side illumin-
ated, they were put on the stage of the light-grader in an aquarium
containing water only about two millimeters deep. ‘Then the
intensity of a small field of light thrown into the middle of the
aquarium was reduced until the animals when passing from
darkness to light no longer responded with the motor reaction.
382 S: On Mast
The movements of the animals were practically restricted to the
plane of the bottom, so that when the animals passed into the
held of light their sides only were fully illuminated.
Three methods were used to ascertain the lowest intensity to
which animals, under the above conditions, react.
First Method.—A field of light of uniform intensity throughout
was thrown into the aquarium and the intensity reduced by
decreasing the functional width of the lens. The reactions of
the animals as they passed into the light area were then studied.
While theoretically this method, as well as those which follow,
should yield accurate quantitative results, practically the results
must be considered as at best only rather gross approximations,
for in the first place it was impossible to prevent fluctuations in the
light intensity; in the’second place Stentors are very readily accli-
mated to light of low intensity; in the third place it was difficult
to see at times if a response was given or not, owing to the low
intensity to which many responded; and, finally, the individual
Variation was so marked that without statistical methods the
point of lowest intensity to which a definite number responded
could be only roughly determined. The threshold also varies
greatly in different cultures and in the same culture under different
conditions. For example, a culture was tested March 3, at 3
o’clock, and definite reactions to a change in intensity of 1.6
candle-meters were obtained, although only a small proportion
of the whole number reacted. The same Stentors were tested
again after having been in the light-grader go minutes in dark-
ness undisturbed, and then only an occasional animal responded
to a change in intensity of 4.8 candle-meters.
It was found that the movements of the animals could be
followed best by studying their shadows cast on a piece of white
paper held about 20 cm. below the aquarium. ‘The paper, if
unglazed, diffuses the light, and therefore reflects it very nearly
equally over both the dark and the light area, so that the intensity
difference is not appreciably affected.
A large number of Stentors from different cultures were tested
for their threshold to light stimuli, as described above, on the
following days: February 20, 21, 22 and 23; March 4, 10 and 16;
Light Reactions in Lower Organisms 383
also August I, 2, 3 and 4. In all the cultures experimented with
animals were found that gave unquestionable reactions when they
passed from darkness into a light intensity of 1.5 candle-meters
and in some culture when they passed into an intensity of 1.2
candle-meters. But no definite reactions were obtained under
any conditions from animals which passed from darkness into
light of an intensity lower than 1.2 candle-meters.
Second Method.—In this method, as in the one described above,
the field of light in the aquarium was uniformly illuminated,
but in place of studying the reactions directly, the animals were
left undisturbed for a given time (15 to 60 minutes) after the light
was turned on, and then their distribution ascertained by counting
those in the field of light and those in dark areas equal in size to
that of the field of light. The results of thirty such experiments
were recorded. “There were, however, a considerable number more
in which the results were so evident that the distribution was not
studied by actual count and no record was made of them. ‘These
tests were performed on the following days: February 20, 21, 22
and 23, and on March 3 and 4, the same days on which expert-
ments described under “First Method’’ were performed, and
the animals used were taken from the same cultures in both
cases. The light intensity of the field in these tests varied from
less than 1.2 to 3.2 candle-meters. In five tests it was 3.2
candle-meters; in four of these tests there were decidedly fewer
Stentors in the field at the close of the test than in an equal area
outside; in the other one the number was about equal in the two
areas, but these animals had been used in the preceding experiment
and were therefore probably acclimated to the light. In twelve
of the tests, the light intensity was 1.6 candle-meters; in eight of
these there were definitely fewer animals in the light area than
elsewhere; in the remaining four the distribution was about uni-
form throughout the aquarium. In eight tests the intensity was
1.2 candle-meters; in four of these there were fewer Stentors in
the light area than elsewhere, but the difference was not very
marked, while in the other four it was questionable which con-
tained the greater number. In the five remaining tests the light
intensity was slightly less than 1.2 candle-meters; in all but one
384. S. O. Mast
of these, the light did not effect the distribution and in this one the
outcome was questionable. ‘These results agree very well with
those obtained by the first method, in which, as here, it was found
that the least change in intensity which would produce any re-
action under most favorable conditions was 1.2 candle-meters.
The tabulated results of these experiments, as well as of those
that follow, will be found in the Appendix (pp. 394-399).
Third Method.—A field of light graded in intensity from one
end to the other was produced by means of a screen containing a
triangular opening placed over the cylindrical lens. ‘The point of
least light intensity along the side of the field to which the animals
responded when passing into the light was then ascertained,
either directly by observing the movements of the animals, or in-
directly by studying the effect of the light on the distribution of
the animals after having been exposed to it for a given length of
time. ‘The intensity of any area in the field could readily be
calculated by measuring the functional width of the lens as
determined by the opening in the screen at the point through which
the light passed to produce the given area. A large number of
experiments on animals from various cultures and under various
conditions were carried out in accordance with this method, the
result being that the threshold was found to vary from 1.2 to 4
candle-meters. In no instance was there any definite evidence of
reaction to a change in light intensity of less than 1.2 candle-meters.
In all of these experiments individuals were met with which
were apparently not affected in the least in passing from darkness
into a light intensity as high as 125 candle-meters. ‘The threshold
of the most sensitive animals also varied considerably in different
cultures, but I think we may safely conclude that the threshold
for Stentors from the cultures tested under the most favorable
conditions was not less than 1.2 candle-meters when the sides of
the animals were illuminated.
b. With the Animal’s Anterior End Illuminated
Let us now consider the threshold of Stentors from the same
cultures under similar conditions, but with the anterior end illumi-
Light Reactions in Lower Organisms 385
nated in place of one side. Stentors are approximately conical
in form, the posterior end being somewhat pointed while the
anterior end is an almost flat surface approximately perpendicu-
lar to the longitudinal axis of the animal. If, then, the light rays
are perpendicular to the longitudinal axis, they must be parallel
with the anterior end, and this end therefore will not be exposed
fully to light, but the posterior end being pointed will be practically
as highly illuminated as if it were turned toward the source of
light. In the experiments discussed above, the light rays were
practically perpendicular to the longitudinal axis of the animals,
therefore the only portion of the body not fully exposed to light
in these experiments was the anterior end. If, however, the
movements of the Stentors in an aquarium are not restricted in
relation to the source of light, it is evident that sooner or later,
in random swimming, the anterior end, as well as the sides
and posterior end, may be turned toward the source of light and
thus fully exposed. If, now, the animals leave that part of the
aquarium nearest the light, it may be due to motor reactions
induced by stimulation of any part of the surface. But if the
anterior end of the animal is more sensitive than any other portion
of its surface and the light intensity is gradually reduced, it 1s
clear that a condition of illumination will be reached in which the
motor reaction will be induced only when the anterior end is turned
toward the source of light. If, then, the threshold is found to
be higher when the movements of the animals are restricted to
a plane perpendicular to the light rays than when they are not
thus restricted, we may conclude that the anterior end is more
sensitive than any other portion of the surface. The following
experiments prove this to be true.
In order to ascertain the threshold when there was no restriction
of movements, the Stentors were put into an elongated aquarium,
one end of which faced a Nernst glower that was on a level with
the bottom of the aquarium. The aquarium was then moved
from the source of light until a place was reached at which the
animals no longer left the end nearest the light. “Theoretically
this method seems very simple, but practically it is quite other-
wise; as in the case of the experiments described above, only
386 S. O. Mast
rather gross approximations can be looked for. We have here
to deal with the fluctuations in the intensity of the source of
light, with reflection of light from the ends and sides of the
aquarium, with absorption of light by the water in the aquarium,
and with the difficulty of determining, without statistical methods,
the lowest intensity to which the animals respond, a difficult prob-
lem because of the marked individual variation of the animals
and the ease with which they become acclimated.
These difficulties were controlled as far as possible in the fol-
lowing way. ‘The variation in light intensity in the Nernst
Jones. is due largely to variations in voltage. Having ascertained
die intensity for a given voltage, the intensity for any other
voltage can be approximately calculated, so that by means of
a volt-meter in the circuit it was possible to ascertain the
approximate light intensity at any given time. Four per cent of
the light which reaches the end of the aquarium nearest the
source is reflected and again four per cent of that which passes
through the opposite end; moreover, there is considerable re-
flection from the sides of the aquarium and from the surface of
the water. Owing to such reflection and to the absorption of
light, the variation in intensity in an aquarium illuminated from
the end by light, even from a single point becomes exceedingly
complicated. With sucha complicated field it 1s of course utterly
impossible to do quantitative work worthy of the name. For use
in the following experiments an aquarium was constructed
accordance with the plan shown in Fig. 6. ‘The construction
will be readily understood by referring to the figure.
In these experiments the light was placed on a level with the
bottom of the aquarium, so that there was no reflection from this
surface. ‘he space between the walls was filled with water to
prevent reflection from the sides. ‘The velvet between the end
walls absorbed most of the light which passed through the
aquarium and consequently prevented most of the reflection from
the end farthest from the glower. Reflection from the surface of the
water was prevented by cutting off the light which would other-
wise reach it by means of a screen in front of the aquarium. ‘The
Nernst glower was entirely surrounded by screens, only one of
Light Reactions in Lower Organisms 387
which contained an opening. Through this, light escaped pro-
ducing a field just large enough to cover the front end of the
aquarium, and since the whole apparatus was in a dark room no
other light reached the aquarium. ‘The heat rays were cut out
by passing the light through 7 cm. of distilled water! in a vessel
with parallel sides.
ee a AS EL]
Fig. 6. Plan of aquarium, natural size. a, Screen; b, piece of black velvet; c, vertical walls 2.5
cm. high. This aquarium was made by gluing glass slides together with balsam. It was contructed
for the purpose of eliminating as far as possible the effect of reflected light (see text).
The aquarium was filled to a depth of five millimeters with
water taken from the culture jar and thoroughly filtered, so as to
eliminate absorption as much as possible. ‘Then, after properly
1To those who still use alum solutions for absorbing rays, the following statements by Nichols
(793, p- 15) may be of interest:
““Melloni (Thermochrose, p. 165), using an Argand lamp with a glass chimney as a source, found
that a layer of alum solution 9.21 mm. thick transmitted 12 per cent of the total incident radiation
and that distilled water transmitted only 11 per cent.”
“<Shelford Bidwell (Nature, Vol. xliv, p. 565), using a paraffin lamp as a light source and the thermo-
pile, obtained the following results:
Solutions Diathermacy
HBsraniys yg cell lierercbey ferctostoter ct atetof¥ ets iofet-\ayo! =o «1cveh «i ishajal eJehe/alotels¥shsvel sla) e)2)ato 1000
\Watiar, Cieailice!,ooononsspacododubocodo ese soaucoGoDobDos0oaGL 199
MAID SAUER a5 co cb aodacoedn andandoOO UDG EESoooDDU gE soAdGC0ae 200
MMumbesatinated esOlm tion's :a\s\clere aise <i- </cie'ele/elareioc\ sae chelelalelelsiela\aie) ole 204,
‘‘Neither potassium alum nor ammoniumalum in solution alters thediathermacy of distilled water
in the region studied,” 7. e., wave lengths 0.776 to 1.414.
Nichols and Coblentz (03, p. 272) state that water transmits 67 per cent at wave lengths 14 and
becomes completely opaque only when wave lengths 1.8 is reached. The upper limit of wave length
in red is usually considered to be 0.76 ys.
388 S. O. Mast
arranging the aquarium with reference to the light, quite a
number of Stentors taken directly from the culture jar were in-
troduced and evenly scattered. ‘The animals were then left un-
disturbed for some time, 10 to 60 minutes, before their distribution
was studied. It was soon found that Io minutes was sufhcient
time to produce an effect, if the light intensity was not below the
threshold. If, on studying their distribution, it was found that
there were definitely fewer within an area, about five millimeters
wide at the end of the aquarium nearest the light than elsewhere,
and that those in the remaining area were still practically equally
scattered, the intensity at this end was considered the threshold.
It will be noticed that owing (1) to reflection from the end, (2) to
absorption, (3) to the difficulty in deciding whether a change in
distribution has occurred, and (4) to possible acclimation, the thresh-
old as read would tend to be slightly too high. Sometimes, if the
threshold was not found at the first trial, as was usually the case, the
animals were thoroughly stirred up and a second trial made with the
same animals, but it was soon found that the threshold of such
animals was higher than that of animals fresh from the culture
jar, even if the light on the first exposure was not above the
threshold in intensity, so that in nearly all experiments fresh
Stentors were introduced for each trial.
In all, 62 exposures were made in accordance with the method
just described (see Appendix, pp. 397-399). These experiments
were performed, on February 25, 26, 29; March 1, 2, 3, 8, 11, 13, 15,
16, 17, and August 1, 2, 3 and 4. The Stentors used were taken
from the same cultures from which those used in ascertaining
the threshold in the light-grader had been taken. Many of the
experiments in the two series were performed on the same days,
or nearly so, and in the same room, so that the conditions in both
were practically the same with the exception of illumination.
But, as already stated, the threshold in the light-grader was found
to vary from 1.2 to 4.8 candle-meters, whereas in these experi-
ments it was found to vary from 0.235 to 0.646 candle-meters.
This contrast is brought out more strikingly in the following tests:
During the period from August 1 to 4, inclusive, Stentors fresh
from the culture jar were repeatedlytested under various conditions
Light Reactions in Lower Organisms 389
in the light-grader and no definite reactions were obtained to
intensities less than 1.78 candle-meters, while animals from the
same culture jar tested at different times during the same period
in the elongated aquarium illuminated from the end, reacted defin-
itely to an intensity as low as 0.287 candle-meters.
It will be remembered that in the first of these series all portions
of the surface of the animals excepting the anterior end were
subjected to illumination, while in the second series the entire
surface, including the anterior end, was exposed to light. The
difference in the threshold in the two series must, then, be due to
the exposure of the anterior end in the second series; and since
the threshold is lower in this series than in the first, we can safely
conclude that with reference to stimulation by light, the anterior
end is the more sensitive portion of the surface of the animal.
The difference in sensitiveness between the anterior end and
other portions of the surface of Stentor was further tested in the
following experiments. In these experiments the light was placed
on a level with the animals, which were hanging from a cover
glass floating on the surface film in a small aquarium. A mirror
was fastened above the aquarium and another below it, each at
such an angle that the light reflected from above fell on the
posterior end, and that from below, on the anterior end of the
animal. Then, by properly screening the light and using the
mirrors independently, the threshold was obtained when the
animals were illuminated on either end. While the results thus
obtained indicate a threshold of lower intensity when the anterior
end is illuminated than when the posterior end is illuminated, I
cannot consider these indications conclusive, since, on account of
the extreme individual variations and the high intensity of light
required, it was very difficult indeed to locate the threshold.
Jennings (’04a) demonstrated by direct observation that ciliary
action in Stentor, Paramecium, Oxytricha and other organisms
produces currents which carry liquid from some distance ahead
of the animals to the oral groove. Now, if the stimulating agent
can be carried with the liquid, “The result is a stimulation on
the oral side of the body, not elsewhere.’’ In giving the motor
reaction, these organisms always turn toward the aboral surface,
390 S. O. Mast
1.e., away from the side stimulated, whenever the stimulating
agent is such that it can be carried in the current, 7. ¢., chemical
or thermal. Roesle (’02) showed that the peristome region is
more sensitive to mechanical stimuli than any other portion of the
surface of the body, and it is also probably true that the same
region is more sensitive to chemical and thermal stimuli. If so, it
is clear that when animals are put into a chemical solution which
acts as a stimulus, or into a liquid at a temperature above the
threshold, the oral side is more strongly stimulated than the
aboral, and we should expect precisely what is found to be true,
1.e.,the animals under these conditions respond with the motor
reaction and in so doing turn the anterior end away from the oral
side, which is more strongly stimulated than the aboral.
The effect of a stimulating agent on a given surface depends
upon the sensitiveness of the surface and upon its degree of
exposure. Mechanical and radiant-energy stimuli are produced
by agents which are in general not affected by currents and thus
can be applied to any desired portion of the surface. It is,
however, found that the animals always turn toward the aboral
side regardless of the surface to which these agents are applied.
Can these facts be explained f
In Euglena the eye spot is in all probability more sensitive to
light than any other portion of the body. It is located in the
dorsal lip near the surface of the body. ‘These organisms are
positive to light of moderate intensity. “hey swim with their
anterior end facing the light, and in giving the motor reaction
they turn toward the dorsal lip, in which the eyespot is situated,
i. €., toward the region most sensitive and consequently most
strongly stimulated, just as Stentor, Paramecium and Oxytricha,
all of which are negative, turn from the side most strongly stimu-
lated in case of thermal or chemical stimuli. But it will be re-
called that here the location of stimulation is not so much due to
Variation in sensitiveness as to variation in exposure of different
regions of the surface, owing to currents. We have demonstrated
that the anterior end of Stentor is more sensitive to light than any
other portion of the surface. Roesle showed, as stated above,
that the peristomal region in certain Infusoria is more sensitive
Light Reactions in Lower Organisms 391
to mechanical stimuli than any other surface area. Is it not
probable, then, that this region in Stentor is also more sensitive
to light than any other? If this is true, then in stimulating the
anterior end with light the oral region will be more strongly
affected than any other, and, in turning from the oral side when
the motor .reaction is given, the animals turn from the part most
strongly stimulated precisely as in response to thermal and
chemical stimuli. This would likewise be true if they were
stimulated by light striking any other part of the surface of the
body than the anterior end, for these animals are translucent,
so that if they were illuminated from the side, for example, light
could reach the oral region by passing through the animal. As
a matter of fact, we have as yet no experimental results with
regard to light reactions in Stentor which cannot be explained
by assuming a given structure in the region of the oral groove
to be the only portion of the body which is sensitive to light.
In givingthe motor reaction to all light, chemical, or thermal,
stimulations, Stentor probably turns always from the side most
strongly stimulated, but apparently not so with regard to mechani-
cal stimulations, forin such stimulations the motor reaction can
be induced by touching any part of the surface. Does this mean
that a certain physiological change induces a given definite reac-
tion, which has become fixed in the race, possibly by “survival
of the fittest,’’ and that all local stimulations are effective only
in so far as they tend to cause general physiological changes? Or
are the local stimulations produced by some agents (chemicals,
change in temperature, light) still in a measure local signs which
induce reactions in harmony with them, as well as cause a definite
physiological change, while in those produced by other agents
(mechanical) the local sign is lost and the response is induced
because of a given physiological state? Or may not the effect of
stimulating mechanically any point on the surface be transmitted
to the region of the peristome and there produce changes greater
than were produced at the point actually touched and thus call
forth a local sign which may regulate the reaction ?
Whatever the final answer to these questions may be, the facts
thus far established by experiment seem to indicate that the motor
392 S. O. Mast
reaction, 7.¢., the turning toward a structurally defined side,
originated in response to stimuli which produced local signs.
4. SUMMARY
1. Stentors free to swim in all directions orient and swim from
the source of light.
2. They orient by means of motor reactions, 1.¢., by turning
toward a structurally defined side and then proceeding on a new
path which forms an angle with the old one. If a single reaction
does not result in orientation, it is repeated until the anterior end
of the animal happens to become directed from the source of
light.
3. The motor reaction is induced by a sudden increase in light
intensity regardless of the relation between the direction of the
rays and the direction of movement of the animals at the time the
intensity 1s increased.
4. If a source of light to which Stentors are oriented is in-
creased in intensity, the animals respond with the motor reaction
and are thus thrown out of orientation, but by repeating the motor
react.on they soon become oriented again.
5. The anterior end of Stentor is more sensitive to light than
any other part of the surface of the body. “The minimum thresh-
old in animals stimulated by rays perpendicular to the longi-
tudinal axis is 1.2 candle-meters, but in those stimulated by light
striking the anterior end it is only 0.25 candle-meter.
6. The threshold varies greatly in individuals under the same
conditions, and in the same individuals under different conditions.
Stentors readily become acclimated to light, but much more
readily under some conditions than under others.
7. Stentors once oriented remain oriented, if the light intensity
is not too high, because they are least sensitive to light when the
rays strike the posterior end.
8. Attached Stentors respond to increase in light intensity by
contracting or by swinging about their point of attachment. If
the increase is great and sudden, they contract; if it is not very
great nor sudden, they swing about their point of attachment.
Light Reactions in Lower Organisms 393
These reactions are independent of any relation between the posi-
tion of the animals and the direction of the light rays.
g. Attached Stentors do not orient, for if the light intensity is
above the threshold when the anterior end is turned from the
source of light, the stimulation induces the motor reaction, which
prevents orientation; andif it is below the threshold, the tendency
to take a position such that their longitudinal axis is perpendicular
to the surface on which they are attached, again prevents it.
10. [he variation in the threshold to light stimuli in attached
Stentors is much greater than in free-swimming ones. In some
physiological conditions they respond definitely to a light intensity
of 1.20 candle-meters, whereas in others they respond very inde-
finitely when exposed to an intensity of 4000 candle-meters.
11. The threshold in attached Stentors is probably lower when
Ight strikes the anterior end than when it strikes the posterior
end.
12. The light reactions of Stentor, both free-swimming and
fixed, cannot be explained by the application of the tropism theory
as defined by either Loeb, Verworn, or Holt and Lee.
5. BIBLIOGRAPHY
CHMIELEVSKY, V., ’04.—Ueber Phototaxis und die physikalischen Eigenschaften
der Kulturtropfen. Beihefte z. Bot. Centralbl., Bd. xvi., pp. 53-
Go, lat, i. .
Hott, E. B., anp Ler, F. S., ’01.—The Theory of Photatactic Response. Amer.
Jour. Physiol., vol. iv, No. 9, pp. 460-481.
Jennincs, H. S., ’04a.—Contributions to the Study of the Behavior of Lower
Organisms. Carnegie Institution of Washington, Publication No.
16, 256 pp., 81 Figs.
’°04b.—The Behavior of Paramecium. Additional Features and Gen-
eral Relations. Jour. Comp. Neurol. Psychol., vol. xiv, No. 6,
Pp. 441-510.
Nicuots, E. L.,’93—A Study of the Transmission Spectra of Certain Substances
in the Infra-red. Phys. Review, vol. i, No. 1, pp. 1-18, pl. 1.
Nicuois, E. L., anD CosBLentz, W. W.,'03—On Methods of Measuring Radiant
Efficiency. Phys. Review, vol. xvii, No. 4, pp. 267-276.
Otrmanns, F., ’92.—Ueber die photometrischen Bewegungen der Pflanzen.
Flora, Bd. Ixxv, pp. 183-266, Taf. 4.
394. S. O. Mast
Peters, A. W., ’02—Metabolism and Division in Protozoa. Proceed. Amer.
Acad. Arts and Sci., vol. xxxix, No. 20, pp. 439-515.
Rogste, E.,’02—Die Reaktion einiger Infusorien auf einzelne Induktions—
schlage. Zeitschr. f. allg. Physiol., Bd. u, Heft 1, pp. 139-168.
RotuHeErtT, W., ’03—Ueber die Wirkung des Aethers und Chloroforms auf die
Reizbewegungen der Mikroorganismen. Jahrb. f. wiss. Bot., Bd.
xxxix, Heft.1, pp. 1-70.
STRASBURGER, E., ’78—Wirkung des Lichtes und der Warme auf Schwarmsporen.
Jena. Zeitschr., Bd. xii, pp. 551-625.
Tow e, Evizaneta W.,’00—A study in the Heliotropism of Cypridopsis. Amer.
Jour. Physiol., vol iii, No. 7, pp. 345-365.
Yerkes, R. M., ’03—Reactions of Daphnia pulex to Light and Heat. Mark
Anniversary Volume, article 18, pp. 359-377.
6. APPENDIX
TABLE I
Light Intensity, 222-Volt Nernst Glower
Photometer to Photometer to | Candle per Standard
Date Glower Candle Hour in Voltage Candle-
in cm. in cm. Grains power
Rebs 25 200 52.78 ? ? HO} —=
Feb. 26 200 52.25 ? P 19+
Feb. 26 200 54.1 ? 211+ 18+
Reb.) 27 201 63.89 165.72 QM ie 13.62
Mian 07 200 56.5 154-3 204-209 16.15
TABLE II
Light Intensity, 110-Volt Nernst Glower
| |
Photometer to | Photometer to | Candle per Standard
Date Glower Candle Hour in Voltage Candle-
in cm. incm. Grains Power
July 15 200 47-8 138.87 108 . 5-110 20.27
July.15 200 44.89 154.3 IIO-III 24.42
July. 27 200 47-8 154-3 IIo 22.48
Aug. I 200 44.17 157.38 LDI5 5118 26.88
Aug. 3 200 48 .32 163.56 109.5-I1I 23.35
Aug. 27 200 45-2 140.96 IIO-III.5 23.00
Light Reactions in Lower Organisms 395
It will be noticed that the light intensity of the 110-volt
glower is higher than that of the 222-volt clower. This is due
to the comparatively low voltage. Later, in comparing the in-
tensity of the 110-volt glower with that of the 222-volt glower
directly, with a voltage of 110+ and 222+, respectively, it was
found that the candle power of the latter was 1.62 times as
great as that of the former.
TABLE It
Light Intensity at ee in Light-grader,222-Volt Nernst Glower.
Photometer | Photometer Candle per Functional Standard
Date to Candle at Hour in Voltage width of Candle-
in cm. Stage Grains | Lens meters
Mar. ? 106.78 es 161.86 206-208? o.75mm. 1.193
Mar. ? 71.6 | S 161.40 | 206-208? I.5 mm. 2.52
Aug. 2 60.37 4 157-39 | 221-223 1.5 mm. 25557
Aug. 27 42.41 | n | 138.87 | 224-225 3.0mm. 6.5+
The light 1 intensities given in the tables were obtained by taking
30 successive readings at intervals of one minute. The distance
from the photometer to the candle is in every case an average of
30 readings. A Lummer-Brodhun photometer was used.
TABLE IV
Threshold for Stentors with the Side Illuminated
First Meruop (see p. 382)
Reaction in Light-grader as Stentors pass from Darkness into Light
Date Voltage of the following Intensities in Candle-meters
re 1.2 1.6 | geo
Feb. 20 ; no no ? | marked
Feb. 21 ? no no definite, but not in all
Feb. 22 ? no P large majority
Feb. 23 ? no no about rin §
Mar. 4 208+ no definite
Mar.10 205-207 no about 1 in 10 many
Mar.16 209-210 no ? a few
Mar.16 206-208 no ? definite, but only a few!|
1after these Stentors had been in the aquarium in the light-grader 1.5 hours, no reactions were
found to lower intensity than 4.8 candle-meters.
396 S. O. Mast
August I to 4, voltage 220-222. During this period Stentors
fresh from culture jars were tested several times under various
conditions and no definite reactions were seen to intensities
lower than 1.78 candle-meters.
TABLE V
Threshold for Stentors with the Side Illuminated
Seconp Meruop (see p. 383)
Time of Expo- | Light Intensity | No. of Sten- | In Equal In Equal
Date sure in in Candle- tors in Field | Field to Field to
Minutes meters of Light | Right Left
Feb. 20 48 Rue definitely definitely +
Feb. 20 33 Buz 4 16 18
Feb. 20 7° 32 6 12 16
Feb. 21 63 1.6 7 13 12
Feb. 22 ? 1.6 B 8 9
Feb. 22 56 1.6 8 15 16
Feb; 22 53 12 II 15 9
Feb. 22 38 | 1.2 10 16 15
Feb. 23 go 1.6 9 9+ 9+
Feb. 23 60 1.6 12 18 24
Mar. 3 65 | 1.6 19 30
Mar. 3 65 Ta, 22 27
Mar. 3 65 | ae 32 31
Mart. 3 go T6- 17 30
Mar. 3 go Tez 14 23
Mar. 3 go i2— 28 32
Mar. 3 65 1.6 30 24
Mart. 3 65 1.2 29 32
Mar. 3 65 tea 44 44
Mar. 3 60 1.6 24 18
Mar. 3 60 1.2 27 32
Mar. 3 60 I.2- 22 32
Mar. 4 120 1.6 9 24
Mar. 4 120 se 16 | 16
Mar. 4 120 1.2- 24 | 16
The temperature varied from 19° C. to 24° C. in these experi-
ments, but this change in temperature produced no apparent
effect on the reactions.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
Feb.
27
27
2)
By
233)
29
=)
2
2)
ay
29
29)
Mar. 1
Mar. 1
Mar. 1
Light Reactions in Lower Organisms
TABLE VI
Threshold for Stentors, the Anterior End Illuminated (see p. 384)
Time of
Exposure
in Minutes
60
7°
48
20
60
Tempera-
ture in
Degrees C.
21
Voltage
209
208
397
Condition
of
Stentors
|
used for
fresh
used for
fresh
fresh
fresh
fresh
fresh
fresh
fresh
fresh
fresh
fresh
fresh
fresh
some time
some time
same again
same again
same again
same again
same again
same again
same again
same again
same again
same again
same again
same again
| same again |
same again |
Distance
from Light} Reactions
in cm.
588 | none
588 | marked
588 | none
588 marked
650 | quite definite
806 none
806 | none
806 none
806 none
723 none
723 | none
670 none
670 slight
670 | marked
( | very few within
J | 5mm. from
779.) | tight end of
| | aquarium
goo | none
goo | none
goo none
806 none
806 marked
825 none
825 none
Rae if | very few within |
\| 3 mm. of light
| end
800 definite
800 definite
836 none
336 if | marked; many |
836
836
U
at dark end
marked; many |
at dark end
none
Intensity
in Candle-
meters
0.26
0.209
0.26
0.24
0.265-+
0.265
0.265+
398
Date
onl
i)
A
_
2
Mar. 2
Time of
Exposure
in Minutes
135
20
60
40
60
60
25
Tempera-
ture in
Degrees C.
22
21
20
22
S. O. Mast
TABLE VI—Continued
Voltage
212
213
211
210
Condition
of
Stentors
same again
fresh
same again
fresh
same again
fresh
fresh
used before
fresh
same again
same again
fresh
used before
used before
used before
fresh
used before
fresh
fresh
fresh
fresh
fresh
fresh |
fresh |
used before |
used before
fresh
used before |
fresh
used before
in cm.
800
800
835
835
850
850
goo
goo
goo
825
75°
75°
800
goo
800
goo
800
800
800
800
600
600
500
300
500
500
500
500
500
500
Distance
from Light
(a
Reactions
none
very few near
light end
few near light
end
many at dark
end; marked
none
marked
very few within
1 cm. of light
end
very many near
dark end
quite definite
>
marked; few
| near light end
none
quite definite
| definite,but not
| very marked
marked
none
marked
marked
| ?
| very marked
marked
?
very definite
none
few within 1 cm.
of light end
| definite
Intensity
in Candle-
meters
0.235
0.209
very low;
light filter-
ed through
thin paper
ditto
0.209
0.265
0.265
0.446
0.646
0.646
0.646
0.646
0.646
0.646
Light Reactions in Lower Organisms 399
TABLE VI—Continued
Time of | Tempera- Condition | Distance Intensity
Date | Exposure | ture in Voltage of from Light} Reactions jin Candle-
in Midutes|Degrees C.| Stentors in cm. meters
| ( |very few within
Aug. 1 ; ? III-113 fresh 800 { | rem.oflight 0.365
end
Aug. 4 ? ? IIO-III fresh 800 definite 0.365
Aug. 4 ? ? TIO-112 fresh goo f See) 0 call 0.287
\
very evident.
A 222-volt Nernst glower was used in all the experiments in this
series excepting the last three, in which a 110-volt glower was used.
For intensity of light, see table on intensity, p. 395.
side ¥. i
cr rf
: Gas
mh SRT ig musty ge pm
CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE
MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE,
E. L. MARK, Director—No. 183.
THEANFLUENCE OF LIGHT AND HEAT ON THE
MOVEMENT OF THE MELANOPHORE
PIGMENT, ESPECIALLY IN LIZARDS
BY
G. H. PARKER
Wirth Turee Ficures
It is now generally recognized that, of the various factors con-
cerned in the integumentary color changes in lizards, none is so
important as the migration of the pigment granules in the large
pigment cells of the derma, the melanophores, erythrophores,
etc. These cells are situated as a rule in the deeper part of the
derma, and their bodies are either embedded in the more or less
opaque guanine layer or lie proximal to this layer. Their pro-
cesses extend distally between the guanine particles, and, as finely
divided branches, form a rich arborization on the proximal face
of the epidermis. When these distal processes are filled with
pigment granules from the bodies of the cells, they form a dark
covering on the distal face of the guanine, and the skin in conse-
quence is dark-colored. When, however, the pigment migrates
fromthe distal processes proximally into the bodies of the cells, the
guanine layer becomes exposed to the light, and, since this layer
is made of reflecting particles, the skin under these circumstances
has a lightish appearance. ‘Thus a distal migration of the pig-
ment results in a dark-colored skin, a proximal migration in a
light-colored one.
The migration of the pigment is influenced both by internal
and external factors. Not only do the emotional states and
other nervous conditions of the lizard make themselves evident
Tue Journat or Experimentar Zoéiocy, Vor. 11. No. 3.
402 G. A Parker
in the color changes of its skin, but external factors, such as heat
and light also induce these changes. It has been pointed out
recently by Parker and Starratt (’04, p. 464) that, in all lizards
on which they could get records, a high temperature is accom-
panied by a proximal migration of the pigment, whereby a light-
colored skin is produced, and a /ow temperature by a distal move-
ment, thus giving rise to a dark coloration. Light on the other
hand seemed to affect different lizards differently. Thus in
Chameleon and Anolis light induces a distal migration of pigment,
while in Stellio, according to Filippi (’66), and in Varanus and
Uromastix, according to Thilenius (’97), light calls forth a proxi-
mal migration. After having studied Anolis and become familiar,
in this lizard, with one in which light produced a distal migration
of pigment, I was naturally desirous of examining a species in
which the reverse took place. Unfortunately the lizards studied
by Filippi and by Thilenius were all old-world species and hence
were not readily accessible to me in a living condition. I was,
therefore, obliged to seek for a representative of this type of color
change among more available forms. Hoffmann (’99, p. 1353)
states on the authority of Wiedersheim, that the horned toad,
Phrynosoma orbiculare, on cool overcast days is dark colored,
and that it changes to silvery gray when sunlight falls upon it.
Wiedersheim believed that this change was caused by a difference
of temperature and not by illumination, but as no proof of this
view was given, Keller (’95, p.132) was free to assert that these
changes were due in his opinion to light. In that case Phrynosoma
would show agreement with Stellio, Varanus and Uromastix,
in that a strong light would induce a proximal migration of the
pigment and a weak light, or none, a distal movement. Since
Phrynosoma is abundant in the southern and western parts of the
United States and since its color changes gave promise of being
the reverse of those in Anolis, so far’as their relation to light was
concerned, | decided to make it an object of study.
Preliminary trials made at my suggestion by Mr. A. S. Pearse
showed that horned toads obtained oo dealers were not usually
ina very satisfactory condition for work of this kind, and further
that the color changes, though present in Phrynosoma, were not
Influence of Light and Heat on Melanophores 403
of a very pronounced type. ‘The subject was, therefore, aban-
doned till through the kindness of Miss S. R. Armington I came
into the possession of a large vigorous specimen of Phrynosoma
blainvillei Gray, from San Diego, California. This specimen
had been well fed, had recently shed its skin, and showed in a
clear and unmistakable way a series of color changes that had
been only faintly indicated in other specimens. Btn results
recorded in this paper refer in the main to the reactions of this
one specimen, though they have been checked by observations on
other horned toads in the laboratory. I am under obligation
to Miss Armington for the privilege of working with this animal.
Fig. 1. Dorsal view of Phrynosoma blainvillei, showing the dark coloration due to exposure to day~
light. <2.
Since both light and heat influence the migration of the pigment
in the melanophores of lizards, 1t was bvieus that in experi-
menting on Phrynosoma, both factors were to be kept in mind.
In a room at 19° C., after an exposure for several hours to bright
but diffuse daylight, the horned toad was deep brownish, mottled
with black and white (Fig. 1). The head was brownish gray.
Down the middle of the back from the head to the tail ran a
broad brownish streak, which was much lighter on the neck than
elsewhere. Right and left of this lighter portion and covering
most of the neck were two large patches almost black in hue.
The trunk was marked by four chestnut-brown transverse bands
404 GH. Parker
alternating with darker bands. ‘The most anterior of the brown
bands was immediately behind the neck and crossed the trunk
from foreleg to foreleg. “The most posterior one was on the
pelvis. ‘The tail was also marked by transverse bands, five dark
ones alternating with four brown ones. On the lateral edges
of the trunk the color was chestnut-brown, and the claw-like
scales of the edge, numbering about twenty to a side, had dark,
almost black bases with white edges and tips (Fig. 2). The legs
were banded transversely dark brown and black. ‘The ventral
side of the animal was light-colored, the trunk being yellowish
with numerous gray splotches.
After the horned toad had been kept in the dark at 19° C. for
several hours, it became distinctly lighter excepting on the ven-
tral surface, which remained unchanged. ‘The head assumed a
light yellowish-gray tint, the brownish dorsal line and the brown-
ish transverse bands on the trunk and the tail also became lighter,
as did the chestnut-brown edges of the body. But the most
marked change was in the lateral claw-like scales; these lost their
dark bases and became yellowish white (Fig. 3).
Since 1t was not always easy to follow the color changes in the
complicated pattern of this animal, [ selected for further study
the lateral claw-like scales, in which these changes went on in a
clear and conspicuous way. Each scale is Soe wene triangular
in form with its claw-like apex turned posteriorly. As already
stated, these scales in bright diffuse daylight at 19° C. are deep
gray or even black except on their free edges and tips, which are
yellowish white (Fig.2). After an hour or so in the dark at 19° C.
the whole scale became yellowish white (Fig. 3). These scales
have a well developed guanine layer and a deep-seated layer of
melanophores whose pigment when in the distal branches pro-
duces the dark central areas and when withdrawn leaves the
whole scale whitish. In some of the experiments the dark central
areas did not disappear entirely even after the animal had been
in the dark for some time, but they remained visible as faintly
grayish or greenish spots. On examining these spots under a
hand lens they were found to contain scattered, minute, dark
points, evidently particles of pigment which had not yet been
Influence of Light and Heat on Melanophores 405
carried proximally far enough to be hidden by the guanine layer
and which thus gave rise to the grayish or greenish tint.
S
ds
Fig. 3
Figs. 2 and 3. Dorsal views of the right side of Phrynosoma blainvillei, showing (Fig. 2) the dark
coloration due to exposure to very bright daylight and (Fig. 3) the light coloration due to retention in
the dark. The negatives from which these figures were made were taken from the same anima)—Fig.
3 immediately after the animal had been taken from the dark, and Fig. 2 after exposure for an hour and
a half to bright daylight. The illumination for photographing was diffuse daylight and the exposure
in each instance was 7 seconds. The two plates were then developed for the same length of time in
the same fluid. In Fig. 3 the very faint bands and patches on the lateral claw-like scales are shadows.
These also occur in Fig. 2; otherwise the dark color on these scales in Fig. 2 is due to the melanophore
pigment. The effect of this pigment can be most fully appreciated by comparing the last three lateral
scales in the two figures. The magnification is only slightly over natural size.
406 G. H. Parker
The complete proximal migration of the pigment is carried
out at a somewhat different rate from the distal migration.
At 19° C. the distal migration was accomplished in about fifteen
minutes and the proximal one ina little over half an hour. ‘Thus
in Phrynosoma, as in Anolis (Carlton, ’03), the distal migration
takes less time than the proximal one.
I next tried the effect of temperature changes on the migration
of the pigment. At 15° C. in bright diffuse daylight, the lateral
scales had very pronounced dark centers, much as at 19° C. under
similar illumination. ‘These dark centers were partly but not
completely lost when the animal was kept in the dark at 15° C.
As these centers usually disappeared completely at 19° C. in the
dark, it is clear that a low temperature favors a distal position
for the pigment.
At 32° C. the animal when in the dark was light-colored and
the lateral scales showed no traces of dark centers. ‘Their tint
was a clear ivory white. When the lizard was transferred to the
light at this temperature, it became rapidly darker, the lateral
scales always showing gray centers, which sometimes became
almost black, but not so much so as at 19°C. Hence a high
temperature favors a proximal position for the pigment.
Similar results, both as respects light and temperature, were
obtained from two other horned toads in the laboratory, but
these animals were much less satisfactory for experimental pur-
poses because of the conditions of their skin. In both the epi-
dermis was filled with fine particles of dirt that rendered the
pigment changes visible only with difficulty, nor were these two
lizards as quick in response as the first one was, a fact that may
have been due to their lack of proper food and surroundings.
From these observations it is evident that both heat and light
influence the pigment migration in the skin of Phrynosoma
blainvillei, a bright light and a low temperature calling forth a
relatively rapid distal migration of the pigment and the absence
of light and a high temperature inducing a proximal migration.
Bright light is antagonistic to high temperature and no light to
low temperature. Since the animals were dark-colored in a
bright light at a high temperature and light-colored in the dark
Influence of Light and Heat on Melanophores 407
at a low temperature, it follows that between 15° C. and 32°C.
light or its absence is a more effective stimulus than heat or cold
and in the main controls the resulting color. I believe, therefore,
that the dark coloration noticed by Wiedersheim (Hoffmann
’90, p.1353) in Phrynosoma orbiculare on sunless, cool days was
probably a light reaction, though the low temperature doubtless
favored it. It seems to me improbable, however, that Keller
(95, p-132) is correct in assuming the reaction to be due exclu-
sively to light. The blanching of ae animal, which is stated by
Wiedersheim to occur when sunlight falls upon it, is not so readily
explained. My three specimens of Phrynosoma remained quite
dark in-full sunlight even after several hours of exposure. Had
they turned light, I should have suspected that it was due to heat,
but in all my experiments with these animals the effect of heat
has invariably been found to be subordinate to that of light and
the animal has always been dark-colored in daylight. How we
are to explain Wiedersheim’s observation, I am at a loss to say,
but it is not impossible that Phrynosoma orbiculare differs from
P. blainvillei in its relation to light and heat, and that like Anolis
it may react to a high temperature by withdrawing its pigment
even when illuminated. In such a case the blanching in sun-
light would be a temperature effect and not due to light. That
such a condition is not improbable may be inferred from the
observations of de Grijs (’99, p-51) on Phrynosoma cornutum.
In this species the individuals are said to be dark-colored in an
unheated cage and light-colored in a warm one, even though
illuminated.
If the light color assumed by some species of Phrynosoma in
bright sunlight is not due to a reversal of the ordinary light
reactions, such as are seen in Chamzleon and Anolis, but to a
temperature reaction, may it not be that the blanching of the
other supposedly exceptional cases, such as Stellio, Uromastix
and Varanus, is also to be explained in this way? Unfortunately
there is not sufficient evidence at hand to settle this question, but
1[n this respect my results agree with the observations of Gadow (or, pp. 510, 521), who states that
Agama stellio when basking and geckos when in broiling hot sunlight are almost black.
408 G. H. Parker
in two of the best known cases, Uromastix and Varanus, this
interpretation is not impossible.
Uromastix, according to Thilenius (’97, p.536), 1s dark colored
at night, but whitens after the morning sunlight has been on it
for an hour or more, and remains so till evening, when it again
becomes dark. No conclusive evidence is given as to whether
this reaction is due to heat or to light, but if, as I suspect, tempera-
ture is the controlling factor, these changes are precisely what
should be expected, and the fact that Uromastix has been observed
by de Grijs (99, p. 51) to become light-colored in a warm cage
favors this view.
The relations of Varanus to heat and light have been stated by
Thilenius (’97, p. 536) more fully than those of Uromastix.
Varanus is said to be dark-colored in the shade at a temperature
of 45° C. to 50° C., but to blanch in the morning sunlight before
the thermometer had reached 30° C. Such an instance might
seem to be a conclusive case of reversed light action, but in my
opinion the dark color in this instance is due, as in Phrynosoma
blainvillei, to the action of the diffuse daylight (shade) irrespective
of the relatively high temperature. The blanching in sunlight,
however, notwithstanding the statement as to temperature made
by Thilenius, I believe to have been due to heat. It is true that
Thilenius states that the temperature of the morning sunlight
had not yet risen to 30° C. when the change took place, but it
seems to me hardly possible that such could have been the case.
Even the early morning sunlight usually has much more heat in
it than this. Possibly TUhilenius took the temperature with an
ordinary mercury-bulb thermometer, and it may be that the
reflection from such a bulb would be sufficient to allow so low a
reading to be recorded, but with a black-bulb thermometer I feel
conhdent that a higher temperature would have been found, and
such a bulb would have imitated much more nearly the conditions
in the dark absorbing skin of the lizard than the reflecting surface
of a glass-and-mercury thermometer would. I, therefore, believe
it probable that the blanching of the skin of Varanus in sunlight
is a temperature reaction, such as de Grijs has observed in other
lizards, and not a reversed light reaction.
Influence of Light and Heat on Melanophores 4.09
If these conclusions are correct, the reactions to light and to heat
of the melanophores and other like cells in the skins of lizards can
be stated very simply, as follows: Light causes a distal migration
of the pigment granules of these cells; its absence a proximal one.
A high temperature causes a proximal migration of the pigment;
a low temperature a distal one. So far as | am aware, there are
no real exceptions to these rules. Light causes a distal migration
of pigment and its absence a proximal one in Chameleon (Briicke,
°52; Keller, ’95, etc.), Anolis (Carlton, ’03; Parker and Starratt
’o4), and Phrynosoma blainvillei; and I know of no lizard of
which the reverse is true. Heat causes a proximal migration of
pigment and cold a distal one in‘Chamzleon (Briicke, ’52; Keller
95); Eumeces schneideri, [arentola annularis, Uromastix, Scelop-
orus undulatus, Crotaphytus collaris, Phrynosoma cornutum,
Amphibolurus barbatus, Agama mossambica, A. stellio, A. iner-
mis and Cachryx defensor (de Grijs, ’99, pp. 51-54), and Anolis
(Parker and Starratt, ’04). Here, too, | know of no exceptions.
At unusually high temperatures, such as obtain in the full
sunlight of torrid deserts, the light reactions of the melano-
phores of certain species of lizards are apparently subordinate to
their temperature reactions, and hence these forms may appear
light-colored in full sunlight. Thus Amphibolurus barbatus,
according to de Grijs (’99, p. 54), darkens in the early morning
sunlight but changes to light gray as the heat of midday comes
on, and Anolis (Parker and Starratt, ’04) becomes green, 1. €.5 1tS
melanophore pigment migrates proximally, at 40° C. to 45° C.
irrespective of illumination. The same is probably true, for
reasons already given, of Stellio (Filippi, 66) Phrynosoma orbicu-
lare (Hoffmann, ’90), Varanus and Uromastix (Thilenius, °97)
Calotes emma (Gadow, ’or, p. 519), and most desert-inhabiting
agamids and iguanids (de Grijs, ’99, p. 54). “Thus, in my opinion,
the apparently exceptional cases of light reactions in melanophores
are really instances of normal temperature reactions and do not
show any reversal of the regular processes. I believe, therefore,
that the simple rules already stated as to the relation of heat and
light to the pigment migration in the melanophores of lizards
will hold for all those lacertilians in which such changes occur.
AIO | G. H. Parker
Not only do the melanophores of lizards show uniformity in
the migration of their pigment under the influence of light, but
the rule that holds in these cases appears to be of much wider
application. It is well known that in the eyes of most verte-
brates the pigment of the retinal pigment-cells migrates back
and forth under varying illumination, and here, as in the mela-
nophores, light causes a distal migration and its absence a proxi-
mal one. ‘The same has been shown to be true by Hess (’05, p.
421) for the retinal pigment in the eyes of cephalopods. ‘The
compound eyes of many arthropods show similar changes. Ac-
cording to Exner (’91) and others, the dark pigment of these
eyes is often divided into two layers, one distal and the other
proximal. ‘The proximal layer consists of dark pigment gran-
ules within the retinular cells and these granules migrate back
and forth under changes of illumination. The distal migra-
tion is made in the light and the proximal one in the dark exactly
as with the melanophores. Not only is there agreement in the
direction of the migration but the relative rates in the few cases
known show a certain similarity. ‘Thus, in the retinular cells in
Palzemonetes (Parker, ’97), the distal migration is more quickly
accomplished than the proximal one, a condition parallel with
that seen in the migration of the pigment in the melanophores
of Anolis (Carlton, ’03; Parker and Starratt, ’04) and of Phry-
nosoma.
In all these instances the direction of the migration is in
strict relation to the source of light and is not determined by such
obvious structures of the cell as the nucleus. Thus in the
melanophores of the lizards, in the retinal pigment cells of the
vertebrates, and in the retinular cells of certain crustaceans,
Gammarus (Parker, ’99), for instance, the distal migration of
pigment is in a direction away from the nucleus, while in the
retinular cells of most crustaceans and insects (Exner, ’91; Parker
’97) it is toward that organ. ‘Thus the position of the nucleus
seems in no way to influence the direction taken by the migrating
pigment.
As the pigment particles of the melanophores are within cell
limits and, when illuminated, move toward the source of light,
Influence of Light and Heat on Melanophores 4Il
the phenomenon may be described as a form of intracellular
phototropism positive in character. It is, however, highly im-
probable that the pigment granules take any active part in this
operation; in my opinion they are simply transported by the
protoplasm, which in some instances certainly receives directly
from the light the stimulus to motion. That the pigment par-
ticles are not necessarily concerned in the motion is seen in the
retinular cells of Gammarus, whose distal pigment, which is most
exposed to light, fails to migrate, whereas that which is some-
what more proximal in the same cell performs the characteristic
migration (Parker, ’99).
It is probable that the forms of pigment migration already
discussed are more or less adaptive in their character. “The dark
color of the lizard’s skin in moderate illumination at a moderate
temperature insures, possibly, among other things, a certain
degree of warmth which would be superfluous, if not dangerous,
at a higher temperature, and in consequence the skin becomes
light-colored in hot sunlight. The movement of the retinal
pigment in both vertebrates and arthropods is well calculated
to protect the receptive organs of their retinas from overstimula-
tion by light and to improve the sharpness of their retinal images.
Thus these pigment changes are not without adaptive character.
Admitting such to be true, it might be supposed that if a case
arose in which a reversed migration of pigment would be of service
to the organism, such a form of migration would be evolved and
a set of pigment cells in whichthe pigment granules under illumi-
nation would migrate away from the source of light instead of
toward it would be produced. In this connection it is interesting
to observe that such a reversed movement of pigment does occur
in the distal pigment cells of the compound eyes of many arthro-
pods, but that where this has been studied with fullness, as in
Palzmonetes, it has been shown that these cells, though they
contain the same kind of pigment as the proximal cells do, have
a pigment change based upon a wholly different principle; the
cell as a whole migrates distally and proximally and the pigment
granules within show no intracellular rearrangement (Parker,
’97). Hence it seems probable that the melanophores, retinal
412 G. H. Parker
pigment cells, and other like structures in which dark pigment
eranules exhibit migratory movements, are restricted as to these
possibilities, and that in light they always transport their pigment
toward the source and never in the reverse direction.
~ Whether heat is as uniform in this particular as light seems
to be, cannot be stated with certainty. Parker and Starratt (’04)
have pointed out the uniformity of its effects on the melanophores
of the vertebrate skin and it is possible that this uniformity also
extends to the retinal pigment, for Herzog (’05), following up
certain observations made by Kuhne (’79, p. 334), has shown
that in the frog for a range of temperatures between 18° Cyand
o° C. the retinal pigment migrates distally with a decreasing
temperature and proximally with an increasing one, as the pigment
of the integumentary melanophores does, but in ranges above
18° C., according to Herzog, the reverse takes place. However,
the relation of the retinal pigment migration to temperature has
been so little studied that further investigation will be necessary
before safe general statements can be formulated.
SUMMARY
1. Phrynosoma blainvillei can change the color of its skin from
a light yellowish gray with dark bands and spots to a dark chestnut
a mottled with black.
The light coloration is produced by the proximal migration
of the pigment granules out of the processes of the melanophore
and other like cells into their bodies, thus exposing to the light
the reflecting guanine layer.
The dark coloration is produced by the distal migration
of these pigment granules from the cell bodies into their processes,
whereby the guanine layer becomes covered and cut off from the
light.
4. In Phrynosoma blainvillei the proximal migration is favored
by heat and the absence of light, and the distal one by cold and
light.
5. In Phrynosoma blainyillei between 15° C. and 32° C. light
or its absence 1s more effective as a stimulus to color change than
heat or cold.
Influence of Light and Heat on Melanophores 413
6. The blanching of certain lizards in strong sunlight, which
has been supposed to be evidence of a reversed light reaction,
is probably not a light reaction at all, but a temperature reaction
normal in direction.
7. As inthe retinular cells of Palzmonetes, the distal migra-
tion of the melanophore pigment of lizards is more quickly accom-
plished than the proximal migration.
Swe is probable that in all melanophores in which there 1s
a migration of pigment, light or a low temperature will induce
a migration toward the source of illumination and the absence
of light or a high temperature a migration in the reverse direc-
tion.
BIBLIOGRAPHY
Bricke, E., ’52.—Untersuchungen tiber den Farbenwechsel des afrikanischen
Chamialeons. Denkschr. kais. Akad. Wiss., Wien, math.-naturw.
Cl., Bd. iv, Abt. 1, pp. 179-210, 1 Taf.
Cartton, F. C., ’03.—The Color Changes in the Skin of the So-called Florida
Chameleon, Anolis carolinensis Cuv. Proc. Amer. Acad. Arts
and Sci., vol. xxxix. No. 10, pp. 257-276, 1 pl.
Exner, S.,’91.—Die Physiologie der facettirten Augen von Krebsen und Insec-
ten. Deuticke, Leipzig und Wien, vi +206, pp., 7 Taf.
Fiurept, F. pe.,’66.—Sulla struttura della cute dello Stellio Caucasicus. Mem.
Accad. Sci., Torino, ser. 2, tom. 23, pp. 363-373, I tav.
Gavow, H., ’o1.—Amphibia and Reptiles. The Cambridge Natural History,
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Grys, P. pr, ’99.—Einiges iiber Farbwechsel-Vermogen bei Reptilien. Zool.
Garten, Jahrg. 40, No. 2, pp. 49-55.
Herzoc, H., ’05.—Experimentelle Untersuchungen zur Physiologie der Bewe-
gungsvorgange in der Netzhaut. Arch. f. Anat.u.Physiol., Physiol.
Abt., Jahrg. 1905, Heft 5-6, pp. 413-464, Taf. 5.
Hess, C., ’05.—Beitrige zur Physiologie und Anatomie des Cephalopoden-
auges. Arch. f. ges. Physiol., Bd. cix, pp. 393-439, Dat. 5-8:
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Ordnungen des Thier-Reichs, Bd. vi, Abt. 3, Reptilia 2, pp.
443-1399, Taf. 49-107.
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anderer Reptilien. Arch. f. ges. Physiol., Bd. Ixi, pp. 123-168,
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Handbuch der Physiol., Bd. ii, Theil 1, pp. 235-342.
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Bull. Mus. Comp. Zool., Harvard Coll., vol. xxx, No. 6, pp. 273-300,
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’99.—The Photomechanical Changes in the Retinal Pigment of
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pp- 515-544, Taf. 17-18.
SOME REACTIONS OF CATERPILLARS AND MOTHS
BY
ALFRED G. MAYER AND CAROLINE G. SOULE
It is believed by many naturalists that the larva of Danais
plexippus is warningly colored, for it is conspicuously ringed in
black, white and yellow; often displays itself openly upon the
leaves of its food plant, and it is not known to be subject to the
attacks of birds or other vertebrates. “Vhe larva normally feeds
only upon species of milkweed (Asclepias), although in captivity
it may sometimes be induced to feed upon the leaves of the carrot.
Each individual larva commonly spends its entire life upon a
single plant. It usually lives upon or near the upper half of the
plant, and only rarely does it descend to a point below the middle
of the plant.
In fact, were the larva to crawl down the stem and escape from
the plant it might possibly starve before finding another Asclepias.
We have frequently placed larve upon the lower part of the stem
of a milkweed (Asclepias) and in all but one instance they crawled
up the stem and remained for days at or near the top of the plant
feeding upon the leaves. In one case, however, the larva crawled
upward but moved away from the milkweed upon some blades
of grass which touched it.
It must be borne in mind, however, that the caterpillar of
D. plexippus is sometimes found upon very small or immature
milkweed plants; too small to provide them with sufficient food
upon which to mature. Under these conditions it would seem that
they must either find other milkweeds or starve. If this be the
case their success in finding other milkweeds must be facilitated
by the fact that these plants usually grow in clusters, but we have
no evidence leading us to conclude that the larva of D. plexippus
has the ability to direct its course to a milkweed rather than to any
other vlant.
Tue Journat or Experimenta Zootocy, Vor 111, No. 3.
416 Alfred G. Mayer and Caroline G. Soule
The larve of Argynnis, Catocala, and some other lepidoptera
feed only at night, and crawl a considerable distance away from
their food, and remain hidden during the day, and these cater-
pillars must in some way be guided pack to their food.
That the larva of D. plexippus may also have this ability, seems
probable from the fact that if the caterpillar be repeatedly dis-
turbed it finally curls up and drops off the milkweed.
Our experiments show that under normal conditions the cater-
pillar of D. plexippus is constrained to remain upon the milkweed
in obedience to two reactions which the creature displays with
almost machine-like regularity. It is negatively geotactic, and
positively phototactic. If the larva be placed head downward,
in darkness, upon a vertically suspended string, it will usually
turn at once, and crawl upward. If nowthe string be reversed so
as to cause the larva to be again head downward, it again reverses
its direction and crawls upward. This may be repeated time
after time with the same result. Often when the string is reversed
the larva continues to crawl downward for a short distance, but
it is restless in so doing, and shows a tendency to frequently stretch
its anterior segments outward at right angles to the body, and to
sweep widely to and fro with its head. ‘This is sure to result soon
in the head being bent completely back upon the body, and in
consequence the larva crawls upward. In crawling upward the
creature 1s more at ease and rarely stretches its head horizontally
outward. Thus in its travels the larva is almost certain to go
farther in an upward than in a downward direction. ‘This is
especially true if the larva be in ordinary daylight but it also takes
place in the dark. Larvz were placed head downward upon a
vertical string which was placed in an absolutely dark chamber,
and at the eal of three minutes the string was observed. If the
larva had turned and crawled upward the string was reversed, and
observed again at the end of another three minutes. ‘Thirty-seven
experiments upon three larve gave the following results. “The
larvee, at the expiration of three minutes, had turned and crawled
up to a point above their original position in twenty-seven cases.
In four cases the larve continued downward, and were found head
downward and still descending, while in six other cases the larvae
Some Reactions of Caterpillars and Moths 417
had ascended above the original position, but had again turned
and begun to descend. It is apparent that the larva shows a
tendency to crawl upward against the force of gravity, and this
reaction alone would tend to retain it at or near the top of its food
plant. No difference is apparent in the reactions of well-fed or
hungry larve, excepting that the latter are more active.
In another series of experiments a milkweed was planted in a
flower-pot and suspended upside down. Larve placed upon the
young leaves, at what was now the lowest part of the plant, crawled
upward, and sooner or later they went up the stem of the plant
and crawled off upoh the flower-pot. One larva crawled up the
entire length of the plant (nineteen inches) and off upon the flower-
pot in four minutes and twenty-seven seconds. Upon being again
replaced upon the young leaves, it again crawled off the plant in
about six and one-half minutes. ‘The plant was then reversed and
placed in its normal upright position. ‘The same larva was then
placed at the base of the stem, and promptly crawled upward to
the top of the plant where it remained feeding for fifty-three hours,
when it was removed for further experiments. Frequent repeti-
tions of this experiment showed that the larve do not long remain
upon a plant which has been turned upside down
We may conclude that the larva has no inherent tendency to
seek out the young leaves, and that its remaining near the top of
the plant it not a matter of “judgment” or conscious preference,
but is due to negative geotaxis, and may be a wholly unconscious
reaction. It is worthy of note that the tendency to turn and crawl
upward only affects the larva when in motion. — It will often remain
at rest head downward for hours at a time, but upon beginning
to move 1s almost certain soon to turn upward.
But, another influence contributes to cause the caterpillar to
remain at or near the top of the plant. ‘This is due to the fact
that the creature is positively phototactic, and is particularly sensi-
tive to the ultra-violet rays. Ifthe larva be placed in the middle
of a horizontally placed opaque tube, one end of which is stopped
with a cover impervious to light, while the other end is open to
admit diffused daylight, the larva always turns and crawls toward
the open, lighted, end of the tube. Experiments were made upon
AI8 Alfred G. Mayer and Caroline G. Soule
six larvae under the above conditions, using a pasteboard tube
eleven inches long and one and one-half inches in caliber, and they
crawled fifty-one times to the open end of the tube and never to the
dark closedend. ‘The larvae were pushed back into the center of the
tube after each trial and the experiment so arranged that the head-
end of the creature faced the dark end of the tube but invariably
the larva turned and crawled toward the daylight. ‘This positive
heliotropism in caterpillars is well known, and Loeb (’05, p. 42)
states that it was shown by all of those upon which he experimented
even by the larvae of the willow-borer which live in the stems of
willows where they are not exposed to light. When this experi-
ment with the horizontal tube is tried, substituting the light of a 36-
candle power kerosene lamp, four feet distant, for ‘pylon a very
different reaction takes place, for under these conditions the larvze
crawled to the light end of the tube twenty-eight times, and to the
dark end twenty-one times. The light of a kerosene lamp is
deficient in ultra-violet rays, and it seemed probable that the larve
might be sensitive to these rays and hence practically indifferent
to the light of kerosene, while they always crawled toward diffused
daylight. In order to test this the diffused daylight was obliged
to pass through a layer of bisulphide of carbon one and one-half
inches thick before entering the light end of the tube. Experiments
were made upon four larve under these conditions. One of them
crawled five times to the light end, and eight times to the dark end
of the tube; another larva went twenty-one times to the light and
one time to the dark end, another went once to the light and ten
times to the dark end, and the fourth larva went three times to the
light end and never to the dark end of the tube.
Thus in forty-nine trials the caterpillars went nineteen times to
the dark end of the tube, and thirty times to the end which admitted
diffused daylight deprived of its ultra-violet rays. Now, if ordi-
nary diffused daylight be admitted at one end of the tube the larve
always crawl toward it, and are also much more active than when
under the influence of the daylight deprived of the ultra-violet
rays. We see, then, that they are attracted chiefly if not wholly
by the ultra-violet rays and are but little if at all influenced by the
rays that constitute, to us, the visible spectrum.
Some Reactions of Caterpillars and Moths 419
Even hungry larve in diffused daylight deprived of the ultra-
violet rays move with extreme slowness, and often remain for hours
at a time motionless; and apparently unstimulated. ‘They act
similarly in the dark. In ordinary diffused daylight, however,
they are very active, and rarely remain quiescent more than a few
minutes at atime. Intense sunlight renders the larva very restless
but it becomes less active when in the shade. When hungry the
larve are much more active than when well fed.
At night when its negative geotropism is apparently the only
factor tending to maintain the caterpillar near the top of the plant,
the sluggishness of its movements in the darkness must be a safe-
guard, preventing its moving to any considerable distance.
The negative geotropism of the caterpillar is, however, more
potent than the influence of the diffused daylight of an ordinary
room, as is shown by the following experiment. If we place the
larva in the middle of a vertical pasteboard tube having the upper
end closed with a cap so as to prevent the entrance of light, while
the lower end remains open admitting the diffused daylight of the
room, the larve show a greater tendency to go upward into the
darkness than downward toward the light. Thus two larve,
placed in such a tube, went fifteen times to the dark (upper) end
of the tube, and eight times to the light (lower) end, although they
were invariably placed head downward, nearer the lower than
the upper end, and facing the light at the beginning of each
trial.
It will be remembered that Loeb showed that the starving larvae
of Porthesia chrysorrhcea are positively phototactic, while they
become practically indifferent to the influence of light when well fed.
Larve of D. plexippus are, however, always positively phototactic
to moderate daylight, whether well fed or starving. Lubbock
(Avebury) (83) showed that ants are always sensitive to the ultra-
violet rays, reacting strongly against them, and Loeb found that
nocturnal moths are attracted mainly if not wholly by the more
refrangible rays of the spectrum.
In 1903 Parker published a beautiful series of observations,
showing that when the mourning-cloak butterfly, Vanessa antiopa,
alights in sunlight it comes to rest with its head turned away
420 Alfred G. Mayer and Caroline G. Soule
fromthe sun, and with its body in line with the rays of light. On
cloudy days or in shaded places this reaction is not displayed,
and even in brilliant sunshine it is overruled by the chemotropic
response to food. ‘This negative phototropism is seen only in
intense sunlight, and then only after the butterfly has been upon the
wing; 7. ¢., after a certain state of metabolism has been established.
The butterfly will creep or fly toward a source of moderate light,
such asa lamp in a darkened room, or a window admitting light;
and it is even attracted toward intense sunlight until it has begun
to fly about, when it becomes negatively phototropic. When the
eyes are blackened no reaction to light occurs, and the butterfly
flies upward.
It appears that positive phototaxis and negative geotaxis are all
that are required to maintain the larva of D. plexippus upon the
young leaves of its food plant, and to practically prevent its wander-
ing away from the plant itself. The larva certainly displays no
“judgment” in finding its most nutritious food, and probably its
reactions are almost if not quite unconscious, for they are dis-
played with almost machine-like regularity at every recurrence of
the stimulus, however as Jennings (’04) has shown, animals are
not machines for their method of behavior is often that of trial and
error and internal as well as external factors modify their behavior.
Another criterion of consciousness is the presence of associative
memory, but all of our attempts to detect the presence of such
memory have failed.
A large number of experiments were made upon larve which
will eat only certain definite kinds of leaves, and will starve to
death without taking a single bite of other sorts of leaves. These
larve can be induced to eat sparingly of previously uneatable leaves,
however, if the sap of their proper food plant be pressed into the
previously distasteful leaves. For example, the larva of D.
plexippus can be induced to eat sumach (Rhus) leaves if the latter
be thickly covered with the milky juice of Asclepias. ‘This only
occurs, however, when the larva has become very hungry.
Conversely, milkweed leaves can be rendered uneatable by
covering them thickly with the milky juice of sumach. ‘The larva
appears not to be guided by any sense of color in seeking its food,
Some Reactions of Caterpillars and Moths 421
for it will eat Asclepias leaves which have been painted over with
aniline red.
We find that any caterpillar can be induced to bite at or devour
any foreign substance, if one proceed as follows: Allow the larva
to begin eating its proper food plant, then slide up in front of
it a distasteful leaf, sheet of paper, piece of tinfoil, etc. “The larva
will then take a few bites out of the foreign substance, but will
soon withdraw its head, often snapping its mandibles and thrash-
ing from side to side. Very soon, however, it reeommences to
devour its proper food in a normal manner. Under these condi-
tions if the foreign substance or distasteful leaf be presented to the
larva at intervals of not more than one and one-half minutes, about
the same number of bites are taken at each presentation. The
instinctive mechanism of eating once set in motion continues as if it
possessed momentum. ‘This may be taken to show that the larva
does not retain the memory of its disagreeable’ experience for an
interval of a minute anda half. If, however, we make the interval
about thirty seconds, the larva will take fewer and fewer bites of the
distasteful object and will soon refuse it altogether, and stop eating
whenever it is presented.
The following table gives the results of observations of the above
sort upon larve of D. plexippus; the intervals of presentation of
the distasteful leaf being as nearly as possible one and one-half
minutes apart. The table shows the number of bites taken by the
larve at successive presentations of a leaf of the Virginia Creeper,
Ampelopsis.
An inspection of the table shows that the successive reactions
in any single series of experiments are very irregular, but that
these irregularities tend to disappear when we take the sum of
the corresponding reactions in a large number of experiments.
For example, in the column headed “Summation of series show-
ing at least nine reactions,’ we see that the numbers are relatively
more nearly equal each to each than are those of any one of the
1 The words “‘ disagreeable,” “‘agreeable,’’ ‘distasteful,’ etc.,are used in default of better expressions,
We must not forget that the larva may be quite unconscious, and may act by reflexes, which may be
very complex and controlled by varying internal and external conditions, thus being capable of much
modifiability, although possibly unconscious.
TABLE I
Showing the Number of Bites taken by Larve of D. plexippus at each Presentation of a Leaf of Ampelopsis, the Ampelopsis
being Presented at Intervals of 13 Minutes
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*From the twenty-first to the fortieth trial, inclusive, this series gave the following for the number of bites at each trial
6, 0, 2, 5, 9, 7, I, O, O, O, O, O, I, O, O, O, 2, 0, 4, O.
Some Reactions of Caterpillars. and Moths 423
component series. [he fact comes to light that there is no sensible
falling off of the reaction, the number of bites taken at the ninth
reaction being fully equal to those taken at the first. It can hardly
be affirmed that the larve must take a certain definite number of
bites from a foreign leaf before being able to “recognize” or react
against its “injurious” or “distasteful” nature, for when larve
are placed in a chamber surrounded with these same leaves they
will starve to death before taking even a single bite. ‘The instinct
to eat must first be initiated by the presence of food proper to the
larva, but once the mechanism of this instinct is called into play,
it continues as if by its own momentum. ‘The phenomenon might,
therefore, be termed the ““momentum of reaction.”” As the value
of this momentum remains the same, and does not decrease with
successive reactions it probably represents an unconscious reflex
on the part of the larva. ‘The presence of its proper food plant
causes the eating reaction to come into play, and this reaction con-
tinues for a constant and apparently unmodified time against a
regularly recurring succession of counteracting stimuli.
Larve do, however, become accustomed to certain stimuli, and
if such a stimulus be repeated, it finally ceases to produce any reac-
tion. If one blows a current of air upon a moving larva, or raps
sharply upon the surface on which it is crawling, the creature con-
tracts and remains motionless for some time. If this stimulus be
repeated every time the larva begins to move, it finally loses all
effect. For example, a single blow of the breath sufficed to halt a
caterpillar of D. plexippus every time it started for four successive
times; at the fifth start, however, two blows were necessary to cause
It to cease moving; then, however, one blow sufficed until the nine-
teenth attempt to start, when eleven blows were necessary to stop
it. [hen one blow sufficed until the forty-seventh attempt at start
when five blows were necessary; on the forty-eighth attempt to start,
one blow still sufficed to stop it; but when it started for the forty-
ninth time, one hundred blows had no effect upon it whatsoever.
Usually, however, the results are not so irregular; for example,
one blow sufficed to stop a caterpillar in thirty-seven attempts to
start, but at its thirty-eighth attempt one hundred blows had no
effect and it moved steadily onward.
A24 Alfred G. Mayer and Caroline G. Soule
When the larva of D. plexippus has become insensitive to blows
of the breath, it still reacts to mechanical shocks, stopping when
the twig upon which it crawls is sharply struck, mechanical shocks
can, therefore, be distinguished from other mechanical or chemical
stimull.
Davenport (97), Loeb ('00), Massart (’01) and Jennings (’02,
’o4) have studied this final failure to react to a repeated stimulus.
The studies of Jennings upon various fixed Infusoria are especially
instructive, and he concludes ‘that the phenomenon is not neces-
sarily due to fatigue either of the muscles or of the sensory or per-
ceptive power, but as Davenport has expressed it, “When an
organism has been stimulated by contact for some time it becomes
changed so that it no longer responds as it did at first.” In other
words, internal factors come to modify its behavior. We must
bear in mind that in some cases this failure to respond to repeated
stimuli may be due to fatigue as has been shown to be the case in
the rejection of pieces of filter paper by the tentacles of Metridium
(see L. F. Allabach (’05); Biol. Bulletin, vol. x, p. 35).
In another series of experiments designed to test the presence
or absence of associative memory, larve of Pyrrharctia isabella
were placed in a wooden box sixteen inches long, five inches wide
and four inches deep, which was divided into two compartments
by means of a central wooden partition. ‘This partition was
pierced by a small opening. The chamber on one side of the
partition contained moist earth and growing food plants while the
chamberon the other side was barren. ‘The larvz were placed inthe
barren chamber and after wandering about they found their way
through the opening into the food chamber. ‘Then after being
allowed to eat for a few minutes they were thrust back through the
opening into the center of the barren chamber. Apparently, how-
ever, they failed to learn the direct path to the food, but always
wandered about upon successive trials. For example, one larva
was placed in the barren chamber twenty-one times; during the
first ten times it found the food in the average time of 50.8 minutes.
The average of the next ten times was, however, 101 minutes and
the final trial consumed 434 minutes. Another larva which was
placed thirty-eight times in the barren chamber appeared to do
Some Reactions of Caterpillars and Moths 425
better for it made an average of 88.8 minutes for the first ten, 66.1
minutes for the second ten, 52.8 for the third ten, and 129.2 minutes
on the last eight trials. Another larva made twenty-five trials
giving an average of 29.3 minutes on the first ten, 17.3 minutes
on the second ten, and 18.8 minutes on the last five. Another
larva made the following averages in forty-two trials. For the
first ten 58.7 minutes, second ten 58.1, third ten 39.1, fourth ten
32.2. On the whole it appears that the larve do not learn, even
after repeated trials, to shorten their paths to the food.
It was evident that the larva are attracted by the presence of
the food, however, for when the food chamber contained earth with
no plants the larve entered it at much more protracted intervals
than when food was present. For example, when the food-cham-
ber contained growing plants, a larva entered it forty times, the
average time for each trial being forty-seven minutes. When the
plants were removed leaving only the moist earth the larva crawled
into the chamber less frequently, the average time in nine trials
being 228 minutes, and when both earth and plants were removed
it made an average during seven trials of 417 minutes.
It will be remembered that Yerkes (02) and Yerkes and Hug-
gins (’03) found that the green crab and the crawfish are able to |
learn simple labyrinth habits.
A study was made of the instinct to spin the cocoon, in order to,
if possible, reduce it to a set of reactions to definite stimuli. ‘The
experiments were carried out upon Samia cynthia and Callosamia
promethea. In both of these moths, however, the larvae accommo-
date themselves to very varied conditions. For example, we have
seen several cocoons of S. cynthia which were in all respects similar
to normal cocoons excepting that they were much larger than the
average. These large cocoons, however, contained each a single
large inner chamber in which there were two pupe. Apparently
two larve had started to spin side by side at about the same time,
using one and the same leaf and had mutually accommodated them-
selves to the conditions so as to contribute each its share toward
the spinning of a single cocoon large enough to accommodate both
of them. If all leaves be removed, the larvae of C. promethea and
S. cynthia will still spin cocoons even upon a perfectly flat surface.
426 Alfred G. Mayer and Caroline G. Soule
These larvae normally start the cocoon by covering the leaf-stalk
with a layer of silk. “hey then spin down the middle of the upper
side of the leaf and draw the edges of the leaf together, forming a
tube. After spinning an outer case of moderately loose silk, the
inner case of densely spun silk 1s constructed. ‘This inner case is
ellipsoidal, and its innermost surface smooth and polished. ‘The
silk at its upper end is, however, loose and the strands are longi-
tudinal so as to allow for the exit of the moth. ‘The lower end of
the inner case 1s densely woven and its innermost surface polished.
The cocoon hangs vertically downward and the larva always
pupates with its head turned upward. We find that this upward
turning of the head of the larva 1s in response to gravity, for if the
cocoon Tbe reversed in the dark immediately after the outer envelope
is completed, the larve often become reversed and still pupate head
upward, but facing what would under normal conditions be the
lower end of the cocoon. Miss Caroline G. Soule first tried this
experiment in 1900 with twenty-eight larva of S. cynthia, reversing
the cocoons of nineteen immediately after the formation of the
outer envelope, and of nine a few hours after it had been completed.
In all of the cocoons which were turned upside down immediately
after the formation of the outer envelope, the larva were reversed
in their cocoons and pupated head upward. In thirteen of these
cocoons the silk of the inner chamber was loose at both ends. In
one cocoon the larva had bitten a hole in the lower end when it
became the uppermost, and had spun the other solid. Five cocoons
had no exit arranged, and their inner capsules were solid at
both ends. In all of the nine cocoons which were turned upside
down, a few hours after the completion of the outer envelope the
larve pupated head downward, and the cocoons were normal in
all respects.
In 190102, a similar experiment was tried upon twenty-seven
cocoons of C. promethea. “Twenty-one of these were not affected;
the larve pupating head downward. In six other cocoons the
larve pupated head upward, thus being reversed in reference to
the cocoon. In one of these cocoons the inner case was loosely
spun at both ends, while in the five others it was spun as in the
normal cocoon and thus the head of the pupa faced the densely
Some Reactions of Caterpillars and Moths 42
“SI
woven end of the inner capsule which apparently allowed of no
ready exit. We tried enclosing larve in capsules of glass and of
cotton network resembling the inner capsules of the cocoon in
shape but the larve still spun an inner capsule of silk in a normal
manner. We then opened a normal cocoon and after removing
the pupa placed a spinning larva within it. The larva spun a new
inner capsule which was nowhere attached to the cocoon within
which it was made. Evidently the instinct to spin the cocoon
may be adjusted to widely different environmental conditions,
some of which are never met with in nature. It cannot be reduced
to a simple reaction of thigmotaxis. ‘That is to say, the larva does
not cease to spin merely because it feels itself pressed upon by the
confined space of the cocoon, for even if it be placed with a cocoon
already spun, it re-spins it. he larva does, however, orient itself
in reference to gravity and normally pupates head upward.
Darwin (71) supposed that the peculiar and often brilliant
coloration of male Lepidoptera had been brought about as the
result of long continued selection upon the part of the females.
Studies were carried out upon the mating instinct in order to
determine whether the females exercised any selection in the choice
of their mates.
In 1900 Mayer studied the mating instinct in Callosamia pro-
methea in which it appeared that the female exercised no choice
in the matter. It seemed desirable to repeat these experiments
upon a large scale and with this in view more than 1500 cocoons
of C. promethea were collected during the winter of IogI—o2.
Large numbers being gathered by our friends Messrs. Clarence
Riker and William F. Patterson. “The cocoons were hung under
trees and the moths allowed to fly about unconfined. It is im-
portant in such experiments to maintain the conditions as close
to those of nature as possible for confinement often produces
remarkable alterations in behavior. For example, larvz which feed
only during the night, in a state of nature, will often eat during the
entire day in captivity. About six hundred males emerged from
the cocoons and the wings of about one-half of them were painted
with scarlet or green ink while the others were allowed to remain
normal in color. It was evident that the males whose wings were
428 Alfred G. Mayer and Caroline G. Soule
scarlet or green succeeded fully as well in their attempts to mate as
did the normal males. More than three hundred attempts to
mate were observed. Seventy per cent were successful and thirty
per cent gave rise to no visible resistance on the part of the female
although no mating occurred. In one instance only was a normal
male resisted successfully by the female, while in another case the
male succeeded in mating although the female made some show
of resistance. ‘l'wo of the green colored males were successfully
resisted by two females, although even a slight showing of resist-
ance on the part of the female was too rare and exceptional to be
of any moment in selection. ‘Therefore, the peculiar black colora-
tion of the male appears not to have been caused by sexual selec-
tion on the part of the female, or at any rate the female promethea
moths of the present time show no dislike for abnormal coloration
in the male.
In Porthetria dispar the male is brown and the female white,
and it 1s possible that the peculiar coloration of the male may have
been developed under the influence of sexual selection on the part
of the female. The experiments of A. H. Kirkland (96) upon
the mating habits of P. dispar, show that in common with C. pro-
methea the males are attracted solely by the odor of the female. In
C. promethea, however, Mayer found that old females are more
attractive than young, whereas the reverse is the case with P.
dispar. Moreover, the severed wings or scales of the females of
C. promethea do not attract the male while those of the female of
P. dispar are highly attractive.
Experiments were made to determine whether the females of P.
dispar exercised selection against maimed or abnormally colored
males. “The wings of an equal number of males and females were
clipped off, and the success achieved by the wingless males in their
attempts to mate was much less than that of the perfect males, as
will appear from the following table which records the results of
one hundred attempts to mate. ;
Some Reactions of Caterpillars and Moths 429
TABLE JI.
Showing the Reactions of Females of P. dispar whose Sight 1s Ncrmal
}
|
|
shen =o 2 Ah Hey tS 80 4 rs)
| 3 te ye) et coe eels 2 oe
a a z Salfakee: o& =
aw © mone pens af Hive
= ryercl is oy 0s S .2 0 =) 1S
| Be ra) 7~e “ig ei es wen
| a Y = es o Sad o & OS
moe 2 -a QO Seas | seas 4s
a4 oe “ee ait Om, ° 5) ey
D Oya tuelen ow q ros oe re) o
ance oS 8 0 -= + Do ay Wt
4 aoe geo |eesF | gas as
3 o 38 038 Sn ~ Sz 5
i net YAg eet) ONS = OW a 0
= 30 & ana ke 82S .o m Fo uO
= ve o o
= = om ey Ay
Perfect G + perfect Q 19 3 7 17 65.5 34.5
Wingless o’ + perfect Q | 4 2 II | eters 76.5
A |
Perfect G' + wingless 9 | 10 I 3 4 yea 28.6
Wingless G'+ wingless Q | 6 ° 6 | 2 | oe: ixXe)
The above table shows that the perfect males were much less
apt to meet with resistance from the females than were the wing-
less males. Some further experiments were made to discover
whether the selection against the wingless males was determined
by sight. The females were blinded by painting their eyes with a
thick layer of quick-drying asphaltum. The results are shown
in the following table:
TABLE III.
Showing the Reactions of Blind Females of P. dispar
|
|
|
een
Soe | ga eR aren) feces 82
~ Sir 5m a8 | 3 8 as
. = o won LO), || silery
Ss =o) = —o ives St | S475 4) 7S)
qo 8 oO > one TB) a o¢ rs
av » — — os 2 2 ist =
po} > ~ | 5 5s | Swe o wk
2g Ss 22 = m | 8 3
ISS a Ow 2 wn ay a | iS ee
2 1 On tal (S| tre 2 3 y ° =)
G Mon ~~ Y Be OS aos 2 o's
z PASE One Pre cohad ECs teen emi bees
a #59 2o% a Scat) eeaee onan
= fy S a= Sree | ks heer
2 cs a a
Pertectag) = pertect 9) — 10 2 2 15 (hie |) 2305
Wingless o + perfect 9 7 2 4 apea 2283
Perfect Go + wingless Q — I. 6 | 9) 50
. ; | |
Wingless G' + wingless 9 4 I 4 ||» 80 | 20
It appears from the above tables that when the female is blinded
the wingless males succeed fully as well as do those which are
normal. Selection on the part of the female is therefore conditional
upon the possession of sight.
430 Alfred G. Mayer and Caroline G. Soule
Another series of experiments was carried out in which the
females were normal in all respects while the wings of the males
were painted scarlet or bright green. ‘This abnormal coloration,
however, failed to affect the results, and the colored males suc-
ceeded quite as well as did normal males. Apparently the female
selects against wingless males but not against those showing
abnormal colors.
These experiments upon P. dispar would have been made more
extensive had it not been that they simply confirm the excellent
and very extensive experiments of Crampton upon Saturnide.
Crampton shows that normal males are more apt to mate with
normal than with abnormal females, and also that abnormal males
mate less readily than normal males. Moreover, abnormal
females lay fewer eggs. In a word the actual basis of elimination
is correlation, not sexual selection exercised by the female in
respect to color.
The mating instinct in the males of C. promethea and P. dispar
is a phenomenon of chemotaxis. Sexual selection on the ground
of color alone does not affect it, and there is no associative memory
connected with it. We frequently placed a normal male among
females, and after mating had taken place, the antennz of the male
was covered with flour paste. Under these conditions the male
never again mated, but often did so immediately when the flour paste
was dissolved with water. It appears, therefore, that the mating
instinct can only be called forth through the sense of smell, and not
through associative memory.
‘The males fly toward the females against the wind. Frequently
we have observed a male flying up against the wind until he passed
by the side of and beyond the female. Under these conditions he
would often remain poised on his wings, and the wind would drifthim
back until he came to leeward of the female, when a few vigorous
strokes of his wings would bring him more or less toward her
again. Inother words, the male pursued the method of trial and
error so ably shown by Jennings ('04) to be prevalent in the ani-
mal kingdom.
Miss Soule found that if the wind be allowed to blow from a
female toward a male Saturnid moth both of the same species, the
Some Reactions of Caterpillars and Moths 431
male may be induced to mate with a female of another species
confined in a cage with him, thus demonstrating that the mating
instinct is called forth through chemotropism.
SUMMARY
1. The caterpillar of the milkweed butterfly, D. plexippus
is positively heliotropic to the ultra-violet rays, but almost if
not quite unresponsive to the rays which to us constitute the visible
spectrum. This caterpillar is also negatively geotropic. These
two reactions serve to maintain it near the upper part of its food
plant, and to lessen the risk of its wandering down the stem and
starving before being able to find another milkweed.
2. The caterpillar has no inherent perception of the proper form
or color of its food but is guided by a chemical sense. Once the
eating reaction be set into play it tends to continue so that the larva
may then be induced to eat substances which it would never have
commenced to eat inthe first instance. ‘This tendency to continue
its activity in the face of a non-stimulus, we have called the
momentum of its reaction.
3. After a larva D. plexippus has commenced to eat a milkweed
leaf, we may cause it to bite at leaves which it would not normally
eat. If this “distasteful” leaf be presented to the feeding larva
at intervals of one and one-half minutes, the caterpillar takes
about the same number of bites each time, showing that it 1s not
influenced after one and one-half minutes by the events of its past
experience. If, however, the distasteful leaf be presented at
intervals of about thirty seconds the larva takes fewer and
fewer bites at each successive presentation and soon refuses it
altogether, and ceases to eat the “distasteful” leaf when it 1s
presented.
4. No associative memory of more than one and one-half minutes’
duration can be demonstrated in larve of lepidoptera.
5. A constantly repeated stimulus loses its effect, and this may
be due not to fatigue, but to internal changes which express them-
selves in modified behavior.
4:32 Alfred G. Mayer and Caroline G. Soule
6. The caterpillar of Samia cynthia and Callosamia promethea
are negatively geotropic when about to pupate, and always pupate
head upw ard. Ifthe cocoon be turned upside down after the outer
case has been spun they will often pupate head upward, thus facing
what in nature. would have been the lower, closed part of the
cocoon.
7. The mating instinct of Porthetria dispar is, on the part of the
male, a reaction of chemotaxis, but the normal females display a
decided selection against wingless males, although not against
abnormally colored males. When the female is blinded she does
not select against wingless males.
AUTHORS ‘CITED
ALLABACH, L. F. ’05.—Some Points Regarding the Behavior of Metridium, Bio-
logical Bulletin, vol. x, p. 35-43.
Crampton, H. E., ’04.—Variation and Selection in Saturnid Lepidoptera, Biolog-
ical Bulletin, vol. vi, pp. 310-311. Also: Science, ser. 2, vol.
xix, p. 459. Also: On a General Theory of Adaptation and
Selection. Journal of Experimental Zodlogy, vol. 11, pp.
425430.
Davenport, C. B., ’97—Experimental Morphology, pt. 1; Effect of Chemical
and Physical Agents upon Protoplasm, pp. 294, London.
Jennincs, H. S., ’02.—Studies on Reactions to Stimuli in Unicellular Organ-
isms, IX. On the Behavior of Fixed Infusoria (Stentor and
Vorticella). Amer. Journ. Physiol., vol. viii, pp. 23-60. Also:
Contributions to the Study of the Behavior of the Lower
Organisms. Carnegie Institution of Washington, Publication
No. 16, pp. 256, 1904. Also: 1905; The Method of Regu-
lation in Behavior, and in Other Fields, Journal of Experi-
mental Zodlogy vol. 11 pp. 473-494.
KirKLAND, A. H., ’96—Assembling of the Gypsy Moth: In Forbush and
Fernald’s Report on the Gypsy Moth. Mass. Board of Agri-
culture, pp. 345-357, Boston.
Lors, J., 00.—The Physiology of the Brain and General Psychology, pp. 309,
New York. Also, ’05: Studies in General Physiology, 2 vols.
Decennial Publications of the University of Chicago.
Massart, J., ’01.—Annals de |’Institut Pasteur, August 25.
Some Reactions of Caterpillars and Moths 433
Lussock, J. (Avebury), ’83. Ants, Bees and Wasps. International Scientific
Series.
Mayer, A. G.,’00.—On the Mating Instinct in Moths. Annals and Mag. Nat.
Hist., ser. 7, vol. v, pp. 183-190. Also: Psyche, vol. ix, pp.
14-20.
Parker, G. H., ’03.—The Phototropism of the Mourning- cloak Butterfly,
Vanessa Antiopa, Linn. Mark Anniversary Volume, pp. 453-
470, pl. 33, New York.
Soute, C. G., ’02.—Notes on Hybrids of Samia cynthia and Attacus prome-
thea. Psyche, vol. ix, pp. 411-413.
YerKES, R. M.,’02.—Habit-formation in the Green Crab, Carcinus granulatus,
Biological Bulletin, vol. iii, pp. 241-244.
YERKES, R. M., and Huccins, G. E., ’03.—Habit-formation in the Crawfish,
Cambarus affinis. Harvard Psychological Studies, vol. i, pp.
565-577.
_
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i ie Pe
MODIFIABILITY IN BEHAVIOR
II. FACTORS DETERMINING DIRECTION AND CHARACTER OF
MOVEMENT IN THE EARTHWORM
BY
H. S. JENNINGS
Fohns Hopkins University Baltimore, Md., U.S.A,
When an organism moves in a certain direction, what are the
various factors by which this direction is determined? ‘This is the
question with which the present paper deals, using the earthworm
asatype. The problem, as will appear, is a complex one, even for
so low an organism.! ;
According to certain theories of tropisms, much held up to recent
times, the direction of movement in any lower organism is always
determined in a precise and simple way by the impact of external
agents. For a brief statement of this view we may quote the
words of Davenport and Perkins (’97) in the introductory para-
graph of a paper on the directed reactions of the slug:
“Tt is almost an axiom in modern zodlogy, that whenever an
organism, or any mass of protoplasm whatsoever, migrates in a
definite direction, it does so because it is guided from without by
the direction of impact of an irritant.”
Just how the external irritant acts in guiding the organism has
likewise been formulated in a simple way. The formulation most
commonly accepted has been that which represents the result as
due to the local action of the stimulating agent on the motor organs
1The author had in hand a more extensive piece of work on the behavior of the earthworm, when
the excellent paper of Harper (’o5) appeared. This made a statement of many of my results super-
fluous. But while the work of Harper went farther in certain directions than my own, I have brought
out certain facts, and especially have dealt with certain general relations which Harper did not take
up. It seems worth while, therefore, to publish a summary of my results, bringing out the general
relations in which I was chiefly interested.
Tue Journat or Exprrimentat Zootocy, Vor. 111, No. 3.
430 H. 8S. fennings
of the region on which the stimulus impinges, as set forth especially
by Loeb and by Verworn. Davenport again has made a con-
venient summary of this theory, in direct application to the earth-
worm, in a discussion of the way in which this animal reacts to
light:
“Represent the worm by an arrow whose head indicates the head end (Fig. 1, 4).
Let solar rays SS fall upon it horizontally and perpendicularly to its axis. Then
the impinging ray strikes it laterally, or, in other words, it is illuminated on one side
and not on the other. Since, now, the protoplasm of both sides is attuned to an
Ss S
Low light attunement.
A
Low light attunement.
Fig. 1. Diagram to explain a tropism in a muscular organism, such as the earthworm. (After
Davenport.)
equal intensity of light, that which is the less illuminated is nearer its optimum
intensity. Its protoplasm isina phototonic condition. ‘That which is strongly illu-
minated has lost its phototonic condition. Only the darkened muscles, then, are
capable of rormal contraction; the brightly illuminated ones are relaxed. Under -
these conditions the organism curves toward the darker side; and since its head
region is the most sersitive, resporse begins there. Owirg to a continuance of the
causes, the organism will cortinue to turn from the light until both sides are equally
illuminated, 7. ¢., until it is in the light ray. Subsequent locomotion will carry the
organism in a straight line, since the muscles of the two sides now act similarly.
‘Thus orientation of the organism is effected. “The same explanation, which is modi -
fied from one of Loeb (’93, p. 86), will account, mutatis mutandis, for positive pho-
totaxis” (Davenport, ’97, p. 209)."
Both this axiomatic view that the direction of movement is
always precisely determined by external agents, and the theory that
this determination is due to direct local action of the agent in ques-
tion, have begun to give way before a careful examination of the
4In making the above quotations, there is no intention of representing Davenport as a sponsor or
defender of the views set forth. In his valuable general works, Davenport has summed up in an
unsurpassed way the general views prevailing at the time, and our quotations are given merely as such
summaries.
Modipfiability in Behavior 437
facts as to just what organisms do. A thorough acquaintance with
the behavior of Paramecium, or of any other typical free-moving
lower animal, renders the above quoted axiom quite unten: ble.
If the animal is placed in such a position that the externzl condi-
tions are uniform in all directions, so that there is nothing in them
to guide it, it does not remain quiet. Paramecium when plzced
in such a situation simply goes straight ahead, and the sz me is
true of most other lower animals. In other words, the differentia-
tions of the animal’s body are sufficient to determine the direction
of locomotion, provided there is nothing else to do so, and often
this factor prevails even when there are external factors which
work decidedly against it. An animal often goes in a certzin
direction merely because there is nothing to prevent its forward
movement. ‘The animal is a going machine, and need not be
prodded up by a present outside stimulus in order to move; it
carries within itself both the impulse to move, and factors deter-
mining the direction and character of the movement. ‘The move-
ment of such a creature as Paramecium is indeed defined in mzny
ways, even in a uniform environment.
If such relations are evident in examining even so low an animal |
as the infusorian, it will not be surprising if we find them to hold
in even higher degree of so complex an animal as the earthworm.
Of course external agents frequently do determine the direction
of movement. But thorough study shows that their action 1s
rarely of the simple and direct character set forth in the summz riz-
ing statement above quoted; it is usually, on the contrary, very
indirect and complex. For the earthworm, this was perhzps first
made evident by the work of Miss Smith (02). “This work showed
that the directive action of light, heat, chemicals, etc., on the e: rth-
worm Allolobophora is far from being of the precise, unequivocal
character that the local action theory would require. She found
that in place of orienting itself by bending steadily to one side under
the unilateral action of a stimulus, the earthworm frequently
alternated movements in various directions, till finally one direction
was selected for further progress. ‘This direction was by no means
usually one of precise orientation. This result was confirmed by
Holmes (’05), who finds that “random movements” play a large
438 | H. 8. ‘fennings
part in determining the direction of locomotion in the earthworm,
as well as in various other lower organisms, under the action of
light and other stimuli. Holmes’ observations inclined him even
to doubt that the earthworm has any localizing power in the nega-
tive reactions to light, in the sense that it is anywise more likely to
turn first directly away from the lighted side.
In essential agreement with such results are the statistics of
Parker and Arkin (01), who found that in reacting to one-sided
illumination the earthworm usually (in 65.5 per cent of all cases)
merely starts ahead, and that when it turns, it may turn either
toward or away from the source of light, the number of turns away
from the light being only 26 per cent greater than the number of
turns toward the light. The large overplus of movements that are
not as required by the theory of direct local action of course require
explanation. Harper (’05) showed clearly that under strong light
the earthworm Perichzta may turn directly away from the light
and he brought out some of the factors on which depend the other
movements, that are not determined in direction by the impact of
the light rays. It is a further inquiry into the determining factors
of movements that do not depend directly on the localization of the
stimulus, that concerns us in the present paper.
In the paper of Holmes (’05) the movements that are not in
relation with the direction of the source of stimulation are charac-
terized simply as “random.” But, of course, the movements are
random only with relation to the external stimulus considered; it 1s
evident that they must be determined in some way. It cannot
be admitted in a scientific treatment of the matter that any move-
ments are random in the sense of undetermined. If we leave the
matter here, calling the movements merely random, we leave our
treatment open to the charge of inexactness, and of the use of non-
scientific concepts; the movements are left without a determining
cause. Why does the animal turn sometimes to the right, some-
times to the left, sometimes go straight ahead, or perform other
activities, even though the external conditions remain the same?
We are forced then to take up the fundamental question, What
factors determine the direction of movement of the given organism
at a given moment? This question the local action theory of
Modipability in Behavior 439
tropisms tried to settle in an axiomatic, a priori way, taking into
consideration only external factors. What is required is that the
question shall be taken up as a problem for objective investigation,
attempting by analytical experimentation to differentiate the
various factors involved, both internal and external. ‘The paper
of Harper made incidentally a first advance in this direction; the
present paper endeavors to deal with the problem more explicitly,
and to advance the analysis farther.
As will appear, the problem is really an extraordinarily complex
one, instead of being of the simple character assumed by the local
action theory, so fie the present essay can give only a general out-
line of the various classes of factors involv ae taking into considera-
tion situations where the external conditions are made as simple
as possible.
EXPERIMENTS
The common earthworm, Lumbricus terrestris L. (L. herculeus
Say.), was used in the present experiments. ‘The method of work
was the simplest, not to say crudest, possible. “The earthworm
was placed on a large sheet of wet filter paper in a dimly lighted
room, pains being taken to keep it moist; then the simplest possi-
ble stimuli, precisely localized, were applied to it. ‘The stimulus
chiefly used was a touch with the tip of a finely pointed glass rod.
Precisely localized chemical and thermal stimuli were also used
but essentially the same result were obtained with all. It is evident
that in these experiments (as in those of other recent authors) the
animals are under most unnatural conditions, and for certain pur-
poses this would of course be a serious disadvantage. For our
present object, however, this is not the case, since these circum-
stances must be taken into consideration as well as all others, in a
general investigation of the factors that determine the direction
of movement.
When a localized stimulus is applied to one side of the earth-
worm, does it respond by a simple contraction or relaxation of the
muscles of the side of the region affected, thus turning the head
from or toward the stimulated side?
Analysis of the results of a large number of experiments shows
440 H. S. “fennings
that when a local stimulus is applied to one side of the anterior
part of the earthworm—-say to the sixth metamere—any one of the
following varied methods of action may result:
1. There may be merely a slight swelling of the region stimu-
lated.
2. The worm may turn the anterior end away from the side
stimulated.
3. It may turn the head toward the side stimulated.
4. It may creep backward.
5. It may creep forward.
6. It may creep first backward, then forward.
7. The head may be merely retracted.
8. The animal may make a sudden right-about-face, interchang-
ing the position of anterior and posterior ends, in the way to be
described later.
g. The anterior fourth of the body of the worm may be raised
in the air and waved wildly about.
From this list it is clear that the reaction must depend on many
factors, since we can distinguish at least nine different methods of
behavior which may follow on the stimulus. Of course the dif-
ferent actions performed in different cases are not matters of chance
but are determined in some way. What are the determining
factors?
So far as I have been able to determine these factors by ana-
lytical experimentation, we can distinguish at least the following
groups:
I. External Factors
1. Intensity of the Stimulation. This is, of course, universally
recognized as a determining factor in behavior. Other conditions
remzining the same, in the earthworm a weak stimulus may induce
a mere swelling of the body at the region stimulated (this varying in
extent in different cases); a stronger stimulus may cause a slight
turning of the head away from (or sometimes toward) the point
stimulated; a still stronger one a retraction of the head, without
turning; a very strong one an immediate rapid crawling backward.
In connection with variations in the other conditions an almost
Modipfiability in Behavior 441
infinite variety of combinations of reactions may be produced by
varying the intensity of the stimulus.
2. Localization of the Stimulus. The different effects of dif-
ferently localized stimuli are of course universally recognized;
this is indeed one of the corner stones of the orthodox tropism
theory. Certain of the relations of reaction to localization of the
stimulus in the earthworm are of interest.
Stimulation first of one side, then of the other, may induce in
the two cases movement in opposite directions. But this is by no
means always the result; the direction of turning depends, as we
shall see later, on a multiplicity of factors. Other conditions
remaining the same, a stimulus of a certain intensity on the right
side of one of the anterior twenty metameres usually causes a turn
to the left; the same stimulus on the left causes a turn to the right.
It is thus clear that there exists the possibility of an immediate
relation of the direction of the movement to the side stimulated, as
Harper showed. ‘This is the factor which the local action theory
of tropisms takes into consideration, though there seems to be no
evidence that the turning is due merely to direct local action.
But in any case it is only one factor out of many. In the region
back of about the anterior third of the body, this factor plays little
or no part, as we shall immediately see.
Comparing stimuli applied to anterior and posterior parts of
the body, very different results are reached. A rather strong
stimulus on the dorsal surface of the anterior third of the body
(to just behind the clitellum) usually causes the animal to creep
backward. This backward movement is usually soon followed
by a forward one. A similar stimulus on the posterior half of the
body usually causes the worm to creep rapidly forward. ‘Between
these two regions there is an area comprising five to ten metameres
in which a strong dorsal stimulus causes an immediate stretching
of the entire worm, the anterior region starting forward, the pos-
terior region backward. ‘The anterior movement soon prevails,
as a rule, so that the worm crawls forward. ‘The final result then
is in all these cases a movement forward; this is preceded in the
case of anterior stimulation by a movement backward, in inter-
mediate stimulation by a movement in both directions, while
442 1. 8. ‘fennings
only in the case of posterior stimulation does the forward movement
take place directly. Under other conditions, to be mentioned
presently, the worm may crawl steadily backward. Further,
which of these three results we get in a given case depends largely
on the intensity of the stimulus and on other factors not yet consid-
ered, as well as upon the localization of the stimulus.
Lateral stimulation on the anterior one-fourth or one-third of the
body may, as we have already seen, cause a turning away from the
side stimulated. Often, however, it causes merely a backward
movement; sometimes a forward one (for the conditions on which
these depend, see later). But lateral stimulation of the posterior
two-thirds of the body never causes turning away from the side stimu-
lated, so far as I have observed. In this region a strong lateral
stimulus causes merely a rapid movement forward. ‘The adap-
tive relation of these facts to the movements of the animal under
natural conditions is evident. An attack in the anterior region is
best escaped by moving backward; in the posterior region by mov-
ing forward. Forward movement is more rapid than backward
movement, so a stimulus at the middle of the body, or even a little
in front of it, is more quickly escaped by a forward than by a
backward movement. Lateral attacks in the anterior region
may be escaped through a sidewise turn, but lateral stimulation in
the posterior region will be much more quickly avoided by a rush
forward. ‘These adaptive relations of course do not show us
why the earthworm moves as it does, but their existence is inter-
esting, and it is possible that in the long run they may have some-
thing to do with determining what movements shall occur.
The fact that strong stimuli on the lateral surface of the posterior
half of the body do not tend to cause turning to one side is one that
needs to be kept in mind in practical experimentation. Thus,
Parker and Arkin (or) found that light directed against the side
of the anterior end of the worm causes turning away, but directed
against the posterior part it does not cause the turning. ‘This, of
course, does not in itself indicate that the posterior half is less
sensitive to light than the anterior half (though this may be prob-
able on other grounds), for even if the light acted most power-
fully on the posterior half of the animal, it would not cause a
Modipfiability in Behavior 443
bending, but merely a movement forward. The argument for
less sensitiveness in the posterior half from the results of Parker
and Arkin rests on the assumption of the correctness of the local
action theory of tropisms for light reactions; this assumption can
hardly be said to have shown itself correct.
A very powerful stimulus, constituting a severe injury, usually
causes the part anterior to the injury to crawl rapidly straight
ahead, while the part behind the injury squirms about violently.
Close examination of this “squirming”’ shows that it is exactly the
movement that is required, in the natural conditions under which
the worm lives, for urging the body forward with great rapidity.
The worm is, of course, usually in a narrow burrow. Under the
circumstances the “squirming” throws first one side of the body,
then the other, against the sides of the burrow, while at the same
time the setz are extended and moved in such a way as to exercise
a powerful leverage against the sides. In this way, by repeated
successive impulses from each side, the worm shoots forward
through its burrow, tending thus to escape from the injurious
agent. The anterior end is not turned about from side to side, but
is held more nearly straight, so that it does not interfere with the
movement induced by the powerful impulses coming from behind.
At the same time it aids the flight by creeping as rapidly forward
as possible.
In a well known paper Norman (97) described the fact that
when the strong stimulusis a cut, actually dividing the worm into
two halves, the parts anterior and posterior to the cut behave
respectively in the two ways above described, and brought this
into relation with the question of the existence of pain in the
lower animals. It was argued that, since only the part behind
the cut shows thé squirming reaction, while the part in front does
not, it cannot be held that there is any indication of pain in the
worm, since it must be supposed that the anterior region is at
least as sensitive as the posterior one. But since the behavior of
both anterior and posterior pieces is of precisely the character that
would under natural conditions most assist the worm to escape |
from the stimulating agent, it may be questioned whether the dif-
ference between the two has any bearing on the probable exist-
444 Hi. S. fennings
ence of pain. There would seem to be no more and no less
reason for supposing the strong lateral movements which urge the
animal forward in the “squirming” to be accompanied by pain,
than the rapid forward creeping of the anterior part.
The remaining more important determining factors in the move-
ments of the earthworm must be classified as
IT. Internal Factors
These are extremely varied, and a complete classification is
impossible at present. Some of the chief ones are the following:
3. The reaction to a given stimulus depends partly on what the
animal has done, and on its position, just before receiving the
stimulus.
‘This factor shows itself as follows: “Che earthworm makes with
its anterior end side to side movements in creeping, turning first to
the right, then to the left. If now we stimulate it Just after it has
turned its head far to the right, it at once as a rule jerks its head
to the left. [his occurs whether the stimulus is applied to the
right side, to the left side, or to the dorsal surface, so that the
direction of turning becomes independent of the localization of the
stimulus. If the animal is lying bent to the right, it is most likely
to bend to the left when stimulated, without regard to the precise
point of stimulation.
4. The reaction to a given stimulus depends partly on a general
tendency of the animal to move in a certain way, namely, forward
rather than backward. ‘This is seen in the different results of
localized stimuli on the posterior and anterior parts of the body.
In the former case the animal moves away from the source of
stimulus (forward), and continues to do so. In the latter case it
first moves away from the source of stimulation (backward), then
1In describing movements that depend upon such a multiplicity of factors as do those of the earth-
worm, it is difficult to treat of any one factor separately without making the effects of that factor
appear more absolute and independent of the presence of others than the experimental results show to
be the case. Thus, while as a rule the results are as given above, different results may be reached
when other factors than those considered in this paragraph become the determining ones. This
remark applies to the discussion of each of the factors taken up separately in the text.
Modipfiability in Behavior 44.5
this movement is as a rule supplanted by movement toward the
source of stimulation (forward).
5. The reaction to a given stimulus depends partly on the direc-
tion in which the animal is crawling at the moment when it is
stimulated. In a quiet worm, posterior stimulation causes move-
ment forward, anterior stimulation movement backward. The same
results are usually observed when the animal is very quietly crawl-
ing forward or backward. But at times, when creeping forward
it is found that stimuli on any part of the body—even at the
anterior tip—merely cause the worm to hasten the forward move-
ment. In the same way, a worm that is creeping backward may
persist in this movement, and even hasten it, in spite of repeated
strong stimulation at the posterior end. ‘The physiological state
seems to have taken a sort of set, causing the worm to obsti-
nately persist in following the direction in which it has started.
Sometimes, when the worm is in a similar condition, a stimulus at
the advancing end of the animal (anterior or posterior) causes a
cessation of the movement for a few seconds, then the worm starts
again in the direction it was pursuing. Stimulation causes it to
suspend operations for a time, but it is not to be easily turned aside
from the course it is following, and it soon resumes this course.
6. The reaction to a given stimulus depends partly on previous
stimuli received. I have not by any means worked out in detail
all the manifestations of this principle; they are very numerous.
A worm which has not received marked stimuli for some time, and
is at complete rest, may not react at all to a slight stimulation.
Two or three similar stimulations, however, rouse it up, and now
it reacts readily. After more stimuli, or more intense ones, its
reaction to any given stimulus changes in character and increases in
intensity. We can distinguish from this point of view a series of
different physiological states, each manifesting itself by a different
reaction. A partial list of these, with the characteristic behavior,
is as follows:
(a) The state of rest, in which the worm does not react readily
to slight stimuli, such as a touch with the tip of a glass rod.
(b) A state of moderate activity, in which a touch at the anterior
end causes movement backward; at the posterior end movement
446 TS. fennings
forward, while lateral stimuli (in the anterior region) cause turning
away from the side stimulated.
(c) A state of excitement, after repeated stimuli, in which the
animal persists in the direction of movement once begun, merely
stopping for a few seconds when stimulated at the end which 1s
advancing.
(d) A state of greater excitement, in which stimuli merely cause
the animal to hasten its movements in the direction in which it has
started, without regard to the localization of the stimulus.
(c) A state of still greater excitement, after long-continued and
intense stimulation. Now the worm responds to a stimulus at the
anterior end, that would in a resting worm cause only a compara-
tively slight reaction, by a rapid “right-about-face.” ‘The body
is suddenly doubled at its middle, so that the anterior and pos-
terior halves become parallel, with the two ends pointing in the
same direction, then the posterior half 1s quickly whipped about,
so that the whole worm is again straight, but is facing the opposite
direction from that in which it was pointed before the reaction.
This peculiar reaction takes place with such rapidity that one can
distinguish the way in which it occurs only after many repetitions.
After this right-about-face the worm usually crawls rapidly in the
new direction.
In the natural condition, within the burrow, this reaction would,
of course, instantaneously direct the animal downward, if attacked
as it creeps toward the surface.
(f) A state of still more intense excitement, after repeated
strong stimulation that is of such a character as to actually injure .
the tissues. “he worm now responds to a repetition of the stimu-
lus (and often when the new stimulus is only slight) by lifting the
anterior fourth of the body into a vertical position, and waving it
about in a frantic manner. ‘This behavior is usually alternated
with the right-about-face reactions, and with persistent rapid
crawling forward or backward. ‘The spectator is involuntarily
inclined to feel that the animal is tormented, and that continuation
of the experiment is cruelty; this may, of course, be due only to the
peculiar constitution of the spectator, and not to that of the worm.
It is beyond question that many other physiological states could
Modipability in Behavior 447
J
—
be distinguished, each with its characteristic method of action. [
have considered only those appearing under relatively simple con-
ditions of stimulation, and such as can be formulated in a more or
less definite way. Many other variations in reaction that may
easily be observed I have not taken up above, because recount-
ing them would add merely a heap of details, the interrela-
tions of which are not clear. I have further omitted all physiologi-
cal states resulting from varied states of metabolism, and have not
attempted to study possible lasting modifications of physiological
state (“habits,” etc.), but have merely dealt with the movements
from moment to moment.
Yet with even this limitation of the field, it is clear that the
cause for movement in a given direction, or of a certain character,
is in the earthworm not simple, but excessively complex. The
present external stimulus is only one of the numerous variable
factors involved. ‘The movement at a given time demonstrably
depends, not alone on present external conditions, but also on
former external conditions, former actions of the organisms, and
present internal physiological conditions that are determined in
many different ways. ‘The direction of movement of one of these
organisms cannot be represented as a simple function of the direc-
tion of impact of some external force, but is the complex resultant
of many different factors.
GENERAL
Cause of Change of Reaction Under Uniform External Stimulation.
In the present and in previous papers I have shown that
even while the external conditions remain the same, the reaction
of the organism changes. Such a statement appears to be looked
upon by some as leaving the movements without determining fac-
tors, and as therefore leading to vitalism and mysticism. But
such an idea results only from a failure to realize what animals are.
They are not static structures, but are bundles of processes, and
it is this that gives us a key to the understanding of changes in
behavior even under uniform external conditions. In every animal
448 IS) feuniaes
processes of the most varied character are occurring: the taking
of oxygen and other substances, digestion, assimilation, dissimila-
tion, secretion, excretion, usually some form of circulation, ete.
Whenever we think of an animal—an earthworm or an infuso-
rian—we need, in order to understand its behavior, to think of it
as a little engine of intense activity. [he movements of the
organism we know to be the results of the production of energy in
these internal processes. We know further that these processes
depend for their normal course upon each other, and upon the
environment, and that this is as true of the movements as of the
other processes. Disturbance of the internal processes we know
to result in changes in the movements. In former papers (05,
’o5a) I have given many cases of this and of the observed
dependence of behavior on the relation of external conditions to
these processes.
What then will happen if some external condition acts upon the
organism in such a way as to modify one of these processes ! ? Sup-
pose, for example,that pressure acts in a given case in such a way as
to interfere with the circulation—of the protoplasm in Paramecium
or of the blood in the earthworm. ‘The animal will perhaps first
respond directly to the pressure as a primary stimulus, giving the
reaction A. ‘The pressure continuing uniformly soon impedes
the circulation. We know that such interference with an internal
process is in itself a cause of reaction; the animal,therefore, now
responds to this interference, giving the reaction B. But circula-
tion cannot be long impeded without interfering with respiration,
and this again is a cause of reaction, giving us perhaps the new
response C. The external conditions meanwhile remain uniform,
but, as we see, the internal condition changes from one state to
another. The interference with respiration is bound ere long to
induce changes in assimilation; these necessarily entrain changes
in dissimiliation, and these changes in excretion. Excretion is
likewise more directly impeded by the stoppage of the circulation.
As a result thus of a uniform external condition modifying prima-
rily but a single bodily process, the internal state of the animal
passes from one change to another, since the internal processes
are bound up in mutual interdependence. We know that the
Modifiability in Behavior 44.9
bodily movements are the result of these internal processes, so
that as one after another of these is blocked, it appears not merely
natural but inevitable that repeated alterations of movement should
result; we Semtnewehicsson reactions 4,5, Gus. 2-2. ~ Lhisvasil
have shown in previous papers, 1s exactly what occurs in many
lower organisms. The action of a uniform external stimulus that
affects any of the life processes 1s necessarily cumulative, since a
change in one process is bound to result in changes in the others.
As a result of the cumulative internal changes we get repeated
changes in movement, even though the external condition remains
the same. There is no difficulty, therefore, in finding determining
factors for such changes of behavior; they are induced by preced-
ing changes in precisely what we know on other grounds to be the
source of movement—the internal physiological processes.
There is thus no reason when we consider the fact that organ-
isms are bundles of interdependent processes, for supposing that
they should always behave in the same way under the same
external conditions. Persistence inthe same movement as one after
another of the internal processes is disturbed would itself be a
puzzle, requiring very precise internal compensatory regulations.
Change of movement is what might be expected, and is what we
commonly find. ‘There is, of course, as much reason for finding
such changes in the lower organisms as in higher ones, and this
again corresponds with the results of experimentation.
“Method of Trial and Error”
In a previous paper (04a) I spoke of the reaction method in
which the organism varies its movements under a given stimulus
as the “method of trial and error,” in order to bring out its simi-
larity to the behavior for which that expression is commonly used
in higher animals. ‘The essential point which I intended to bring
out by using this term in connection with the way in which these
animals move toward or away from certain agents was the follow-
ing: the organism performs varied movements, some features of
450 H. S. fennings
which are not determined! by the localization of the stimulus, but
by other factors; it then continues those movements which bring
it into or toward a certain condition, this condition being usually
either a greater or a less action of the stimulating agents, as
the case may be.? Holmes (’05, p.111) objects to the use of this
expression for some of the cases for which I employed it, as, for
example, the reaction to light in Euglena, while proposing to
reserve it for such cases as the earthworm. His reason for this is
that ‘‘ Euglena does not react by a number of indiscriminate move-
ments until the right one is accidentally hit upon, but by a direct
reflex, whose effect is to bring the organism more nearly parallel
to the direction of the rays.’ He continues, “The phototaxis of
Euglena is not so manifestly the outcome of the trial and error
method as that of the earthworm. In the latter case light does
not cause directly a movement which makes for orientation. ‘The
direct response may or may not have that effect. “The successful
response is accidentally hit upon.” He further suggests that the
expression “trial and error’ be “limited to those cases in which
the adapted movements may be regarded as chance successes.”’
I have, of course, no desire to enter into an academic discussion
as to the proper definition to be given to the hackneyed expression
“trial and error,’ and I should be quite willing to drop that
expression, not only for Euglena but for the earthworm and other
lower organisms; it has perhaps gained in its history implications
of various sorts which find no corresponding factors in lower
organisms. But what does seem to me worth while is to try to make
clear the points on which the various methods of behavior that I
classified together under this name actually agree, these being the
points which impelled me to class them together. It appears to
1The word determine is of use in experimental work only when it expresses a concrete experimental
result, namely, that when one factor varies, another (the determined one) varies in a corresponding
way. The factor A (e. g., localization of the stimulus) is said to determine the factor B (e. g., direc-
tion of movement) when a change in A involves experimentally a change in B. If B remains the same
even when 4 is changed or disappears (as in the case of the movements referred to above), then B is
not determined by A.
?The essential feature of this type of behavior could be expressed more generally, so as to
include other than directed reactions, as follows: the organism performs varied movements, some of
which do not tend to produce the result that is finally brought about by the behavior as a whole; of
these varied actions, those are followed up which do tend to produce this final result.
Modipfiability in Behavior 451
the writer most unfortunate to attempt to make a distinction on
the basis of the emphasis of such terms as “indiscriminate,” “ac-
cidentally,” “chance.” The “random” movements of the earth-
worm are of course no more a matter of chance than are the varied
movements of Euglena. ‘They are merely determined in a dif-
ferent way, and in very complex ways, as I have attempted to
show in the present paper. Where the two agree is in the fact that
they are partly determined by some other factor than the localiza-
tion of the source of stimulus, and that they do not all tend to pro-
duce the finally resulting orientation. In both cases, if the stimu-
lus were differently localized the first movements would still be the
same. ‘This is as true of the movements of Euglena as it is of
those of the earthworm, in becoming oriented to light. Euglena
when stimulated as a result of light coming from one side swerves
more than usual toward its own dorsal side. “This movement is
at first just as likely to take the anterior end away from the source
of light as toward it (see Jennings, ’04). In precisely the same
way, according to Holmes’ account, the earthworm in reacting by
the “random movement”’ method is as likely to turn its anterior
end in one direction as in the other. But in both these organ-
isms, after swerving for a certain distance in one direction, the
creature swerves in the opposite direction. In Euglena this is due
to the fact that the animal revolves on its long axis, so that the
dorsal side, toward which it is swerving, soon reverses its position;
in the earthworm it is due (partly at least) to the fact that a previous
turn to the left itself serves to induce a succeeding turn to the right.
The movement is fully determined throughout, in both cases equally.
Now in both the organisms, since there is successive swerving
in opposite directions, when the light is coming from one side
one of the movements is bound to take the anterior end in general
toward the source of light, the other away from it, and there is no
more accident about this in one case than in the other. In both
organisms, of these movements in different directions, “only those
are followed up which bring the animal out of the undesirable
situation” (Holmes).!| In Euglena “light does not cause directly
1That is to say, in both cases the following up of a certain motion 7s determined by the localization
of the stimulus.
452 H. S. fennings
a movement that makes for orientation,’ any more than it does
in the earthworm. Part of the movement is directly away from
the position of orientation (see Fig. 23, Jennings, ’o4), and this
very phase of the swerving is more pronounced when the animal
is becoming oriented to light than in the usual spiral path when
it is not reacting at all. Movever, this phase of the movement is
a necessary one in bringing about the reaction, if the analysis I
have given in the paper just cited 1s correct. The stimulus of
light causes pronounced movements both away from and toward
the source of stimulation, and the organism follows up more
decidedly those which lead toward the optimum condition, just as
happens in the earthworm.
Thus, while it appears to me immaterial whether we choose to
call either or neither of these cases reaction by “trial and error,”
the essential point lies in the fact that in these as in many other
organisms stimulation causes varied movements, which do not all
lead toward the condition finally attained, and that those move-
ments which do lead toward this final condition (the “optimum’’)
are followed up more decidedly than the others. The behavior
may perhaps be most accurately characterized as “selection from
among the conditions produced by varied movements.” In general
we find that many organisms are so constituted that internal
conditions (permanent or temporary) will produce under stimu-
lation movements that are varied in precisely such a way as to sub-
ject the creature to as varied environmental conditions as possible,
and thus give it an opportunity to select what is nearest the opti-
mum.! Every one of these movements 1s, of course, as absolutely
determined as is the most orthodox tropism, only the determining
factor is not the localization of the stimulus (or other external
factor) alone.
Certain recent writers have seemed to imply that there is a con-
trast between the “‘trial and error’? method, and behavior that is
definitely determined by structural and other internal conditions.
It needs to be emphasized, perhaps, that the behavior which I and
others have characterized by this phrase is very precisely deter-
mined by structural and other internal conditions; indeed, its dis-
1As to how this selection occurs, see the author’s paper on the “Method of Regulation in
Behavior” (05a).
Modipfrability in Behavior 453
tinguishing feature is the fact that it is thus determined by such
conditions, rather than exclusively by the external conditions. It
is, of course, very true, as Harper (’05) remarks, that definitely
localized reaction methods are developed as we rise higher in the
scale, yet it appears to be equally true that if we mean by “trial
and error’ the performance of varied movements, subjecting the
organism to varied conditions, certain of which are selected, then
this also becomes more highly developed and more used by organ-
isms as we ascend the scale. We must not forget that this
expression “trial and error” was originally based on the behavior
of such highly developed organisms as the cat, dog and monkey;
and doubtless there is no organism which uses this method to any
such extent as does man. Whenever the external conditions do
not furnish a precise determining factor for the movements, yet
some sort of reaction is required, any organism is forced to have
recourse to this style of behavior, performing varied movements
till a condition is reached that relieves the organism of the neces-
sity of continuing these movements. In its highest form we call
this experimentation.
In his recent Experimentelle Biologie, v. Uexkiill ('05) is unable
to understand how we gain anything by characterizing certain
behavior as “trial and error” (p. 128). Whether we gain Het by
a certain procedure depends largely on what the problems are in
which we are interested, and we find that v. Uexkill limits the
problems of biology in so extraordinary a way that he could not
possibly be expected to see any value in such a procedure. For
him, “There is thus for animal biology only one point of view—
to understand the purposive structure of every animal on.the basis
of its reflex arcs” (J.c., p.g6). He is, moreover, convinced a priori
that we can never understand the purposiveness of organisms
froma causal standpoint; “ Die beiden Betrachtungsarten [Ursach-
tlichkeit und Zweckmissigkeit] auf einander zuriickzufiihren 1s
unmoglich” (p. 129). When we add that v. Uexkiill holds that
any action of organisms may equally be characterized as trial and
error,’ it is evidently not surprising that he can make nothing of
1We have perhaps here further evidence that this expression is one that lends itself too readily
P P P
to misinterpretation to make it the best characterization for the behavior in question.
4.54 H. 8S. “fennings
the characterization of certain kinds of behavior as trial and error.
On the other hand, some of us are interested in the question as to
how it happens that organisms do those things that aid them in
carrying on their physiological processes and thus keep them in
existence—a matter which does not fall within the field of biol-
ogy at all, as limited by v. Uexkill. To such as are interested in
this problem it may very well appear that we have really made a
certain advance when we recognize the fact that organisms do not
in response to stimulation always perform at once a directly regu-
latory action; do not react merely by a stereotyped reflex, as has
often been represented; but that on the contrary they perform
varied movements, some of which are not directly regulatory, but
which do subject them to varied conditions, certain of which con-
ditions are selected, through cessation of the varied movements,
thus resulting in regulation. If we are further able to find out the
determining factors for setting in operation these varied move-
ments, and for their cessation when a favorable condition is reached
it will naturally appear to those interested in the problem sketched,
that we have made a further advance. Recognition of a physio-
logical law that results finally in the persistence of only the one
movement that is directly regulatory, so that the animal later
reacts by a stereotyped regulatory reflex, and no longer by the
varied movements (“trial and error’’), will seem a further advance
to those interested in precisely the problem of how such definite
regulatory reactions happen to exist. It is not necessary to hold
that these determining factors and laws have been completely
unveiled (see Jennings ’o5a, for a tentative statement of them), in
order to recognize the importance and interest of the line of work
that is concerned with their investigation. On the other hand,
those for whom regulation is a priori an unsolvable problem, and
who limit the field of biology to understanding the purposive
structure of animals on the basis of their reflex arcs, will of course
find these matters outside the field of their interest. But those
whose interests are thus narrowly bounded are certainly in the
minority; the writer does not happen to know of any biologist
aside from Dr. v. Uexkiill that holds to such limitations.
zyr
Modifiability in Behavior 455
Pith ReARURE CEhED
Davenport, C. B., ’97.—Experimental Morphology, vol. 1, 280 pp.
Davenport, C. B., and Perkins, HELEN, ’97.—A Contribution to the Study of
Geotaxis in the Higher Animals. Journ. Physiol., xxii, gg—1Io.
Harper, E. H., ’05.—Reactions to Light and Mechanical Stimuli in the Earth-
worm, Pericheta bermudensis (Beddard). Biol. Bul., x, 17-34.
Hoimes, S. J., °05.—The Selection of Random Movements as a Factor in Photo-
taxis. Journ. Comp. Neurol. and Psychol., xv, 98-112.
Jennincs, H. S., ’04.—Reactions to Light in Ciliates and Flagellates. Contr.
to the Study of the Behavior of the Lower Organisms. Carnegie
Institution of Washington, Publ. 16, pp. 29-71.
?04a.—The Method of Trial and Error in the Behavior of Lower
Organisms. Jbid., pp. 235-252.
’05.—Modifiability in Behavior. I. Behavior of Sea Anemones. Journ.
Exp. Zool., 11, 447-472.
’o5a.—The Method of Regulation in Behavior and in Other Fields.
Tbid., 11, 448-494.
Norman, W. W., ’97.—Diirfen wir aus den Reactionen niederer Thiere auf das
Vorhandensein von Schmerzempfindungen schliessen. Arch. f. d.
ges. Physiol. Ixvii, 137-140.
Parker, G. H. and Arkin, L., ’o1.—The Directive Influence of Light on the
Earthworm Allolobophora feetida (Sav.). Amer. Journ. Physiol.,
iv, I51-157.
SmirH, AMELIA C., ’02.—The Influence of Temperature, Odors, Light and Con-
tact on the Movements of the Earthworm. Amer. Journ. Physiol.,
vi, 459-486.
UexkuLL, J. v.,’05.—Leitfaden in das Studium der experimentellen Biologie
der Wassertiere. Wiesbaden. 130 pp.
a
x ie : id
Pee |
*
ta
e
HE SPEYSIOLOGY OF REGENERATION
BY
T. H. MORGAN
With Seven Ficures
Lest the title mislead someone expecting to find in_ the follow-
ing pages an account of the processes of assimilation and of resp1-
ration, that presumably take place during regeneration, I ought to
state that I shall deal only with the physiology of the growth pro-
cess as shown in the regeneration of a new part. Morphogenesis
does not express my meaning in all respects, for | am not con-
cerned so much with changes in form as with the rate of growth and
of differentiation. If I have taken a liberty in using the term phy-
siology to cover these kinds of changes, my excuse must be that we
are dealing with phenomena that lie on the borderland, where
physiology and morphology overlap, and appear to merge into each
other.
It is generally, if tacitly, assumed that when undifferentiated
cells are supplied with food materials growth must follow, but I
shall try to show, on the contrary, that whether or not growth takes
place depends not so much on the available food sueply as ona
formative influence that regulates both the kind and the amount
of growth. The nature of this formative influence is the most dif-
ficult and problematical factor with which we shall have to deal.
RATE OF REGENERATION IN STARVED AND WELL-FED
SALAMANDERS
If the rate of regeneration is in any way connected with the food
supply, the fact ought to become at once apparent by comparing
the process in well-fed and in starved individuals. I have carried
out such an experiment with the salamander, Diemyctylus virides-
cens. The results show that we must be careful to distinguish,
in the use of the word rate, between a simple increase in size, and
Tue JourNat or ExpPeRIMENTAL ZOOLOGY, VOL. Il, NO. 4.
458 T. H. Morgan
the rapidity of the process of differentiation (or development in
the narrower sense) of the new part.
Zeleny' has recently shown that the increase in size of regener-
ating arms of the brittle-star and of the legs of the crayfish is deter-
mined, in part, by the number of the appendages removed. ‘The
more parts removed, the faster each regenerates. Zeleny discusses
amongst other factors, the possible relation of this result to the
food supply, and points out that the larger the number of append-
ages removed the greater will be the temporary surplus of food,
for the amount necessary to nourish the entire leg may be greater
than that used at first in the growth of the small new part. Whilst
pointing out the possibility of this interpretation Zeleny carefully
refrains from committing himself to it as the only explanation of
his results.
It seemed to me that this question might be tested in the follow-
ing way. Ifthe rate 1s determined by the food supply, then if two
sets of individuals are selected, and one set starved and the other
fed, the latter should be in a better condition for regeneration than
the former. If the same number of parts 1s removed in each, the
well-fed individuals should regenerate faster than the starved ones.
If fewer parts are removed from the well-fed individuals than
from the starved ones, nevertheless the well-fed individuals should
regenerate faster, for, the greater amount of food given ought to
much more than outweigh the surplus in the starved ones due to
the absence of more parts.
‘The experiment was carried out with salamanders collected in the
autumn. ‘They were in excellent condition when caught, although
not so large as they soon became when fed on pieces of beef
The individuals were kept for several weeks, without much food,
before the experiments began. In some individuals one leg was
removed, in others two, in others three, and in still others one, two,
or three legs and also the tail, which was cut off near the base.
Duplicate sets were prepared, each containing several of these dif-
ferent kinds of individuals. One lot was kept without food and
the other fed about every other day on small pieces of raw beef.
1Zeleny, C. A Study of the Regeneration of the Arms of the Brittle-Star. Biol. Bull., vi, 1903.
Compensatory Regulation. Jour. Exp. Zodl., ii, 1905.
T he Physiology of Regeneration 459
As the new limbs developed they were carefully compared and in
some cases measured. It was soon seen that no constant differ-
ence could be detected in the two sets, or between different kinds
of individuals of the same set, if the regeneration of the new legs
is measured by their rate of differentiation. “Therefore, food does
not seem to be the main factor in the result. But another fact also
came to light. ‘The new legs of the well-fed individuals were larger
than those of the starved ones. ‘This difference is correlated with
the great difference in size between the two sets, for the well-fed
animals grew to a large size, while the starved ones dwindled
almost to a skeleton. By actual weight after eleven weeks one
well-fed individual weighed 3.48 gms. and a starved one .67; the
latter having, therefore, only one-fifth the weight of the former.
This difference in weight is not due to the storage of fat; but all of
the organs of the body, heart, liver, pancreas, intestine, skin,
muscles, etc., are larger in well-fed animals. “The new limbs also
partake of the general condition of well being, so far as size is con-
cerned; in other words, they developed in proportion to the size of
the old part. Measurements of the new limbs show that those of
the well-fed individualshave outstripped those of the starved indi-
viduals. ‘The difference in diameter was especially marked, while
the length of the new limb seemed to show less difference.
It has been stated that no difference in the rate of differentiation
was found, but owing to the very considerable individual variation,
small differences, if they exist, might have been easily overlooked,
and while this must be freely granted, the main result was quite
definite that no appreciable difference was seen, while the differ-
ence in size was quite apparent. ‘There is another consideration
in this connection. If the difference in size of the new parts, in
relation to the number of parts removed, depends on the surplus
of food, the detection of the difference might largely depend on the
size of the part removed. In a form like the salamander, where
the legs are relatively very small in proportion to the rest of the
body, the difference in the amount of surplus food would be so
small that we would not expect to detect any difference in the
relative sizes, even if it exists, when one or when three legs are
cut off. In fact, I could detect no such difference in these forms,
460 T. H. Morgan
when individuals lacking one, two or three legs were compared
with each other; neither in the starved nor in the well-fed sets.
When the whole of the tail is removed the loss becomes proportion-
ately greater but still I failed to note any differences. It 1s to be
remembered that Zeleny’s results show only an increase in size of
the new part, and not in rate of differentiation, and my own re-
sults show to some extent the same thing; at least, this difference
was found between the well-fed and starved sets, if not between the
individuals with one or with more parts removed. In the latter
case measurable results might depend, as stated above, on the rela-
tion between the relative size of the body and of the parts removed.
Zeleny’s important discovery, regarding the relation between the
size of the new parts and the number of parts removed, bears a
close resemblance to another curious fact in regard to the rate of
regeneration. If the distal end of the tail is removed it regenerates
more slowly than when more of the tail is cut off. “Thus the more
the material removed the greater the rate of regeneration of the
new part. Stated in this form the two results appear to be identi-
cal. ‘This question may now be considered.
RATE OF REGENERATION OF THE TAIL OF DIEMYCTYLUS AT
DIFFERENT LEVELS
If the tail of one individual is cut off near the base and of another
near the outer end, a great difference in the rate of growth of the
new tail becomes apparent. The nearer the cut to the outer end
the slower the rate of regeneration In general it may be said that
the rate of development of the new tail is directly proportional to
the distance of the cut surface from the distal end of the tail. A
few actual measurements will bear out this statement.
1Spallanzani observed that it takes as long for the toe of a salamander to regenerate as it does for an
entire leg. King found in the starfish that regeneration is more rapid from the base of the arm than
from its tip. The results are in accord with the fact just stated for the tail of the salamander.
The Physiology of Regeneration 461
INCREASE IN LENGTH OF REGENERATING TAIL
Jan. 29, Cut Off at Base Cut Off Near Tip
A —
Guia aN aa =o
IMMER ‘Gn eso acoocbedooogsans BounG RTO BORE y OMUROE 3 4 44 I 2
Veloaa.co ogo combs COO cine Sone trae ener 3 33 4 2 2
li See DUCED OSS Do cacti CRO n aE Onc Ca eeR ete 4 53 6 13 33
Missa bacdes6od HOcd 100d DOGO OOD EAD Roar 4 6 64 2 4
Slee Dy Old aca HATO Dodo ao HetO Con eee aay are 43 6 Gf a, 4
Nell “Gls coco qscoR ode CeOO hak eME ADO R See Temenos 43 6 7 2 4
LAN Pelepetee Setar a aferelorererataters ain eters sioreisaraietnseleo.omel 5 6 7 2 4
ISDS nips BH CRROD OO LOC RE GOE A San ert aeeeesee 6 7 7 — 44
Increase in Lenetu or ReEGENERATING Tait
Feb, 3. Cut Off at Base Cut Off Near Tip
SSS (SS aN
IMPs ahd aoc oct OBE On De aGae Berane 3 3 4 I I 13
nie S athccty BORO ae See ea ee 4 34 5 14 I 14
TO jdtre Sc SORES OS CoE Oe ee me 53 6 7 2 1% 3
EEN RoI DOLE Oph oC HOC ROrt 6 — 8 24 2 —
MICs ey eT Cisiee Sos eses Sissies 7 — 8 24 24 _
ESM raleoat tere ese crop sieleaeseres- Pla Houses saiasen ws 1% — 8 _ 2+ —
1/1 bored Seto poe ine ia ie ee aaa 7 — 7% — 24 _—
ith Sh cdi hale COCA BEAD Care REE 7 -- 1B — 3 _
What is the meaning of this result? Is it due to the larger
amount of food-material available when more of the old tail is
removed? ‘This possibility was tested by starving one lot of ani-
mals. ‘The results are not in accord with the assumption. For
example: In the second half of the preceding table in the last two
columns of the series ‘‘cut off at base” and in the last column of
the series ‘“‘cut off near tip,” the records of individuals are given
that were not fed after March 6. No decrease in the rate of regen-
eration is to be found. In fact these individuals as long as they
lived did even better than the others. “The salamanders were in a
well-fed condition at the beginning of the experiment and the ma-
terials derived from their own bodies sufhiced, during the time of
the experiment, to give sufficient materials for maximum regener-
ative growth. In the experiment in which the legs as well as the
tail were cut off, it was apparent that the new tails in the starved
set were not as large, as in the well-fed set. In the starved animal
the old tail also was very emaciated and much smaller in the ver-
462 T. H. Morgan
tical and transverse diameters than was the tail of the well-fed indi-
viduals. ‘Ihe difference in length was not so apparent, which 1s
probably due to the loss in the bones being less than that in the
other tissues.
The normal tail of Diemyctylus is much bigger at its base than
nearer its distal end, so that the cross-section of the base is larger
than that of the tip. Can this difference account for the difference
in the rate of regeneration from the two levels? At the beginning
of the new growth this relation may account for the difference in
size of the new part, because the amount of material proliferated
from a large surface may be greater than that from a small one.
This, in itself, would not account for the results, if, at each level,
the new part, from beginning to end, wereas broad as the old part,
but such is not the case. “The new part has more the shape of a
somewhat flattened cone, with a broader base in one case than in
the other, but tapering quickly to its apex. Therefore, if more
material were proliferated from a broader base the cone would be
longer 1. ¢., it would be in proportion to the base. On the other
hand the period of proliferation is short, and_ the basal parts soon
differentiate intotheir ofgans. Subsequent growth takes place near
the tip. Hence after the first period is passed, the new tail must,
in both cases, continue to grow in length through its own activity,
and its increase in length must henceforth be due to this activity
and not to proliferation from the base. It may appear that the
difference in rate is due to some initial difference in the material
at different levels of the old tail. If it were simply a question of
material, per se, we should expect the new growth from a basal
surface to be as rapid during the later stages of formation of the
new tail as at first, since the material for both came from the same
level, but this is not the case. Hence, I conclude, that the cause
of the difference observed is not due to a difference in the old ma-
terials that go to produce the new part. The analysis leads here
to the same conclusion as in other cases of posterior growth to be
described, in all of which the result appears to be due to some re-
tarding influence that appears as the growth approaches its natural
terminus. ‘The retardation is the same for the growth at the end
of a new part (that arises from the base) and for the new growth
The Physiology of Regeneration 463
that begins from the old part near its end. A discussion of this
point will be left until other cases have been considered.
THE RATE OF REGENERATION OF THE EARTHWORM AT
DIFFEREND LEVELS
In order to study the rate of posterior regeneration in the earth-
worm, Allolobophora feetida, at different levels the worms were
cut in two at the following places; (1), near the posterior end, re-
moving 20 to 25 segments; (2) near the middle, 7. ¢., at about the
5oth segment; and (3), just behind the girdle at about the 33d
segment. It is not advantageous to cut further forward, for, as I
have shown elsewhere, the power of regenerating a posterior end
ceases rather suddenly about the level of the 15th segment. ‘The
following tables give the results of three experiments of this kind.
Table I is for a set of worms 55 days old (September 30 to Novem-
ber 24). Table IL is for the same set 35 days old (September 30 to
November 4). Table III is another set 57 days old (December
28 to February 23).
TABLE I
September 30 to November 24
Worms Cur Near Posterior Enp, at Mipp1e anp Back or GirDLE, ANTERIOR Enp InTAcT
Posterior End Middle Behind Girdle
Old New Old New Old New
83 7 49 34 35 45
84 7 53 27 33 54
67 12 49 38 37 37
73 12 32 fr)
64 17 31 25
81 6 32 °
65 16 34 30+
If we consider the data given in these tables we find in the first
series, Table I, that from the posterior /evel the number of new seg-
ments regenerated is small, the maximum number being 17. In this
case there were 64 old segments, showing that the regenerating end
was not very near the posterior end, since about 36 segments must
have been removed, or what is more probable some of the posterior
segments pinched off after the operation. In the other cases where
464 T. H. Morgan
25 were absent six or seven new segments regenerated. From the
middle region the number of new segments is 27, 34, 38; a much
larger number than in the last case. From the region behind the
girdle the number of new segments is 25, 30, 37, 45, 543 which is
on the whole a still larger number than the last, although the dif-
ference 1s not very great. ‘he highest number, 54, is higher than
the greatest number for the middle region which is 38.
In ‘Table Il only one worm is recorded for the posterior level,
and this has only 8+ new segments. In the middle region the
numbers for the clearest cases are 26 -+, 34+, 34+. Inthe region
behind the girdle the only worm that regenerated normally had
52 new segments. ‘The results in this table are few but they agree
with those of the last one.
TABLEII
September 30 to November 4.
Worms Cut as in Taste Tl. Intact Anterior Enps
Posterior End Middle Behind Girdle
Old New Old New Old New
77 8+ 50 26+ 38 52+
55 34+
47 34+
51 eS
53 18+
In the series recorded in Table III, the number of new segments
for the posterior region is variable, owing in part to the fact that
the levels from which the regeneration occurs are somewhat dif-
ferent, as shown by counting the old segments. ‘There is one case
with a very large number, viz: 24 (with 72 old segments) which
gives almost the complete number, but in the other cases where
the cut was made at about the 80th segment, only from 5 to II
new segments regenerated. From the middle region the number
of new segments is greater than for the last level, giving a maxi-
mum of 51, but most of the other cases produced about 30 to 40.
From the region of the girdle the numbers are still larger, with a
maximum of 61, the others varying from 31 to 40, or thereabouts.
In this table also the data show the same relation between the rates
at anterior and posterior levels as do the other two.
The Physiology of Regeneration 465
TABLE III
December 28 to February 23
Twenty-Five Worms IN £AcH Set were Cur (A) 1N Front or Girpte; (B) Beninp Girpre;
(C) ar Mippte; (D) near Posterior END
Posterior End (D) At Middle (C) Behind Girdle (B) In Front of Girdle (A)
Old New Old New Old New Old New
72 13 about 50 42 about 34 61 19 °
76 8 about 50 35 37 19 °
74 bf) about 50 33 31 20 oO.
75 II about 50 34 19 (abn) 21 22 (abn)
78 8 about 50 38 36-40 (abn) 23 +17-20 (abn)
88 5 about 50 «= 42 55 (abn) 26 23
71 17 about 50 ~=s«<II Abn. 25 50
68 4 about 50 551 40+ (abn)
123 24 about 50 40
about 50 24
about 50 30
There is also another series, Table IV (56 days old), including
only two levels. From the middle level of the worm the maximum
number of new segments is 58 (with only 41 old segments present),
while the other individuals have between 23 and 43. From the
region in front of the girdle (22 to 26 old segments) the numbers
are very variable; 40 new segments being the maximum. Com-
parison of these results with those in the other tables shows again
that in the middle and anterior regions the number of new-seg-
ments is large, and much greater in number than when the worm
is cut in two nearer the posterior end.
TABLE IV
January 6 to March 3
Intact ANTERIOR END,
ANTERIOR Enp Intact; Cur at A Few Anterior SEGMENTS Cut Orr Cur 1 Front or
Mippte or Worm Axso Cur at Mippie or Worm. GIRDLE
Old New Old New Old New
56 39 about 50 30 22 °
55 43 about 50 45 24 °
41 58 about 50 35 24 12-+
47 44 about 50 18 24 3+
23 about 50 50 24 40 (about)
32 about 50 45 26 30 (about)
38 about 50 26
30 about 50 42
about 50 48
30 about 50 30
30 about 50 48 48
24 about 50 23
about 35 55
466 T. H. Morgan
What 1s the cause of this difference in the rate of regeneration
at different levels? “The amount of food available at different
levels might appear to furnish the most probable explanation of
such differences. For instance, if the food is digested in the an-
terior part of the body, let us say in the stomach and the anterior
part of the stomach-intestine, there will be the same amount
present in the worms cut at the three levels; but since on the hypoth-
esis the posterior end uses up more food than it digests, the surplus
for regenerative purposes will be greater the less there remains of
this posterior region. Hence at he level of the girdle, the regen-
eration will be more rapid than at the middle, and at the cell:
more rapid than at the posterior end. I tried to test this possible
interpretation in thefollowing way. ‘The head ends of some of the
worms were cut off, and also at the same time the posterior ends at
the same three levels as before. For two or three weeks, or more,
the worms were unable to obtain food, hence if the same difference
in the rate of regeneration at the three levels were to occur the
assumption that the difference is due to a food relation is dis-
proven. Such was found to be the case, as the following data
show. ‘The series were made at the same time as were those given
in the preceding tables with which, therefore, they are to be com-
pared. Thus Table V should be compared with Table I; Table
VI with Table I]; and Table VII with Table IV.
TABLE V
September 30 to November 24
Worms Cur Near Posterior Enp, at Mippie AND BeuIND GirpLE; Atso A Frew ANTERIOR
SecMents REMOVED
Posterior End Middle Behind Girdle
Old New Old New Old New
69 9 60 39 34 29
84 7 64 29 37 40
83 9 54 42 35 48
76 13 46 38 34 33+
87 5 33 35 (about)
64 17 34 49
79 16 34 35
As shown in this table there is a marked difference in rate of
regeneration between the posterior and middle levels, but not be-
tween the middle and the girdle levels; in fact, rather in favor of
The Physiology of Regeneration 467
the latter, but since the same difference in rate is found between the
girdle level and the tail level as is found in intact worms, the
results show that the difference in rate is not due to a difference
in the food supply.
In the next series, Table VI, the only survivors were those cut
at the level of the girdle (except one worm cut at the middle which
did not regenerate at all).
TABLE VI
September 30 to November 24
Worms Cut BenInp GirpDLE AND A Few ANTERIOR SEGMENTS Cut Orr
Old New
35 58+
29
fo)
_— abnormal
= very short
Owing to the difficulty in counting the segments in these worms
the results are unsatisfactory; but so far as they go the results show,
when compared with those of Table II, that the rate is about the
same in both, yet while confirmatory the number of cases is too
small to be of much value. In the middle columns of Table IV
there are recorded other cases of worms cut in two at the middle,
and at the same time some of the head-segments were also re-
moved. ‘The number of posterior segments that regenerated is
about the same as in the corresponding set for the same level with
intact anterior ends as recorded in the first columns of Table IV.
These results, taken in connection with those given above, seem to
show that whether the worms are with or without food for a con-
siderable period of the time the number of the segments produced
is about the same. Suppose we reverse the argument and assume
that, since in the starved worms the materials for regeneration
must be supplied by the reserve materials in the worms themselves,
then the longer the piece the greater will be the sum total of the
reserve supply and hence we should expect more regeneration; but
the facts contradict this assumption, for the longer the worms the
slower the regeneration. Here again we find that the results can-
not be explained as due to the food factor. If we assumed that
the anterior end is the storehouse for the reserve, and that the
468 T. H. Morgan
posterior end merely uses up the food, and, therefore, the longer the
piece the less material available for posterior regeneration, we
might appear to offer a formal explanation of the results, but there
are no facts in favor of this assumption, and the experimental results
that are next to be described negative such a conclusion.
In order to examine the relation between the rate of regeneration
and the size of the piece the following experiment was made. ‘The
worms were cut near the middle and at about the 20th segment
from the posterior end in one series and in the other series at the
middle and in front of the girdle. ‘The pieces between these levels
were used in both cases. In the former only a very few posterior
segments regenerated (only one or two), and in the latter cases only
6,7 and 12. ‘The results are shown in Table VII. This isa dis-
tinct difference, to be sure, although the number of segments in
both cases falls below those of the check series. “The small num-
ber of cases is no doubt partly responsible for the few new seg-
ments at the middle region, as shown by other experiments to be
described, but that this 1s not the whole question is also shown by
the next series.
TABLE VII
January 6 to March 3
Cur 1n Two at Mipp1Le AND IN FRONT OF
AxnouTt 20 PosTERIOR SeGcMENts Cut Orr; ALso Girpie; New SeGMENTs From MIDDLE
Cur at Mippte or Worm. LEVEL
New Segments New Segments
° 6
I 12
I 7
°
°
I
°
°
2
I
I
°
°
In order to obtain a series of still shorter pieces, the worms were
cut into five pieces, each in the following way. Each worm was
o>
The Physiology of Regeneration 469
first cut in two in the middle (about the 50th segment). ‘The an-
terior end was then cut into three pieces, by one cut behind the
girdle and by another halfway between the anterior end and the
girdle. [he posterior end was also cut into three pieces; thus, 20
segments were cut from the posterior end and thrown away, the
remaining part was then cut in two. All of the pieces of one kind
were kept together, and were killed and examined after two months
(January 6 to March 3). The check set of longer pieces will be
found in Table IV.
TABLE VIII
January 6 to March 3
Worms Curt into Six Pieces (Tam Piece THrown Away). For Derarts, srr TExt
1p D Cc B A
Old New Old New Old New Old New Old New
13 15 12 18 14 14 26 15 (about) 19 knob
17 4 II 18 13 17 23 18
16 5 12 17 15 14 22 °
I5 2 21 6 8 14 19
15 ° 19 20 15 8
17 ° 16 14 15 knob
17 ° TOenE SG 14 14
17 re) II 15 14 18 (about)
17 fo) = fo)
21 5 — °
12 8 = °
For convenience the pieces will be called A, B, C, D, E; the first
being nearer the anterior end, etc. If we examine the piece in the
reverse order, beginning with E, we find relatively few new pos-
terior segments in these pieces, although in one case the surprising
number of 15 segments have developed. It seems not unlikely that
in this case a piece was misplaced. In the D series the number of
segments is very variable, ranging from 5 to 35, the latter number
giving nearly the entire number lacking at the posterior end. In
the C series, whose posterior end is at the middle of the worm,
the number of segments is about the same as in the last case, except
there is no such extreme case as that mentioned. In the B series
only one piece was alive (of the 25 cut off) with about the same num-
ber of new segments as in the last cases. In the A series only one
470 I. H. Morgan
piece had regenerated (from the posterior end of the 23d segment)
with 18 poorly developed segments.
With the exception of the E pieces this series does not show any
very marked difference for the different levels. “There is a possible
source of error in the pieces from the posterior region, for I have
sometimes found these breaking up into smaller pieces, or pinch-
ing off pieces from the posterior end. ‘The small number of the
old segments in some of these pieces, where the expectation is
about 15, may be due to this; but granting the possibility of such
occurrences the main results cannot be due to this factor as will be
seen by examining the number of old segments present in the
pieces. |
If we compare this table with the longer pieces of Table IV, com-
paring, for example, those cut at the middle of the worm in the two
cases, we find that fewer segments regenerate as a rule in these very
short pieces. It will be recalled that in none of these pieces does a
head develop at the anterior end; on the contrary, most of the more
posterior pieces develop a heteromorphic tail on the anterior end.
How far this heteromorphic development may affect the result 1s
not clear, but that the small number of posterior segments cannot be
be due to this factor is shown by comparison with somewhat longer
pieces that may also develop heteromorphic tails. The great
mortality of these short pieces indicates that they are not under
very favorable conditions, although the death rate is especially
high at first, and while later the small pieces appear to be in a
healthy condition and may live for several months, yet after two
or three months they all die of starvation. One point, however, 1s
fairly clear that although the small pieces lack the power to produce
new parts at the maximum rate for a given level, yet the retarda-
tion is far from being proportionate to their small size. From this
fact we may safely conclude that the amount of the food supply
in the piece is not the main factor in its rate of regeneration,
although when it has decreased belowa certain point regeneration
may stop or be much retarded for want of materials. “These nega-
tive conclusions in regard to the rate of growth are useful in so far
as they clear the way for the discussion of the main problem of:
what factors regulate the rate of growth, for, if it is not due to
The Physiology of Regeneration 4A7E
food the road is clear to search in some other direction for the mean-
ing of the facts. Before attacking this fundamental question I
should like briefly to bring into relation with the foregoing results
certain other facts that I have already published.1
I have shown that if 10 to 12 posterior segments are cut from
the posterior end of a worm, and then the next Io to 12 segments
are cut off, the piece lying between these cuts does not as a rule
produce any new posterior segments, even after three and a half
months. Whole worms, however, that lack the last Io or 12 seg-
ments regenerate a few segments in this time. In another experi-
ment, long pieces from the middle of the worm, having the ante-
rior end also removed, were compared with similar pieces with the
head end intact. The rate of posterior regeneration was the same
for both. Again, some worms with only a few posterior segments
removed were compared with similar ones which lacked also the
head end. ‘The regeneration at the posterior end was the same
in both after a month showing that the results do not depend on
the question of the taking in of food. ‘These and a few other
experiments are in harmony with the results described above.
So far the results have been judged by the number of the
new segments produced; in other words, by the differentiation of the
new part. If, however, we test the results by the size of the new
part certain differences are apparent. [he most general result 1s
seen at once when well-fed worms are compared with starving
pieces.
The new part in the well-fed individual is larger, and this is
especially noticeable in the diameter of the piece, that often
approaches that of the old part. The individual segments are
also larger in the well-fed worms, so that for an equivalent num-
ber of segments the new part is longer. In the starved worms the
new segments are often very small, especially when the old part ~
is a very short piece. In striking contrast to this difference in size,
which is so apparent that it is not necessary to take measurements
to discover it, the number of segments is, as we have seen, approx-
imately the same in a well-fed and ina starved individual. ‘These
‘Morgan, T.H. Regeneration in Allolobophora feetida. Archiv f. Entw. Mech., 1897.
472 feel is Morgan
results show that while the size of the new part 1s dependent on
the food supply, the growth and differentiation of the new part is
to a large extent independent of this factor. ‘The difference in the
size of the new part in the two cases shows, nevertheless, that the
new part is affected by the conditions of the food supply, and it is
probable that the smaller number of posterior segments regener-
ated by very short pieces is the result of the lack of food for further
growth, hence the pieces from different regions show approxi-
mately the same number of segments, but the difference in rate
of regeneration of larger pieces at different levels cannot be ac-
counted for in this way as the experiments first described have
shown. So long as there is enough food material in the blood or
other fluids of the body to allow growth to take place at all it goes
on at a rate determined by the peculiarities of each level, and largely
independent of the food supply.
If my conclusions from the data are correct, and the difference
in the rate of development of the new parts at different levels is not,
in the main, due to differences in the food supply, to what is the
difference due? The answer that first suggests itself is that the
difference must be due to differences in the materials of the worm
at different levels. If by differences at different levels we mean
the differentiation of the parts, or the kind of material that
exists at each level, then the conclusion may express a part of the
truth, but if we mean that each level possesses limitations in its
powers of growth, not possessed by other levels the answer can be
shown to be inadequate. For example, if the rate of regeneration
from the middle of the worm were determined by certain peculiari-
ties of its material, we should expect the same rate to continue
until the entire missing part was replaced; and if we were then to
cut off the tip of the new tail after it had completed itself it ought
to regenerate as rapidly as does the new part from the middle of the
worm, because its material has come from that level. While I
have not actually carried out this experiment there can be no doubt
that the newly regenerated tail would show at its tip the same
retardation shown by the unregenerated tail. Furthermore, if
this view were correct how could we explain the termination of the
growth process when the normal number of segments has been
The Physiology of Regeneration 473
replaced? Why should not the new part continue indefinitely to
grow? If we can find the explanation for the cessation of growth
at the proper terminus we can probably find also an explanation
for the difference in the rate at different levels, for, as can be shown,
the two things appear to be one and the same. In other words, as
the new part grows longer its materials change, and this change
is of such a kind that it leads to the cessation of growth. Hence
starting under different conditions at different levels the same end
result will be reached in all cases, and when the terminus is reached
the growth should slowly decline as we find in fact that it does.
It is this idea that will be developed later.
There is another point that must be mentioned here. The dif-
ference in rate 1s not so much due to an initial difference in the
appearance of new material at different levels, as to the relatively
greater slowness of the growth of a terminal part after it has been
once started. [his seems to mean that the stimulus for the form-
ation of the new part is so much greater than the difference in the
rate for each level, that the bicrer’ becomes entirely overshadowed
in the formation of the first new tissue. Moreover, the prolifera-
tion of new material as the result of an injury may depend on a
different set of factors than the subsequent rate of growth of a new
part after the original stimulus that led to the proliferation has dis-
appeared.
In order to compare the rate of regeneration at the beginning
and toward the end of its period of growth some of the same lot
‘of worms that gave the records of Table IV were kept alive for
another month (January 6 to April 6) in order to see how much
further the regeneration of new segments would continue. The
results are given in the following Table IX.
TABLE Ix
Ste CuT NEAR
Cur at MippLe Cur Bexinp GirDLE Posterior Enp
Old New Old New Old New
48 52 33 13 (abn) 75 10
59 32 32 72 75 8
41 53 36 60 63 17
52 36 34 52
gt 40 34 53
1 46 23 45
A74 T. H. Morgan
The number of new segments from the middle region is about
the same as it was a month earlier; the full number having been
nearly reached, although not quite in a few cases. More new seg-
ments are found in the worms cut just behind the girdle; the num-
ber approaching completion here also. In the cases where about
25 segments had been cut from the posterior end, the number
removed had not yet been made good, and the same holds for
the single case where about 37 old posterior segments had been
removed. The results show a much smaller proportionate
increase during the third month than during the two preceding
ones. In other words, as the new part approaches completion its
rate of regeneration declines.
REGENERATION OF THE CAUDAL FIN OF FUNDULUS
Experiments on the regeneration of the caudal fin of Fundulus
were carried out during the summer of 1905, while occupying the
Table of Columbia University at the Marine Laboratory at Woods
Hole, and continued throughout the present winter at the New
York Aquarium, where, thanks to the admirable arrangements and
to the opportunity for scientific work extended by the Director,
Mr. Chas. Townsend, it has been possible to carry on the work
under the most favorable conditions.
In two former papers! dealing with the regeneration of the fins of
Fundulus and of some other species of fish I have described the
differences in rate of regeneration from the basal and distal parts of
an oblique cut made through the tail. ‘This difference I referred
correctly, as further results have shown, to the retardation of the
growth on the more distal parts. ‘This retardation, however, did
not seem to me to belong to the same category of facts as the
retardation from a more distal squarely-cut surface, and it was
primarily in order to account, if possible, for this difference that
Tagain undertook a re-examination of the facts. “The new results
have cleared up this point satisfactorily, and I have gone further
and been able to obtain data that bear on the more fundamental
question of the rate of growth from different levels.
‘Morgan, T. H. Regeneration in Teleosts. Archiv f. Ent. mech., x, 1900. Further Experiments
on the Regeneration of the Tail of Fishes. Archiv f. Ent. mech., xiv, 1902.
The Physiology of Regeneration 475
This problem of the rate of growth at different levels would seem
to be a comparatively simple one, but I have found it somewhat
baffling, owing to the great individual differences in the rate of
growth in different fish. “Iwo methods of study have been fol-
lowed. In the one, thetails were removed from a number of fish
at two or three different levels, and the rate of regeneration meas-
ured from time to time in the living fish, or a few fish were killed
at intervals and then measured. In the other case the records of
the rate of growth of the same fish were kept which gives more
accurate data. :
The results may be briefly summarized in the following state-
ments. [he rate of growth 1s, at first, nearly the same whether the
cut is made near the base or near the outer end of the tail. This
period covers that of the first proliferation of new material. Very
soon, the new parts grow more rapidly from the basal than from
the more distal cut surface. In general, the nearer the new part
approaches its completion, the slower its regeneration, so that the
new part from a distal cut surface very soon grows with extreme
slowness, while that from the basal cut continues for a longer time
to grow, but it, too, as it gets longer, shows an ever increasing
retardation in its growth.
In regard to obliquely cut surfaces, ] can confirm my former
statements, namely, that regeneration from the more distal part
of the oblique surface is much retarded after the first period of pro-
liferation is over. Moreover, this retardation is far greater than
that seen in a cross-cut surface at the same distance from the end
of the tail, and consequently is not due to the same factor. I can
now give some further experiments that throw a great deal of light
on this retarding factor in oblique regeneration.
If the tail of a fish is cut off in such a way (Fig. 1) that two
cross-cut surfaces a and b are exposed at different levels, it will be
found that on a, regeneration progresses much more slowly (after
its first beginning) thanon b. The rate of growth on 5 is as fast or
faster than that on a cross-cut surface of the whole tail at this level,
while that on a is very much slower, and seems almost to come to
an end after a time unless the new part from / catches up to the
476 T. H. Morgan
new part ona, after which the two new parts continue to grow
forward together until the tail is completed.
In the reverse case the cut: surface a is made the broader of the
two,as shown in Fig.2._ Under these circumstances the retarda-
tion is smaller on a, while that on b is the same as in the former
case, which is the same as that of an entire cross cut for this level.
The results show that the retardation of the distal surface 1s in pro-
portion to its height, while the growth on the basal cut surface
is the same as the regeneration for an entire cross-cut surface at
this level. “The resultsare the same whether the distal partial sur-
face is at the top or at the bottom of the tail.
FiG.et , Fic. 2 Fic. 3
It might be supposed that the retardation on the outer partial cut
a is due to this part lying at one side of the tail, where it might
be supposed to receive less nourishment. I tested and disproved
this possibility in the following way. The tail was cut off as shown
in Fig. 3. Here the middle portion has a small and independent
cut surface. Regeneration takes place from all three surfaces, but
the growth from the outer one a was markedly less than that from
the other two. After a time the new upper and the lower parts,
from the upper and the lower basal cut surfaces, catch up to the
middle part, and after this has occurred the three parts, com-
pletely united, grow forward as a single organ to complete the
length of the tail. In order to prevent this latter result I have cut
away after a time the new parts above and below, leaving the
middle part intact, and in some cases this was done two or three
times before the upper and lower parts had caught up to the middle
part. In this way I hoped to discover whether the middle part
would finally grow out to its full length, or whether it would not go
nr
The Physiology of Regeneration 477
beyond a size proportionate to the width of the piece from which
itarose. It was found that the new middle part continued to grow,
but with extreme slowness. Whether it would finally reach its full
length I do not know, but it is not improbable that it would do so. -
These results explain, in a way, the regeneration from an oblique
surface. If we consider any part of the outer oblique surface by
itself alone, it is in the same condition as the outer part in the pre-
ceding cases of partial cut surfaces, and the retardation of the
growth in both would seem to be due to the same factors, whatever
these may be. The outer part of an oblique surface cannot com-
plete itself, except very slowly if at all, until the new tissue from
the more proximal parts of the same cut surface has caught up,
and set it free, so to speak. ‘This is, in fact, what occurs. The
nature of the retarding influence will be discussed after some fur-
ther facts have been considered.
The details of the experiments in which the rate of regeneration
at different levels was examined may now be given.
The first experiment was with Fundulus majalis. ‘The opera-
tion was carried out on July 7, and the first measurements were
made July 26. The same lot of living fish gave all of the measure-
ments. The tails were cut near the outer or distal end, near the
base, and obliquely between these two levels. From the basal cut
surface the new parts measured 14, 2, 2, 24, 23, 24, 3, 3. From
the distal cut surface the new parts measured 2, 2, 1%, I¥, 14, 2.
Already at this time some of the basal cuts had regenerated faster
than the distal ones. The oblique cut surfaces were measured
near the top and near the base where the length was greatest. The
two measurements for each fish are bracketed, the distal one stand-
ing above. They are iG ee The basal measure
ments correspond with those of the same level given above. The
distal measurements are about half as much as the distal measure-
ment given above. Five days later, August 1, the following meas-
urements were taken.
Basal, 33, 45 35 3) 3h: 43) 3% 45 4 44
Distal, 4, 33, 345 3, 34 32
Oblique, : {
478 T. H. Morgan
There seems to be little or no difference at this time between the
basal and the distal cut surfaces. “The oblique surfaces are much
shorter at the distal part than at the basal, as in the last case.
Eight days later the following measurements were taken:
Basal, 7, 5, 6, 7, 5, 6, 6, 7, 6
Distal; 0,055°502,.4564
B tes care ae ane
Bene: 8 f \5 B (4
The new parts from the basal surfaces are noticeably ahead in
most cases of those from the distal surfaces. ‘The oblique cuts
show the same relation as before.
Eight days later, August 17, the results were as follows:
Basaly: 7... 0,05. 7.055510 050
Distal, 5, 7, 45 55 445 4
2
Oblias 1619 18 18 13
The relation is the same here as before. It is noticeable that
even at this time the distal cut surfaces had not regained their full
length. Eleven days later the measurements were about the same,
no appreciable increase in length being noted. As the fish were
not in as good condition as at first the last results are no doubt due
to this. Other results, to be described later, where the fish were
under better conditions, show that the new growth continues after
this time.
The next series of experiments were made with Fundulus hetero-
clitus. The tails were cut off on July 7 and the first measurements
made July 20.
Basal, 2 +, 1, 14, to 12, I} to 14, 2, I
Distal, 14, 12, 2, 14, 14 to 13, 14, 14, 14, 14
CeO Ta eT an Parca Ma Ter
eta ee palace ae a ee hr rae 8
Ts cies | aie sea aet
2ei\eg ee | ones,
At this time, 13 days after the operation, there is nothing to show
that there is any difference between basal and distal rates of growth.
In the measurements from the oblique surface the basal growth
is noticeably greater than the distal, and even also greater than
the basal growth from the basal cut surface. Whether this is a
The Physiology of Regeneration 479
real difference, or one due to unintentional cutting nearer to the
base in oblique cuts, is difficult to decide, but the latter view is the
more probable. The following experiments were carried out on
Fundulus heteroclitus in the New York Aquarium between Decem-
ber 8, 1905,and January 30,1906. [he waterin whichthe fish lived
was warmed to about 69° F. Measurements of the amount of
old material cut off were made. ‘They are given below:
Basal, 10, 10;.94,'9, 9; 9, 9,95 8
Distal, 53, 535 55 5 5) 59 43
Large fish were used in this experiment. The distance from
the rounded end of the scales covering the base of the tail to the
end of the tail measured, as shown above, about 10. Hence the
basal cut passes just at the edge of the scales. It was found better
not to cut closer than this level to the base; for further forward
there is soon reached a region from which regeneration is abnormal
and often delayed. ‘The first measurements (of the tails of a few
fish that were killed and put into formalin) after 21 days (December
29) were as follows:
Basal, 33, 3, 3, 3
Distal, 34, 34, 24, 24
The rate at the two levels seems to be nearly the same at this
time. The next measurements were made after 38 days (Jan-
uary I5).
Basal 54,54, 5, 4
Distal, 4, 4, 34, 32, 34
The new part from the base has now outstripped that from the
distal level, although the latter has not yet reached its full growth.
The third and last set of measurements were made after 58 days,
January 30.
Basal, 6, 6, 54, 5, 5
Distal, 33, 3
By this time the basal surfaces have regenerated nearly twice as
fast as have the distal ones, although the latter still fall short of
their full length, which they would have attained had they regener-
ated as fast as the basal cut surfaces.
In the last series the new tails had not regained their full length
after 58 days. In order to obtain later stages for comparison a
480 T. H. Morgan
new series was started February 16 and continued until May 11, a
period of 84 days. ‘The results are given in the following tables.
For comparison I give a few measurements of the lengths of the
old pieces cut off February 16. The pieces cut off from the distal
end measured in I0 cases 3, 3, 34, 33, 34, 34) 34) 33) 4, 44; and
from the basal end 7, 7, 74, 74, 8, 8, 8, 9. Subsequent examina-
tion showed that some of the distal cuts had been unintentionally
made nearer the middle part of the tail (those measuring 4 mm,
above, for instance) and these have been included in the tables
in the column marked “Middle.”
Basal Middle Distal
Marchicg' is iitpme muita crete tes Ibe? 25 ee 132 1% 1%
UAH E mE CE eae 2ege Bar ak 14 I 2
Bi aerials td eda 44 4h 22 24
April arG cca cams iuaes seen ae 4t 4% 43 pie Lei arey 2a 2 ae oe
BT agen trata ste ore Cites at enat ete BE 3k 4 23 ihc 2 ark roe
Minty Gets ce conceit ieee Ay 3h Fgh at 3 -I 24 24 2
The results show that even after 84 days the new parts from the
distal end had not reached their full growth, neither had the new
part from the basal cut, although it was longer than the former, and
enough had been produced to have completed the new parts of the
distal cut had it regenerated as fast as the basal. ‘There is for both
levels a marked decrease in rate in the later stages which is the
greater the more distal the cut surface..
The slow rate of regeneration from distal cut surfaces, shown in
the preceding results, led me to examine this question further. A
terminal piece was cut from the tip of the tail much shorter than
in any of the preceding cases in order to see whether the same delay
‘would manifest itself in this case, or whether the initial growth
would replace at once so small a part. On March 3 a very
narrow piece measuring 2 mm. was cut from the tip of the tail.
On April 6 the new part measured $ mm.; on April 14, 13;0n April
28,1 mm.~° Ina control, in which as much as 7 mm. was cut off, the
new part measured 2 mm. on April 6, and 3 mm. on April 28. In
both cases the fish had been kept in dishes in the laboratory and
were under rather poor conditions, lacking food and sufficient
air. Nevertheless the regeneration went on at about the same rate
as when the conditions are better.
The Physiology of Regeneration 481
In another case, 2 mm. was cut off from the distal end of the tail
on February 17. On March 1 the new part measured 1 mm.; on
March 17 it measured 3 mm.; on March 24, 1} mm.; on April 6,
14, on April 31, 2mm. Thus it took two and a half months to
reproduce 2 mm. at the distal end, an amount that could be pro-
duced in two weeks from a basal cut. In another case 14 mm. was
. cut off on February 17. On March 17 the new part measured
about I mm.; on March 24 it still measured 1 mm.;and on April 31,
Imm. Thus at the end of this time the part cut off had not been
completely restored.
In the same series as the one of December $ to January 30,
other kinds of operations were also carried out. In several cases
cross-cut surfaces were made at two levels, as shown in Figs. 4 and
5. The rate of regeneration from the two levels is shown in the
following figures, with the numbers attached. ‘The first series is
from December 8 to December 29.
1 13
} Cy © ee A I ©
4 3- ) :
ety Me aw ly 5
2 Fie: 4 Fic, 5
It will be seen if these results are compared with those of the pre-
ceding tables, in which entire cross-cut surfaces are involved, that
the regeneration from the distal partial surface in these cases
is greatly inhibited, being not more than one-third as much as
from an entire surface at the same level. [he statement holds
both when the partial surface is at the edge and when it 1s in the
middle of the tail. The next series (December 8 to January 5)
gives further cases of this sort.
at
nn
tol
nN
wal
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iS)
v
ne
wv
wv
wn
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we
an
wal
482 T. H. Morgan
During the interval of 38 days between these two series the new
part from the outer cut surface has grown very little, but that from
the inner cut surface has grown as fast, and apparently a little
faster, than from the entire cut surface. Here also the greater
regeneration from the base is probably due to unintentionally
cutting the partial basal surface nearer the base of the tail as
examination showed. ‘The third series (January 30) gave the.
following measurements:
~~ YS
Fic, 4b Fic. 5b
The new part from the outer surface is longer in some cases,
and this is undoubtedly due to the new growth from the basal sur-
faces having reached and passed the level of the distal cut. Other
series show that in this way the tail may finally complete itself.
Three other series of experiments gave essentially the same
results, and, therefore, need not be cited. One point of some addi-
tional interest is furnished by these other series. ‘The rate of
regeneration from the outer partial cut surface is greater the
broader, 7. ¢., the higher dorso-ventrally, the cut surface. ‘This
result shows that the retardation is directly connected with the
height of the cut surface, and only secondarily with its distance
from the base of the tail.
REGENERATION OF THE TAIL OF THE GOLD FISH CARASSIUS
AURATUS
This fish has a forked tail which introduces some new factors
into the problem. ‘The most important fact connected with the
regeneration of a forked tail is that the new part becomes forked
very early, before, in fact, the new part has grown out to the level
of the old notch. ‘This shows that the distal end of the new tail
The Physiology of Regeneration 483
is very early laid down, although the later growth must take place
very near the outer edge of the new tail; the tail retaining its forked
form during the whole of the subsequent growth. ‘The results for
three cases in which the tails were cut squarely off, one near the
base, the other two nearer the distal end, are shown in Fig. 6.
The distance of the first from the notch is 8 divisions (= 4 mm.),
and of the latter 2 divisions (=I mm.), and of a third 4 (=2 mm.)
The measurements give the rate in growth in the middle and at
the upper and lower parts where the lobes grow out.
The results show that the growth is rapid at first, especially
when we consider that the healing over of the cut surface and that
the arrangement of the new materials take place during this time.
The notch soon appears in the new tail owing to less rapid growth
in this region. It will be noticed that the middle region is nearer
its definitive end than the upper and lower parts of the tail and
this may account for its retardation. It also seems, as far as |
could judge, that the notch appears sooner when the regeneration
is from a distal than from a more basal cut surface, and this is in
accord with the idea just expressed. [tis not improbable, however,
that the appearance of the notch is due to other factors. The
upper and the lower parts continue to grow longer after the
middle part has reached or nearly reached its goal. At least
this seems to be the case, although the middle part may also
continue to grow, but so slowly that its progress was not observed
in the somewhat rough measurements that I have made.
In another experiment with gold fish, measurements were not
taken but sketches of each fish were made at intervals, and as the
results illustrate some other points, they may be briefly mentioned
here. In one set the exceedingly slow growth of the middle part
was noted, the notch had not appeared after 45 days, although the
middle had nearly reached its outer level. ‘The upper and the
lower parts had grown faster, but had not reached their limit at
this time. By January 13 the notch was present, although the
level of the old notch had not been reached. Its appearance is
due to the greater growth above and below. In another set the
notch appeared soon because the cut was made nearer to the old
notch. ‘The upper and the lower parts had grown past the level
JAN
27
= THT. Morgan
2%
; L
DEC =
2
: =i)
H
JAN al a ; d+
3 > 22
‘lL 3
JAN
12
Fic. 6
LV Se Oe ee
~
The Physiology of Regeneration 485
of the old notch. In another case a small outer and a large inner
surface was left. “The new growth on the latter was removed once.
The development was delayed, and even on February 15 it had not
reached its full length. At this time the notch was present and
the upper and the lower parts were of the same length.
AN EXAMINATION OF THE RELATION BETWEEN THE INCREASE IN
SIZE IN NORMAL AND IN REGENERATING SALAMANDERS
The rate of growth at neat stages of embryonic and larval
development and its cessation in many species when a certain size
or condition has been reached furnishes some of the most perplexing
and important problems of morphology and physiology. It has
proven difficult in the case of normal development to attack this
problem except by measuring the different rates of growth at dif-
ferent stages of development, but this offers little opportunity
to test experimentally any conclusions that the measurements may
lead one to draw. It would seem that a chance to study the
problem experimentally is afforded in the remarkable power
to regenerate shown by many animals. ‘The surprising fact has
never, I believe, been sufficiently appreciated, that regeneration
means a sudden and rapid renewal of the growth process, which
takes place not only in those animals that have unlimited powers
of normal growth, but also in those whose normal growth is limited
within rather narrow boundaries. ‘The fact that an animal that
has ceased to grow larger will replace a lost part shows that its
growth has come to an end not because of the loss of the power to
grow, but because of some retardation of normal growth that has
taken place.
It is commonly pee in the case of an animal that has
reached adult size that an equilibrium has become established be-
tween the amount of food that can be digested and the body weight.
Suppose, if this is true, that a part of the body of appreciable
weight is suddenly removed without affecting the surface of absorp-
tion of the digestive tract, will the animal quickly regain its lost
weight before the missing part has regenerated? In the case
of an animal that has not reached its upper limit of adult size, and
486 T. H. Morgan.
in the case of one having no well defined upper limit the problem
is more complicated, but if the size at any one time is due to an
equilibrium between the food digested and the body weight, we
might anticipate on the theory that an animal lacking a part of the
body would increase more rapidly in proportion to its weight than
an intact animal. The salamander that I have used to test
this point belongs to the latter class. “The experiment was as
follows: ‘The tails of a few salamanders were cut off at the base,
the animals being weighed both before and after the operation.
They were then weighed at intervals of a week during the period
of regeneration of a new tail. Control, intact animals were also
kept under the same conditions and their increase in weight also
recorded. ‘The tailless animals have had their body weight sud-
denly decreased by the loss of the tail, while the organs of digestion
remain as before, hence, since the tailless animals have less body
weight to keep up, they might be expected to increase in propor-
tion to their weight faster than the check series. If both sets
increase in weight, as the results show to be the case, the body
weight of both would be expected to increase at the same rate, but
the amount of material that goes to nourish the tail in the tailed
animals might go as additional increase to the body of the tailless
ones. Hence, as I have said, we might expect at most only a
greater increase in the tailless set, in proportion to the amount
removed in the tail which is about one-seventh to one-tenth of the
total weight. Unless the weights show a very constant although
small difference it would not be possible to detect the supposed
influence. Unfortunately, the outcome has shown that the increase
is too variable to furnish very definite information on this ques-
tion.
There is a further possible complication in the results. The
presence of a regenerating stump may in itself react on the rest
of the body, and cause the general growth to take place faster,
in somewhat the same way that the presence of young in the uterus
of a guinea pig, itself not fully grown, does not interfere with the
normal growth of the parent,’ but on the contrary, the powers of
‘Minot, C.S. Seriescence and Rejuvenation, Jour. Physiol., xii, 891-
The Physiology of Regeneration 487
assimilation seem to be increased, since under the conditions the
mother digests enough material not only for her own normal growth
but also for that of her rapidly growing progeny as well. In the
case of the guinea pigs the result is complicated by the amount
of food taken by the mother, which may be greater during preg-
nancy since the amount does not appear to have been regulated;
but in the case of the salamanders the amount given was carefully
controlled.
The salamanders were kept in flat dishes containing half an
inch to an inch of water, and were fed on small pieces of raw beef
about every other day. As they take food from the forceps the
amount that each takes can be regulated, which would not be the
case if the food were simply left in the dishes. As the animals grow
larger they can swallow larger pieces, hence for some time their
weight can be steadily increased by increasing the size of the piece
of beef given to each.
The animals were not young, so that their increase in size is not
to be ascribed to the increase from youth to age. ‘The life history
of this species insures the maturity of all the individuals, since
Gage! has shown that they pass two years on land before under-
going the final changes that transform them into the aquatic Die-
myctylus. ‘The susceptibility of the animals to the amount of food
is shown not only in their rate of increase, but also by the rapid
loss in weight when food is scarce or absent, although they may be
kept alive for several months deprived of food. Hence in weigh-
ing the animals care must be taken to weigh them at corresponding
times from the last feeding. Failure in this respect is at once
shown in the weight, asa few cases in the tables indicate. Asa rule
the last feeding was 24 hours before the time of weighing. ‘The
average of the five heaviest animals that I obtained by a steady and
abundant feeding for three months was 3.14 grams and the mini-
mum average weight for five starved individuals kept ina similar
dish wast.oo. Inthe well-fed set there were considerable individ-
ual differences. “[husthe largest individual in the same set weighed
3.25 and the smallest 1.62 grams or about half. I isolated one of
"Gage,S.H. Life History of the Vermilion Spotted Newt. -Am. Natural., xxv, 1891.
488 T. H. Morgan
the smallest individuals in the hope of determining the cause of
this difference and found that it took less food than the others.
No doubt its smaller size is directly connected with this condition.
The most important fact connected with the increase in size of
Diemyctylus is that the increase is not due to the storage of fat,
as the weight of a man who had reached adult size might be in-
creased by over feeding, but to an actual increase in size of nearly
all of the organs of the body. The increase 1s, therefore, to be
ascribed to growth, not the storage of reserve material. The
starved animal may live for a long time at the expense of its formed
tissues. [his possibility indicates that an animal of average size,
kept without food, may still be able to supply a regenerating part
with materials for growth—the interesting point being that one
part is rapidly growing at the expense of the rest of the body. ‘The
simplest interpretation of this result is, I think, that there must be
present in the blood at all times a certain, although perhaps vari-
able, amount of nourishing materials, and that a ‘newly regener-
ating part has the power to take from the blood the materials that
it needs for growth, even when the 2mount present in the blood
has fallen so low that the rest of the tissues cannot mzintain them-
selves, but break down to supply the blood with a certzin amount
of nutriment. If this idea expresses approximately the relation
that exists, it follows that while the new part requires a certain
amount of food in order to continue growing, it can take advan-
tage of a condition that the older or differentiated tissues cannot
make use of; in fact, when the latter slowly lose ground. ‘There
is apparently a similarity in this respect between an embryo and
the newly regenerating part. Since in regeneration the new part
is formed directly out of the old tissues we may assume that this
property of young parts is something connected with their lack
of differentiation, which is lost when differentiation takes place,
and is regained again when the differentiation 1s lost.
In the first experiment the tails of six salamanders (three males
and three females) were cut off near the base. In another set (also
of three males and three females) the animals were intact. ~The
former weighed 1.46 grams a piece (average weight) and the tails
cut off weighed about 0.16 gram. Owing to a loss of blood, etc.,
,
]
\
The Physiology of Regeneration 489
the tailless animals were weighed again the next day, and their
weight (1.28 grams) was found to be in this case about the same
or a little greater, due perhaps to the absorption of water by the
cut surfaces. The control set were at the beginning a little heavier,
weighing 1.85 grams. The following table gives the average
individual weight of these two sets of animals from December 12
to February 3. On January 5 one of the tailless animals died, and
on January 10 another.
TABLE SHOWING THE INCREASE IN WEIGHT OF TAILLESS AND INTACT SALAMANDERS
TABLE A
Rateof | Control Rate of Rate of
Tailless Tailless
Increase | Intact Increase Increase
MDC Ge 2 a yaya ta -totss21=)s%0)6)otele olor: <1- 1.46 | 1.85
(intact)
MD lercvecarcsersic es oe orate alels <a eo
(tailless)
IOs Ag So toos oe GOnC Ou aEne 1.28 .06
IS cecécaobbqaododdEeoge 1.53 25 1.74 sii
Pirlo ea oe ERE Das .O4 1.65 =09. | {2:28
[EtS) BE eee ee epee ee 1.88 31 1.98 533 | (intact)
Wooésccopseaacs beneaeson 1.95 -O7 2.06 08 || 2.08
DONA ee eer ae niar: 1.96 .O1 2.13 .07 [ (tailless)
7 |p POS SORE CLD Exe OOO 2.13 5G 2.36 23 [Feb.4
ihGis /<ligeaaon sees saanec sane 2.08 =9)5 2/323 ait \2.01
Gln oueattdidea a abe caeoe oes 2.18 -I0 2.30 -O7 Die diges ai ili
1/6 BUGS BO ORC ore an 222 04 2.34: 04 2533 Bi
DY ental Abode 8 De cer ena 2.36 14 2.45 II 2ER4 -O1
Wt eooae bocca ooonaeosoee Pieyn —.02 2.56 II 2.41 .O7
MOM nae eres Bae ae eeerereas 2.45 sii 2.43 51 je (5 .10
11 fe ie Dae Bee eet SLA 2.38 ey 2.41 16? 2.45 —.0o6
DAP A eet ata eae ah ehs ape ete as eats 2.64 .26 2.78 37 Dielfe) 28
Hilsoge oton Bota aoa eee 2.66 .02 2.66 | —.12 2.80 -O7
PN tl i eanrteya iar sees oPereas erste hovers sway 2.83 Aa) 2.78 12 2.84 04
From this table it appears that both sets steadily increased in
weight, but the increase was greater in the tailless set. ‘The dif-
ference in rate during the first two weeks, before the new tail has
appreciably developed is much greater in the tailless set. “There
is a gain of .35 for the tailless animals and a loss of .20 in the
intact set. If the next week is taken into account when both sets
made great gains the balance remains nearly the same. ‘Their
490 T. H. Morgan
differences may be due to uncontrolled factors—and in part I
suspect this must be so, for there is no assignable cause for the
decrease in the intact series—but if the difference is directly con-
nected with the absence of the tail in one set it is obvious that the
entire loss was made good, which isa greater increase than we
should anticipate since the expectation would only be a propor-
tionately greater increase in the tailless set.
The tables also show that after four months the tailless set had
doubled the weight while the intact ones had increased only one
and a half times.
The greater increase in weight in the tailless set might be sup-
posed to be due to the stimulus of the growing part on the body asa
whole, as said above; orthe difference may be a purely fortuitous
one, the tailless animals happening to be a faster growing set. By
January 27 the new tails were about one-half to one-third of an
inch long, and their increase in length continuously adds new
weight to the tailless set. In order to see if the same result would
follow if some of the intact individuals had their tails removed, they
were separated in two lots; one lot kept intact and the other cur-
tailed. The last two columns of the table give the rate of growth
of the tailless set. The average weight before the operation was
2.28 grams; afterward 2.08 and the following day 2.01. ‘The
data are controlled by those of the middle columns. It will be
noticed that the control, February 3, weighed a little less than
the animals whose tails were to be cut off. During the two weeks
following the operation the tailless salamanders gained .32 gram
while the intact ones gained only .11 gram. If the third week
is also taken into account the tailless animals gained .33 gram,
and the intact ones .22 gram, which gives still a difference in
favor of the former. When the experiment closed the tailless ani-
mals had gained .83 gram and the intact .55 gram.
Still not convinced that the difference in these two cases might
not be due to uncontrolled conditions, I started two new sets on
March 2. The results are given in the next two tables. ‘The ani-
mals used had been kept all winter in the laboratory, and were
under-fed, but for ten days before the operation they had been well-
fed. ‘Iwo of the animals in this set had had their legs cut off
The Physiology of Regeneration 491
some time before and were regenerating new ones, which intro-
duces, perhaps, a disturbing factor in the result.
Tasies SHOWING THE INCREASE IN WEIGHT OF TAILLESS AND INTACT SALAMANDERS
TABLE B TABLE C
PS Paess: 4 Controt SS Sie pulstarsreseh | ContTROL
Rate | | Rate Rate | | Rate
Ole lok Of s| of
sl. ing | | inc. | ince} inc.
ies | | (Intact 1.24
1.46 | | | | 1.68 = = a
atch 2 \tailless : | WEEE oe es .OI 1.40 16
Aes | 2-05 1.48 on 1.53 13
Al 1.30 — | — | _ BOA BE G6 .20 1.63 -20
Hel] Lez S65 2.41 .36 10 1.57 -35 1.67 04
17 |e k= 40 EN |) Baie | .29 17 eis 7 04 1.67 -00
Mion || UXty rad eee 10 2 Ari LO .O2 ep +04
Z1..| 1-96 -29 cee We = oti! BWiSol| eG yu |
one sick | one died | April 7..| 1.96
April7..| 1-93 OC ez i57 | 24 14..| 1.98
one died |
igo! Uae SOO 22.70) | 1g |
The left hand table (B) shows that during the first two weeks
the tailless set again gained faster, but there is a strange rise and
fall in the control set that probably makes the result of little value.
If we take another week into account when the disturbance may
have had time to subside the rate of increase of the tailless set is
still double that of the intact ones.
In the right hand table (C) the control animals increased in
weight at first much faster than the tailless, but later the tailless
ones gained much more rapidly than the control.
Our examination of the tables shows that it would be hazardous
to ascribe the greater initial gain (in three of the former cases)
in the tailless animals to the loss of the tail, although this may be
the case. ‘The difference when it occurs seems too great to be due
to a proportionately greater increase as the result of the loss of a
part; and if not due to variable factors, 7. ¢., accidental, it may
mean that the changes taking place at the cut surface incite the
digestive tract to greater activity or the cells of the body to greater
assimilation.
In the first series (Table A) there is not only an initial greater
492 T. H. Morgan
gain in the tailless set but an actually greater increase in weight
throughout the series. This might be attributed to the influence
of the regenerating tail on the growth of the rest of the body, but
as the difference is not found in the other three series, and in one
set, in fact, the intact animals grew faster, we must conclude that
there is no clear evidence in favor of the view that a regenerating
part has in its later stages at least an influence on the digestive or
assimilative changes that take place in other parts of the body.
The great powers of growth in a regenerating part may be local in
their influence and not transferable to other parts. “The question:
is, however, worthy of further examination.
CONCLUSIONS
In connection with the description of the experiments a partial
analysis of the results has been attempted, and much of the ground
gone over need not be traversed again here, but the more general
bearings of the facts may now be discussed. ‘The problems of
special interest are those connected with the rate of growth at differ-
ent levels, the rate of growth from partial as compared with entire
cut surfaces, and the rate of growth on different parts of the same
oblique surface.
The question whether the differences in rate can be explained as
due to the amount of food available at each level has been suf-
ficiently examined. Ample evidence was found showing that the
differences in rate of growth are not due to differences in the avail-
able food supply. It would be erroneous to conclude from this
that the available food supply has no influence on any of the phe-
nomena of regeneration, for it has been shown that the size of the
new part, for example, is affected by the amount of food, in the
same way as the rest of the body, and it has also been shown that
when starvation has gone beyond a certain point, even the forma-
tion of new parts may be delayed, or stopped before the animal
perishes from hunger. But despite these effects the experiments
show that the rate of formation of new parts as seen in the regen-
eration of the limb of Diemyctylus, and in the growth in length of
the tail of the earthworm and of the salamander takes place at the
same rate, whether the animal is fed or starved, provided there
The Physiology of Regeneration 4.93
still remains enough food for the formation of new material. The
meaning of this relation seems to be that the greater power of
assimilation of a young part makes it possible for this part to draw
the necessary nourishment from the blood, although the amount
present in the blood is below that which is necessary to maintain
in statu quo the differentiated tissues. “These slowly decrease in
size and in the number of their cells, while the new part is increas-
ing in size and in the number of its cells. “The ultimate physio-
logical-chemical basis on which this difference between differ-
entiated and undifferentiated materials rests is entirely unknown
at present. [he most important consideration in this case is that
the material of the new part is derived directly from that of the old,
so that the difference is one of condition only, and is, therefore, a
reversible process. In other words, because a tissue has become
differentiated it has not lost the potentiality of becoming young
again, provided it gives up its differentiation. “This consideration
has a bearing on the problem of the difference of rate at different
levels, as will be apparent later on.
It has been shown in the fish that the rate of growth is retarded
on a partial surface, provided the surface does not connect at one
or at both ends with the rest of the tail. For instance if the tail is
cut off, as shown in Fig. 7A, the outer free cut surface a regen-
erates more slowly than does an entire cross-cut at the same level;
but if the partial surface is continuous with the rest of the tail at
the same level as in Fig. 7A at d, it regenerates at the same rate as
does the entire cut surface at this level. Results similar to the last
are found when a square piece is cut out of the middle of the tail,as
inFig.7B. The proximal, partial cross-cut surface }, continuous
both above and below with the rest of the tail, regenerates as fast
(or faster than) an entire cross-cut at the same level. It is to be
remembered that the longitudinally exposed edge connecting the
cross-cut surface a and J, does not proliferate in a vertical direc-
tion except in so far as to cover the exposed surface and to com-
plete the structure as far as the next fin ray.!. Additions to the
1The explanation of this seems to be that fin rays cannot develop new ones except from the cut
ends of the old ones. If one were split lengthwise it would probably complete itself, but not produce
new ones,
4.04. T. H. Morgan
new part from the basal cut surface are not made from this source,
and the rate of growth of the basal part is not, therefore, increased
in this way, but the growth from the base seems to be faster, never-
theless, along the line of the longitudinal cut surface, as seen for
example in Figs. 7A and B, than at the opposite edge where the
new part is free. In some way the presence of the new material
along the longitudinal edge accelerates in its vicinity the growth of
the new part from the base.
1)
Fic. 7
What factor retards the development of a free, partial cut sur-
face? The retardation is not due to the level, for it occurs at all
levels alike. It is in proportion to the height of the free cut sur-
face, hence the retardation must be in some way connected with
the height of the base from which the new part arises. One might
be inclined to interpret this result as due to a proportionate devel-
The Physiology of Regeneration 495
opment, by which I mean that the regulation of the growth is such
that its rate is proportional to the base from which it arises. But
even if this were the case, it would give no causal explanation of
the results, unless it be assumed that proportionate development
is in itself a vitalistic explanation and a causal one in that sense.
But the facts do not seem to bear out this interpretation, for, while
the growth on a free partial surface is undoubtedly delayed, there
are some indications that it might continue slowly toward the
natural terminus of its development. At least in the gold fish I
have obtained evidence of this sort in two cases, where the lower
lobe grew out slowly to its original length. In Fundulus the evi-
dence is not so clear, but mainly on account of insufficient material.
Now the factor that seems to be responsible for the retardation
in the growth from a free partial surface appears to be one involving
the pressure relations in the new part. Observations show that
the new part from a partial surface is rounded at the sides (as 1s
also the new part from an entire surface), and this condition at the
sides appears to be responsible for the retardation of the rest of
the new part, for, since the rounding 1s nearly the same for a new
part from a partial and from an entire surface, the retarding
influence will be the greater the shorter the base. In contrast with
this retardation from a free partial surface we have seen that when
a partial surface is not free but continuous at one or at both ends
with the rest of the tail, as at b in Fig. 7A, there 1s no retardation.
In this case the side of the new part, that is continuous with the
old part, is not rounded, but even with the rest of the new growth,
hence the rate of growth is not retarded on this side, while on the
free side the relation is the same as when the growth takes place
from an entire cut surface. In fact, the growth of the new part on
the side in connection with the old tail often seems to be accelerated,
especially along the longitudinal cut surface that 1s also proliferat-
ing alittle new material, and this condition is in accord with my
interpretation.
The new part from an oblique surface also shows a retardation
of growth on the more distal parts of the cut surface, and the differ-
ence between the rate of growth on the more distal and the more
basal parts is in proportion to the degree of obliqueness of the
4096 T. H. Morgan
cut surface, as seen in Figs. 7C and D. ‘The retardation in this
case seems to call for the same explanation as that observed on a
partial surface, for, if we consider by itself alone any part of the
more distal surface it may be treated in the same way as a partial
cut surface, because, despite the fact the parts lying next to it at
a lower level grow faster than the part in question, yet owing to
the fact that the part below is still behind that of the lower end of
the postulated part. there will be a retarding influence onthe growth
of the latter. The pull or tension that exists will, on the theory,
hold back the parts just above it. If we imagine this same
influence existing throughout the whole of the new growth on an
oblique surface, we can get an insight into the factor at work, and
see how this case falls under the same head as that of the growth
froma partial surface. Our analysis and experiments with oblique
cut surfaces lead, therefore, to the conclusion that the slower growth
over the more distal part of the oblique surface is not due to its
more distal position, for comparison with cross-cuts at the same
level disprove this interpretation, but the result is due to another
factor. This factor is a formative one in the sense that the fail-
ure of the maximum potential of growth over the more distal part of
an oblique surface is due directly to the new growth below it not hav-
ing reached the same level, and owing to this difference there arises
a pull ortension on the part that retards its maximum possible rate.
Before taking up again for further analysis the principal ques-
tion of the retardation of growth at different levels, I should like
to clear the way still further by referring briefly to the kind of
regeneration from the anterior cut surface of the earthworm (or of
Lumbriculus) and from the oraland basal ends of a piece of the
stem of Tubularia. Both of these cases may appear to stand in
contradiction to the conclusions so far reached in regard to poste-
rior growth in Diemycytlus, Fundulus and Lumbricus. In reality,
as I hope to show, there is no contradiction between the interpre-
tation of the two classes of facts.
When one segment is cut from the anterior end of the earth-
worm, it is replaced by one; when two are cut off, two regenerate;
when three are removed, three regenerate, and so on up to five,
although for five sometimes only four come back. When more
_ The Physiology of Regeneration 497
than five are cut off only five (or sometimes only four) regenerate.
This rule holds only for the anterior end. As the region of the
gizzard is approached (about the 15th segment) the head, if it
develops at all, is abnormal; and behind this level there regenerates
a heteromorphic tail from the anterior end. In Lumbriculus,
when from one to seven anterior segments are removed, the same
number is regenerated; when more than seven, six or seven come
back; and this rule holds for the greater part of the length of the
worm, since there is no such regional limitation in this species for
head formation as in the earthworm.
In the regeneration of the anterior parts in these two species
there is to be observed no such difference in rate from different
levels as seen in posterior regeneration, and this is the relation
referred to above that may seem to be in contradiciton to the con-
clusions reached in the case of posterior growth. But it is to be
recalled that we are dealing here only with the part that is first
laid down, and not with subsequent growth after the terminal or-
gans have been formed. ‘The comparison should properly be made
in the two cases only between the terminal organs. ‘The forma-
tion of the head segments that are all laid down at the same time
is comparable with the formation of the terminal posterior seg-
ments that are formed in allcasesof posterior growth. I have no
observations in the earthworm showing that there is any difference
in the time of formation of the posterior terminal segment from
cuts at different levels, and in the salamander I could detect no
such difference. If such exists the difference is slight and in this
respect the conditions are similar to the formation of a head at
different levels, which also seems to take place at the same time,
although the possibility of slight differences that were not detected
must be granted for both cases. In both head and terminal seg-
ment the centripetal influences seem to be the predominating ones.
These considerations show that, in principle, there is no conflict
between anterior and posterior regeneration. The difference
found in the latter case is due to later growth in the posterior end,
and no such growth takes place in the anterior end.
It has been shown in Tubularia that the time required for the
formation of a new hydranth depends on the distance of the cut
498 T. H. Morgan
surface from the old hydranth.t' ‘The nearer the cut surface to the
oral end the quicker the regeneration. “Thesame law holds also for
the development of the aboral hydranth from the aboral end of a
piece. In both of these cases we are again dealing with the devel-
opment of a terminal organ in the formation of which the centri-
petal influences predominate. ‘Therefore, the difference in the rate
of appearance of the hydranths at different levels involves only the
appearance of a terminal organ, which is, as I have tried to show,
a different problem from that of the growth of an organ as it ap-
proaches the terminus of its growth. This difference in the forma-
tion of the hydranth of ‘Vubularia is due, I believe, to the amount
of stem differentiation at different levels. ‘The gradation of this
differentiation is from the oral to the aboral end of a piece of the
stem. Whether after the hydranth has emerged, the stalk grows
faster from a cut surface nearer the base remains to be examined.
Let us return now to the main problem of the factors involved in
the growth of the new part in the posterior regeneration of the
salamander, fish and earthworm. As a result of removal of
the posterior end there is a proliferation of new materials, and this,
as we have seen, appears to take place at about the same time for
all levels. “The exposure itself may appear to give the stimulus .
that calls forth the proliferation, but it seems improbable that this
is the immediate cause, since the greater part of the proliferation
takes place after the closure of the skin over the wound. It seems
more probable that the real stimulus is to be sought for in the loss
of the connection with the old parts; in other words, to the loss of
the normal pressure relations essential for the normal equilibrium.
I base this inference mainly on the results of grafting experi-
ments in hydra where, when dissimilar regions are united, each
part completes itself at the line of graft, although the actual cut
surfaces are completely united, and the subsequent changes may
not take place for a week or more after the operation, when the
effects of the injury as such must have long since passed away.
The terminal part is quickly formed in the proliferated mate-
rials. Between the terminal part and the old part there is also laid
1This has been shown by Driesch, Stevens and Morgan.
The Physiology of Regeneration 499
down a growing zone that is a normal structure for the posterior
end. I have already given my reasons for supposing that the
growing region has the same potentialities for all levels,t and that
it continues to grow until some retarding influence delays, and
then prevents its further growth. We have also seen that the
retarding influence is connected with the completion of the normal
form, hence it is in the nature of a formative influence. I have
also compared the retardation of a regenerating part to the retarda-
tion seen in the growth of the whale organism. The growth of
many animals slowly decreases as the typical form or size 1s
approached.
In the case of the posterior growth under consideration, the clue
to the solution of the manner of growth is to be found, I think, in
the relation of the new segments or parts to the parts lying prox-
imal to them. At first this i is the old part, and the first segment
develops in relation to this part, the next one develops in relation
to the first new one, and so on for the whole stries. But what, it
may be asked, is the nature of this relation that determines the
formation of the successive parts? ‘The old part has a certain dif-
ferentiation as well as the potentiality of forming the whole of the
distal or other regions. ‘The relation in question must depend
in some way upon differentiation, but differentiation in itself can-
not be assumed to be a formative factor, since we know of no such
influence extending from cell to cell. If, however, the differentia-
tion is an expression of certain pressure relations that have deter-
mined the differentiation and which since they still remain, deter-
mine the pressure relations of the neighboring parts, and determine
the kind of new differentiation that will take place, the new part
thus formed will, in turn, influence the differentiation of the next
new part that develops, and the process will continue until the
completion of the typical form has been accomplished.
The new growth will come to an end when the last formed part
has developed, whose differentiation is of such a kind that the
resulting pressures, thereby established, no longer act as a stimulus
on the growing region to produce another new part. In the forma-
‘An exception for tail formation must be made for the most anterior end, and for head forma-
tion in the region behind the gizzard of the earthworm.
500 TY. H. Morgan
tion of a new tail the pressure relation is a gradually decreasing
quantity, and along with this decrease there goes a decrease in the
stimulus to further growth that ultimately comes to an end. This
analysis shows why there should be a gradual slowing down of the
regeneration as the normal form 1s approached, and it 1s apparent
thet this retardation will be the same whether it occurs near the
end of an old part, or, as a new part approaches completion; for,
on the hypothesis, the conditions will be the same in each. ‘The
hypothesis gives at least a formal explanation of the facts,an@l
can find no other that will. The most problematical part of the
hypothesis is, I think, the assumption regarding the nature of the
influence of the formed part upon the unformed part. I have
assumed this to be a pressure relation of some kind. Possibly
some other condition may be found that expresses this relation
more correctly, but the remainder of the argument may stand even
if it be found that the nature of the influence is different from that
which I have assumed. My assumption has, however, the advan-
tage that it puts into the same category the influences that deter-
minate the formation of a terminal part, and the subsequent
growth of a posterior end, namely, a condition of pressure or ten-
sion. My pressure hypothesis has also the advantage, I think,
that it involves only a known quantity. It appeals on the
whole to phenomena that are known to occur in living things;
for, response to pressure, or stereotropism in adult animals and
plants is well known. ‘That growth is influenced by pressure
is also known. Less familiar perhaps is the assumption that dif-
ferentiation is itself a response to a pressure relation rather than
due only to the kind of material contained in a cell, although the
latter also may be a factor that enters at times into the result.
I have expressed elsewhere the idea that polarity is an expres-
sion of the gradation of differentiated materials. We may now push
the analysis further and refer the polarity to a gradation in the
pressure relations, since these are the dynamical expression of the
eradation of the materials, as shown in their differentiation.
These differences can be traced to the egg where the differences
in the pressure relations of the cells give rise to the later
differentiation.
HYDRANT FORMATION: AND° POLARITY IN
TUBULARIA
BY
T. H. MORGAN
The two problems of hydranth formation and of polarity in
Tubularia have much in common, yet, as | shall try to show they
may sometimes involve fundamentally distinct factors. Failure
to distinguish the proper field of each has led, I believe, to unfor-
tunate and unnecessary confusion.
By polarity we mean in Tubularia the formation of a hydranth
on the oral end, and of a stolon on the basal end of a piece. But
since in Tubularia a hydranth not infrequently develops also on
the basal end of the piece, it may appear that our definition of
polarity has no real significance. I do not think that this is the
case, although unquestionably the problem needs further analysis.
Inasmuch as a hydranth may develop at every level, it follows
that every part has the capacity to produce this organ. Why then
does it not develop equally on an oral or on a basal end? ‘Theo-
retically both ends have the possibility of forming a hydranth, but
there are two facts that show that the conditions are not the same
on these two cut surfaces. First, when an oral and a basal end of
halves of the same piece are exposed at the same level (the other
ends respectively of the pieces being tied) the cut oral end develops
its hydranth before the cut basal end. Second, while a stolon often
develops from the basal end, and can easily be called forth, it is
the rarest occurrence for a stolon to develop from the oral end,
even when a hydranth 1s present on the basal end and a new cut
is exposed near the oral end. Hence I| believe we are warranted
in using the term polarity in Tubularia, although the distinction
between the two ends is less apparent than in other cases in which
axial heteromorphosis does not occur.
JOURNAL OF EXPERIMENTAL ZoOLoGy, Vou. 111, No. 4.
502 T. H. Morgan
In my last paper’ on regeneration in Tubularia I gave a number
of experiments to show that when a piece is tied at its oral and its
basal end, and is then cut in two in the middle, the oral cut end of
the basal half develops its hydranth before the basal cut end of the
oral half. I have repeated this experiment again on a much larger
scale. “There was one difference in the conditions of the two ex-
periments, viz: in that in the present case the basal piece was not
tied at its basal end because another experiment had shown that
a basal ligature has no influence on the rate of development of
the oral hydranth. ‘The basal piece developed in practically all
cases its oral hydranth before the basal hydranth of the oral half
appeared. ‘The result is, therefore, the same as when the basal
end of the basal piece is ligated.
THE CONDITIONS ON WHICH THE DEVELOPMENT OF THE ABORAL
HYDRANTH DEPEND
In a former paper? I suggested that the suppression of the hy-
dranth at the basal end of a piece, open at both ends, is due to the
more rapid development of a hydranth at the oral end. If this
hypothesis is correct we should expect the converse to be true and
anticipate a delay in the formation of the oral hydranth if the basal
hydranth could be made to develop first. This can be done by
tying the oral end of a piece first, and then, as soon as the
basal hydranth has begun to develop, or even after it has emerged,
by cutting off the oral end of the piece below the ligature. Under
these conditions I find that the development of the oral hydranth
is delayed or suppressed for a time.
It would seem, therefore, that something 1s used up in the form-
ation of the hydranth that is necessary for its development, and
that whichever hydranth develops first uses up this something so
that the other hydranth fails for the time to develop. But the con-
ditions seem to be more complicated than this, as I pointed out,
and as the following considerations will show:
“Polarity” considered as a Phenomenon of Gradation of Materials. Jour. Exp. Zodl, ii, 1905.
?Morgan, T.H. An Attempt to Analyze the Phenomena of Polarity in Tubularia, Jour, Exp.
Zool, i, 1904.
Hydranth Formation and Polarity in Tubularia 503
If a long piece is cut into a number of pieces 2 to 5 mm. in length,
each will develop its oral hydranth in the same time that a long
piece cut off at a corresponding level develops its hydranth.
Therefore, a long piece must be capable of forming a far greater
amount of the something necessary for hydranth formation than
required for its oral hydranth alone. Why then does not the basal
hydranth also develop?
By means of a separate experiment I found that a short piece
(cut off below the hydranth) and a very long piece (cut off with
its distal cut end at the same level) develop their oral hydranths in
the same time. It might seem, therefore, that the effect produced
involves only the immediate region near the cut surface, and the
breaking down of the ridges in this region during the time of
hydranth formation suggests that the active factor is the material
of the ridges that is thrown into the circulation, but I have come to
question whether this material is the something demanded; for as
I have already pointed out, it is not obvious why the basal end
might not also break down, and develop its hydranth by the use of
the material thus set free, at the same time in which it develops
its basal hydranth when the oral end is tied. In fact, in a certain
small percentage of cases an oral and a basal hydranth develop on
a plece at the same time—or very nearly so—and, more rarely, a
basal hydranth may develop while the oral hydranth is delayed
or suppressed. In very short pieces, less than the length of the
normal primordium of the hydranth, double hydranths and par-
tial double hydranths are of common occurrence. ‘Therefore, I
conclude that the basal hydranth fails to develop because it does
not receive the stimulus necessary to start its development what-
ever the nature of this stimulus may be. Driesch has assumed
that the stimulus calling forth the hydranth is the action of the salt
water on the cut end. [I tried to test this view by sticking the basal
ends of long pieces into sand with only the basal part of the
pieces surrounded by water. ‘The oral ends were an inch or two
above the water and surrounded by air. The air was kept satu-
rated with moisture by covering the dish. Under these conditions
the oral hydranth developed as rapidly, possibly more so, than
when surrounded by water. The result does not conclusively
504. Terie Morgan
show, however, that the action of the water may not be an impor-
tant factor, since the surfaces above water were of course moist; yet
the results do seem to show that we must be cautious in accepting
Driesch’s explanation as the only interpretation.
Another experiment bearing on the question under considera-
tion is the following. ‘The oral end of a piece was tied and then
after twelve hours, before the basal hydranth had developed, the
oral end was cut off below the ligature and the basal end tied distal
to the region that produces the hydranth. The oral hydranth did
not develop any sooner (1. e., in actual time) or at most very little
sooner than it does when simply cut off. “The result shows that in
a tied piece the oral end does not undergo the changes prepara-
tory to hydranth formation (as I thought probable at one time),
and the more rapid development of the basal hydranth when the
oral end is tied cannot be due to materials set free from the oral
end that act as a stimulus for the basal development.
I must here go over again two experiments described in my last
paper that appear to have a further bearing on this problem. I
found that if pieces were cut off and allowed to remain with both
ends open for four, six, eight or twelve hours, and then the oral
ends were tied, the basal hydranths often developed as fast, or
nearly as fast, as the control pieces tied at once. It might ap-
pear that changes are taking place in the aboral end of such a
piece, open at both ends, that are leading to hydranth formation,
and that ordinarily the changes are only retarded by the more rapid
development of the oralend. That this is not the real explanation,
plausible as it may seem, is shown by another experiment. If, as
before, pieces are cut off and allowed to stand for several hours,
then tied at the oral end as before, but at the same time several
millimeters of the basal ends are cut off, these pieces produce their
aboral hydranths in the same time, or nearly so, as do those whose
basal end is not cut off after tying. The result shows that the ac-
celeration of the aboral hydranth is not due to preliminary changes
in that end, or at most only in a minor degree. Of course, if too
much time is allowed to elapse before the oral end istied, the con-
trol develops first, but a distinct hastening of the aboral develop-
ment 1s nevertheless observable. I have found, for instance, that
Hydranth Formation and Polarity in Tubularia 505
if the tying is delayed for twelve hours, the aboral hydranth is
generally later in appearing than that of the control, but not twelve
hours later, and in very favorable cases I have found them develop-
ing at the same time.
Evidently then the results cannot be ascribed to changes in the
aboral end, and probably not to changes in the oral end, but must
be due to something that takes place throughout the entire piece.
On this “something” depends the more rapid aboral development
when the oral end 1s tied.
Another experiment, designed to test this supposition, 1s impor-
tant, and although I have repeated it time after time I still feel the
same doubts as to the result that I spoke of in my last paper.
Pieces of the same length were cut off near the oral end of the stalk.
It is of much importance to have the pieces of the same length znd
from the same region, since the rate depends on the distance of the
aboral cut-surface from the cut end. For comparison, therefore,
the cut ends must be at the same level; but in addition to this the
rate differs in pieces of different ages, and although I have
attempted to pick out similar pieces, it is practically impossible to
determine accurately the age of the piece. A small piece that has
newly arisen as a bud develops more promptly than one of the same
diameter that has failed to develop fully owing to crowding by the-
larger pieces. Pieces of the latter kind are unfavorable for com-
parison. With this understanding concerning a source of error the
experiment may now be described. After four, six, eight and twelve
hours, or at other intervals, ligatures are tied around the pieces;
in some of them near the oral end, in others near the middle, znd
in others near the base. “The development of the basal hydranth
in these three kinds of pieces was then compared. ‘The results
indicate that the basal hydranths in the three kinds of pieces
develop at nearly the same rate, although the shorter bzsal pieces
are often behind the other two, as I found in previous experiments
of this kind, but the delay when it occurs does not seem to be in
proportion to the relative lengths of the pieces between the liga-
ture and the basal end. In my former paper | stated that the
experiment might show whether the materials set free in the circu-
lation affect the development at the baszl end, because in the longer
506 1. H. Morgan
piece tied near the oral end there would be more of such material
shut off than in the shorter pieces tied nearer the base. ‘This inter-
pretation of the experiment now seems to me erroneous. ‘There
would be quantitatively (7. ¢., absolutely) more material in the
longer piece, yet the relative amount is the same and it is the
relative amount to a given volume that must act as a stimulus
on the basal end. Consequently we should not anticipate quicker
stimulation in one case than in another. On the other hand if the
substance 1s notgiven off equally fromthe entire wall of the piece and
if relatively less is given off from the basal region than from the oral
region some difference in the time of basal development might
follow. It may be questioned, however, whether the difference,
sometimes found, is due to this rather than to some other factor.
It seems to me that more emphasis should be laid on that side of
the results that shows clearly there is no proportion between the
length of the shorter piece and the basal development, rather than
on the apparently slight difference sometimes observed in the dif-
ferent cases.
Before coming to closer quarters with our problem a few addi-
tional results must be briefly given:
In a few cases a series of ligatures were tied at intervals around
the oral end of a piece. If the changes induced by closing the
anterior end introduces a factor that accelerates the development,
a number of successive ligatures might be imagined to accelerate
the aboral hydranth, but no acceleration was observed.
It has been stated that a single ligature at the basal end does
not hasten the oral development. Also a series of successive liga-
tures, several hours apart, does not accelerate the oral develop-
ment.
If the development of the oral end retards the basal develop-
ment, it might be supposed, conversely, that, if any changes take
place at the basal end, successive removals of the basal end might
accelerate the oral development; but no such result was found.
This observation is in accord with other experiments that seem to
show that as a rule no development, or very little, takes place for
a time at the aboral end.
On the other hand, removal of short millimeter pieces from the
e) oa
Hydranth Formation and Polarity in Tubularia 507
oral end at intervals delays the development of the oral end but
only a very little provided the intervals are not too long. For
example, 1 in one case a short piece was cut from the oral sel of a
long piece after five hours and another from the same piece after
eight hours. ‘The primordia appeared thirty hours after the stems
had been removed from the colony, and at the same time (or so
nearly so that little or no difference could be detected) in the con-
trol and in the pieces twice operated upon. In another experi-
ment short pieces, I to 3 millimeters in length, were cut from the
oral end of different pieces at different times, 7. ¢., not from the
same piece, successively as before, at different times. ‘The tips
of some pieces were cut off after six hours, from other pieces after
nine hours, and from other pieces after twelve hours. In the con-
trol a short piece of the same length was cut off at once. It was
found, when the primordia appeared that the six and the nine hour
pieces developed at nearly the same rate; the twelve hour pieces
were somewhat behind, but not, apparently, twelve hours behind.
The controls were like the six and the nine hour pieces, but pos-
sibly a little ahead of them. ‘Thus it appears that some effect is
produced by cutting off the oral parts, out of which the hydranth
develops, but not in proportion to the intervals. ‘There was
another series of operations in the same experiment. Long pieces,
similar to the last ones, were removed at a much greater distance
from the oral end, entirely beyond the region of polyp-formation.
It was to be expected that the new oral ends would develop
mote slowly, because the cut surface is more basally situated. It
was found, in fact, that these pieces were somewhat behind the
preceding ones, but the six and the nine hour pieces developed at
nearly the same rate, while the eleven hour pieces were a little
retarded. There was need of a control here that was not made, so
that any deduction from the results is unsafe; but it did not appear
that the six and nine hour operation had much delayed the devel-
opment within the period of thirty hours.
From these last experiments it seems probable that a change
takes place in the whole piece that leads up to the formation of the
hydranth, as well as changes at the oral end itself. An accelera-
tion in the formation of the hydranth results even when the oral
508 T. H. Morgan
end out of which the hydranth develops is removed, provided the
removal is not too long deferred. ;
The rate of development in most of these cases had been judged
by the time of the first appearance of the primordia as indicated
by the end of the piece becoming red. ‘There is allowed here
some leeway, unfortunately, for personal judgment. In all cases
the time of emergence of the polyps was also recorded, and in gen-
eral the two results are found to coincide. ‘The latter method is
more exact in some ways, but since the polyp may not emerge until
twelve to twenty-four hours after the first appearance of the pri-
mordia, it has been found better, on the whole, to judge the rate
by the appearance of the primordia rather than by the emergence
of the polyp, since there is a shorter time between the operation
and its result, so that the effects are less likely to be complicated
by other conditions.
It has been stated that in a small percentage of cases, when the
pieces were left open at both ends, there was, in this species, an
almost simultaneous appearance of oral and basal primordia. We
must assume that pieces of this kind were also present in the experi-
ments and these may tend to confuse the results. It is, therefore,
unsafe to rely on the result of one or two pieces in a series and a
larger number of cases must be recorded. In practice this source
of error is difhcult to control, but I do not think it has vitiated the
results seriously.
When pieces are cut at intervals from the oral end there is. a
noticeable, and often a marked, increase in the number of basal
hydranths, especially if the pieces are not too long. If very long,
the time required for the basal end to produce its hydranth is so
much greater than that of the oral end, that the latter is stimulated
to produce a new hydranth before the aboral end can begin, hence
the latter is kept incheck. Very short pieces, especially those from
the oral region, often produce “double hydranths,” I have com-
pared the development of such pieces with that of similar pieces
producing only the oral structure, and, so far as I could make out,
both developed at the same rate. In other words, the simulta-
neous development of the basal polyp does not seem to hold in
check the oral polyp, provided both start at the same time. I
Hydranth Formation and Polarity in T ubularia 509
have also observed in longer pieces that when both oral and _ basal
primordia appear at the same time that such pieces produce their
polyps as soon as do pieces that make only oral polyps. ‘The re-
sult seems somewhat paradoxical, but goes to show, I think, that
the retardation of the basal polyp is ordinarily due to its failure to
receive the proper stimulus to development, rather than to any in-
herent lack of latent food-producing properties in the piece. “The
stimulus once received, however, the development can go on simul-
taneously with that of the oral polyp, neither suffering retardation.
If this interpretation is correct it brings us a step nearer the solu-
tion of our problem.
Such is the evidence that I have been able to collect. The ap-
proach of warm weather has prevented further experimentation,
since the Woods Hole Tubularia develops poorly above a certain
temperature. Enough has been gathered, however, to throw some
further light on the two questions of polarity and hydranth forma-
tion.
CONCLUSION
The polarity of Tubularia has something to do, I think, with the
differentiation and stratificationof the materials as shown bythe dif-
ference in the behavior of the two cut ends of a piece. ‘The entire
stem at every level has the potency to produce hydranths and
stolons, but the kind of structure produced, and the rate of appear-
ance of the hydranths at the oral and the aboral ends shows clearly
that there is something in the sequence of the layers or in the direc-
tion that is a factor in the result. “This influence may be overcome
by more powerful factors as when a hydranth develops from a
basal end, or, as occurs more rarely, when a stolon develops from
anoralend. ‘This reversal does not, however, mean that no polar-
ity exists. The question arises as to the nature of the postulated
stratification. At one time it seemed to me not improbable that
it might relate simply to the relative age of the stem at different
levels. The growth in length of the stem takes place apparently
just below the hydranth so that the younger parts are always the
more distal parts. Hence it might appear that the age of the ma-
terial gives the stratification. I soon abandoned this idea because
510 T. H. Morgan
Stevens and I found that when a newly formed stolon, whose grow-
ing end is its newest part, is cut off the new hydranth appears on
the end originally nearer the old part; in other words on its oldest
end. The new stolon shows, therefore, the same polarity as the
stem; in fact, the stolon is only a continuation of the stem, and is
a root only in the sense that it sticks to the substratum.
That younger pieces regenerate more quickly than older pieces
was shown in an experiment in which old and young pieces
from the same colony were compared. I also removed some long
pieces with short lateral branches that arose near the base. ‘The
polyps were cut from the old stems and from the young branches.
‘The young branches formed their primordia much sooner than the
old stems. Each had been cut off just below the hydranths. This
result shows that the old tissue of the stem becomes young again
when it produces a new branch, at least so far as the material of
the branch itself is involved.
The youthfulness of the stems is, therefore, an important influ-
ence in determining its rate of regeneration, but will it explain the
phenomena of the polarity of the stem? I have suggested that the
stratification is due to the relative amount of hydranth forming
material at every level, without attempting to define more precisely
what this material may be. I can now, I think, give a more sat-
isfactory definition of this relation. ‘The farther the level of the
stem from the hydranth the greater its differentiation as stem,
hence its gradation of differentiated materials and hence the longer
road it must retrace to produce another structure, the hydranth.
This differentiation into stem means that the latent capacity to
form a hydranth can be less easily called into action. ‘That it
can be awakened, however, is shown by the regeneration of a
hydranth at each level when the stem 1s cut; and also by the form-
ation of a bud, which means the local awakening of a hydranth
at the expense of the old differentiated material. In the new
branch, therefore, we also get a quicker response in hydranth
formation than in the old stem at the same level.
It may appear that the behavior of pieces of the stolon, men-
tioned above, contradicts my hypothesis, because the part of the
piece that develops its hydranth, while nearer, it is true, to the
Hydranth Formation and Polarity in Tubularta 511
old hydranth, is a new formation not connected with hydranth
development at all, but with stem formation. In reality there is
no contradiction here, because the tip of the stolon is a structure
sui generis, and its stratification is from its tip inward. ‘The part
nearer the old stem has, therefore, less developed the stolon making
qualities and more those of stem, hence the hydranth is more easily
developed at this oral end where the conditions that call forth stolon
formation are less active.
Loeb stated that when a ligature is tied at the oral end and an
abora] hydranth develops the polarity of the whole piece has been
reversed. ‘That this is not the case was shown, convincingly, I
think, by an experiment of Stevens and myself. We tied a ligature
around the oral end, and then, when zn a2 sboral hydranth had evel
oped, we cut the piece into small parts kept carefully oriented.
Nearly all the small pieces produced new hydranths at the original
oral ends, and the only exceptions were those from the region near
the new basal polyp. In other words, the polarity has been
changed only in the immediate vicinity of the new basal polyp, and
the rest of the stem retains its original orientation or stratification.
That its polarity might in time become changed is patent, but
that it is not immediately changed by the presence of an aboral
hydranth is shown by the experiment. In other words, the devel-
opment of the basal polyp in a piece tied at the oral end is not due
to a reversal of the entire polarity, but due to local conditions at the
basal end, calling forth the development there of a hydranth, which
leads to local changes in the material involved.
Loeb’s theory? that polarity as an expression of the direction of
the current in the digestive tract has been fully considered by
Stevens and myself in an earlier paper. Loeb’s idea that the red
pigment is a hydranth forming substance has also been there con-
sidered. His rejoinder to our criticisms Is that it is inconsequent
on our part to imply that some of the red pigment may not be
hydranth forming stuff because a large part of it 1s thrown away
but this reply fails to showin the least that the red pigment assumed
‘Loeb, I, Concerning Dynamic Conditions which Contribute Towards the Determination of the
Morphological Polarity of Organisms, University of California Publications. Physiology, i, 1904.
512 T. H. Morgan
to be remaining hashad sucha function. ‘The reply of Loeb ignores
also a number of other results that we obtained that indicate that
the red pigment has no such role, for our criticism was not based
on the ejection of the red pigment alone.
Ihave tried not to lose sight of the possibility that the polarity may
be an expression of a fundamental stereometrical arrangement of
the ultimate structure of the cytoplasm. ‘To imagine a network of
this sort running through the differentiated organs might form an
attractive speculation, but would be simply fanciful in the present
state of our knowledge. If we imagine a stereometric network as a
part of the specialized structure, we must be prepared to admit that
it changes at each level as the structure changes. ‘Therefore, it
seems to me simpler to base our hypothesis of polarity on the differ-
ence in differentiation itself, and not on an imaginary polarized
system associated with the living materials.
The other question, with which the present experiments are
more particularly concerned, relates to the factors that hold in
check the development of the aboral polyp. This may seem a
trivial question in itself, yet in principle it involves some of the
most obscure points in regeneration, and for this reason I have
studied it in detail, for it seemed to me that if we could give an
answer to this question we have made a step in advance in the
study of regeneration in general. While I do not pretend to have
solved this problem, still the experiments permit us, I think, to push
the analysis further than was possible before.
Without going over the ground already covered in the preceding
account of the experiments let us attempt to scrutinize the results
more closely. It has been shown that the development of the oral
polyp is responsible for the retardation of the basal polyp. Con-
versely if a basal polyp is caused to develop first 1t may temporarily
hold in check the formation of the oral polyp. Our problem has
narrowed itself to the determination of the nature ofthisfactor. The
analysis seems to show that something must be set free in the stem
that is necessary to stimulate the formation of the polyp, and also
that the something is used up by the developing polyp. ‘The
stimulus is internal not external. If it were external we could not
explain why the basal polyp 1s delayed when the oral polyp develops,
Hydranth Formation and Polarity in Tubularia 513
or why it begins to develop as soon as the oral end is tied. It seems
plausible that the stimulating agent is some material, set free either
at the ends or by the entire wall, that is a storehouse of reserve
materials. ‘To call it hydranth forming material begs the question,
and introduces an unnecessary assumption since there 1s no need
to postulate such a substance, inasmuch as the cells of the stem in
every part are themselves capable of developing into a hydranth,
the more rapidly the less they are differentiated in other directions.
If we assume that a stimulating substance is set free, we must then
assume that it 1s used up in the development of the hydranth,
which after all is exactly the sort of thing a food substance is
expected to do.
When the oral end is tied no hydranth develops there, hence the
food substance accumulating soon starts the aboral development.
Once started the development continues without further need of
the stimulus, because possibly in the changes that have been initi-
ated enough material has been set free to give all that is needed for
development, or possibly because a process of this kind once started
can and must continue even if the stimulus that started it is removed.
This view is necessary in order to explain the simultaneous
development of oral and aboral hydranths that sometimes occurs.
It may be asked whether the stimulating material is set free only
near the cut ends or throughout the piece. Since the piece has
potentially the power of starting a dozen or more polyps, as shown
when it is cut into many pieces, | think that it is more probable
that the materials are set free throughout the piece, although possi-
bly more near the cut ends. The material must be soluble and
pass into circulation, for otherwise the basal hydranth would
develop irrespective of what is taking place at the oral end. The
experiment of cutting off short pieces from the oral end after certain
intervals shows that the region involved must be more extensive
than that occupied by the primordium of the hydranth. The
greater frequency of basal hydranths under these conditions shows
that initial changes have taken place at the basal end also, but that
they are not very great is shown by the experiment of cutting off
the basal end, and finding that the basal hydranth of a piece tied at
the oral end still develops almost as fast as when left uncut.
514 T. H. Morgan
Why, it may be asked, does the basal hydranth begin to develop
when the oral end is tied? Must we suppose that whether it
develops or not (as when the oral hydranth is developing), it is still
setting free materials? “To answer this question we must turn to the
experiments that involve tying off different lengths of basal ends.
These experiments show that a longer tied-off part produces a basal
hydranth only a little sooner than a shorter tied-off part. If
these results are confirmed on a larger scale with more abundant
and favorable material they would seem to mean that the
material set free, that acts as a stimulus involves a greater part
of the stem than that of the immediate hydranth forming region.
That the stem has the capacity to set free much more than this
has been indicated. It appears, therefore, that we are dealing
here with one of those characteristic cases of organic equilibrium,
not uncommon in growth phenomena and starvation periods. ‘The
stem, isolated from its feeding organ, the hydranth, slowly sets free
in the fluid food materials from its reserve supply. ‘This material is
drawn upon by the first hydranth to develop, usually the oral one.
The balance must be continually made good by the stem until the
hydranth is finished. Should two hydranths start at the same time
double the material is used up, and in order to maintain the
equilibrium, double the amount must be set free by the rest of the
stem. But if the amount set free is used up at the same rate by the
oral hydranth, the aboral hydranth does not get the stimulus
necessary to begin its development. Should it once begin, how-
ever, it proceeds without regard, or with little regard, to the
amount set free by the stem, which will tend nevertheless to become
greater, the greater the difference between the amount in reserve
and that in the circulating fluids.
A number of experiments were made to test whether when a
piece (open at both ends so that the oral end begins to develop) is
tied after a time, the basal development is hastened even more than
when the oral end is tied at once. This seems to be the case, but I
do not think that it can be due to the materials set free at the oral
end. Whatever materials are set free must be used up by the oral
end as soon as it is in excess, otherwise the basal end would start.
Hyrdanth Formation and Polarity in Tubularia 515
It seems much more probable that the basal acceleration is due to
changes having been initiated in the stem that leads to the rapid
formation of materials that have been taken from the fluids by the
oral end. ‘That end being suddenly closed the surplus becomes
quickly sufficient to stimulate the basal polyp. But, as explained,
the difficulties and uncertainties of this experiment make it unde-
sirable to lay too much stress upon its results.
My analysis leads, therefore, to the following interpretation of
polyp formation. ‘The regeneration is due to changes set up in the
stem resulting from the separation of theold polyp. Thestimulusis
largely internal, although another factor, the presence of an open
end, is also essential, as shown by closing the end by means of a
ligature in which case no polvp develops.t_ The oral end develops
first, both because it is a younger part (1. ¢., less differentiated
stemward), and because it has the direction of differentiation for
hydranth formation. Its development holds in check for a time
the basal hydranths, because the hydranth that first develops uses
up or may even deplete the circulating fluids of its surplus food
supply. Itis well known in other cases of regeneration that a grow-
ing part will growat the expense of old parts. Whenthe oral end is
tied the food supply in the fluids of the stem soon rise to a point
sufficient to start the basal development. The growth process
once started is powerful enough to draw from the common body
fluids or other sources sufficient material for its further develop-
ment.
‘If tied very near the old polyp, where the cuticle is thin, an oral polyp sometimes develops
behind the ligature,
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PRUE aK
SlUDIES ON THE DEVELOPMENT OF THE STARFISH
EGG
BY
D. HoH: TENNENT AND M. J. HOGUE
With Five Pirates
INTRODUCTION
The studies made in the preparation of this paper have led to
the view that a conjugation of sperm and egg chromosomes takes
place soon after fertilization in eggs which have been treated with
CO, and subsequently fertilized. “Thee de process takes place in
normally fertilized eggs is suggested by the similarity in shape of
chromosomes in eggs “oe these two classes, but no detailed study of
the processes occurring in normally fertilized eggs has been made.
This interpretation is made with caution and it is recognized
that its truth can be determined only by a reinvestigation of the
processes occurring in normally fertilized eggs, or better, by a care-
ful study of the changes taking place during the formation of the
germ cells.
Inasmuch as most of the accounts of the cytological processes
occurring during artificial parthenogenesis have been based on
experiments performed on eggs which had given off their polar
bodies while the egg was still within the ovary, it seemed that
further observations on an egg which might be subjected, either
before or after the extrusion of the polar bodies, to influences cap-
able of causing parthenogenetic development might be of interest.
'The experimental work on starfish eggs, described in this paper, was done by the senior author
in the summer of 1905, at the Marine Biological Laboratory, at Woods Hole. Some of the
material then obtained has been studied during the year 1905-06 in the Biological Laboratory of
Bryn Mawr College. Miss Hogue has studied the eggs developing as a result of treatment with
CO, sea-water and has written the account of the nuclear changes, seen in sections of these eggs,
given in section 1 of this paper. For the remainder of the paper the senior author is responsible,
THE JouRNAL OF ExPERIMENTAL ZOOLOGY, VOL. III, NO. 4.
518 D. H. Tennent and M. fF. Hogue
The eggs of the starfish (Asterias forbesii) lend themselves to
such eigen ations. These eggs, as is well known, if ripe when
removed from the ovaries and allowed to remain in sea-water, soon
mature, and further, these eggs, like the eggs of other Echinoderms
and unlike the eggs of Molluscs and Annelids, may complete their
maturation phenomena before the entrance of the spermatozoon.
Delage’s accounts (02, ’04) of the use of CO, in the treatment of
the eggs of Asterias glacialis naturally suggested that his conveni-
ent method might be found useful in experiments on the eggs of
Asterias forbesil.
Delage ('02) made use of a siphon in which sea-water was
charged with CO, by means of sparklet bulbs. His best results
were obtained with eggs which were subjected when in the “‘stade
critique”’ to the action of charged water for an hour, after which
the eggs were removed to ordinary sea-water.
Doses the work of which this paper is an account it was soon
recognized that the duration of immersion mentioned by Delage as
most favorable for the eggs of Asterias glacialis is much too long
for the eggs of Asterias forbes. The eggs of this starfish when
allowed to remain in CO, sea-water' for more than half an hour
were apparently killed, as they disintegrated without undergoing
development.
Eggs in various stages of maturity were subjected to the action of
CO, sea-water for varying periods of time. ‘The details of these
trials are unnecessary. It is sufficient to say that the egg of
Asterias forbes, like the egg of Asterias glacialis, is in its most
favorable condition during the time that elapses between the
extrusion of the first and of the second polar body. With the eggs
of Asterias forbesii it was found that uniformly the best results
were obtained when the eggs were subjected to the action of the
CO, sea-water immediately after the appearance of the first polar
body as a protrusion from the surface of the egg.
The best length of time of immersion was about five minutes.
Good results were obtained by the immersion of the eggs in CO,
‘Throughout this paper the term “CO, sea-water” will be used instead of the longer phrase
“sea-water charged with CO,.”
The Development of the Starfish Egg 519
sea-water for from three to ten minutes, but five or six minutes gave
uniformly the best results. However, in one lot of eggs in which the
time was ten minutes, fully 95 per cent of the eggs segmented
regularly and gave rise to normal swimming embryos which were
kept alive and under observation for more than a month.
It seemed that the best results were obtained when the sea-water
was charged and allowed to stand in the siphon for from ten to
twelve hours before using.
Method of Treatment
The eggs were shaken from the ovaries into large dishes of sea-
water, in which they were allowed to remain until the first polar
body had made its appearance. ‘They were then drawn with a
pipette from the bottom of the dish in which they had partially
matured and transferred gently and with as little water as possible
to finger-bowls. “The CO, sea-water was then run slowly into the
finger-bowl until the bowl was about half-filled. During the whole
operation, care was taken to avoid violent agitation of the eggs.
The eggs settled to the bottom of the bowls in about three
minutes. At the end of the desired period of immersion, the CO,
sea-water was withdrawn and replaced by ordinary sea-water, this
in turn being changed for fresh sea-water as soon as the eggs had
again settled.
The method is convenient in its application and for use with the
starfish egg offers an ideal reagent.
T he Scope of the Investigations
Since the method is so sure in its action, and since many of the
developmental processes reproduce so faithfully the processes
occurring in the normally fertilized egg, it seemed that we might
have in the starfish egg treated with CO, the means of imitating
the processes of normal parthenogenesis occurring among rotifers,
crustaceans and insects.
Delage (01) as a conclusion from experiments in which the
eggs of Asterias glacialis were subjected while in the critical
stage (in this case the time when the germinal vesicle loses its
membrane and prepares for the emission of the polar globules),
520 D. H. Tennent and M. Ff. Hogue
to the action of a solution of KCl, expressed the idea that arti-
ficially parthenogenetic eggs, like those in which parthenogen-
esis occurs naturally, emit but one polar body, and that the agent
producing parthenogenetic development acts by the inhibition
of the formation of the second polar body, the second polar body
playing the role of a spermatozoon.
Delage in 1902 found that eggs submitted to the action of CO,
during the time between the disappearance of the nuclear mem-
brane immediately preceding maturation and of the return of the
nucleus to the resting condition ordinarily preceding fertilization,
developed independently of the polar globules. Parthenogenesis
(02, p. 231) resulted whether the egg had given off neither, or
one, or both of the polar globules.
Subsequently (04), by a somewhat more complicated method
of treatment, he was able to produce parthenogenetic develop-
ment, using CO, as a reagent, in sea urchin eggs in which he
knew that iherh polar bodies had been given off.
In my work on the eggs of Asterias forbesii it became apparent
that although the sean polar body could not be seen in many
of the eggs, it made its appearance in many, and that the eggs
developed into swimming embryos in both cases. It was found
later that the second polar body was formed in every case,
although it might remain within the egg membrane in a cup-
shaped depression in the surface of the cytoplasm.
But, although both polar bodies were extruded, thus removing
the possibility of imitating exactly the processes of normal par-
thenogenesis, there was as yet no evidence that part of the
chromatin normally extruded in the second polar body was not
retained within the egg and that this might later assume the
functions of the sperm nucleus.
In consequence of this possibility, it seemed that if it were
possible to fertilize the egg after its subjection to the action of
CO,, this retained chromatin might be rejected or at least some
series of changes might be caused that would be of interest when
compared with the normal maturation and fertilization stages
and with those occurring in the egg which had been induced to
develop by treatment with CO, sea-water.
Fal
2
The Development of the Starfish Egg 521
This idea involved the question of the comparative effective-
ness of the CO, solution and of the starfish spermatozoon on the
egg of the starfish.
After determining that it was possible to fertilize eggs after
they had been immersed in CO, sea-water, the question quite
naturally arose: What will be the result of treating fertilized eggs
with CO, sea-water ?
The results of the investigations may be discussed to the best
advantage in three sections.
Section 1 embodies the observations on living eggs after their
treatment with CO, sea-water and the data obtained from a
study of the sections of the eggs so treated.
Section 2 contains the data obtained from a study of eggs
which were treated with CO, and subsequently fertilized.
Section 3 gives the result of the examination of eggs that were
fertilized and subsequently subjected to the action of CO, sea-
water.
I. UNFERTILIZED EGGS TREATED WITH CO, SEA-WATER
a. Observations on the Living Eggs
The time required for maturation varies, as is well known,
with the condition of the egg, the surrounding temperature, etc.,
so that any facts which were observed as to variations of this
kind are without value in this connection, but it is of importance
to notice the influence of CO, sea-water in delaying the comple-
tion of maturation.
In one lot of eggs the first polar body was given off one hour
and ten minutes after the removal of the eggs from the ovary.
In the eggs of this lot treated with CO, sea-water the second
polar body appeared one hour and fifteen minutes after the first
had been extruded or two hours and twenty-five minutes after
removal from the ovary.
In the stock dish a quantity of the same lot of eggs were allowed
to complete their maturation undisturbed and the second polar
body appeared thirty-five minutes after the first or one hour and
forty-five minutes after the eggs were removed from the ovary.
pv D. H. Tennent and M. f. Hogue
The CO, sea-water is thus seen to have delayed the process of
maturation forty minutes.
In some lots of eggs, but not in all, a distinct membrane, some-
what thicker than the ordinary fertilization membrane, was
formed and pushed out from the surface of the eggs when these
were transferred from the CO, sea-water to ordinary sea-water,
this activity recalling the facts observed by Lefevre (’05, ’06)
on eggs of Thalassema treated with acid solutions. ‘This mem-
brane carried the first polar body out with it, the second polar
body being extruded into the space between the surface of the
egg and the membrane.
In the average lot of eggs the series of changes preceding
the first cleavage follows very closely, those described by Wilson
(01) as occurring in Toxopneustes eggs, the important difference
being that in the eggs of Asterias forbesii that have received the
best degree of treatment with CO, sea-water no cytasters were to
be observed. In eggs which had received too prolonged a treat-
ment, numerous cytasters might be seen.
In about three and a half hours after removal from the CO,~
sea-water the cytoplasm becomes coarser and looser in appear-
ance, apparently becoming more fluid in character in the region
of the nucleus. Radiations appear stretching out from the
vaguely defined clearer area into the denser cytoplasm. As a
result of these changes the nucleus becomes very distinct.
These primary radiations then become fainter and new radia-
tions growing in from the sides of the nucleus are seen, the nuclear
membrane breaks down and the definite mitotic figure is seen to
be forming, its centers gradually enlarging as the figure becomes
definitely established. ‘The division of the egg into two cells
is completed in about four hours after removal from the CO,
sea-water. On one or two occasions, the temperature being
low, the first division was completed only after five hours.
It is of interest to notice that in the eggs of Asterias glacialis
observed by Delage (’02) segmentation commenced about three
hours after removal to the ordinary sea-water, the eggs having
remained in the CO, sea-water for an hour, that is, segmentation
commenced four hours after the beginning of the treatment, while
a
The Development of the Starfish Egg 523
in the eggs of Asterias forbesii, observed by me, the segmentation
began in approximately the same time, about four hours from
the beginning of the treatment, although the eggs had remained
in the CO, sea-water for but five or six minutes.
b. A Study of the Nuclear Changes as Seen in Sections
Delage in his work on Asterias glacialis did not make a detailed
study of these phenomena. ‘The purpose of his work being finally
(04) to raise the larve until they would metamorphose.
Morgan’s (99) observations on the eggs of Asterias forbesil
when treated with solutions of NaCl, MgCl and KCl (’99, p.
499) although brief, since the eggs were immature, are of interest.
He mentions the appearance of areas of cyanoplasm and the fact
that in some unfertilized eggs that had been in a solution of mag-
nesium chloride for three hours, stars with delicate rays made their
appearance in these areas of cyanoplasm.
Kostanecki’s work on the eggs of Mactra (04) is of especial
interest in the present connection, since in this egg, which nor-
mally does not extrude its polar bodies until after the entrance of
the spermatozoan, he traced a series of phenomena analogous to
those observed in the starfish egg.
The eggs of Mactra were treated with solutions of KCI, NaCl,
CaCl or with concentrated sea-water. ‘The most normal develop-
ment was obtained in eggs which remained in a weak solution of
KCl thirty minutes. Here both polar bodies were given off
(04, Fig. 34) and from the chromosomes remaining in the egg a
nucleus was formed quite as in the fertilized egg, although it
contained but twelve chromosomes, one-half the normal number
(twenty-four), as was later shown in the first segmentation spindle.
The polar spindles developed deeper within the cytoplasm
and the polar bodies were larger than those of fertilized eggs. In
the manner of division of the segmentation nucleus and in the
absence of centrosomes and centrosome-like structures in these
divisions, the eggs of Mactra differ from those of Asterias forbesil.
Scott (’06), in his observations on the parthenogenetic develop-
ment of Amphitrite eggs after treatment with salt solutions,
shows that the ripeness of the eggs, the strength of the solutions,
524 D. H. Tennent and M. ‘fF. Hogue
etc., were the factors determining whether development would
be nearly normal or very abnormal. If the Amphitrite eggs are
ripe, normal polar bodies are given off in a weak solution of
calcium nitrate, although the subsequent segmentation 1s abnor-
mal. Here, again, little comparison can be made between these
eggs and those of Asterias forbesii, the most important point of
agreement being that both polar bodies are given off in each
case.
Lefevre’s (’06) work on artificial parthenogenesis in the eggs
of Thalassema shows many points of agreement with my obser-
vations on starfish eggs, the formation of the “fertilization mem-
brane,” the extrusion, as a rule, of both polar bodies may be
mentioned here and further comparisons reserved for a later
mention.
Inthe present experiment the eggs, after the extrusion of the first
polar body, were covered with sea-water charged with carbon
dioxide. ‘They remained in this four minutes and were then
transferred to ordinary sea-water.
The series consisted of twenty-five stages, the earlier numbers
of which were fixed at five minute and the later at ten minute
intervals in Boveri’s picro-acetic acid.
The eggs were cut in sections 3 microns thick and stained with
Heidenhain’s iron-hzematoxylin, long method. Eosin, erythrosin,
and Bordeaux red were tried as counter-stains but the iron-
hematoxylin gave the clearest and best effects. “Total mounts
were stained by Conklin’s hamatoxylin method.
Throughout the series a few eggs were found with the germi-
nal vesicle intact, which is due to the fact that the nuclear mem-
brane had not begun to fade when the eggs were treated with
the CO: sea-water, 7. e., theywere‘not in Delage’s “critical stage.”
After the first polar body is given off, a cone of cytoplasm is
often seen projecting from the surface of the egg at the side of
the first body during the time that the second polar body is forming.
This cone frequently persists until the first segmentation spindle
is well formed (Figs. 21 and 26) and is occasionally seen in the
two-cell stage at the edge of the cleavage plane.
The polar bodies are of the same size as those given off in fer-
The Development of the Starfish Egg SpE
tilized eggs, differing in this respect from those observed by
Kostanecki in the artificially parthenogenetic eggs of Mactra.
The cleavage is normal, dividing the egg into halves and the
second forming four equal-sized blastomeres.
Shortly after the extrusion of the first polar body the chromo-
somes are found lying on the spindle fibers, which have already
begun to degenerate at their polarends (Fig. 1). In a later stage,
eighteen chromosomes may be counted (Fig. 2), and with them
are seen a few spindle fibers. ‘These fibers disappear and the
chromatin is left free in the cytoplasm (Fig. 3), more or less massed
together. In the cytoplasm directly beneath the first polar body
is a darkly staining region which is not represented in the draw-
ings. It does not take the chromatin stain.
The formation of the second polar body proceeds slowly and
the time of the division of the chromosomes varies. In an egg
from stage 5 (Fig. 4) the chromosomes, undivided, are still attached
to the fibers of the first polar spindle. In stage 11 (Fig. 5) the
chromosomes are divided for the second polar body although
they are still attached to the fibers of the first polar spindle.
From a study of the preserved material, it seems evident that
the second polar spindle is formed tangentially to the surface
of the egg (Fig. 6) and that it later revolves until it has taken a
radial position (Figs. 7 and 8). The centrosomes, single and
double, may be seen clearly in these spindles. (Figs. 6, 7 and 8.)
In the early anaphase of the second polar spindles (Fig. g) the
chromosomes are scattered irregularly over the whole spindle.
In the late anaphase (Fig. 10) they are collected in plates at the
two poles of the spindle, one or two of the chromosomes being
later than the others in taking their positions.
After the second polar body is formed, the chromatin is more
or less free in the cytoplasm, some of it lying on the astral rays
as though passing down these into the cytoplasm (Fig. 12), and
the remainder forming five or six vesicles which fuse to form the
female pronucleus. At each division of the nucleus, chromatin
is thrown out into the cytoplasm. Occasionally the chromatin
is found lying free in the cytoplasm, without a trace of astral
radiation or of vesicle formation (Fig. IT).
526 D. H. Tennent and M. ‘f. Hogue
The newly formed nucleus now moves to the center of the
egg and at the same time begins to divide. ‘The walls of the
vesicle become indented (Fig. 13), and on the side nearest the
surface of the egg two centrosomes appear and move around the
nucleus until he: lie one at each end of the slightly elongated
nucleus. This process differs from that described by Lefevre
(06) for Thalassema in which the cleavage asters with their cen-
ters arise simultaneously at opposite poles of the egg nucleus.
At the same time the astral fibers are forming in the cytoplasm.
This growth apparently begins at the nuclear membrane and
extends into the cytoplasm of the egg as described by Wilson
( ‘a1) and Morgan (’99) for Echinodermeggs. Later, these radia-
tions collect at the two ends of the nuclear vesicle whither the
centrosomes have migrated.
Following this stage the nuclear membrane breaks down and
particles of chromatin, seen in Fig. 15 on the astral rays, pass into
the cytoplasm.
As the nuclear membrane disappears (Fig. 16), the conspic-
uous nucleolus unravels and its chromatin, together with that
derived from the chromatin network of the nucleus becomes
broken up into short threads, the discharge of chromatin into the
cytoplasm continuing meantime. The study of the sections
has not shown whether all of the chromosomes of the equatorial
plate are derived from the nucleolus. ‘The spindle fibers grow
into the nucleus and may be seen stretching from the poles to the
center of the nucleus.
At this stage the nuclear sap has been poured out into the
cytoplasm and a difference in staining reaction of the areas
indicated by the dotted line may be seen.
Figs. 14 and 17 represent later stages of the first segmenta-
tion spindle.
In the late anaphase the chromosomes become enclosed in
vesicles (Fig. 18), which fuse to form the daughter nuclei. ‘The
nuclear membrane forms as the astral rays are disappearing.
During this process chromatin rejection continues, the rejected
chromatin being seen enclosed in little vesicles lying in the cyto-
plasm (Fig. 22).
The Development of the Starfish Egg Saf
After the daughter nuclei are formed they move apart and the
constriction appears which divides the egg into halves. It is
interesting to note that the cytoplasm does not begin to divide
until the daughter nuclei are completely formed. Figs. 23 and
25 represent the spindle and equatorial plate of the second segmen-
tation.
Mathews (’95) was unable to find centrosomes in the seg-
mentation spindles of Asterias forbesii. In the material from
which these sections were made two kinds of centrosomes were
found; one granular, containing several small deeply staining
bodies (Figs. 17 and 23); the other, sometimes a single body,
more often two, from which the radiation extend (Fig. 16). In
Fig. 18 the double centrosomes appear in the cytoplasm while
the astral rays are disappearing.
It has been impossible to count the chromosomes in the seg-
mentation spindle as they do not take definite form (Fig. 14)
until they line up in the equatorial plate, ready for division.
Here, again, it was useless to try to count them since they were
massed together and of irregular shape. Often part of one
chromosome is in one section and part in another. Again,
some divide before others and frequently when the majority of
the chromosomes have separated, a long chain of chromatic
material, as yet unsegmented, extends down the middle of the
spindle. A few chromosomes seem to have a. characteristic
form.
It will be remembered that in the second polar spindle there
were eighteen chromosomes (Figs. 1,2 and 8). From Figs. 9g
and Io it seems evident that at least eighteen chromosomes will be
left in the egg after the second polar body is given off. Fig. 21
is drawn from a section showing the two polar bodies at the sur-
face and the equatorial plate at the center of the egg. This
is shown again drawn under higher magnification in Fig. 20.
While it is impossible to count the number of chromosomes
exactly, it is evident that there are at least eighteen here.
Another egg (Fig. 24) shows the two polar bodies at the surface
and the equatorial plate at the center, with the spindle fibers in
cross-section. This equatorial plate is shown again in Fig. 19.
528 D. H. Tennent and M. fF. Hogue
In this the chromosomes have divided for the daughter nuclei
of the first division.
Delage maintained a doubling in the number of chromosomes
in the parthenogenetic eggs of Strongylocentrotus lividus. ‘The
eggs had given off both polar bodies when treated with the solution
and yet later they contained the normal somatic number of chromo-
somes. He held that at some time, as yet undetermined, the
chromosomes divide again and so establish the normal number
(autoregulation), these in turn dividing to form the chromosomes
of the daughter nuclei. Boveri has since shown that eighteen
is not the somatic number of chromosomes for Strongylocentrotus
lividus, but the reduced number.
In the parthenogenetic eggs of Mactra, Kostanecki (’04)
found the reduced number of chromosomes in the segmentation
spindle when the egg had given off both polar bodies. ‘These
eges did not segment many times.
Boveri (’04) experimenting with the fertilization of nucleated
and non-nucleated fragments of sea-urchin eggs, found that when
a non-nucleated fragment is fertilized it contains less chrom-
atin; z. e., the nuclei are smaller than the fertilized nucleated
fragments. Morgan (’95) performed similar experiments with
similar results.
Stevens (02) while working on the eggs of Echinus micro-
tuberculatus which were cut into pieces while in the anaphase of
the first division, found that fragments containing a centrosome
and a small number of chromosomes may divide five or six times
without the chromosomes returning to the constitutional number.
Wilson (’o1) showed that in parthenogenetic Toxopneustes
eggs the number of chromosomes is eighteen, one-half the number
occurring in fertilized eggs.
The evidence at present, seems in favor of the permanence of
the number of chromosomes occurring in the unfertilized egg
which is caused to develop parthenogenetically after two polar
bodies have been given off.
Another idea of Delage’s, that the presence of two polar bodies
might be due to the division of the first polar body, seems incor-
rect. In Asterias forbesii the formation of the second polar body
The Development of the Starfish Egg 529
has been traced and its actual cutting off has been noted. The
second polar body is slightly smaller (Figs. 21 and 24) while the
first polar body contains the greater amount of chromatin (Fig.11).
One egg was found in stage 24 (Fig. 24) in which the second
polar body had been formed but not extruded from the egg
before the nucleus began to divide. Here it is certain that the
polar body does not again enter the nucleus. ‘This retention of
the second polar body within the egg is not the usual method of
procedure but may be accounted for by the tardiness with which
this egg began development. ‘There is, of course, a possibility
that later the chromatin of this retained polar body might mix
with that of the egg nucleus in a manner similar to the behavior
of the sperm nucleus in partial fertilization as noted by Boveri.
One cannot but be impressed with the normal procedure of
development in the eggs treated by Delage’s CO, method. In the
thousands of eggs examined in the study of these sections, there
were not more than a dozen abnormal structures. (It is to be
remembered, of course, that this series which has been studied
with greatest detail was selected primarily because of its perfec-
tion.)
Two cases of multipolar spindles were observed, one with
three poles, the other with four. A few eggs, undivided, con-
tained three nuclei, each with its amphiaster ready for division.
There were also two or three eggs which had divided into three
blastomeres. The cleavage in the early stages was normal.
No cytasters were formed in the eggs.
SUMMARY
1 Two polar bodies are given off.
2 Eighteen single chromosomes are left in the egg after the
extrusion of the second polar body. (The number counted varies
within slight limits.)
3 Centrosomes are present in the polar and segmentation
spindles.
4. Cleavage is normal though slower than in fertilized eggs.
530 D. H. Tennent and M. fF. Hogue
II. OBSERVATIONS ON EGGS WHICH WERE FIRST TREATED WITH
CO, SEA-WATER AND SUBSEQUENTLY FERTILIZED
Description of the Experiments
Since it was not known whether the eggs were capable of
fertilization after removal from the CO, sea-water, some pre-
liminary work was necessary in order to determine, first, whether
it was possible to fertilize such eggs, and, second, the optimum
time for such fertilization after the eggs had been removed from
the CO, sea-water.
A set of eggs was subjected immediately after the appearance
of the first polar body, to the action of CO, sea-water for six
minutes and then transferred to a large dish of sea-water and
used as stock from which at five minute intervals during a period
of two hours, eggs were taken and treated with active sperm.
A series of these eggs (24 stages in all), each stage taken ten
minutes after the addition of the sperm, was fixed for sectioning
and the remainder of each stage were allowed to continue their
development. The study of these sections is now but partially
completed.
Some data of this work may be of interest. I give brief notes
of observations on the stock and four stages from the twenty-four
which were under observation.
Stock—Eggs remained in sea-water for one hour and eleven
minutes when the first polar body was seen pushing out from
the surface of the majority of the eggs (9:15—-10:26).
(1) Eggs subjected to CO, sea-water for 8 minutes (10:26—
10:34)
(2) 11:15, still but one polar body.
‘(3) 11:30, two polar bodies and irregular membrane.
(4) 3:00, ten per cent in two-cell stage.
Stage A—Stock eggs treated with sperm at 10:40 (six minutes
after removal from CO, sea-water).
(1) 10:43, fertilization membrane distinct.
(2) 11:35, two polar bodies, one inside, one outside of fertili-
zation membrane.
The Development of the Starfish Egg 531
(3) 12:00, beginning two-cell stage.
(4) 3:00, twenty per cent past eight-cell stage.
Stage C—Stock eggs treated with sperm at 10:50 (sixteen
minutes after removal from CO, sea-water).
(1) 12:08, beginning two-cell stage.
(2) 3:00, twenty per cent past eight-cell stage.
Stone D—Stock eggs treated with sperm at 10:55 (twenty-one
minutes after removal from CO, sea-water).
(I) 12:10, some segmented.
(2) 3:00, thirty-five per cent segmented.
Stage H—Stock eggs treated with sperm at 11:15 (forty-one
minutes after removal from CO, sea-water.)
(1) 12:15, beginning two-cell stage.
(2) 3:00, thirty per cent eight-cell stage.
In A eggs began to divide one hour and twenty minutes after
fertilization.
In B eggs began to divide one hour and eighteen minutes after
fertilization.
In D eggs began to divide one hour and fifteen minutes after
fertilization.
In H eggs began to divide one hour after fertilization.
In this experiment the highest percentage of dividing eggs was
obtained from those treated with sperm between twenty and
forty-five minutes after removal from CQO, sea-water. After
11:15 through the four succeeding stages 11:20, 11:25, 11:30,
11:35, the percentage of eggs dividing in approximately one
hour after fertilization rapidly diminished, being practically
zero at 11:35 (5 minutes after the second polar body had been
given off in the CO, stock).
In all of the succeeding stages, segmentation began between
2:20 and 2:40 or roughly four hours after treatment with CO,,.
From these observations it seemed possible to conclude that eggs
treated with CO, sea-water were capable of fertilization during
the hour immediately succeeding removal from CO, sea-water.
After that time had passed the spermatozoa, although still exceed-
ingly active, were either not able to enter the egg or entering it
produced no effect and the eggs proceeded to their parthen-
532 D. H. Tennent and M. f. Hogue
ogenetic division. Fresh spermatozoa were tried but without
avail.
For the sake of comparison, a table showing the percentage of
eggs developing from a set allowed to mature for one hour and
forty minutes (10:00-11:40), in sea-water (until the extrusion
of the first polar body), and then fertilized at the intervals noted,
is given. These eggs were not treated with CO, but were simply
fertilized with sperm.
Fertilized 11:40, ninety-five per cent segmented.
Fertilized 12:03, ninety-five per cent segmented.
Fertilized 12:25, ninety per cent segmented.
Fertilized 1:40, thirty per cent segmented.
Fertilized 3:00, no eggs segmented.
Fertilized 4:00, no eggs segmented.
In this set of normal eggs a considerable percentage was
capable of fertilization for more than two hours after the extru-
sion of the first polar body.
Comparison of these sets of results, then, show that apparently
treatment of the eggs with CO, sea-water shortened the time
during which the sperm might be effective.
From the observations mentioned it was thus found that the
most normal results were obtained by fertilizing the CO, eggs
from twenty to forty minutes after their removal from the CO,
sea-water. These eggs when successfully fertilized commenced
their segmentation in approximately one hour and thirty minutes
after fertilisation: The ‘treatment with sperm shortened the
time elapsing between the treatment with CO, sea-water and
the beginning of segmentation, or, stating it more concretely :
The time usually required for segmentation of CO, eggs was
about four hours from the extrusion of the first polar body.
The time required for the beginning of segmentation of CO,
eggs subsequently fertilized was about one and one-half hours.
The eggs that had been treated with CO, and subsequently
fertilized gained in time, then, two and one-half hours. ~
In work with other lots it was found that the greatest percentage
of fertilized eggs was to be obtained, if the CO, eggs were treated
The Development of the Starfish Egg 528
with sperm twenty or thirty minutes after removal from the
CO, sea-water.
Several sets of eggs were thus treated and from these, series
were fixed which have served as the basis of the cytological study.
It is of considerable interest to notice that the thick vitelline
membrane pushed out from the egg, did not prevent the entrance
of the spermatozoan, and that in eggs fertilized after the appear-
ance of this membrane, a second membrane, somewhat thinner
than the first, was formed, so that the eggs were surrounded by
concentric membranes.
In several cases, the actual passage of the spermatozoan through
the outer membrane was observed, while in another lot of par-
thenogenetic eggs which were treated with sperm when in the
two- and four-cell stages, the spermatozoa were seen swimming
actively between the blastomeres.
The sections of these eggs have not been studied so that it 1s
impossible to state whether or not the spermatozoa entered the
blastomeres, or their effect on subsequent developments.
The Study of Sections of the Eggs
The facts determined by the study of sections of eggs treated
with CO, sea-water and later fertilized, show that the processes
occurring are so like the normal that a detailed description is
unnecessary. I shall, therefore,content myself witha description
of the figures and later make a comparison between the facts
brought out by Miss Hogue in her study of the parthenogenetic
development and those determined in my own study of the eggs
which have received the double treatment.
In some of the eggs sectioned, the first polar body was in process
of formation. Here, there seemed absolute evidence of the long1-
tudinal division of bivalent chromosomes.
Fig. 30 shows a section through the remains of the first polar
spindle immediately after the extrusion of the first polar body.
In the polar body and in the egg the dumb-bell-shaped bivalent
chromosomes are seen (Fig. 31). In some cases a tetrad effect
(Fig. 32) due, I believe, to the grouping of the double chromo-
somes was to be seen.
534 D. H. Tennent and M. ‘f. Hogue
In some cases the chromosomes of the second polar body
remained within the egg (Fig. 33), clustered beneath the first
polar body where they apparently degenerated, since in slightly
later stages they were seen to be broken into small fragments, and
in still later stages a few slightly stained granules were found in
this position. ‘There was absolutely no evidence that as chromo-
somes they took any part in subsequent nuclear transformations.
The best evidence shows that in the second maturation division
the bivalent chromosomes are divided to form univalent chromo-
somes (Figs. 34-37) as has been described by Mathews (’95).
The division is transverse. I have been unable to find evidence
of the double longitudinal division which has been described by
Bryce (02) for Echinus esculentus.
In many cases abnormalities occur (Figs. 38-42). In some
cases tripolar spindles are seen, in which event it seems possible
that both polar bodies are being formed in the same division.
The chromosomes remaining within the egg soon form five or
six vesicles which are drawn together and fuse to form the female
pronucleus which lies surrounded by some of the degenerating
fibers of the second polar spindles (Figs. 44, 48, 49 and. 59).
Meantime the sperm nucleus, which has become vesicular, has
been moving, accompanied by its aster toward the egg nucleus, the
centrosome and aster dividing as the two approach ( Figs. 45 and
46), the two nuclei finally fusing to form the segmentation nucleus.
In a very few cases the egg nucleus seems to be provided with
an aster of its own (Fig 44). In most cases it seems simply lying
among some of the fibers remaining from the second polar spindle.
The segmentation nucleus remains in a resting condition for
some time, during which the progression of the asters to opposite
sides of the nucleus may be observed. From their inner sides
fibers may be seen projecting into the nuclear membrane (Fig. 58).
and in slightly later stages these fibers may be seen within the
nucleus although the membrane seems intact. ‘The nucleus has
meantime become decidedly elongated in outline.
Succeeding this stage the conditions represented in Figs. 59-
61 rapidly succeed one another. The nuclear membrane appar-
ently dissolves first at the poles and finally dissolves throughout,
The Development of the Starfish Egg 535
during which process the achromatic figure increases greatly in
size.
The chromatic reticulum and the nucleolus break down into
coarse threads which in succeeding stages becomes finer, these
threads ultimately becoming broken up into short rods. (Figs. 54—
56). These rods become rounded (Fig. 57) and during the for-
_ mation of the equatorial plate are replaced by bivalent structures
(Figs. 62-68), which lie with their long axis at right angles to the
long axis of the spindle.
All of the observations point to a conjugation of rounded unt-
valent chromosomes to form elongated bivalent chromosomes.
In Figs. 54 and 55 the chromosomes are seen to be rod-like.
In Fig. 57 the form has changed, all of the chromosomes having
‘become rounded, a few showing the bivalent form. In Figs. 62
and 63, 66 and 67, it is seen that the bivalent form has become
more common while the actual number of chromosomes has
diminished, while in Fig. 68, which represents all of the chromo-
somes of the equatorial plate, it is seenthat the number of chromo-
somes has been still farther reduced and that with the exception
of six univalent chromosomes all of the chromosomes show the
bivalent form.
These chromosomes retain their bivalent form during the seg-
mentation divisions (Figs. 64 and 65 and 61 and 69). In Figs.
61 and 60 it is seen that some of the chromosomes are still of the
univalent form. ‘This was true of all of the segmentation stages
examined.
The division of the chromosomes in the segmentation is longi-
tudinal, the chromosomes which are at first placed with their
long axes at right angles to the long axis of the spindle becoming
pulled out so that their long axes lie parallel to that of the spindle
(Figs. 64, 65 and 53).
The daughter nuclei are formed by the fusion of chromosomal
vesicles.
Granules of chromatin are thrown out into the cytoplasm from
the time of the breaking down of the nuclear membrane preced-
ing segmentation until the formation of the daughter nuclei is
completed.
536 D. H. Tennent and M. ‘fF. Hogue
Further reference will be made to some of these changes in the
general considerations at the end of this paper.
III. EGGS FERTILIZED AND SUBSEQUENTLY TREATED WITH CO,
In these eggs the sperm was allowed to act for ten minutes
and the eggs were then transferred to CO, sea-water for five
minutes. I give the data of but one lot.
Fertilized eggs placed in CO, sea-water (12 :28—12:33).
No eggs segmented until 3:40 or three hours and seven minutes
from the time of removal from the CO, sea-water.
Since the sections of these eggs have not yet been studied it is
impossible to say definitely whether the eggs developed as a
result of the CO, treatment or as a result of fertilization by the
sperm.
It is probable that since segmentation began within three hours
after the eggs were removed from the CO, sea-water the sperm
fertilization was effective, in which event the action of the CO,
sea-water was simply to delay development.
SUMMARY
The observations on the behavior of eggs treated with CO, may
be summarized as follows:
1 Unfertilized eggs subjected to the action of CO, sea-water
for from 3 to 10 minutes commenced segmentation about four
hours after treatment.
2 (a) Unfertilized eggs subjected to the action of CO, sea-
water for from three to ten minutes and fertilized twenty to
thirty minutes after removal from the CO, sea-water began seg-
mentation about one hour after fertilization.
(b) Unfertilized eggs subjected to the action of CQ, sea-
water for from three to ten minutes when treated with sperm
one hour or more after removal from the CO, sea-water segmented
four hours after removal from the CO, sea-water.
3 Fertilized eggs subjected to the action of CO, sea-water
segmented three hours after removal from the CO, sea-water.
A
The Dev lopment of the Starfis cb ik Egg 537
GENERAL CONSIDERATIONS °
As to the cause of the parthenogenetic development of eggs
treated with CO,, whether it is by reason of a change of osmotic
pressure, or because of agitation, or of the exposure to some
specific chemical substance, or is due to the presence in the sea-
water of new compounds formed in reactions which may take
place between the CO, or impurities which may be present in the
materials used and substances present in sea-water, this paper
has nothing to say.
The simple fact remains that eggs treated with CO, as has been
described, segment regularly and develop into embryos which in
form and structure cannot be distinguished from embryos obtained
from fertilized eggs.
Whether the reagent acts as a stimulus, or a shock, or a poison,
the author cannot say. The evidence, however, is sufficient to
show that Delage was correct in his view that maturation is
arrested temporarily by the action of the CO,,.
It is in the comparison of sections of CO, eggs with those
of CO, eggs which were subsequently fertilized and with those
of normally fertilized eggs that the most interesting facts come
to light. All behave in essentially the same manner in the
maturation processes, these processes apparently agreeing with
those described by Mathews (’05) but which he has not figured
in detail. In the material studied, which it is to be remembered
must be regarded as having received somewhat artificial treat-
ment from the beginning, the number of chromosomes remaining
in the egg, after the extrusion of the second polar body, varies
slightly, the number ranging from eighteen to twenty-three,
although eighteen seems to be the common number.
It is seen that the maturation processes in both the CO, eggs
and in the CO, eggs which were subsequently fertilized, differ
remarkably from those observed in compressed eggs by King
(06). Here, although the first polar body may be extruded, the
second is retained, all of the retained chromatin going into the
formation of one or several vesicles which unite to form the egg
nucleus, but “the retention of chromatin that is normally extruded
538 D. H. Tennent and M. F. Hogue
in the polar bodies does not lead to a parthenogenetic develop-
ment of the egg. ” In compressed eggs which were fertilized,
great differences in size in the two pronuclei were noted, a phenom-
enon which does not occur in CO, eggs subsequently fertilized.
The polyspermy observed in fertilized compressed eggs has been
noted in CO, eggs subsequently fertilized which did not receive
the treatment which results in normal development.
It is worthy of note again that in the CO, eggs which were sub-
sequently fertilized and in ordinarily fertilized eggs the chromo-
somes during the maturation divisions are bivalent (Mathews,
double chromosomes) and at the close of the maturation divisions
eighteen univalent (Mathews, seventeen [ ! | single) chromocnni
remain within the egg.
It will be noticed that the straight CO, treatment ou has
some influence on the maturation processes, for while the number
of chromosomes remains the same as in the fertilized eggs, the
shape of the chromosomes varies.
‘The process of reconstruction of the egg nucleus is by the forma-
tion and fusion of chromosomal vesicles in eggs of the three
classes mentioned.
The appearance of the segmentation nucleus in the CO, eggs
and in the CO, eggs which were subsequently fertilized shows no
difference. ‘This nucleus breaks down in the same manner in
both cases, the nuclear reticulum becomes threadlike and breaks
up into fragments, the mass of chromatic material being the same,
so far as the eye can judge, in the one case as in the other.
In neither case can the exact number of fragments be counted,
but it is clearly to be seen that they are not to be regarded as
individual chromosomes since the numbers counted are more
than double those found at any earlier or later stages when the
count may be made with reasonable certainty, and since many of
these fragments may be seen in various stages of withdrawal into
the cytoplasm.
But at the time of the completion of the equatorial plate, funda-
mental differences between the chromosomes of the CO, eggs and
the eggs which were subsequently fertilized may be seen. In the
one case the chromosomes are irregular and in the other, of a
The Development of the Starfish Egg 539
distinct bivalent form; a form which is preserved during the longi-
tudinal splitting of the chromosomes as it divides to form the
daughter nuclei, a form which persists in the later divisions of the
egg so far as observed and a form which is again seen in the
maturation divisions until the final separation of bivalent into
univalent chromosomes during the formation of the second polar
body.
These facts, it seems to me, are to be explained only by the
suggestion that a conjugation or synapsis of egg chromosomes
and sperm chromosomes takes place immediately before the
formation of the equatorial plate of the first segmentation spindle.
That this process takes place seems very probable when, after
comparing Figs. 19 and 20, representing sections through the
equatorial plate in CO, eggs, with Figs. 54, 56 and 57, repre-
senting sections through the equatorial plate of CO, + sperm eggs,
one follows the series of changes shown in Figs. 62, 63, 66,
67 and 68, in which the rounded chromosomes are shown, some
lying free, some drawn closely together in pairs and still others
showing the completed bivalent form. ‘This form is retained
from the equatorial plate stage (Fig. 68), through the division
hgures ( Figs. 64, 65, 69, 61) of the CO, + sperm eggs and may be
seen in the = ee figures of the normally fertilized eggs ( Figs.
28 and 29).
As a result of such a conjugation the number of chromosomes
would remain the same in the parthenogenetic as in the fertilized
eggs, with the difference that in the parthenogenetic eggs there
remain eighteen univalent chromosomes and in the fertilized eggs
eighteen bivalent chromosomes.'
1During the summer of 1906 the experiments which have been described in this paper were repeated
and some further observations made. Inthis work it was demonstrated satisfactorily that the equa-
torial plate in the eggs from some individuals contained, within slight variations, thirty-six chromo-
somes. The Woods Hole starfishes thus show a variation as to the number of chromosomes similar to
that pointed out in Echinus microtuberculatus by Stevens (’o2). In such eggs as in the eggs that have
been considered in this paper the number of chromosomes remains the same in both fertilized and in
parthenogenetic eggs. A point of disagreement between these eggs and those obtained during the pre-
vious summer is that dumb-bell-shaped chromosomes were found in parthenogenetic eggs. Little de-
pendence, therefore, as has been suggested by several authors recently, can be placed upon the shape
of the chromosomes as an indication of valency.
540 D. H. Tennent and M. F. Hogue
It is possible that the objection will be made that the activity
of the CO, sea-water is such as to change the shape of the chromo-
somes in the equatorial plate of the first segmentation spindle,
since we have seen that the shape of the chromosomes in the
maturation divisions was thus influenced.
In replying to such an objection, it can only be pointed out that
the number of chromosomes, although these chromosomes vary
in size, shape, etc., remains apparently the same in both the CO,
eggs and in the CO, eggs which were subsequently fertilized, and
this in spite of the fact that we should expect that the number in
the fertilized egg to be doubled.
An additional point of interest comes out in the comparison of
these eggs, and this in respect to the cleavage asters. “The
cleavage asters with their centers have the same appearance in
both the CO, eggs and in the CO, eggs which were subsequently
fertilized. In eggs which were fertilized at the optimum time
these were the only asters formed, while in eggs fertilized later
than this time many cytasters and additional sperm asters were
formed, the subsequent divisions being exceedingly abnormal.
The study of these phenomena has been partly completed and
its results will be submitted in a later contribution.
Bryn Mawr College
June, 1906
The Development of the Starfish Egg 541
LITERATURE
Boveri, [H., ’04.—Ergebnisse iiber die Konstitution der Chromatischen Substanz
des Zellkerns. Gustav Fischer. Jena, 1904.
Bryce, T. H., ’02.—Maturation of the Ovum in Echinus esculentus. Quart. Jour.
Mic. Sci., vol. xlvi.
DeacE, YVES, ’o1.—Etudes Expérimentales sur la Maturation Cytoplasmique
et sur la Parthénogénése artificielle chez les Echinoderms. Archiv.
Zool. Exp. 3d series, vol. ix, 1901.
’02.—Nouvelles Recherches sur la Parthénogénése Expérimentale chez
Asterias glacialis. Archiv Zool. Exp. 3d series vol. x, 1902.
’04.—Elevage des Larves Parthénogénétiques D’Asterias glacialis.
Archiv Zool. Exp. 4th series, vol. ii, p. 27-42. 1904,
’04.—La Parthénogénése par L’ Acide Carbonique obtenu chez les Oeufs
aprés l’émission des Globules Polaires. Archiv Zool. Exp. 4th
series, vol. 11, p. 43-46.
’05.—Nouvelles Expériences de Parthénogénése Expérimentale. Archiv
Zool. Exp. 4th series, vol. iii, Notes et Revue, clxiv-clxviii.
KostaneckI, K., ’04.—Cytologische Studien an kiinstlich parthenogenetisch sich
entwickelnden Eiern von Mactra. Archiv f. Mikr. Anat., vol. xiv.
Kina, H. D., ’06.—The Effects of Compression on the Maturation and Early
Development of the Eggs of Asterias forbesii. Archiv fiir Entwick.
der Organismen, vol. xxi.
LEFEVRE, GEORGE, ’05.—Artificial Parthenogenesis in Thalassemamellita. Science
P- 379» 1905-
- ’06.—Further Observations on Artificial Parthenogenesis. Science, pp.
522-524, 1906.
Morean, T. H., ’96.—The Fertilization of non-nucleated Fragments of Echino-
derm Eggs. Archiv fiir Entwick. der Organismen, vol. ii.
'99.—Action of Salt Solutions on the Unfertilized and Fertilized Eggs
of Arbacia and of other Animals. Archiv fiir Entwick. der Orga-
nismen, vol. vill.
Scott, J. M., ’06.—Morphology of the Parthenogenetic Development of Amphi-
trite. Jour. Exp. Zodl., vol. ii.
Stevens, N. M., ’02.—Experimental Studies on Eggs of Echinus microtubercu-
latus. Archiv fiir Entwick. der Organismen, vol. xv.
Witson, E. B., ’01.—Experimental Studies in Cytology. A Cytological Study of
Artificial Parthenogenesis in Sea Urchin Eggs. Archiv fiir Ent-
wick. der Organismen, vol. xii.
Witson, FE. B., and Matuews, A. P., ’95.—Maturation, Fertilization and Polarity
in the Echinoderm Egg. Jour. Morph., vol. x.
DESCRIPTION OF PLATES
The drawings were all made from camera lucida sketches. Zeiss compensating ocular 12, with 2 mm.
oil immersion objective, giving a magnification of 1500 diameters, were used for all of the figures except
those hereinafter noted. All of the drawings are reduced one-half in reproduction.
Fig. 1.
Fig. 2
Fig.
egg.
Fig.
Fig.
Fig.
Fig.
Fig.
(Figs 1-27 from co, eggs)
Prate I
First polar body formed. Remains of degenerating spindle. Eighteen chromosomes present.
. Later stage of degenerating spindle. Eighteen chromosomes.
. Complete disappearance of spindle. Additional chromosomes were in next section of the
4. Chromosomes remaining on a half spindle.
5. Same as Fig. 4, except that the chromosomes have divided.
6.
7
8
Second polar spindle tangential to surface of egg.
. Radial position of second polar spindle as seen in later stages.
. Second polar spindle with eighteen chromosomes.
Figs. 9 and 10. Early and late anaphase of second polar division.
Fig. 11. Two polar bodies given off; chromatin lying free in cytoplasm.
Fig. 12. Formation of female pronucleus through fusion of vesicles.
Fig. 13. Female pronucleus preparing to divide; chromosomes and fibers appearing.
Fig. 14. Late prophase of first segmentation division.
Figs. 15 and 16. Early prophase of first segmentation. Ocular 4, 2 mm. oil immersion objective.
ee
DEVELOPMENT OF THE STARFISH EGG PLATE I
D. H. Tennent anv M. J. Hocur
16
Tue Journat or ExperiMENTAL ZOOLOGY, VOL, III, NO. 4 M J.H. del.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Prate II
17. Anaphase of first segmentation.
18. Vesicle formation preceding formation of daughter nuclei.
1g. Equatorial plate of Fig. 24.
20. Equatorial plate of Fig. 21.
21. Section of entire egg through equatorial plate. Ocular 4, 2 mm. oil immersion objective
22. Formation of daughter nuclei.
23. Metaphase of second segmentation.
24. Section of entire egg with chromosomes of equatorial plate divided in first segmentation.
Ocular 4, 2 mm. oil immersion objective.
Fig.
Fig.
25. Section through equatorial plate of second segmentation spindle.
26. Second polar body retained within the egg. Nucleus dividing. Ocular 4, 2 mm. oil immer-
sion objective.
DEVELOPMENT OF THE STARFISH EGG
PLATE II
D.H. TENNENT AND M. J. Hocure
Tue JourNat or ExperiMENTAL ZOOLOGY, VOL, III, NO. 4
Pirate IIT
Fig. 27. Second segmentation, slightly earlier than Fig. 23. .
Figs. 28 and 29. Adjoining sections of normally fertilized egg in anaphase of first segmentation.
(Figs 30-69 from CO, eggs which were subsequently fertilized)
Fig. 30. Section through portion of egg immediately after extrusion of first polar body.
Fig. 31. Chromosomes lying free in cytoplasm.
Fig. 32. Tetrad effect due to grouping of bivalent chromosomes.
Fig. 33. Second polar body not extruded. Chromosomes of second polar body lying in cytoplasm
beneath the first polar body.
Fig. 34. Second polar spindle. Chromosomes in process of transverse division.
Figs. 35 and 36. Adjoining sections through second polar spindle. Chromosomes in transverse
division.
Fig. 37. Second polar spindle. Chromosomes separated into univalent elements.
Fig. 38. Univalent chromosomes remaining in egg.
Fig. 39. Section through long axis of second polar spindle which was lying tangentially to surface
of egg.
Figs. 40 and 41. Sections through long axis of multipolar spindle.
Fig. 42. Three sections in series through chromosomes remaining in egg after.extrusion of second:
polar body.
DEVELOPMENT OF THE STARFISH EGG PLATE III
D.H.TEnNeEnT anv M. J. Hocure
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Fig. 43.
Prate IV
Second polar spindle formed near center of egg.
Fig. 44. Female pronucleus formed by fusion of chromosomal vesicles. Part of second polar
spindle lying in cytoplasm beneath first polar body.
Fig. 45.
Fig. 46.
Fig. 47.
Fig. 48.
Male and female pronuclei. Aster divided.
Male and female pronuclei. Centrosome divided.
Segmentation nucleus.
Male pronucleus approaching partially formed female pronucleus. Ocular 4, 2 mm. oil
immersion objective.
Fig. 49.
Fig. 50.
Bigs 51.
membrane.
Fig. 52.
Fig. 53.
Same as Fig. 48.
Vesicles which are to fuse and form female pronucleus.
Segmentation nucleus elongated; a chromatic fibers are to be seen within the nuclear
Ocular 4, 2 mm. oil immersion objective.
Male pronucleus.
Portion of first segmentation spindle.
Figs. 54 and 55. Adjoining sections showing thread broken into short, rod-like segments.
Fig. 56.
Fig. 57.
somes,
Part of section through nuclear material. Three bivalent chromosomes present.
Rod-shaped chromosomes rounded up into spherical structures. Some bivalent chromo-
DEVELOPMENT OF THE STARFISH EGG PLATE IV
D.H. Tennent anv M, J. Hocur
Tue JourNat or ExperiMENTAL ZOOLOGY, VOL. 111, NO. 4 D. H. T. del.
Pirate V
Figs. 58, 59 and 60. Stages in breaking up of segmentation nucleus and of formation of first
segmentation spindle.
Fig. 61. Anaphase of first segmentation.
Figs. 62 and 63. Equatorial plates. Univalent chromosomes pairing.
Fig. 64 and 65. Adjoining sections of chromosomes of same spindle showing chromosomes drawn
out in first segmentation division.
Fig. 66. Equatorial plate. Chromosomes pairing.
Fig. 67. Same as Fig. 66.
Fig. 68. Equatorial plate just before division, nearly all of the chromosomes being of bivalent
form.
Fig. 69. Late anaphase of first segmentation. Same as Fig. 61.
PLATE V
D. H. T.del,
DEVELOPMENT OF THE STARFISH EGG
D. H. TENNENT AND M, J. Hocue
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THe JourNAL OF ExPERIMENTAL ZOOLOGY, VOL. III, NO. 4
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AS; i in iy _
From the Anatomical Laboratory of the Johns Hopkins University
SOME EXPERIMENTS ON THE DEVELOPING EAR
VE Wee OF FHE TADPOLE WITH RELATION
TO LOUMIBRATION:
BY
GEORGE L. STREETER, M.D.
Associate, Wistar Institute of Anatomy
With Twe ve Ficures
The eventual object of the experiments reported in the following
paper was the rearing of some tadpoles which had been deprived
of their auditory vesicle and acoustic ganglion, either on one side
alone or on both sides; that is to say, an artificial production of a
unilateral and bilateral absence of the acoustic apparatus. ‘This
was done in the expectation that it might be possible to trace the
central acoustic path, in this new way, and perhaps throw further
light upon its course and relations. ‘The absence of these sense
organs, however, produced such definite abnormalities in the
behavior of the growing larve and in the development of their swim-
ming «bilities that it became at once apparent that I was dealing
with valuable evidence in respect to their function and its bearing
on the mechanism of equilibrium. It is, therefore, deemed advisable
to restrict the following paper to the physiological features of these
experiments, and reserve the study of the central nervous system
of the reared specimens for a later communication.
What we already know concerning the function of the vertebrate
ear is based principally on experimental sectioning or stimula-
tion of the semicircular canals, or the nerves to their ampullz, in
adult birds and fishes?
‘Read in part before the Section of Anatomy of the British Medical Association, at the meeting held
in Toronto, August 21-25, 1906.
? For experimental work on fishes we are for the most part indebted to Lee (’93 and °98) and Lyon
(00), both of whom carried on their experiments at the Woods Hole Laboratories. Further work on
fishes has just been completed at the same place by Professor Parker, whose paper I am told is now in
press and will appear in the Bulletin of the U, S. Fisheries Bureau. An abstract of part of his
work was read before the American Zodlogical Society (Parker, ’o5). A voluminous literature exists
concerning experiments on higher vertebrates, particularly the pigeon, but it need not be considered
here.
THE JourNAL oF ExPERIMENTAL ZOOLOGY, VOL. III, NO. 4.
544 George L. Streeter
The fact that it is possible to experiment on the embryo and to
produce at will practically a congenital absence of thisorgan, besides
serving as a control over the experiments on adult animals, intro-
duces a direct advantage both as regards the ease with which the
operation is performed and as regards its completeness and per-
manence and freedom from injury of adjoining structures, the latter
point beingof particular importance to those who are still in doubt
as to how much 1s due in the experiments on adults to injuries and
stimulationsassociated with the operation and how much is purely
the result of the cessation of the stimuli which normally originate in
thelabyrinth. Furthermore, since the labyrinth is removed during
the early formative period at a time when it may be presumed that
the various organs possess their greatest adaptability, it will be seen
that such embryonic interference affords a most complete test
of the power of functional compensation on the part of other
organs.
Behavior of Normal T ad poles
In analyzing the behavior of operated specimens it was found
necessary to make a preliminary study of control tadpoles, in order
to determine the normal development of motor reflexes and their
coordination and the consequent establishment of equilibrium.
This was done by removing the larve from their gelatinous cap-
sule shortly after fertilization and following their development in
tap water. In this way it was seen that in the process of learning
to swim they pass through three periods, which may be named as
follows:
1. Stage of non-motility, first three days.
2. Stage of spinal reflexes, fourth to sixth days.
3. Stage of equilibrium, sixth day to maturity.
The first stage, with a favorable temperature, lasts from the
time of fertilization to the third or fourth day. During this time
the larve, aside from the movement due to cilia, lie motionless on
their side on the bottom of the dish and do not respond to stimuli.
The second stage begins at the time when they first respond to
2
Experiments on the Developing Ear Vesicle 545
mechanical stimuli by flexion of the body and tail. These reac-
tions consist of simple motor reflexes at first, but they soon become
combined and coordinated so that by a series of such body flexions
they are able to wiggle rapidly forward on the bottom of the dish.
This manner of progression evidently consists entirely of spinal
cord reflexes and is not controlled by higher centers. In order to
perform it, it is necessary for the tadpoles to touch the bottom or
Fic. I
Fic. 2
Fig. 1, Outline drawing of normal tadpole (Rana sylvatica) of the second stage or stage of spinal
reflexes. Enlarged 8 diameters.
Fig.2, Outline drawing of normal tadpole (Rana sylvatica) at the beginning of the third stage. This
specimen had the power of equilibration, although sections of the ear vesicle showed that the
development of the semicircular canals was not yet complete. Enlarged 8 diameters.
side of the dish; when they are driven up into the free water with
a pipette, where there is no contact with solid objects, they make
no effort at movement, but sink inertly to the bottom; on striking
the bottom they run forward again. ‘The third stage begins when
they are first able to move freely about without touching solid
1T have been informed by Dr. R. G. Harrison that it is just at this time that the motor nerve
roots make their appearance, and this may determine the onset of the second stage. According to
his observations the power of muscle contraction follows almost immediately after the development
of the motor roots; but it never precedes their development, as is maintained by some. He has
found the motor root present in specimens that had not yet moved.
546 George L. Streeter
objects. At this time a new controlover their movements is devel-
oped, in virtue of which they become able to leave the bottom of the
dish and swim up into free water with maintenance of what may
then be called equilibrium. ‘The form of the tadpole during the
latter part of the first stage 1s shown in Fig. 3. The second and
third stages are shown in Figs. 1 and 2.
The correlation between the histological development of the
labyrinth and the development of the power of equilibrium was
studied by selecting specimens of the second and the beginning of
the third stages, carefully noting their behavior, and then cutting
them in serial sections.!
From these series it could be seen that shortly before the animal
enters the stage of equilibrium the labyrinth consists of a closed
epithelial sac incompletely subdivided into compartments and
possessing differentiated nerve endings which are connected with
the brain by the acoustic nerve and ganglion. ‘That at least one
such apparatus is essential for equilibrium will be seen when I
describe the behavior of tadpoles that have been completely
deprived of the same. As regards the semicircular canals it 1s a
different matter; they can already be seen in the process of
development, but are not completely pocketed off until after equili-
brium is already established. Consequently the semicircular canals
as such are not an essential factor in equilibration.
Method of Operation
Larve of Rana sylvatica measuring about 3 mm. long were
selected as being most suitable for the operation. ‘Their general
form at this time 1s shown in Fig. 3. There is a distinct tail bud,
and on the head the eminences caused by the optic cup and head
ganglia are visible. ‘he structure that is to form the future laby-
rinth is situated just dorsal to the ganglionic eminence and is shown
The correlation between the histogenesis of organs and the development of their functional activity
forms a fruitful field which has been explored by comparatively few investigators. It may be approached
both through ontogeny and phylogeny. Prentiss (o1) by this means worked out important facts
regarding the crustacean otocyst. Many details concerning the verte brate ear which do not belong to
the scope of the present paper could doubtless be learned in the same way.
Ex periments on the Developing Ear Vesicle 547
in Fig. 3 by the mark +. It consists of a cup-shaped mass of
cells (auditory cup) which have differentiated themselves from
the deeper layer of epidermis, and are just
in the process of closing in at the edges to
a
form the completed ear vesicle. In size eo
this ear cup or ear vesicle is about one- :
half that of the optic cup.
For performing the operation it 1s not
necessary to anesthetize the specimen as
itis stillin the non-motile stage and does aiaicemor cone
not respond to stimulation. After remov- _ operation, just at the end of the non-
inethe lakvatromiits eelatinous capsule it motlestage. The tail budis present
is placed under a binocular microscope Sele oe eS
5 nences due to the optic cup and head
and anincision made nearthe place indi- ganglia. Above the latter is the
cated in Fig.3. The edge of the incision _ point of operation shown by a cross.
is then raised a little and the auditory cup PE
is picked out witha needle. After a little
practice one learns to make the incision directly at the edge of the
cup so that it comes away easily and intact, resembling somewhat
a thimbleberry. Lying just in front of it is the acoustic ganglion
which is not so sharply outlined. This is also removed and,
in order to make sure that it is all taken out, the surrounding
mesoderm is cleaned out as far in as the brain. Where but one
vesicle is to be removed the operation is then complete, and the
specimen is left to proceed in its development. The wound
immediately closes of itself and heals in the course of a few hours
leaving no trace of the operation. Where both sides are operated
on, the same procedure is carried out on both sides. The ear
vesicle never regenerates following complete removal.
The ear vesicle was removed on one side from thirty specimens
and on both sides from twenty specimens. ‘The animals were then
kept under observation and their behavior recorded through the
whole larval period and until the completion of metamorphosis.
The following notes were selected from these records.
Fig. 3. Outline drawing of Rana
548 George L. Streeter
Removal of One Ear Fesicle
Twenty-four hours after operation: Specimens are 5.5 mm. long
and show presence of gill buds. In appearance and behavior, no
difference can be detected between them and normal tadpoles.
They lie on their side and on stimulation flex their body, but make
no attempt at swimming.
Forty-eight hours after operation: Specimens are 7 mm. long,
gills are branched and the blood can be seen circulating through
them. In appearance and behavior they still show no departure
from that seen in normal control specimens. While at rest they
lie on theit side. On stimulation (sunlight, jarring the dish, or
touching with needle) by a rapid flexion of the body and tail from
side to side they swim forward, 5-10 cm., on the bottom of the
dish in a straight or slightly curved line, and then come to rest on
their side, and remain so until a new stimulation excites another
such excursion. ‘Their course is directed either by the side or
bottom of the dish. When forced up into free water the flexions
stop and they sink inertly to the bottom.
Third day after operation: Specimens average about 8 mm.
long, abdominal epidermis differentiated from that of the dorsal
parts of the body by being less pigmented. Appearance and be-
havior is still practically normal. ‘They begin to show a tendency
to assume the upright position while at rest, but no great impor-
tance can be attached to this feature as throughout the early days
of the tadpole period, preserved specimens lie in the same positions
as living ones. ‘Their posture in water may be entirely determined
by their body proportions. ‘Their movements remain of the spinal
cord type seen on the previous day, the response being more prompt.
Fourth day after operation: Specimens 9-9.5 mm. long. In
appearance the operated specimens are the same as the normal
ones, but in behavior they present a difference. ‘The normal ones
still confine their movements to the bottom or side of the dish;
when stirred up into free water, though most of them still roll about
inertly, some of them are able to maintain a direct course. On the
other hand the operated ones, as soon as they are driven from the
bottom, swim in a spiral or circular manner as shown in the
Experiments on the Developing Ear Vesicle 549
accompanying Fig. 4. “The tendency is to swim with the operated
side under, and in the rolling movements around the long axis of
the body it is from the operated side under to the opposite. When
these same specimens touch the bottom they are able to direct their
course as on the previous two days. Evidently, a functional union
is normally established at about this time between the ear vesicle
and the spinal cord reflex centers, upon which the individual is
dependent for maintaining its position in free water, and it 1s not
until this occurs that the removal of the ear vesicle causes any
symptoms.
Fig. 4. Sketch showing three typical swimming movements made by specimens on the fourth day
after removal of their left ear vesicle.
Sixth day after operation: Specimens about 12 mm. long, and
have commenced to nibble at food and pass faeces. The charac-
teristic movements which first appeared on the fourth day have
become stronger and stand out in more marked contrast to the
behavior of normal specimens which at this time can swim easily
up into free water with accurate maintenance of equilibrium.
Seventh day after operation: ‘The operated specimens show
distinct improvement in swimming ability; many of them are now
able to maintain a fairly direct course in free water, but on excita-
tion they renew the spirals and circles which characterized the
fourth, fifth and sixth days.
550 George L. Streeter
Eighth day after operation: nearly all the specimens now
swim freely and directly in all parts of the water, and irregularity
of swimming is only elicited by excitement.
Tenth day after operation: Swimming 1s practically normal.
Their movements are under such control as to enable them to sup-
port themselves in free water and nibble at floating stems and
leaves. It can be seen, however, that in swimming they lean
Fig. 5. Photograph of a frog whose left ear vesicle was removed when a tadpole 3 mm. long. The
only asymmetry noticeable is the absence of the ear elevation on the left side normally caused by the
labyrinth and its cartilaginous capsule; the lateral line on that side is straight from the eye back, while
on the right or normal side it is deflected. The posture is normal. Enlarged 34 diameters.
g gece 32
slightly toward the operated side, a sympton which persists through-
out their larval period.
Twelfth day after operation: Specimens are normal as regards
size, nourishment, and symmetry, except for the absence on the
operated side of the elevation which is caused normally by the
labyrinth and its cartilaginous capsule. In behavior they differ
from the normal only in the slight leaning toward the operated side
Experiments on the Developing Ear Vesicle 551
and a momentary loss of equilibrium which can be elicited by
excitement.
Three months after operation: ‘The specimens passed through
a normal metamorphosis at the end of the third month. A photo-
graph made of one of them a few days after the completion of the
process is shown in the accompanying Fig. 5.
As long as they continued as swimming tadpoles the slight lean-
ing toward the operated side persisted and it was possible through
excitement to cause a momentary disturbance in equilibrium, but
the latter became gradually more difhcult to demonstrate. As
soon as they commenced to make use of their legs the character
of the swimming changed; it then became a series of leg strokes
instead of the sinuous flexions of the body and tail. After that it
was no longer possible to detect the leaning toward the operated
side; both when swimming and when at rest their behavior was to
all appearance normal. When taken out of water they jumped
normally and came to rest in a normal posture. When turned
over on their backs they righted themselves promptly.
The fact that the slight disturbance of equilibrium, which could
be still detected in the tail-swimming tadpole, could no longer be
seen in the leg-swimming frog, a change completed within four or
five days, probably does not signify the cure of the condition, but
rather that under the latter circumstances a slight defect 1s more
difficult to recognize. ‘The corollary of this sould be that equili-
brium in the swimming tadpole is a more delicately balanced
mechanism than in the Ticking and jumping frog.
Removal of Both Ear Vesicles
During the first three days after the operation the appearance
and behavior of these specimens are the same as seen in the normal
ones, and in those from which one ear vesicle was removed. The
response to stimuli 1s perhaps a trifle less prompt, but otherwise
they could not be distinguished one from the other.
Fourth day after operation: It was seen that in one-sided oper-
ations the specimens commenced about this time to make excur-
552 George L. Streeter
sions into free water, and in doing it they departed from the nor-
mal by swimming in spirals and circles. “Tadpoles with both ear
vesicles taken out make no such excursions and show decidedly
less activity. Occasionally they flex their body and tail from side
to side producing a snapping effect which does not result in any
forward progress. Like the other specimens they are, however,
able to wiggle along in contact with the side and bottom of the
dish.
Seventh day after operation: The specimens are smaller and
are retarded in development as compared with the normal and
one-sided specimens. ‘They are, however, symmetrical in form
and are normal as regards the appearance and movements of the
eyes, mouth, heart and intestine. ‘They are decidedly less active
and stimuli produce irregular attempts at swimming, sometimes
somewhat spiral in character but usually nothing more than a
series of awkward flexions of the body. ‘These flexions also
occasionally occur with no apparent stimulus. They make a
partially successful effort at nibbling on the bottom of the dish.
Twelfth day after operation: Absolutely no improvement in
swimming; any attempt at it results in a series of somersaults.
they throw their body up into the water and then promptly sink
to the bottom in almost any position. When at rest, they lie on
their side, back, or normally on their belly, depending apparently
on whether their intestine is filled with sand, etc., to properly
balance the body. ‘The intestine is very apt to be empty because
of the difficulty they experience in feeding. ‘They do not wiggle
along on the bottom as well as they did on the fourth and fifth
days.
‘Two months after operation: The specimens could not be
carried much beyond this point, the difficulty apparently being
starvation from inability to wander around and collect food.
Perhaps also the respiration was involved, for they were unable
to go to the surface for oxygen as the normal tadpole does.
In behavior they showno improvement. For the most part they
lie stiff and inert in various positions on the bottom, and their
occasional attempts at swimming have never developed into anything
more successful than was described on the seventh and twelfth
Experiments on the Development Ear. Vesicle 553
days after operation. ‘Their appearance departs from the nor-
mal principally in the small contracted character of the abdominal
region. In volume they are about one-third as large as the normal
specimen, varying from2.5 to 4.cm. in length. They have a hind
leg bud 2.5 to 3 mm. long. As some of them commenced to
die at this time the rest were put in preserving fluids for micro-
scopial purposes.
A summary of the above notes on the operated individuals may
perhaps be best formulated by making the following comparison
with the three stages of normal behavior.
First stage: [he operation was performed during the latter
part, while the animals were still non-motile.
Second stage: During this period they behave exactly like
normal specimens, both those having one vesicle removed and
those that have been deprived of both vesicles. They respond to
stimuli and learn to wiggle along in contact with the bottom of the
dish in the normal manner.
Third stage: It is at the beginning of this period that they
depart fromthenormal. Itcanbe plainly seen from their conduct
that something has happened to that controlling influence from
above, which they require in order to leave the bottom and to swim
and maintain their position in free water. In case but one ear
vesicle is gone they swim in spirals, circles, or straight while roll-
ing around their long axis. ‘This, however, lasts only a few days
and then it is gradually overcome. From then on they swim
almost perfectly; there may be a slight tilting toward the operated
side and on excitement a momentary loss of equilibrium, but this
would only be seen on careful examination. It is a different mat-
ter where both labyrinths are absent; the animals in that case are
completely and permanently incapacitated for swimming. ‘There
Is no apparent sense of equilibrium and they never develop any.
The animals were kept alive about two months, at the end of
which time their movements were as irregular as at the beginning.
Transplantation of Ear Vesicle After Bilateral Removal
From the above experiments it became evident that a tadpole
having but one labyrinth proceeds in its general growth and
554 George L. Streeter
develops swimming abilities about as wellas the normal animal; but
specimens deprived of both ear vesicles never learn to swim and
never develop any sense of equilibrium. ‘The next step was to
see if it would be possible to remove both vesicles and atthe same
time transplant one of them into a new position, having in mind
the successful results obtained by Lewis (04) in transplantation of
the optic cup.
After that operation if the tadpole succeeded in developing
equilibrium and the power of swimming then it would prove that
a transplanted ear vesicle could establish new connections with the
central nervous system and develop its normal functions; the ship
would simply be sailing with its compass set up in a different place.
Fig. 6. Tadpole showing elevation in front of eye caused by the transplanted left ear vesicle, the
right ear vesicle having been entirely removed. Drawing made three months after operation. Enlarged
4 diameters.
The operation was one that could be performed without diff-
culty. A tadpole about 3 mm. long is selected and the ear
vesicle taken out on one side in the manner described above. ‘The
specimen is then turned over and the opposite ear vesicle is
uncovered and loosened from the epidermis. Before actually
removing it a straight incision 1s made with scissors or needles in
front of the eye and a pocket is created by gently spreading the sub-
jacent mesoderm apart until the brain is exposed. The loosened
ear vesicle is then lifted from its natural place and slipped into
this pocket. If the incision is carefully made the edges of the
wound close at once and on the following day there is no trace of
the operation left. Nine operations of this kind were made and
seven of the tadpoles successfully reared. While they were grow-
ing it could be seen from a surface view that the transplanted
vesicle was developing and causing a corresponding elevation in
Ex periments on the Developing Ear Vesicle 555
front of the eye. A sketch of one of these at the end of the third
month is shown in Fig. 6.
Their behavior during the first week following the operation was
identical with that of specimens deprived of both vesicles as was
to be expected. ‘Toward the fifth and sixth days they could
make progress while touching the side or bottom of the dish, but
any attempt at swimming in free water resulted only in irregular
flexions of the body and somersaults. It was hoped that the trans-
planted vesicle might then begin to function and make it possible
for them to perceive their position while in free water, but this did
not occur. They continued to behave in all respects like tadpoles
having no labyrinth and never gave evidence of possessing any
trace of equilibrium.
At the end of the third, fourth and twelfth weeks specimens
were killed in preserving fluid and prepared in serial sections.
Examination of the sections showed that in six out of the seven
specimens which were cut, the transplanted vesicle had developed
to a greater or less extent, and it was these vesicles that formed the
surface elevations that had been macroscopically visible in front
of the tadpole’s eyes. Graphic reconstructions of them are repre-
sented in Figs. 7 to 12. It will be seen that none of these consti-
tute a perfect labyrinth, but on closer study it 1s found that they
all possess certain features which are characteristic of it. Inthe
first place, that which was transplanted in the form of an open
auditory cup developed after the operation into a closed vesicle
containing endolymph. This did not then remain a simple vesicle,
but exhibited the tendency to subdivision into two or more com-
partments, the utriculus and sacculus, as seen in Figs. 7,8, 12. Inthe.
walls of these compartments there are areas of specialized epithelium
representing the maculz acustice. In Fig. 7 there opens out of
the more dorsal compartment a distinct endolymphatic appendage.
A typical semicircular canal is not present in any of them; but
what may be called a canal tendency is seen in Fig. 8, where
there is a tube uniting the two principal compartments. The
small blind pouches leading off the main vestibule, three of which
are present in Fig. 10, doubtless represent abortive canals. In
transverse section they are perfectly round and look like typical
556 George L. Streeter
canals. It may be recalled that Ridinger (’88) described the
semicircular canals as developing in the form of blind tubes sprout-
ing out from the general vesicle. It is quite possible that he was
dealing with an abnormal embryo and had the same form of canals
that we see in Fig. 10.
The ear vesicles are more or less completely enveloped in con-
nective tissue membranes and they are partly incorporated in
masses of cartilage, some of which belongs to the normal cartilagi-
nous cranium and some of it is the regular cartilaginous capsule
of the labyrinth, the two fusing together in some places.
In four cases (Figs. 7, 8, 9 and 10) a group of ganglion cells and
nerve fibers are attached to the median side of the vesicle near its
caudal end and extend toward the central nervous system. In
one instance (Fig. 7) the nervous connection between vesicle and
brain at the junction of olfactory lobe and fore-brain, is complete,
though it 1s only a few fibers that actually enter the brain. As the
acoustic ganglion at the time of the operation is attached to the
auditory cup some of its ganglion cells are undoubtedly carried
along with it, and it is probable that it is these cells that furnished
the nerve connections just described. At the time the transplanted
ear cup was slipped into its pocket the adherent ganglion cells
must have been lodged in various positions as regards the ear cup
and the fact that they all come finally to lie on the median side of
the vesicle and lead toward the brain must be explained by some
theory of an attraction existing between brain and nerve.
When we have to deal with a transplanted labyrinth that has
reached a development equal to those that function in young tad-
poles, and has established communication with the central ner-
vous system, we might expect that it would show some sign of
physiological activity. The failure of it to do so is perhaps best
accounted for by the fact that the point of entrance into the brain
is so far away from the hind-brain centers and the spinal cord that
connections with these are not established. If the experiments were
varied and the vesicle transplanted to some point in the neighbor-
hood of the occipital nerves this difficulty would be obviated.
Fig. 7 Fig. 8 Fig. 9
Fig. 10 Fig. 11 Fig. 12
Figs. 7 to 12. Graphic reconstructions showing the form and relations developed by transplanted ear
vesicles, one to three months after the operation. In all six cases the right ear vesicle was removed and
the left vesicle transplanted into a subdermal pocket between eye and nostril. In Figs. 7,8, 9 and 10
the acoustic nerve and ganglion extended from ear vesicle toward brain; in Fig. 7 the connection was
complete, the fibers entering at junction of fore-brain and olfactory lobe. Central nervous system,
shaded; ear vesicle, solid black.
558 George L. Streeter
Conclusions
In the tadpole the ear vesicles are essential for the development
of the power of equilibration, but the study of normal specimens
shows that well developed equilibration may be present before
the completion of the semicircular canals; the latter as such are
therefore not essential.
When both vesicles are removed no other organ compensates
for their loss and the animal is completely and permanently help-
less as regards the maintenance of equilibrium. When only one
ear vesicle is taken out the remaining vesicle 1s capable of perform-
ing the work of both so perfectly, that the casual observer would
mistake them for normal individuals.
Transplantation of the ear vesicle shows that the group of cells
forming the auditory cup or primitive ear vesicle is specialized to
that degree that although removed from their natural relations
and placed in a new environment they still continue to differentiate
themselves into a structure approximating the normal labyrinth.
A nerve and ganglion develops, and complete nervous connection
may be established between the transplanted vesicle and the brain
at an abnormal place. Where the latter occurred it did not give
evidence of any functional ability.
LITERATURE
Lee, F.S.,’93.—A Study of the Sense of Equilibriumin Fishes. Journ. of Physiol.,
vol. xv and xvil.
”98.—The Function of the Ear and Lateral Line in Fishes. Amer. Journ.
of Physiol., vol. i.
Lewis, W. H., ’04.—Experimental Studies on the Development of the Eye in
Amphibia. Amer. Journ. Anat., vol. u1.
Lyon, E. P.,’00.—A Contribution to the Comparative Physiology of Compensatory
Motions. Amer. Journ. of Physiol., vol. 11.
Parker, G. H., ’05.—The Skin, Lateral-Line Organs and Ear as Organs of
Equilibration. Proceedings of Amer. Zo]. Soc., Science, vol. xxi.
Prentiss, C. W., ’o1.—The Otocyst of Decapod Crustacea; its Structure, Develop-
ment and Functions. Bull. Museum Compar. Zool., Harvard
College, vol. xxxvi.
RupinceER, ’08.—Zur Anatomie u. Entwickelung desinneren Ohres. Berlin, 1888.
asain
iaiE RELATION BETWEEN FUNCTIONAL REGU-
LATION AND FORM-REGULATION
BY
C.-M. CHILD
The phenomena of regulation in the organic world have received
much attention in recent years and it has become more and more
evident that a consideration of these phenomena involves some of
the most fundamental problems of biology. It is perhaps not
going too far to say that the solution of the problems of regula-
tion will constitute a solution of most of the other problems which
underlie physiological biology. Moreover, we have, in the possi-
bility of analyzing and modifying regulatory processes by experi-
mental conditions, a means of attacking the problems involved,
which is more exact and has already proven more fruitful than
other efforts directed toward the same end.
Inorganic life structure and form are to the observer perhaps
the most salient features and so constitute the most available
criterion for distinction and recognition not only of the compo-
nent parts of theorganism butof differentorganisms. Any method
of procedure which permits the control, analysis, and modification
of the processes which give rise to structure and form is, therefore,
of the greatest importance, since it affords an insight into some of
the phenomena most characteristic of and peculiar to organic life.
The experimental method as applied in the field of form-regula-
tion is of this character, and it has already demonstrated that
many of the formative processes are dependent upon conditions
which can be altered experimentally. Thus we are able in many
cases to modify and alter the form and structure very widely.
We are at present only on the threshold of this field of investiga-
tion but the outlook for the future is most promising. As our
methods improve and as we come to comprehend more clearly
the character of the phenomena with which we are dealing the
THE JourNAL oF ExprrIMENTAL ZOOLOGY .VOL. Il, NO. 4.
560 CoM -Ghid
field within which these methods are applicable will extend andthe
value of the results obtained will increase.
Certain workers in the field of form-regulation have, it is true,
reached somewhat disappointing conclusions. Some maintain
that the data are as yet insufficient to permit any interpretation,
others that the phenomena of regulation belong to a totally differ-
ent category from those of the inorganic world and that, therefore,
we cannot hope to interpret them in terms of physics and chemis-
try. he reason for these conclusions is, the writer believes, to
be found in the fact that these authorities assume the formative
processes to be a special series or complex of processes in the
organism differing in nature from other so-called functional pro-
cesses. The organism is regarded as possessing two groups or
complexes of activities, the one giv ing rise to structure, the other
concerned with the dynamic activities in the structure. Any
such distinction seems to the writer entirely artificial and without
basis in fact. Numerous examples of the dependence of struc-
ture for its existence upon dynamic or functional conditions are
before us, and the only reason why the data are not still more
abundant is that those already obtained have failed to attract
due attention.
It becomes increasingly evident that the organism 1s primarily a
dynamic or functional complex and the structure and form are
merely visible expressions of the dynamic conditions. The
writer has attempted in various papers during the last three years
('02—’o6a) to develop this idea and apply it to specific cases of
form-regulation but it seems worth while to present in more gen-
eral form some of the conclusions reached, together with some of
the facts on which they are based, and to show how the ideas
involved may be applied to other cases.
The process of form-regulation is commonlyregarded as consist-
ing essentially in the replacement of a lost part—hence the term
regeneration is used by some authors as synonymous with form-
regulation. But form-regulation includes not only the replacement
of lost parts: it may also involve hypertrophy or atrophy of parts
remaining, the substitution of one part for another, the develop-
ment of a part widely different from that lost, partial replace-
Functional Regulation and Form-Regulation 561
ment or no replacement at all. ‘The recognition of the fact that
these various phenomena, some of which are widely different from
regeneration in the original sense are fundamentally similar justi-
fies the use of a term which has not the disadvantage of possessing
another and different meaning. Moreover, the term regulation
serves or should serve to call attention to the fact that there is
something similar in these processes to what has long been known
as regulation in functional processes.
In the following paragraphs some of the writer's conclusions
are presented, aaditias often in somewhat positive fashion, but the
positive form of statement has its value in the presentation of
hypotheses.
T he Initial Factor in Replacement of a Lost Part
Driesch (’01) maintains that the initial factor in regulation is a
disturbance in function or in constitution. While the writer can-
not agree with Driesch in regard to the sharp distinction which he
makes between function and constitution, it is sufhciently obvious
that the initial factor in the process of replacement consists in
some change in that part of the dynamic system which remains.
Have we any data which afford a clue as to the character of
this change and the regions involved? In this connection
certain experiments on flatworms may be mentioned first. In
Stenostoma (Child, ’02) or Cestoplana (Child ,’05b) after removal
of the posterior end of the body at any levels except immediately
posterior to the cephalic ganglia the posterior portion of the
remaining part is used by the animal in much the same manner as
the posterior portion of the part removed. ‘The functional activ-
ities of this region which are involved in locomotion are readily
observable since it forms the chief region of attachment and 1s
employed almost constantly during progression. ‘These forms
possess means of adhesion along practically the whole length of
the ventral surface but under normal conditions the posterior end
is most used and the organs of adhesion are most highly devel-
oped there. ‘This functional substitution is often very imperfect
at first, but rapidly becomes more perfect and after a few hours
562 C. M. Child
the posterior end of the piece is employed in much the same
manner as the original posterior end, though adhesion is apparently
not as firm.
In short, the animal has altered its behavior and in response to
the new conditions has ‘‘learned”’ to use this part in place of the
part removed. ‘This altered function involves not merely the
portion immediately adjoining the cut surface, but other parts for
a greater or less distance anterior to this (Child, ’o5b). In other
mane. a portion of the piece becomes functionally posterior
even though its original position may have been far from the pos-
terior end. If we follow the process of form-regulation which
succeeds this change in function we find that this part becomes in
the course of time by a process of redifferentiation structurally as
_ well as functionally a posterior end. In Stenostoma the process
of formation of the new posterior end is very rapid, and if we pre-
vent the piece from attaching itself in the characteristic manner
form-regulation is delayed (Child, ’03a). In this latter case then
we have demonstrative evidence that the functional conditions
connected with the use of the part in a certain manner are impor-
tant factors in form-regulation. Moreover, facts which cannot be
cited here lead us to the same conclusion in regard to Cestoplana.
In some of the flatworms, e. g., Planaria maculata (Morgan,
’98, etc., and others) a new head region can be formed at any level
of the body, in others, like Dendroccelum, only in the more
anterior regions (Lillie, ’or), and in still others, Leptoplana
(Child, ’o4c), Cestoplana (Child, ’o5b), etc., only from levels
immediately posterior to, through, or anterior to the cephalic
ganglia. Lillie was first to note that the pieces of Dendroccelum
capable of producing a new head react much more like the normal
animal than those incapable of such regulation. ‘The writer has
observed the same difference in reaction in Leptoplana, Cesto-
plana, and many other Turbellaria. Undoubtedly in these cases
the character of the reactions is dependent in large measure on the
physiological character of those portions of the nervous system
which are present. In all of these pieces where formation of a
new head is possible the anterior end of the piece behaves in some
degree, often very slightly it is true, like a head. We find, more-
Functional Regulation and Form-Regulation 563
over, that this ““head-likeness”’ increases as time goes on and long
before the new head is fully formed the behavior of the growing
parts is very distinctly head-like. In this case the piece is “learn-
ing’’ to use the region as a head and the morphological develop-
ment of the head follows.
In these cases as well as in many others the behavior of the
pieces and the use of the parts affords us valuable evidence as to
what is really taking place. It must not be supposed that the
movements themselves are the only or most important factors
involved. They may and undoubtedly do play a part in many
cases where particular regions are subjected to particular mechan-
ical or other conditions in consequence of characteristic move-
ments (Child, ’02, ’03a, ’o4a, ’o4b, ’o4c, ’05a, ’05b), but their
chief value is that they serve as an index to internal conditions.
and changes.
These cases are perhaps sufficient to illustrate the point of view
and to afford a certain basis in fact forit. Other facts which bear
upon the same point have been cited and discussed in the papers
above referred to. According to this point of view the first step
in the process of form-regulation is functional regulation, an
alteration in internal dynamic conditions in consequence of the
disturbance of what we may call the physiological equilibrium of
the system. ‘The process of functional regulation 1s not, however,
fundamentally a process of “trial and error’ (see pp. 578, 579),
for its nature is in large measure predetermined by the dominant
reactive capacities of he system.
But can we bring such cases as the regeneration Of anvabmm
the starfish, a leg in the crayfish, or the amphibian, into the same
category’ Is there any functional regulation in the direction of
substitution of a part remaining for a part removed.
In such cases as these the first visible change is the closure of
the wound either by a mass of coagulated blood, leucocytes, etc.,
or by cellular material and in all cases sooner or later by a mass of
tissue composed of cells capable of division and growth. This
process of wound-closure, while regulatory in nature in that it is
the result of reactions in response to new conditions 1s not directly
connected with the replacement of the lost part, but is primarily
564 C. M. Child
the result of purely local conditions. As 1s well-known, it occurs
not only where replacement takes place later but also in those
cases where there is no approach toward replacement. It is evi-
dent then that the closure of the wound is not, strictly speaking,
the first step in replacement of the lost part, although it may be
the first morphological step in regulation This distinction is
important, for, as was pointed out above, regulation and even
form-regulation involves much more than the replacement of lost
parts.
But the small mass of growing, physiologically plastic tissue
which closes the wound may stand, as soon as it forms, or strictly
speaking as soon as it begins to form, in a relation to the whole
system or organism more or less similar to that in which the part
removed or some portion of it originally stood. “This region must
be subjected to many conditions—internal and sometimes exter-
nal—similar in greater or less degree to those to which the part
removed or some portion of it was subjected. ‘The nerves which
formerly led to the part or some of them now lead to this region
and the regeneration of the nerves begins very early. It stands in
the same or somewhat similar relations to other parts of the body
as the part removed and must, as it forms, use a certain amount of
energy which is derived from other regions in much the same
manner as the part removed derived its energy. In short,
this region may by virtue of its relations to the whole become the
physiological representative in greater or less degree of the part
removed. In case this physiological substitution takes place up
to a certain degree further regulation occurs and the region begins
to develop into the part removed. In case the substitution does
not occur or is insufhcient in degree to bring about further
growth no replacement of the lost part occurs. Ths growth pro-
ceeds the dynamic conditions are more and more modified so that
the substitution becomes more and more complete until the miss-
ing part 1s fully formed.
In many cases the growth of the regenerating part may proceed
at least for a time without actual exercise or use of the part,
though complete differentiation apparently does not occur with-
out some degree of use. ‘The regenerating arthropod appendage
Functional Regulation and Form-Regulation 565
is coiled beneath the closed end of the stump until the first moult
following the operation. It is clear, therefore, that the conditions
which determine the earlier stages of the growth and differentia-
tion of the part are not identical with those to which the part 1s
subjected later though they are just as truly functional. The
growth of the new leg is not the result of the attempt to use the
leg which is missing. ‘The growing tissue begins to develop into
a leg because its relations to the other parts of the system are in
some degree similar to those of the leg removed. As it grows, the
conditions approach more and more nearly those to which the
normal leg is subjected, 7. ¢., there is a gradual return of the
functional conditions to the normal.
This condition, functional substitution in the tissue closing the
wound, constitutes the one extreme as regards the process of
replacement and results in the formation of the lost part wholly
by the outgrowth of new tissue. At the other extreme are the
cases similar to those first mentioned where the functional sub-
stitution involves a considerable portion of the piece and the
process of replacement is one of redifferentiation of this region
without the outgrowth of new tissue except that closing the
wound. Between these extremes are found the various interme-
diate forms of form-regulation.
T he Common Methods of Form-Regulation
If the functional substitution for the part removed is accom-
plished within the part remaining, the region involved undergoes
a process of ‘‘redifferentiation”’ (Child, ’06a) and the conditions
determining the growth of new tissue from the cut surface may be
largely or wholly absent except so far as closure of the wound is
concerned. This is the case in Stenostoma (Child, ’02) and
in posterior regulation in Cestoplana (Child ’osb). If, on the
other hand, the substitution is confined to the region immediately
adjoining the cut surface or to the tissue closing the wound form-
regulation is almost wholly or wholly a process of regeneration.
The substitution in the old part may, however, be incomplete and
the distal portion of the part removed may be formed by the
566 C. M. Child
growth of new tissue, 7. ¢., regeneration, while its basal portion is
formed by redifferentiation as in Planaria maculata (Morgan, ’98,
etc.; Child, ’06b). Again the substitution in the old part may be
more complete at one level than at another and the relative
amounts of regeneration and redifferentiation will vary at differ-
ent levels. ‘This is to some extent the case in Planaria maculata,
for example, where the regenerated anterior region is shorter and
the redifferentiated region is longer in pieces from levels near the
head, while the reverse is true in pieces from the middle regions
(Child, ’06b). The difference is still more marked in Polyche-
rus where the writer has found! that the new posterior end is
formed almost wholly by regeneration in pieces from levels near
the head and almost wholly by redifferentiation in pieces from
levels near the posterior end, and the relative amounts of rediffer-
entiation and regeneration show all intermediate conditions in
pieces between these two extremes. And finally in some cases
form-regulation at the posterior end may be largely or wholly a
process of redifferentiation and at the anterior end a process of
regeneration as is the case in Cestoplana (Child, ’o5b). In all
these cases of ‘‘mixed”’ form-regulation the redifferentiation occurs
in the region which is physiologically similar to the part removed
to such a degree that substitution occurs readily and with relative
completeness, while regeneration occurs where the substitution is
confined to the tissue closing the wound or to regions immediately
adjoining the cut surface.
In short, the greater the physiological similarity between the old
part or a given region of it and the new, the greater the amount of
redifferentiation andthe less the amount of regeneration. The
reverse 1s also true up toa certain point. When the part remaining
is so widely different from the part removed that no substitution is
possible in any region not even regeneration occurs except in
closure of the wound, and the missing part is not replaced. All
intermediate stages between complete regeneration and mere
wound-closure may occur in a single individual at different levels.
This is well illustrated in Leptoplana (Child, ’o4c) and in Cesto-
1 Not yet published.
. ha
Functional Regulation and Form-Regulation 567
plana (Child, ’o5b), where the regeneration of the head is complete
from levels anterior to or through the cephalic ganglia and in
Cestoplana also from levels immediately posterior to the ganglia,
and becomes increasingly incomplete with increasing distance
from the ganglia, until in pieces from the posterior regions scarcely
more than wound-closure occurs. The absence of regeneration
beyond wound-closure in many higher forms is doubtless due
largely to the fact that the physiological specification of the tissues
is so great that no appreciable substitution occurs in any region
after removal of a part.
But form-regulation may occur, nevertheless, even in such cases
and may be represented by hypertrophy of other parts. A good
illustration of this is the hypertrophy of one kidney following the
removal of the other.
A process of destruction often occurs during form-regulation.
This is commonly the case in redifferentiation where the structures
formed and maintained by a given complex of conditions cannot
persist under the altered conditions and so degenerate or atrophy.
In a piece of Cestoplana from the prepharyngeal region, for exam-
ple, the new pharynx appears a considerable distance from the
posterior end of the piece and all the intestinal branches posterior
to the new pharynx disintegrate and are replaced by others formed
anew. It is probable that the movement of intestinal contents
during contraction and extension plays an important part in this
rather remarkable change. ‘The data in this case are as yet un-
published.
The Rate of Form-Regulation and the Limit of Size
The development of a part reproduced by regulation is often
greatly accelerated as compared with normal ontogenetic develop-
ment. ‘This rapid development can be due to nothing but a dis-
proportion between size and intensity of physiological conditions
to which the part is subjected. It is a familiar fact that increase in
intensity of functional conditions brings about increase in size or
hypertrophy of the part involved and decrease in intensity a
decrease in size or atrophy. ‘The new part in regeneration and
often also in redifferentiation is at first much ‘‘toosmall,”’ 2. e., the
568 C. M. Child
intensity of the physiological conditions to which it is subjected
corresponds to a region of much larger size and hence increase in
size takes place very rapidly. Dunne growth the relative inten-
sity of the physiological conditions decreases and so continually
approaches that existing in the original part before removal.
Hence unless other factors prevent, the part will grow with decreas-
ing rapidity until it attains approximately the size of the part re-
moved.
In the flatworms where form-regulation will occur in starving
pieces we find that the increase in size often ceases even before the
new part has attained the proper proportions with respect to the
old. In these cases the new part grows at the expense of the old
and may undergo absolute as well as relative increase in size while
the old part is undergoing absolute decrease. In such cases an
equilibrium between old and new parts different from that existing
in the normal animal is attained. As the new part grows the
conditions for further growth decrease in intensity and as the old
part is reduced its demand for nutritive material becomes rela-
tively more intense and a larger proportion of the material avail-
able is used up in its own activities. Therefore, growth in the new
part must cease before it reaches normal proportions, even with
respect to the reduced size of the old part.
The Relation between Rate of Regulation and the Degree of Injury
Comparison of two series of pieces of Leptoplana, the one set
consisting of the anterior one-fourth or one-fifth of the body, the
other of the anterior four-fifths or three-fourths, both kept without
food, shows that the pieces of the first series regenerate from their
posterior ends very much more rapidly and a much larger amount
of new tissue than the pieces of the second series (Child, ’o4b).
Yet the pieces showing the greater and more rapid regeneration
are only one-fourth as large as the others.
If we compare the behavior of the two sets of pieces during regu-
lation, especially during the earlier stages after the Tegeneration has
begun, we find that in ae first series pie new part is used to a much
greater extent than inthe second. Not only is this the case but the
activity in the old parts also is much greater in the first series than
Functional Regulation and Form-Regulation 569
in the second or in the normal animal. ‘The movements are more
violent and the irritability appears to be greater. In short, to all
appearances, marked modification in the dynamic conditions has
resulted from the removal of the posterior four-fifths and a scarcely
appreciable modification from the removal of the one-fifth.
When the new tissue has appeared it becomes in the first series
the functional representative of the four-fifths removed, and in the
second only of the one-fifth. ‘The early stages of its growth are of
approximately the same size in both cases but in the first series the
intensity of dynamic conditions must be much greater in this
region, since its role in the complex is much more extensive and
important than in the second. It must demand and receive a
much greater proportion of the energy of the complex. Conse-
quently growth or hypertrophy is much more rapid and greater in
amount. Moreover, if it is true that removal of the four-fifths has
brought about an increase in the intensity of dynamic processes in
the old part this will doubtless also tend to increase the rapidity of
growth in the new part, especially during earlier stages, since it
is involved in the various reactions. As the new part develops, the
intensity of activity appears to decrease.
Here as in other similar cases it is not simply the degree of move-
ments or exercise of the part that determines the rapidity and
amount of growth, though this of course may constitute one factor.
It is rather the conditions underlying the motor activity, the nerve
stimuli, the metabolic conditions, etc., of which the motor activity
is an index (Child, ’o4b, ’os5b).
Zeleny has recently obtained somewhat similar results in several
species of decapod crustacea and in a species of ophiurid (Zeleny,
"05a, 05b). In all of these cases the rate of regeneration increases
with the degree of injury up to a certain point. These cases differ,
however, from the case of Leptoplana in that here regeneration
occurs from two or more different regions while there only one
region is involved. Strictly speaking the case of Leptoplana 1s
comparable to removal of larger or smaller parts of a single arm
in the starfish or of a single leg inthe crustacea. Butif we remove
three or four arms from the starfish or several legs from the crayfish
each one of the newprimordia represents, when it appears, no larger
ae C. M. Child
portion of the complex than does the primordium of a single arm or
leg cut off at the same level, yet it regenerates more rapidly.
It was noted above that the short piece of Leptoplana showed
much more intense activity than the long piece. It is perhaps not
too much to say that this increased activ ity is a response or reaction
to the absence of the four-fifths. On receiving a stimulus the piece
reacts but reaction in the normal manner 1s impossible and the
result is ““irradiation”’ of the stimulus and the appearance of other
more or less violent reactions. ‘This fact of ‘‘irradiation” has long
been recognized in nerve physiology and Jennings’ recent experi-
ments on the modifiability of behavior show that it is an important
factor in behavior.
Zeleny has been unable to observe any characteristic difference
in activity connected with the degree of injury. But the reaction to
the injury need not necessarily appear as actual motor activity; It
may take the form of increased rapidity of metabolism or other
forms. In those cases where’ increased motor activity is present
it serves as an index to internal conditions but its absence does not
necessarily indicate the absence of increased dynamic activity of
other kinds.
There can be no doubt that a normal reaction normally carried
out has a certain effect on the individual and that an attempt and
failure to carry out the reaction has another and very different
effect, viz: in the direction of altered character and increased inten-
sity of reaction. [he old experiments upon the reflexes of the
decapitated frog are sufhcient demonstration of the fact. The
larger the number of arms or legs removed in the cases cited above,
the more impossible is the accomplishment of the normal reactions
and consequently the more intense the effect on the animal as
regards other reactions. As the new tissue appears it becomes
a more or less complete functional substitute for the parts
removed very incomplete at first no doubt. In the conditions of
intensified activity resulting from the operation the functional
conditions to which the regenerating structures are subjected must
increase in intensity with the degree of injury. ‘This increase will
be all the greater because their presence is the first step in the
approach to a normal method of reaction to which the system 1s, so
Functional Regulation and Form-Regulation 571
to speak, “‘seeking.”’ As regeneration goes on the functional sub-
stitution becomes more complete, and reactions take place more
nearly in the normal manner, functional conditions become rela-
tively less intense, and the rapidity of regeneration decreases.
Compensatory Hypertrophy
In a number of cases among the Crustacea (Przibram, ’o1, ’02,
05; Zeleny, ’o5a), where an asymmetery of the chelz exists,
removal of the more highly specialized chela 1s followed by a more
or less complete transformation of the other into the more highly
specialized type, and the regenerating chela develops into the less
specialized form. Thus a reversal of asymmetry results. If the
less specialized chela alone is removed no reversal occurs. Zeleny
(05a) has obtained somewhat similar results with certain species
of serpulids where two opercula, one large and functional, the
other small and rudimentary, are present. Removal of the func-
tional operculum results in a reversal of the asymmetry while no
reversal occurs after removal of the rudimentary operculum. When
the whole head region, including both opercula is removed both
regenerate in the form of the functional structure. Wilson (’03)
has found in Alpheus grounds for believing that when the nerve to
the chela is cut the reversal does not occur, but further experiments
along this line are needed.
A satisfactory interpretation of these cases on a functional basis
appears possible. When the specialized chela or operculum is
removed the disturbance of the system thus produced results in
changes in reactions and it seems probable that the easiest and
most natural change will be such that the less specialized or rudi-
mentary organs will receive stimuli and be subjected to conditions
which originally affected the specialized or functional structures.
In fact, it is dificult to see how any other interpretation can be
made to serve. Zeleny (’c5a) postulates for the serpulids the
existence of a ‘‘retardation stimulus” from the functional oper-
culum which, so long as this is present, inhibits the development of
the other. The nature of such a retardation stimulus is highly
problematical. It seems to the writer much more probable that
the failure of the rudimentary or less specialized part to develop
572 C. M. Child
beyond a certain point under normal conditions is due rather
to the absence of adequate stimuli than to a positive inhibitory
stimulus. So long as the other more highly specialized part
is present the animal reacts in a characteristic manner which
involves the less specialized part only to a certain degree; and the
structure of that part expresses the character of the conditions to
which it has been subjected. Removal of the less specialized part
does not alter the reactions to any such extent as removal of the
other, hence the part removed regenerates in the same form.
Removal of both places the new primordia on equal terms and both
may producethe more highly specialized or the functional structure.
Here, as elsewhere, the functional conditions involved are not
primarily those connected with use or exercise of the part but the
sum total of its dynamic relations to the whole system.
Polarity and Axial Heteromorphosts
Physiological polarity in the developed organism may be defined
provisionally as a habit of reaction resulting from past or present
relations. ‘This polarity of the organism may be a consequence of
the polarity of the egg and this in turn a consequence of the rela-
tions of the egg-cell to the body of the parent or of other con-
ditions affecting the egg. At any rate there seems to be no good
ground for believing that polarity is a permanent and funda-
mental property of the cell or of protoplasm, although certain
authorities have urged this view.
Polarity may be rendered visible by structural differentiation
along the axis or it may not, but in any case the structure is prima-
rily aresult not a cause. It may, however, become the determining
condition in that it determines the character of the reactions.
The original polarity is commonly maintained in form-regula-
tion simply because each of the parts reacts most readily in a
manner approaching that in which it has reacted in the past. But
this habit of reaction can be altered in certain cases. In Tubu-
laria, for example, pieces very commonly produce a hydranth at
both ends, the oral hydranth, first the aboral later. The delay in
the appearance of the aboral hydranth represents the time neces-
sary to alter the character of the reaction or in other words to
Functional Regulation and Form-Regulation Ge)
change the habit. Moreover, when the oral hydranth is pre-
vented from forming, the aboral hydranth appears more quickly
than when the oral hydranth is permitted to form. Here we have
an example of the case discussed above in which failure to carry
out a ‘“‘normal” reaction or the reaction most readily performed
results in increased intensity of other reactions. ‘That the habit
is really changed is shown by the fact that a second aboral hy-
dranth develops more rapidly than the first. Undoubtedly struc-
tural changes in the various regions result from the changes in
reaction but these are not visible in this form except in the struc-
tures produced at the cut surfaces.
Axial heteromorphosis can be brought about in various ways in
many of the lower forms. ‘The isolation of very short pieces 1s per-
haps the simplest method. In these cases the piece is not large
enough to represent the whole complex and the reaction in which the
piece has been most intimately involved in the past becomes the
chief or only possible reaction and similar structures are produced
at both ends. We find, therefore, that pieces of this kind often
produce at both ends the structures characteristic of the regions
with which they have been most closely associated in the past.
Sometimes other special factors modify this result, ¢.g., preparation
for fission in Planaria maculata (Child, ’o6b), or as in Tubularia
and some other hydroids the dominance of a particular reaction-
complex throughout the individual. This latter case requires a
word of comment: pieces of Tubularia from any part of the stem
or from the stolons produce hydranths much more frequently than
they do stolons when the ends are not in contact with a surface or
solid. Polarity in this form is commonly indicated by the relative
size and rapidity of formation of the hydranths from different ends
and at different levels of the piece rather than by the formation of
typically different structures, though occasionally stolon-forma-
tion occurs at the aboral end even without contact. It is possible,
however, as the writer has found, to increase experimentally and
without contact the intensity of the stolon-forming reaction to such
an extent that almost every piece will give rise to stolons at the
aboral end. Many of these stolons lb: after a considerable time
produce hydranths at their tips. The writer is inclined to believe
ens C. M. Child
that the hydranth-forming reactions may be interpreted as prima-
rily a reaction to lack of nutriment. ‘This reaction occurs most
readily of course in those parts which have been most closely asso-
ciated with it in the past.
In a number of forms short pieces are not necessary for the
occurrence of heteromorphosis. ‘These include Tubularia and
various other hydroids, some other ccelenterates, the earthworm
and some other forms. In some of these as in Tubularia one form
of reaction is dominant throughout, and in others, like the earth-
worm, this dominance is regional. Inthe earthworm, for example,
pieces from the posterior region commonly produce tails at both
ends (Morgan, ’02). ‘The head-forming reactions can occur only
in the more anterior regions. The very close relation between
head-formation and the nervous system in this as well as in many
other cases indicates that important factors in polarity are situated
in the nervous system.
Heteromorphosis may also occur in various forms when a short
piece is grafted in reverse position on a much longer piece. In
such cases the short piece produces at its free end the structures
characteristic of the end of the longer piece with which it is united.
These are undoubtedly simply cases of physiological dominance of
the larger component. ‘The greater intensity of its reactions alters
the conditions in the small piece so that this becomes functionally a
part of the other. ‘This is doubtless accomplished by the passage
of stimuli from the larger into the smaller piece and it may be by
the actual growth of nerves in some cases.
The regulation of a piece into a complete individual with typical
axial differentiation and maintenance of the original polarity
depends upon the relative difference in reactive capacity in the
different regions. In all such cases the piece must possess the
power to react in some degree like the whole. Its isolation brings
into play potentialities not evident while normal relations were
maintained, and the regions best fitted by past associations, for
particular reactions perform them most readily and thus the origi-
nal polarity persists.
Functional Regulation and Form-Regulation 575
The Relation Between the Nervous System and Form-Regulation
This relation which 1s very general, though of course not univer-
sal, affords the strongest evidence in favor of the view that form-
regulation is a functional process. No good ground has been dis-
covered for assuming the existence of a peculiar trophic or forma-
tive influence originating in the nervous system. Goldstein, in a
recent paper (’04), gives a most interesting discussion of the subject
and concludes that the relation between the nervous system and
growth and differentiation is essentially functional in character.
It must be remembered, however, that the nervous system adds
nothing fundamentally new to the phenomena of life. ‘The trans-
ference of stimuli in definite directions and along more or less
definite paths occurs where no visible nervous system exists. In-
deed the development of the nervous system itself is probably in
greater or less degree the result of such conditions. ‘The problem
of form-regulation is, therefore, not necessarily different in its fun-
damental features in those cases where no nervous system exists or
where regulation occurs in the absence of visible nerves, as com-
pared with those where visible nervous structures are present.
Conclusion
Regulation in general may be defined as a return to physiological
equilibrium after such equilibrium has been altered by external
conditions (Child, ’06a). Holmes (’04) has recently given a very
similar definition which he developed from the idea of sym-
biotic relations or “social pressure.” As the writer has attempted
to show in a previous paper (06a) the idea of symbiotic relations
does not afford a basis for the replacement in form-regulation of a
part similar to that removed since removal of an element or a group
of elements in the symbiotic complex must, according to Holmes’
assumption, alter the condition of the whole in such manner that
the ‘‘social pressure” upon the part which becomes the substitute
for the part removed will be different from that originally exerted
upon this part, and something else rather than a duplicate of the
part removed must be formed. But a characteristic feature of
form-regulation is a return or approximation to the original con-
576 CoM AGhila
dition and any interpretation must recognize this fact. In the
paper above referred to the writer has called attention to the fact
that replacement of the missing part cannot occur unless that
portion of the system remaining 1s capable of reacting in a manner
essentially similar in some degree to the part removed. But mere
capacity to react in this manner is not sufficient in most cases.
‘This method of reaction must be the predominant or characteristic
method, for if it is not, then a structure different from the part
removed or nothing at all is replaced. ‘This idea affords a basis
for understanding why the ability to replace lost parts is limited in
many cases. When by removal of a part the system is altered to
such an extent that the previous method of reaction becomes
impossible or is no longer the dominant method, replacement can-
not occur. In such cases, however, provided the system is not
altered to such an extent that continued existence is impossible,
some other form of regulation may occur and a new equilibrium
may be established differing more or less widely fromthe old. ‘Thus
when we remove the ganglia from Leptoplana and various other
polyclads the processes characteristic of the head-region can no
longer occur and the head 1s not replaced. In Planayia,on the other
hand, removal of the ganglia does not alter the complex to such an
extent as in Leptoplana since the nervous system is not as highly
cephalized here and the headless piece or certain parts of it still
retain the ability to react like a head in sufficient degree to initiate
the process of head-formation. When this process is once started
the ability to perform the characteristic reaction increases and
the return to the original condition is gradually accomplished.
The removal of the part brings into play various potentialities of
reactions which exist in the remaining piece by virtue of its past
relations to the whole and those processes which are predominant
determine the character of form-regulation. But objection may be
raised that a region in the middle of the body of Planaria, for exam-
ple, has not been associated in the past with reactions or processes
characteristic of the head and tail regions. _ If we observe the living
animal, however, we find that the more intense motor reaction of
either end may visibly involve the middle region in greater or less
degree. They must also involve it in many ways not visible. It is
a
Functional Regulation and Form-Regulation 577
probably true that the middle region does not while still a part of
the whole initiate such reactions, because whenever conditions
which give rise to such reactions are present other parts of the body
react much more readily than this. But when we sever its connec-
tion with other parts we find that it is visibly capable of initiating
such reactions, though of course very imperfectly and much less
promptly than the original head and tail regions. ‘The ability to
develop a new head and tail cannot arise from anything else than
this ability. In this particular case—Planaria—this ability evi-
dently depends in large measure upon the nervous system. ‘This
idea does not conflict in any way with that developed in the fore-
going sections, viz: that a functional substitution of some degree
must precede and initiate the process of replacement. It is evi-
dent that such functional substitution cannot occur unless the sys-
tem retains the ability to react to some extent like the part removed.
The substitution may alter the reaction of a larger or smaller part
of the piece but the very fact of substitution depends on the occur-
rence in the system of reactions resembling in greater or less degree
those of the part removed.
In connection with the views expressed above a recent paper by
pe nes (05), and also certain parts of his book (06), are of inter-
Jennings’ recent work has demonstrated the high degree of
modifiability in the behavior even of the lower organisms and
here he attempts to apply the conclusions reached to other fields.
His views on the individual adjustment or regulation of behavior
are based on the following premises.
‘“‘t Definite internal processes are occurring in organisms.
“9 Interference with these processes causes a change of be-
havior and varied movements, subjecting the organism to many
different conditions.
‘“*2- One of these conditions relieves the interference with the
internal processes so that the changes in behavior cease and the
relieving condition is thus retained.”’
The selection of the process or reaction which relieves the inter-
ference depends upon the fact of relief.
Jennings attempts to apply this ‘‘method of trial and error” in
reaction to the phenomena of regulation in other fields, pointing
578 C. M. Child
out the possibility that the selection of the apparently advantageous
reaction is to be found in the fact that it relieves disturbing condi-
tions.
It is perhaps sufficiently evident from what has been said above
that Jennings’ views and the writer’s are somewhat similar in certain
respects. ‘lhe physiological equilibrium which forms the basis of
Holmes’ and the writer’s definition of regulation corresponds to the
condition postulated by Jennings in which the various physiological
processes are occurring without interference: the alteration of equi-
librium is brought about by some interference—in the cases under
discussion in this paper usually by removal of a part. In con-
sequence of this interference a more or less extensive rearrangement
of dynamic processes occurs which becomes manifest in alteration of
the localization and perhaps also of the character of the reactions:
because of the occurrence and continuation of certain of these pro-
cesses a return to equlibrium occurs; but the writer does not believe
with Jennings that the continuation of particular processes is neces-
sarily due to the fact that they “relieve the interference,” their
relation to relief may, however, bring about their intensification.
‘There is one point in connection with form-regulation which
Jennings has not discussed but which is of considerable impor-
tance in this connection and that is the frequency of return or
approximation to the original condition. ‘This is so characteristic
of regulation that certain authors like Driesch (’01) have made it
the basis of their definition. In the regulation of behavior and of
chemical processes it cannot be supposed that only a single method
of reaction exists whereby the interference can be removed or dimin-
ished. In many cases, indeed, this is visibly not the case and the
organism may attain in consequence of the interference an equilib-
rium widely different from the original. It seems probable, for
example, that the formation of a new head at the posterior end anda
tailat the anterior end or of these structures on the right and left side
respectively of the piece in Planaria would remove or diminish the
interference and so constitute a return to equilibrium, but this does
not usuallyoccur. Itisnecessary to recognize the fact thatthe possi-
bility of reaction is limited somewhat strictly by the past relations -
of the part or, in other words, by its reactivecapacity. In short, the
Functional Regulation and Form-Regulation 579
reaction leading to replacement of the lost part 1s usually the reac-
tion which the piece can perform most readily, 1.e., that for which
it is best adapted. The endeavor has been made to direct atten-
tion to this fact in the preceding paragraphs. Hence the process of
trial and error does not occur to any great extent in most cases and
a very high degree of uniformity in the processes of form-regulation
exists. Moreover, a particular reaction-complex must continue
fairly constant for a considerable period of time before it becomes
manifest in such extensive structural development as appears in
form-regulation. If the characters of such reactions were not very
strictly limited we should expect a much greater range of variation
in the processes of form-regulation than occurs. ‘That variation in
the results does occur has been pointed out by various authors.
The writer has called attention to the fact that in Leptoplana
(Child, ’o4c) some individuals are more capable than others of
producing head-like structures in the absence of the ganglia, as well
as to numerous other cases. In a recent paper (Child, ’o6b) a
rather remarkable case of this kind has been noted: in certain
species of Planaria very small pieces from the middle region of the
body may produce any one of five different results, viz: single tail-
less anterior regions, single headless posterior regions, double
anterior regions, double posterior regions, or normal animals.
This region is apparently ‘“ndifferent,” 7.c., neither anterior nor
posterior reactions are dominant in particular parts of the piece
though both are possible in some degree, and the result depends
apparently upon a “chance,” /.e., upon certain internal conditions
not at present recognized. These contribute to determine what
reactions shall become dominant in each piece.
But the variation in processes of form-regulation in general 1s
not sufficiently great to indicate that “trial and error’’ plays any
important role. The reactions which bring about form-regulation
do not continue because they “relieve the interference’’ but because
they are dominant in the part in consequence of its past relations
to other parts. In the regulation of behavior there is doubtless
often a greater range of possibilities, but even here it appears
probable that the character of the regulatory reaction is deter-
mined, in many cases at least, not by the fact that it “relieves the
580 C. M. Child
interference” but by the character of the “ apparatus” or system
under the existing conditions.
All the evidence points us to the conclusion that the phe-
nomena of form-regulation are not essentially different from other
dynamic phenomena in organisms and that they must be inter-
preted on a dynamic basis. As the writer has maintained in
various papers the organism is primarily a dynamic complex and
visible structural foarte: are primarily incidents or by-products of
the dynamic processes. Formative processes, so-called, do not
differ fundamentally from processes of behavior.
Objection to such interpretation, may be made on the ground
that it is not really an explanation but rather a step away from
explanation in that it groups phenomena apparently relatively
simple in the same category with the most complex phenomena of
life. In answer to this it may be said that the first step in true
interpretation is the recognition of the nature of the problem.
With increasing knowledge of vital phenomena it is becoming more
and more evident that we cannot select a single group of these
phenomena and ‘‘explain”’ them without reference to other groups.
The organism is not a mosaic of independent complexes acting in
different ways. ‘There is not a particular group of dynamic pro-
cesses concerned in building the machine and another in keeping it
going. We shall find similar laws governing all whatever may
be our final conception as to the nature of these laws. Semon (04)
has recently proceeded froma somewhat similar idea, in his exceed-
ingly interesting attempt to interpret the phenomena of heredity,
habit and memory on a common basis.
Recognition of the fact that the fundamental problems are the
same in fields before regarded as distinct or only remotely related is
always an advance and when different observers in these different
fields recognize this fact independently of each other, the signific-
acer oF the conclusions is necessarily greater than it aeula be
otherwise.
Hull Zodlogical Laboratory
University of Chicago
May, 1906
Functional Regulation and Form-Regulation 581
BIBLIOGRAPHY
Cup, C. M., ’02.—Studies on Regulation, I. Fission and Regulation in Steno-
stoma, Pts. 1, 1] and III. Archiv fiir Entwickelungsmech. Bd. xv,
H. 2 and 3, 1902.
’03a.—Studies on Regulation, II. Experimental Control of Form-Reg-
ulation in Stenostoma. Archiv fiir Entwickelungsmech. Bd. xy,
H. 4, 1903.
’03b.—Studies on Regulation, II]. Regulative Destruction in Zooids
and Parts of Zooids in Stenostoma. Archiv fiir Entwickelungs-
mech., Bd. xvii, H. 1, 1903.
’o4a.—Studies on Regulation, [V. Some Experimental Modifications of
Form-Regulation in Leptoplana. Journ. Exp. Zodl., vol. i, No.
I, 1904.
’o4b.—Studies on Regulation, V. The Relation between the Central
Nervous System and Regeneration in Leptoplana. Posterior Re-
generation. Journ. of Exp. Zool., vol.1, No. 3, 1904.
‘o4c.—Studies on Regulation, VI. The Relation between the Central
Nervous System and Regeneration in Leptoplana. Anterior Regen-
eration. Journ. of Exp. Zodl., vol. i, No. 4, 1g04.
’05a.—Studies on Regulation, VII. Further Experiments on Form-
Regulation in Leptoplana. Journ. of Exp. Zodl., vol. ii, No. 2,
1905.
‘o5b.—Studies on Regulation, VIII. Functional Regulation and
Regeneration in Cestoplana. Archiv fiir Entwickelungsmech. Bd.
RIK Ld .¥3s LOS.
‘o5c.—Studies on Regulation, IX. The Positions and Proportions of
Parts During Regulation in Cestoplana in the Presence of the Ceph-
alic Ganglia. Archiv ftir Entwickelungsmech. Bd. xx, H. 1, 1905.
’o5d.—Studies on Regulation, X. The Positions and Proportions of
Parts during Regulation in Cestoplana in the Absence of the Ceph-
alic Ganglia. Archiv fiir Entwickelungsmech. Bd. xx, H. 2,
1905.
’o6a.—Contributions Toward a Theory of Regulation, I. The Signi-
ficance of the Different Methods of Regulation in Turbellaria.
Archiv fiir Entwickelungsmech. Bd. xx, H. 3, 1906.
’06b.—The Relation Between Regulation and Fission in Planaria. Biol.
Bull. vol. xi, No. 3 ,1906.
Drtescu, H., ’o1.—Die Organischen Regulationen. Leipzig, 1901.
582 C. M. Child
Go psTEIN, K., ’04.—Kritische und Experimentelle Beitrage zur Frage nach dem
Einfluss des Zentralnervensystems auf die embryonale Entwickel-
ung und die Regeneration. Archiv fiir Entwickelungsmech. Bd.
xvii, H. 1, 1904.
Hormes, S. J., °04.—The Problem of Form-Regulation. Archiv fiir Entwicke-
lungsmech., Bd. xvii, H. 2 und 3, 1904.
Liture, F. R., ’o1.—Notes on Regeneration and Regulation in Planarians, II.
Amer. Journ. of Physiol., vol. vi, No. 2, 1901.
Jenninos, H. S., ’05.—The Method of Regulation in Behavior and in Other Fields.
Journ. of Exp. ZoGl., vol. 11, No. 4, 1905.
’06.—Behavior of the Lower Organisms, New York, 1906.
Morean, T. H., ’°98.—Experimental Studies of the Regeneration of Planaria Macu-
lata. Archiv fiir Entwickelungsmech. Bd. vu, H. 2 und 3, 1898.
’02.—Experimental Studies of the Internal Factors of Regeneration in
the Earthworm. Archiv fiir Entwickelungsmech. Bd. xiv, H. 3
und 4, Igo2.
PrzipraM, H., ’o1.—Experimentelle Studien tiber Regeneration. Archiv fiir Ent-
wickelungsmech. Bd. xi, H. 2, Igo.
’o2.—Experimentelle Studien tiber Regeneration (Zweite Mittheilung).
Archiy fiir Entwickelungsmech. Bd. xi, H. 4, 1902.
’05.—Die “Heterochelie’”’ bei decapoden Crustaceen (zugleich: experi-
mentelle Studien tiber Regeneration. Dritte Mittheilung). Archiv
fiir Entwickelungsmech. Bd. xix, H. 2, 1905.
Semon, R., ’04.—Die Mneme als erhaltendes Prinzip im Wechsel des Organischen
Geschehens. Leipzig, 1904.
Wison, E. B., ’03.—Notes on the Reversal of Asymmetry in the Regeneration of
the Chelz in Alpheus heterochelis. Biol. Bull., vol. iv, No. 4, 19.
ZELENY, C., ’05a.—Compensatory Regulation. Journ. of Exp. Zodl., vol. 11, No.
I, 1905.
’05b.—The Relation of the Degree of Injury to the Rate of Regeneration.
Journ. of Exp. Zodl., vol. 11, No. 3, 1905.
A STUDY OF THE SPERMATOGENESIS OF COPTO-
Sverre AURICHALGCEA AND COPTOCYCLA GUI-
ama LT ESPECIAL. REFERENCE. TO; “THE
PROBLEM OF SEX-DETERMINATION
BY
W. N. NOWLIN
Witu Two Pirates
Very little work on the germ cells of the Coleoptera had been
done until last year, when the form Tenebrio molitor was inves-
tigated by Dr. N. M. Stevens. The results obtained were so
suggestive from the standpoint of sex-determination that she is at
present engaged in examining a number of species of this order
of insects.
The following paper is a contribution to this series of investi-
gations, and I take pleasure in acknowledging my indebtedness to
Dr. Stevens, both for material used and for direction in the work.
I. MATERIAL AND METHODS
The beetles under discussion are Coptocycla aurichalcea and
Coptocycla guttata, both collected from Convolvulus arvensis at
Woods Hole, Mass., September 5, 1905.
The testes were fixed in Flemming’s strong solution, Hermann’s
platino-aceto-osmic, and Gilson’s mercuro-nitric, each of which
gave excellent results.
Heidenhain’s iron-haematoxylin method was used for most of
the preparations. Other stains, employed principally for chemi-
cal tests, were thionin, Delafield’s haematoxylin and orange G, and
with the material fixed in Hermann’s and Flemming’s fluids, the
safranin-gentian stain of Hermann. ‘The iron-hematoxylin slides
were often counterstained with orange G.
Tue JourNat oF ExprerIMENTAL ZOOLOGY, VOL. III, NO. 4.
584 W. N. Nowlin
II. OBSERVATIONS
The testes of these beetles consist of many follicles radiating
from a central duct, the vas deferens, into which are poured the
spermatozoa. We should expect then to find ripe spermatozoa
at the inner end of the follicles and spermatogonia at the outer,
with the intervening stages between. ‘This is true in most cases
but it is by no means an inviolable rule. Cysts containing ripe
spermatozoa may be found mingled with those containing first or
second spermatocytes, and second spermatocytes may occur in the
same cyst with the first. The classification then cannot be made
by the location but purely from the character of the cell itself.
I. Coptocycla aurichalcea
The equatorial plate of a spermatogonium of Coptocycla auri-
chalcea contains twenty-two chromosomes, most of which are V-
shape, and radially arranged with the concave side outward (pl. I,
Fig. 1). Near the center usually, though it may take any other
position, is a very small chromosome, which is the small member
of an “‘idiochromosome”’ group, and while we cannot here be sure
of its mate we are safe in assuming it to be one of the five smaller
chromosomes. An attempt was made to arrange the chromo-
somes in pairs according to form and size, but it soon became
evident that they differed in different plates; the form, whether
rod-shaped or V-shaped, depending largely on position with respect
to the otherchromosomes. As is unusual the behavior of the differ-
ential chromosomes in the spermatogonium is in every respect like
that of the others: each divides longitudinally and passes to the
poles, where condensed masses are formed.
The earliest spermatocyte shows a true condensation stage or
bouquet stage [synizesis of McClung (’05)], in which the chroma-
tin is in threads closely looped together at one side of the nuclear
space (Fig. 2). The number of loops is here very evidently more
than the reduced number and probably each loop corresponds to
a spermatogonial ;chromosome. In a slightly later stage the loops
straighten until many free ends are seen above the condensed mass
(Figs. 3 and 5). Each of these strands appears to be a single
Spermatogenesis of Coptocycla 585
chromosome, and this idea is strengthened when in the next phase
the distal ends are seen to bend toward one another in pairs and
unite (Figs. 5 and 6). Fig. 4 is a cross-section of a nucleus at this
stage. [hus synapsis takes place in the early part of the growth
period soon after the last spermatogonial division. ‘This agrees
with Montgomery’s (’03) description of the synapsis stage in
Amphibia and furnishes more detailed evidence of the process.
Up to this period the idiochromosomes have been indistinguish-
able from the others, but a few very clear sections were obtained
which showed at the base of the loops, close to the nuclear mem-
brane, the larger member of the unequal pair (Fig. 5). “This sug-
gests the probability that the idiochromosomes retain their charac-
teristic form during the greater part if not all of the synizesis and
synapsis stages. All the chromatin has a strong afhnity for stains
during these periods, and the spireme is most distinct when first
formed (Fig. 7), but almost immediately it becomes very faint (Fig.
8). The differential chromosomes still stain deeply, however. ‘The
spireme soon breaks up into separate rods, each showing a longi-
tudinal split (Figs. 9,10), even at this time there seems to be a
characteristic form, one chromosome assumes a U-shape, one a
triangular form, and one that of a nearly closed ring. ‘These show
still more clearly in the later prophase (Fig. 12).
The split disappears (Fig. 11), the chromosomes condense, grow-
ing slightly thicker and shorter (Fig. 12), until we get such forms
as appear in Fig. 13. The centrosomes are large and distinct in
this material, and at this stage two may be seen lying close against
the nuclear membrane. In Fig. 13 we see them nearly 180° apart,
and as yet no spindle fibers have appeared. ‘[hroughout the
spireme stage the idiochromosomes are dense spherical bodies.
In many cases only the larger member is visible (Figs. 7, 8, 9).
In Fig. 11 both are plainly seen. In the later stages while the
ordinary chromosomes are undergoing great changes the differen-
tial chromosomes remain the same (Figs. 12, 13).
An equatorial plate of the first spermatocyte (Fig. 15) shows
eleven chromosomes, and since the spindle fibers are very large,
these may often be seen in cross-section attached to the periphery
of the chromosomes (Fig. 15). When the material is faintly
586 W.N. Nowlin
stained one chromosome stands out prominently, due to its
retention of the stain (Fig. 14). ‘This is the larger member of the
differential group, as may be seen in a lateral view of the spindle
(Fig. 21). The small idiochromosome fails to show here, due prob-
ably to the angle at which the pair is lying; in late prophase (Figs.
17, 22) the pair is easily distinguished. While in numerous meta-
phases the small chromosome 1s difficult to discern, there are afew
cases in which it is clearly seen (Fig. 18). This unequal pair
exhibits no difference in behavior during mitosis, but like the others,
and at the same time, with the exception of one which always
divides early (Figs. 18, 21), it divides transversely, the large mem-
ber passing to one pole and the small member to the other (Fig.
25)
We have in this division one of the clearest cases of reduction
division possible. In the spermatogonium the chromosomes are
V-shaped. During the condensation stage they elongate into
loops, at first closely massed together, but later these straighten and
unite in synapsis to form longer loops. ‘This is followed by the
formation of a continuous spireme (Figs. 7 and 8) which soon
breaks up into segments, each split longitudinally (Figs. 9, 10). In
the prophase of the first spermatocyte many of the chromosomes
are thick rods, which upon examination are seen to be two V’s
joined end to end, giving in profile a typical E-shape (Fig. 16, ).
The spindle fiber is attached at the vertex of each V of the bivalent
chromosome and in anaphase this element separates at the orig-
inal place of union, at right angles to the initial longitudinal split
(Figs. 18, 20). The V-shaped chromosomes observed in the
spermatogonium are thus restored and carried into the second
spermatocyte. Exactly the same thing is true in the separation of
the components of the cross-shaped chromosome (Figs. 16, a—d),
and of the ring-shaped one (Fig. 16, /—o, and Figs. 22, 23, 24).
There is no rest stage between the two generations of sperma-
tocytes, but almost immediately the massed chromatin in the late
anaphase (Fig. 26) separates into chromosomes which begin to
arrange themselves in the equatorial plate (Figs. 27, 28, 29). In
Fig. 30 we see this in progress, the small chromosome being very
distinct. In Figs. 27 and 28, we again see eleven chromosomes
Spermatogenesis of Coptocycla 587
of which one is the small idiochromosome. Having seen that in
the first division the large idiochromosome goes to one pole and
the small to the other, we should expect to find half the daughter
cells containing the large and half the small chromosome of this
pair. ‘his is exactlythe case. Fig. 29 shows a representative of
the cells possessing the large member. ‘This is especially clear in
material very much destained, for here as in earlier stages this
peculiar chromosome holds the color much longer than the others.
In other respects the differential chromosomes are not different,
but divide longitudinally with the ordinary chromosomes.
In early anaphase each V_ shows a distinct split which
lies in the plane of the equatorial plate (Fig. 31). ‘That this is
the original longitudinal split seen in the early prophase
(Figs. 9, 10) there can be no doubt. One chromosome which
maintains its identity from the early prophase through the first
spermatocyte as a cross is sufficient evidence. In the late prophase
and even in metaphase this chromosome (Fig. 16, a—e) may be
seen to be longitudinally split (Fig. 16, d). ‘The line of separation
comes at right angles to this split and the univalent chromosomes
pass to the poles as V’s, each possessing a longitudinal split, though
it closes and cannot be seen again until the beginning of the follow-
ing anaphase. [he chromosomes are typically arranged with the
vertices attached to the fibers and pointing inward. In division
they are drawn apart, at first with the ends of the V’s pointing pole-
ward (Figs. 31, 32) but later the vertices turn toward the poles,
though disorder often reigns until a comparatively late anaphase
(Fig. 33).
In the spermatids there is a great amount of archoplasm, and
in the late telophase of the second division it occupies the greater
part of the cytoplasmic area (Fig. 36). At a slightly later stage
the mass loses entirely its fibrillar structure and, condensed some-
what, lies as a gray sphere (iron haematoxylin) against the nucleus
(Fig. 38). This is the so-called nebenkern of the spermatid. The
entire cell now elongates, and with it the axial filament. ‘The
archoplasm has assumed the form of a large pennant attached to
the nuclear membrane, and the axial fiber runs throughout its
length and often beyond (Fig. 39).
588 W.N. Nowlin
A cross-section of the tail at this time shows that the archo-
plasmic sheath 1s folding about the axial fiber (Fig. 50, a). When
the transformation is nearly complete the two cannot be distin-
guished from each other, the sheath lies close against the filament
with a thin layer of cytoplasm outside (Fig. 50, c). Often in cross-
section the sheath appears to be split (Fig. 50, 5). Since longitu-
dinal views of the tails reveal nothing of the kind we are left to
conjecture that this appearance is due to oblique sections of
the trough-shape sheath. The fact that many of these appear
together does not alter the explanation, for the spermatozoa and
late spermatids lie parallel in large numbers.
‘There is one peculiar but apparently typical stage in the develop-
ment of the tail: while the head is yet round, though the chromatin
is much condensed, the axial filament has assumed a vacuolated
appearance (Fig. 40). ‘This lasts until the head has begun to elon-
gate, and is just over in Fig. 41. This is not to be confused with
the rare occurrences of double filaments seen in Fig. 48. ‘These
are the products of giant cells, due to a failure of one of the divi-
sions of the spermatocytes or the spermatogonia to complete itself.
‘They consequently have two centrosomes and two axial filaments.
Paulmier (’99) found similar conditions in Anasa. As in other
insects the axial filament in the Coleoptera seems to arise in close
relation with the centrosome.
After the vacuolated stage the tail narrows and lengthens much
more and we see it in its final form in Fig. 44. All this time the
head of the spermatid has been changing. The dense chromatin
mass begins to lose its afhnity for haematoxylin until a deep gray
results. Astructure is now revealed in the nucleus, not heretofore
seen, a small densely staining nucleolus-like body (Fig. 36). “The
nucleus now widens its circumference and the chromatin condenses
around the membrane in the form of a ragged border (Fig. 38).
The nucleolus-like formation is not seen at this stage, but the sup-
position is that it is merely obscured by the dense patches of chro-
matin, for in the next stage it again appears and occupies a most
characteristic position: the chromatin is arranged in a crescent
shape and the nucleolus lies in a clear area between the arms of
the crescent (Fig. 37). The chromatin becomes diffuse and min-
Spermatogenesis of Coptocycla 589
gles with the karyolymph until a uniform light gray results (iron
hematoxylin). The nucleolus is unaffected, changing neither in
size nor in staining reaction. It makes a change, however, in
position about this time, moving from a point opposite the cen-
trosome around the periphery of the nucleus until it often lies
very near the centrosome (Fig. 49).
The head of the spermatid lengthens and becomes vacuolated
(Fig. 41), but as the elongation increases the vacuoles disappear
and the deeply staining nucleolus-like body is apparent, lying
closely against the wren membrane (Fig. 45, y). Very soon this
single hae breaks up into two, three or four parts and later, it
disappears (Figs. 46 and 47). The head condenses, lengthens,
stains intensely (Fig. 42), and in its final form is spirally twisted,
resembling the Bacterium spirillum (Figs. 43, 44).
It is impossible at the present time to say just what may be the
function of this peculiar, deeply-staining spermatid element.
It suggests the accessory of Orthoptera which can be seen
in one-half the spermatids (McClung, ’99, ’oo, ’02a), and at
first it seemed probable that it might be one of the idiochromo-
somes in the beetle as it, in one stage, is about the size of the
larger member of the pair. Instead of being found in half the
cells, however, it is without doubt in all, so that this explanation
was relinquished. It is characteristic of the spermatids of all the
beetles thus far studied and of some of the Orthoptera.
2. Coptocycla guttata
In external appearance Coptocycla guttata is very different from
C. aurichalcea, but in their germ cells there are many points of
resemblance. Guttata has the smaller number of chromosomes,
eighteen in the spermatogonia instead of twenty-two, but the
size and form are much the same in the two species (Figs. 1 and
51). The unequal pair is also present here.
The suggestion that synapsis takes place immediately after the
condensation stage in C. aurichalcea is strongly confirmed by gut-
tata. In the condensation or synizesis stage, the chromatin is in
590 W.N. Nowlin
the form of loops with the ends against the side of the nuclear mem-
brane (Fig. 52). [his point Montgomery calls the distal pole of
the nucleus and the opposite point on the nuclear membrane, the
central pole. ‘The process is the same as in aurichalcea; the loops
straighten, thrust the free ends toward the central pole (Fig. 53),
bend toward each other in pairs and unite end to end (Fig. 54).
The end of the chromatin thread either bears an enlargement or
stains more deeply in cross-section for there is at this point the
appearance of a deeply staining bead. This dark spot thus con-
veniently marks the place of union in the bivalent chromosomes,
and we are led to the conclusion that the short loops first observed
in the synizesis stage are univalent chromosomes. ‘The idiochro-
mosome pair is véry distinct at the base of the loops as shown in
Big.952:
The growth stages are not unlike those of ©. aurichalcea. A
spireme (Fig. 55) is formed which varies in its staining reactions
exactly as in C. aurichalcea. ‘The thread finally breaks up into
bivalent elements which stain deeply and exhibit a longitudinal
split, and at last condense into forms which they retain during the
first division. In Fig. 57 are seen two bivalent chromosomes
assuming the form of rings, and in Fig. 58 these have closed
together. ‘This figure also exhibits crosses which later change to
the form found in aurichalcea. Most of the chromosomes of this
species are dumb-bell-shaped in the first spermatocyte mitosis and
of the usual V-shape in the second. ‘The idiochromosomes main-
tain their spherical form throughout all the stages.
An equatorial plate of the as maturation division shows nine
chromosomes arranged usually in the order seen in Fig. 60, with
seven in a circle about the other two. All nine chromosomes may
be seen in lateral view in Fig. 59, where they are just coming into
the spindle. ‘The dumb-bell shape of four is here quite evident
and the unequal pair is conspicuous. ‘Three of the others appear
as straight rods and the other one bent in V-form.
During metaphase the chromosomes arrange themselves with
their long axes parallel with the axis of the spindle, and later they
divide at right angles to their length. In other words, they exhibit
qualitative division, bivalent chromosomes separating at the point
S permatogenesis of Coptocycla 591
of union made during synapsis. Asa result the small chromosome
of the idiochromosome pair goes to one pole while the large mem-
ber goes to the other (Fig. 63). [he equatorial plates of the
second division confirm this, half possessing nine V-shaped chro-
mosomes of approximately equal size (Fig. 71), the other half hav-
ing eight large and one small chromosome (Fig. 65). Fig. 66
shows the second maturation spindle with this small chromosome
in metaphase, and in Fig. 64 it is seen dividing.
Here, then, as in C. aurichalcea and ‘Tenebrio molitor
(Stevens, 05) half the spermatids will possess the small idiochro-
mosome, and half the large.
The transformation of the spermatid is essentially the same
as in aurichalcea. ‘The deeply staining nucleolus-like body is
present but no clue to its function or nature is given. ‘The ripe
spermatozoa are not spirally twisted but resemble that stage of
aurichalcea seen in Fig. 42.
Owing to lack of material it was impossible to study the divid-
ing somatic cells of the male and female forms. ‘That they would
exhibit the same conditions found repeatedly in other beetles there
is no doubt. By permission I have reproduced four drawings
from Dr. Stevens’ originals, which show the chromosome of
(Fig. 67) a somatic cell from the digestive tract of a male pupa
of Tenebrio molitor, and (Fig. 68), a female somatic cell of the
same form found in the egg follicle; (Fig. 69) a female somatic
cell of Trirhabda virgata from the egg follicle, and (Fig. 70) a
male somatic cell taken from the larval body of the same species.
Here, as in other forms that Dr. Stevens has investigated, the
small idiochromosome goes to the male and the large one to the
female.
SUMMARY OF OBSERVATIONS
Coptocycla aurichalcea
1 The spermatogonial number of chromosomes in Coptocycla
aurichalcea is twenty-two, twenty-one of which are V-shaped and
one very small one, spherical in form.
592 W. N. Nowlin
2 Synapsis takes place in a manner, so far not described but
also observed in certain beetles now being investigated by Dr.
N. M. Stevens. The loops seen in the synizesis stage, which
represent individual chromosomes, straighten and unite in pairs
by the free ends which are pushed up into the nuclear space.
Pseudo-reduction, therefore, occurs just after synizesis and_ just
before the formation of the spireme.
3 The first maturation mitosis is a transverse, reducing divi-
sion, the second longitudinal, occurring along a lengthwise split
formed early in the prophase.
4 There is present a typical pair of idiochromosomes [accord-
ing to Wilson’s definition (’05 and ’06)| which, with the others.
divides qualitatively in the first division and quantitatively in the
second, the small member, therefore, going to one-half the sperma-
tozoa and the large member to the other half.
5 The chromosomes show marked individuality from the
beginning of the prophase, one having the form of a ring, two of a
cross, several the shape of an E, and finally the unequal pair.
Coptocycla guttata
1 The spermatogonial number of chromosomes is eighteen,
seventeen large V-shaped chromosomes and one which is small
and spherical.
2 All other observations on this species confirm those on auri-
chalcea.
GENERAL DISCUSSION
Individuality of the Chromosomes
Convincing results have been published in regard to the individ-
uality of the chromosomes by Boveri ('02), who found a difference
in their function; by Sutton (’02), who found a difference in size;
and by Baumgartner (04), and others, who have discovered a dif-
ference of form.
The idiochromosomes indicate so clearly a difference in function
as well as in size that it is unnecessary to go into detail on this
S permatogenesis of Coptocycla 593
point. [he ordinary chromosomes also confirm the size difference;
though no careful measurements were made, it is obvious that
such differences exist at least in most cases (Fig. 19). However,
the difference in form is most evident in Coptocycla aurichalcea,
and furnishes another strong support for the doctrine of the
individuality of the chromosomes.
Of the eleven bivalent chromosomes several possess forms charac-
teristic enough to mark them as the same in different generations;
there are two or three crosses, several E’s,a ring and an unequal
pair (Fig. 16). ‘There is, doubtless, a size difference that separ-
ates those of a group though this is not as obvious in some cases
as in others. ‘The ring form occurs only once in this species, but
twice in guttata and maintains its identity of shape from an early
prophase, being much more expanded then, however, than later.
It is formed by the V-chromosomes of the spermatogonium uniting
by both ends, as is plainly shown in Fig. 16, /, where two ends are
not firmly joined. In a late metaphase the bivalent pair seems
to elongate slightly; this closes the opening and gives the
appearance seen in Fig. 23. In profile the ring assumes an oval
form, with the central opening much smaller (Fig. 16, 7), but this
is distinguished from the actually elongated phase by the length
(lite. 23).
Mendel’s Law
Assuming that the idiochromosomes are sex-chromosomes, or
represent the sex-characters, they furnish excellent opportunities
for speculation on the application of Mendel’s law to chromo-
somes.
If the Mendelian principles of segregation apply to sex, there
should be in the second generation 25 per cent males, 50 per cent
hybrids, and 25 per cent females. Castle (03) concludes that
there are no individuals pure in regard to the sex-character, but
only hybrids are produced. Now to apply this theory to the bee-
tles. We know by actual observation that the male somatic cells
possess the small idiochromosomes, and the female somatic cells
the large one. Below are given in a schematic representation
594. W. N. Nowlin
two kinds of eggs and two kinds of spermatozoa with the dot
above to indicate the idiochromosome and the letter to show the sex
ae
There are four possible combinations here:
er (FERTILE MALE) = 4
—@e (INFERTILE FEMALE) =f
@
ic =@@ ([\WFERTILE MALE) =
:
&
?
— @@e (Fee Br FEMALE ) =4
Now by actual examination of the male and female somatic
cells we know that no such combinations as (2) and (3) exist.
Our observations, therefore, strongly support the theory of selec-
tive fertilization, only gametes bearing opposite sex-characters
fusing.
While the beetle furnishes no explanation why femaleness as
a character often dominates (Castle ’03) yet its chromosomes
indicate why it 1s recessive a part of the time in these insects. In
the 50 percent of spermatozoa that carry the female character in
the form of the small idiochromosome, this is overpowered by the
much larger male idiochromosome in the eggs with which they
unite. Whenthe chance is even, 7. e., when the idiochromosomes
are equal in size, then femaleness invariably dominates.
Spermatogenesis of Coptocycla 595
The Idiochromosomes
But two groups of animals thus far investigated are known to
possess the idiochromosomes; these are the Hemiptera and
the Coleoptera. It is probable that they exist in other forms
and have been wrongly classified as was the case with Paulmier’s
(99) work on Anasa, and Montgomery’s (01) on Coenus delius
and Euschistus tristigmus. Paulmier failed to distinguish between
the accessory and the microchromosomes, while Montgomery saw
the small idiochromosomes in the resting stages and the different
number of chromosomes in different cysts, but misinterpreted
them.
To Wilson is due the correct interpretation of these idiochro-
mosomes in the Hemiptera heteroptera, asa distinct pair of chromo-
somes with a definite and characteristic behavior in the different
generations.
Slightly earlier than this and independently Dr. Stevens work-
ing on one of the Coleoptera, ‘Tenebrio molitor, found an unequal
pair of chromosomes, which proves upon comparison to be essen-
tially the same as those of the Hemiptera.
In details of behavior, however, these chromosomes differ mark-
edly in the two groups. As in Lygzeus and Coenus (Wilson, ’05)
the idiochromosomesof Coptocycla maintain their identity through-
out the growth period. ‘They are first distinguished in the synize-
sis stage as a compact, spheroidal body at the base of the chromatin
loops (Fig. 52). The small member has not been observed at this
stage, but it is doubtless present and is obscured by the larger of
the idiochromosome pair. As this pair appears in the spheroidal
form, so early in the growth stage, it is a question whether it ever
assumes the form of loops with the other chromosomes.
In Lygzeus in the synizesis stage the larger differential chromo-
some is elongated and bent U-shape. In the post-synaptic phase
instead of lengthening and splitting longitudinally as in Lygzus,
both of the idiochromosomes of Coptocycla maintain their com-
pact, rounded form. In early prophase these chromosomes in
Hemiptera exhibit a bipartite structure and sometimes remain
separated while the ordinary chromosomes have fused in synapsis.
596 W. N. Nowlin
In Coptocycla the unequal pair has fused, but neither member
shows any tendency toward being bipartite. In fact, at no time
is there a hint of a longitudinal split, unless the vacuolated
appearance of the large idiochromosome in the early growth stage
may be considered as such.
The later behavior of the idiochromosomes is markedly different
in the two groups. In the Hemiptera they remain separated and
univalent in the first maturation mitosis, but at the close of this
division their products conjugate to form a dyad, which in all but
one form, Nezara, is asymmetrical. Inthe Coleoptera the behavior
during mitosis is exactly like that of the ordinary chromosomes;
having united in the synapsis stage to form a bivalent, they divide
transversely in the first mitosis and longitudinally in the second.
The result is, of course, the same in both cases; the spermatids
are of two kinds as regards the idiochromosomes, half possessing
the small and half the large one. The distribution of these chro-
mosomes to the somatic cells of the two sexes is also the same in
the two groups; the male cells contain the smaller, the females
cells the larger idiochromosome.
Sex-determination
The observations recorded in the present paper add nothing
new to the subject of sex-determination, and their chief value con-
sists in their confirmation of the very suggestive work on Tenebrio
molitor (Stevens, ’05).
Since McClung (00) advanced his theory that the accessory
chromosome of the Orthoptera is a sex-determinant, numerous
investigators have sought evidence for or against it. The first in
its favor was that of Sutton (02), who found the odd chromosome
present in the male somatic cells and absent in the female cells of
Brachystola magna (23 in & cells, 22 in @ cells). Since that
time, however, Wilson has found the reverse true for Hemiptera,
1. e., theadditional chromosome in the female somatic cells (Anasa
21 #, 22 2), and this leads him to question Sutton’s count for the
Orthoptera. Wilson suggests that the accessory chromosome of
Anasa, Protenor and the Orthoptera is the homologue of the larger
Spermatogenesis of Coptocycla 597
member of the idiochromosome group found in Coenus, Lygzus
and certain other Hemiptera, and its missing mate is the homologue
of the small idiochromosome. He thus supports the view of Paul-
mier and Montgomery in regard to degenerating chromosomes.
Knowing from his observations on Anasa that the accessory
goes to the female in the Hemiptera, he, therefore, conjectured
that the larger of the idiochromosomes would be found in the
female somatic cells, and the small one in the male somatic cells,
and he later found positive proof of this.
As stated before Dr. Stevens while investigating a form of beetle,
Tenebrio molitor, found that an unequal pair of chromosomes is
present, the large one of which goes to the female somatic cells,
and the small one to the male somatic cells. Since then she has
confirmed this in other species of Coleoptera, so that at last there
seems to be much indisputable evidence for the chromosome-sex-
determinant theory.
The question arises, what is to be done with such forms as Ther-
mopsis (Stevens, 05) and Banasa (Wilson, ’05). In the former
there are no chromosomes which have any external peculiarity
that would mark them as sex-determinants; in the latter there are
two sets of chromosomes that we interpret as sex-determinants
when they appear separately in other forms.
The solution for Thermopsis is perhaps less difhcult than for
Banasa; one pair of the chromosomes may bear the sex-character
although there are no external differences. ‘This does not conflict
with present views. In Banasa, the situation is different. Here
we have a pair of idiochromosomes as well as a typical accessory.
In other cases Wilson has suggested that the large idiochromo-
some is the homologue of the accessory inasmuch as both are
members of pairs, one member of which is degenerating or has
already disappeared. ‘This seems plausible and if taken from
the standpoint of degeneration only, there is nothing conflicting
in the fact that two pairs of chromosomes even in the same cell
are undergoing the change. In fact, as Wilson suggests, it adds
weight to the idea of degenerating chromatin, for we have no
reason to suppose that degeneration is necessarily limited to one
pair.
598 W.N. Nowlin
However, taken from the standpoint of sex-determination it
seems to offer serious difficulties. While we are perhaps not justi-
fied in assuming that the sex character is confined to one chro-
mosome, or to one pair of chromosomes, all recent observations
point that way; then on a priori grounds it seems improbable that
the same function should be assigned to two pairs of chromosomes
in the same cell. With the numerous characters to be transmitted
it would seem more likely that one pair of chromosomes must be
the bearer of many qualities.
In Banasa there are four classes of spermatozoa which contain:
(1) the small idiochromosome + the accessory;
(2) the large idiochromosome + the accessory;
(3) the small idiochromosome — the accessory;
(4) the large idiochromosome — the accessory.
If the chromosome relations in the male somatic cells of Banasa
correspond to those in Lygzus and Anasa, only one of these classes
of spermatozoa (3) can be used. Any combination of (1), (2),
(4) would refute the idea of homology of these two types of chro-
mosomes. ‘The same thing is true as to the production of females;
but one kind of spermatozoon (2) can be functional.
‘This means that three-fourths of the spermatozoa are function-
less as regards production of males, three-fourths as regards pro-
duction of females and one-half absolutely functionless. Such a
condition seems most improbable, but, of course, the only way of
determining it is to study the male and female somatic cells of
Banasa. ‘Ihis promises interesting results as it will either refute
the homology theory (for this form, at least) or reveal new facts in
regard to selective fertilization and a functional and non-functional
condition of spermatozoa.
While Castle believes there is no hard and fast rule for domi-
nance in the sex character in dicecious individuals, it seems that
in the beetles we have a clear case of female dominance. In cells
where the male and female sex chromosomes are equal in size,
femaleness invariably dominates. Where they are unequal (the
female small, as in Coleoptera and some Hemiptera, or entirely
missing as in Orthoptera) then the female character is visibly
Spermatogenesis of Coptocycla 599
recessive, due merely to its reduced strength or entire disappear-
ance in the male.
The results pointed out for Tenebrio and confirmed in this paper
are briefly as follows: an unequal pair of chromosomes is present
which, we have strong evidence for believing, transmit or deter-
mine the character of sex. The fact that the small one invariably
occurs in the male somatic cells and the large one in the female
somatic cells seems on first thought to mean that the small one
carries maleness and the large one femaleness. But Wilson’s
thorough analysis of the conditions in Hemiptera (’06) has shown
that the larger idiochromosome which alternates between the
sexes must bear the male character, while the small idiochro-
mosome, which is confined to the male sex, must represent the
recessive form of the female character.
While it has been proved beyond doubt that certain chromo-
somes are concerned in the determination of sex among insects,
a general application of the theory cannot yet be made, but it must
for the present be limited to those forms possessing the accessory
or the idiochromosomes. Without doubt further investigation
will reveal either similar differential chromosomes in all forms, or
show something homologous to them. It may not be a difference
of size or shape that will distinguish them from the ordinary chro-
mosomes, but probably one of behavior. Even in case the last
difference is not found it need not disprove the chromosome-sex-
determinant theory, for the sex character doubtless can be carried
like other pairs of antagonistic characters without affecting the
chromosome visibly. ‘The eggs of comparatively few forms have
been investigated, and it is possible that a dimorphism of the
chromosomes in the maturated oocytes may be found in certain
groups.
Biological Laboratory, Bryn Mawr College
April 30, 1906
600 W.N. Nowlin
LITERATURE
BaumGarTNER, W. J., ’04.—Some New Evidences for the Individuality of the
Chromosomes. Biol. Bull., vol. viii, No. 1.
Bovert, Tu., ’02.—Ueber Mehrpolige mitosen als mittel zur Analyse des Zell-
kerns. Vehr. d. Phys. Med. Ges. zu Wiirzburg, N. F., Bd. xxxv.
CastLE, W. E., ’03.—The Heredity of Sex. Bull of the Mus. of Comp. Zool.,
Harvard College, vol. xl, No. 4.
McCuune, C. E., ’99.—A Peculiar Nuclear Element in the Male Reproduc-
tion Cells of Insects. Zool. Bull., vol. ii.
’00.—The Spermatocyte Divisions of the Acrididz. Kans. Univ. Quart.,
vol. ix, No. 1.
’02.—The Accessory Chromosome—Sex-Determinant ? Biol. Bull.,vol. iii,
Nos. I and II.
’o2a.—The Spermatocyte Divisions of the Locustide. Kans. Univ.
Quart, vol. i, No. 8.
Montcom_ery, Tu., ’03.—The Heterotypic Maturation Mitosis in Amphibia
and its General Significance. Biol. Bull., vol. iv, ’02—’03.
Stevens, N. M., ’05.—Studies in Spermatogenesis with Especial Reference to
the Accessory Chromosome. Carnegie Institute of Wash., Pub.
No. 36.
Sutton, W. S., ’°02.—On the Morphology of the Chromosome Group in Brachy-
stola magna. Biol. Bull., vol. iv, No. 1.
Witson, E. B., ’05.—I. The Behavior of the Idiochromosomes in Hemiptera.
Journ. Exp. Zool., vol. ii, No. 3.
’05.—II. The Paired Microchromosomes and Heterotropic Chromo-
somes in Hemiptera. Journ. Exp. Zool., vol. ii, No. 4.
06. III. The Sexual Differences of the Chromosome-Groups in
Hemiptera, with Some Considerations on the Determination
and Inheritance of Sex. Journ. Exp. Zool., vol. tii, No. 1.
DESCRIPTION OF PLATES
All drawings were made with the aid of a camera lucida. A Zeiss oil-immersion 2 mm. objec-
tive and oc. 12 were used throughout, and the drawings have been reduced one-third.
Pirate I
Coptocycla aurichalcea
Fig. 1. Equatorial’ plate of spermatogonial mitosis, 22 chromosomes.
Fig. 2. Bouquet-stage or synizesis, showing univalent chromosomes in loop form.
Fig. 3. Later stage in which loops are straightening.
Fig. 4. Cross-section of the stage in Fig. 2.
Fig. 5. Synapsis. Fusion of univalent chromosomes end to end at y. Large idiochromosome
x, at base of loops.
Fig. 6. Late synaptic stage, showing the relative lengths of the bivalent elements.
Fig. 7. Formation of spireme; x, the large idiochromosome.
Fig. 8. A slightly later stage; spireme very pale, « deeply stained.
Figs. 9,10. Very early prophase, showing the longitudinal split in the chromosomes, idiochro-
mosome excepted.
Fig. 11. Still later prophase. Both members of the unequal pair visible (x).
Figs.12,13. Prophase in which the chromosomes are contracting into their characteristic forms.
Fig. 14. An equatorial plate of the first division showing nine of the eleven chromosomes. The
idiochromosomes retain the stain much longer than the others.
Fig. 15. An equatorial plate of the first mitosis with the full number, eleven chromosomes, pres-
ent. The large linen fibers are seen attached to the chromosomes.
Fig. 16. Three types of chromosomes, found in C. aurichalcea, from various points of view,
a—e, the cross-shaped chromosome; a, between a front and side view; b, front view when the longi-
tudinal split is closed; c and e, profile; d, front view showing split, f/—h, the E-shaped chromosome
f, front view; g, profile, bivalent chromosome dividing; h, profile; /—o, ring-shaped chromosomes:
], the two univalent chromosomes not well fused; m, probably later stage in which union is more com-
plete; 7, ring closed and seen in profile; 0, central opening growing smaller.
Fig. 17. Late prophase of first spematocyte; chromosomes coming into equatorial plate; «, the
idiochromosome pair.
Fig. 18. Equatorial plate formed and one chromosome divided ahead of the others.
Fig. 19. Shows the size difference in some of the chromosomes.
Fig.20. Anaphase of the first division, showing distinctly the form of the univalent chromosomes,
and the manner of separation.
Fig. 21. Side view, first spindle, showing the densely staining idiochromosome when the others
are very pale.
Figs. 22-24. Different views of the ring chromosome.
Fig. 25. Idiochromosomes dividing in first mitosis; /, large chromosome; s, small chromosome.
Fig. 26. Late anaphase of first spermatocyte.
Figs. 27,28. Equatorial plates of second mitosis, showing 10 large and one small (s)chromosome.
Fig. 29. Equatorial plate of same mitosis, showing 11 large chromosomes, /, the large idiochro-
mosome.
Fig. 30. Prophase of second mitosis, showing 10 large chromosomes and the small idiochro-
mosome.
Fig. 31. Anaphase in which the large idiochromosome is dividing.
SPERMATOGENESIS OF COPTOCYCLA
W. N. Now iin
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Journat or ExperIMENTAL ZOOLOGY, VOL. II, NO. 4
PLATE I
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19
Prate II
Coptocycla aurichalcea
Fig. 32. Anaphase of second maturation division, showing the position assumed by the chro-
mosomes when first separated.
Fig. 33. Late anaphase; small idiochromosomes dividing.
Fig. 34. Late anaphase; unusual case in which the form of the chromosomes is not obscured by
massing.
Fig. 35. The usual late anaphase.
Fig. 36. Very early spermatid showing a great amount of archoplasm.
Fig. 37. Nucleus at a slightly later stage, showing the peculiar nucleolus-like body.
Fig. 38. Archoplasmic substance in the form of a sphere applied to nuclear membrane.
Fig. 39. Archoplasm elongated into a pennant form with axial filament running throughout.
Fig. 40. Vacuolated stage in formation of the tail.
Fig. 41. Vacuolated appearance of head c, centrosome.
Figs. 42-44. Later stage in the transformation of the head. Ripe spermatozoa (44) possess a
spirally twisted head.
Figs. 45-47. Later behavior of the nucleolus-like body.
Fig. 48. Giant spermatozoa, with two centrosomes and two axial filaments.
Fig. 49. Shows the migration of the nucleolus-like body.
Fig. 50. Cross-sections of the tail of the spermatid, a—b, at about the stages shown in Fig. 41;
c, in Fig. 44. 3
Coptocycla guttata
Fig. 51. Equatorial plate of spermatogonial mitosis; 18 chromosomes.
Fig. 52. Synizesis stage showing univalent chromosomes in loop form. Large idiochromosome
at base of loops.
Fig. 53. Later stage in which loops are straightening and bending toward each other.
Fig. 54. Synapsis. The dark bead at the highest part of loop marks point of union.
Fig. 55. Spireme stage; chromatin very pale with exception of unequal pair (x).
Fig. 56. Same stage, showing small idiochromosome somewhat removed from the large member
and connected with it by a chromatin strand.
Figs. 57, 58. Formation of ring and cross-shaped chromosomes.
Fig. 59. Late prophase in which all nine chromosomes are seen from a side view. «, the idio-
chromosome pair.
Fig. 60. Equatorial plate of the first maturation mitosis; 9 chromosomes.
Figs. 61,62. Prophase and metaphase, respectively, showing the idiochromosome pair («).
Fig. 63. Anaphase of first mitosis; the idiochromosome pair, dividing qualitatively. s, small
idiochromosome; /, large.
Fig. 64. Very early anaphase of second mitosis, showing division of the small (s) idiochromosome.
Fig. 65. An equatorial plate of the second spermatocyte in which the small idiochromosome
(s) is present.
Fig. 66. Metaphase of second spermatocyte. The small idiochromosome (s) undivided.
Fig. 67. Equatorial plate from dividing somatic cell of male pupa (Tenebrio molitor), showing
nineteen large and one small chromosome.
Fig. 68. Equatorial plate of a dividing cell of follicle of a young egg (Tenebrio molitor), showing
twenty large chromosomes.
Fig. 69. Equatorial plate of a dividing follicle cell of a young egg (Trirhabda virgata), showing
twenty-eight large chromosomes. ;
Fig. 70. Equatorial plate from the somatic cells of a male larva (Trirhabda virgata), showing
twenty-seven large and one small chromosome.
Fig. 71. An equatorial plate of same spermatocyte in which all nine chromosomes are of approxi-
mately equal size.
SPERMATOGENESIS OF COPTOCYCLA
W. N. Now.in
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PLATE II
Mim
o3 of
Journat or ExPeriMENTAL ZOOLOGY, VOL. II, NO. 4,
TORSION AND OTHER TRANSITIONAL PHENOM-
ENA IN THE, REGENERATION OF THE CHELIPED
OF THE LOBSTER (HOMARUS AMERICANUS)
BY
VICTOR E. EMMEL
Wirn Two Pirates
INTRODUCTION
In the course of a series of experiments and observations on
the phenomena of regeneration in the lobster, which necessitated
a detailed study of the external morphological characteristics of
the regenerating cheliped (’05, ’06), it was noticed that in the
successive stages of development there occurred a gradual torsion
of the regenerating chelz.
This and some other closely related phenomena do not seem
to have been hitherto recorded, although the regeneration of the
crustacean limbs has been the subject of extensive investigation
by Herrick (’95), Morgan (’02), Przibram (’o1, 02), Steele (’04),
Schultz (05), Wilson (703), and other writers. “These phenom-
ena offer new material for the study of the phylogenetic signifi-
cance of the regenerative process.
The problems of development are now being approached by
the experimental method, and interpretations are based on
physiological and mechanical princlples. And yet while the
processes of development may be essentially physiological in
nature and affected by mechanical factors, still the important
truth remains that the organization of the cells is an “inheritance
from the past,” and that consequently the method of develop-
ment may still furnish evidence of phylogenetic value. Wilson’s
(’94) valuation of the embryological phenomena may apply
equally well to those of regeneration: “the idioplasm of every
species has arisen through the modification of pre-existing
Tue JourNat or EXPERIMENTAL ZOOLOGY, VOL. III, NO. 4.
604 Victor E. Emmel
idioplasm, and every response that it gives to stimulus is an
expression of its past history. Hence, we need not despair of
ultimate success in the attempt to decipher the meaning of the
embryological record, and to find in ontogeny a real criterion
of homology, and it is here that we find encouragement, if any is
needed, not to relax our efforts to investigate the normal phenom-
enon of comparative embryology on the largest scale and down
to the minutest detail” (p.123). It is with the purpose of contri-
buting something to the facts bearing on these problems that the
following observations are presented.
These observations were made at the Anatomical Laboratory of |
Brown University, and at the Experiment Station of the Rhode
Island Commission of Inland Fisheries, where the lobster hatchery
furnished unsurpassed facilities to obtain material for expert
mental work. I desire here to express my thanks to Dr. Mead
for his interest and helpful suggestions in the present study.
I. ON THE REGENERATION OF THE CHELIPED
a. Method
In order to trace the transformations involved in the regenera-
tion of the cheliped, a series of drawings have been made to
illustrate successive stages in the process.
The material for this was obtained from about 75 fifth-stage
lobsters. ‘These lobsters were practically equal in size and age,
and had all molted to the fifth stage, July 30 and 31. On August 1,
the right and left chelipeds were autotomously removed from
each individual. Every twelve hours, two or three of these
lobsters were killed in corrosive acetic and preserved in alcohol.
This was continued until August 13, when some of the remaining
lobsters had begun to moult to the sixth stage. By this method,
regenerating finbe were obtained in a series, ranging from
minute papillae to fully formed functional structure. These
preserved lobsters were then carefully examined and eight
specimens selected which presented typical stages in the differen-
tiation and development.
Regeneration of the Cheliped of the Lobster 605
The data for these eight specimens may be tabulated as follows:
Removed | a No. of days re- | ;
No. Stage Chetpeds) | Killed montane | Drawing
I Vi |) Aug. 1, 8\p.m. Aug. 2,8 p.m. Tee dayaunta| ieesbie wT
2 Vs |sAug. 1,8 p.m. Aug. 5,8 a.m. 3h days | Fig. IIT
3 V | Aug.1,8p.m. | Aug. 6,8 p.m. 5 days | Figs.I,IV, XV
a Vv Aug.1,8p.m. | Aug. 7,8 p.m. 6 days | Fig.V
5 V | Aug. 1, 8 p.m. Aug. 8 7 days) Vio BigsVil
6 V | Aug. 1, 8 p.m. Aug. 9,8 p.m. Sedaysiy eles ehigeViel
7 Vv Aug.1,8p.m. | Aug. 13,8 p.m. 12 days | Figs, VIII, XVI
8 VI | Aug. 1, 8 p.m. Aug. 15, 8 a.m. 133 days | Figs. IX, XVII.
Camera lucida drawings were made of a right cheliped of each
lobster. ‘The drawings were taken as nearly as possible from a
constant plane (Fig. I), which shows a regenerating bud (reg)
on the right basipodite (bs) of lobster No. 3. Each lobster
was laid flat on its left side upon the microscopic stand, thus
giving in every case, a postero-lateral view of the basipodite and
regenerating cheliped.
b. Torsion of the Regenerating Cheliped (Plate I) .
Kus
Hirst Day CLlobster No. 1, Fig. Il). . During’ the fist and
second days after the removal of the cheliped, only a very
slight regeneration, if any, can be detected over the exposed
surface of the basipodite (bs). ‘The first external indication of
activity among the regenerating cells, is a minute light colored
papilla pushing up through the dark clot of blood in the region (b&).
Third Day (Lobster No. 2, Fig. III). By the third day a distinct
bud has appeared. Seen from the exterior, it is a simple club-
shaped mass enclosed within an epithelial membrane or sac.
The only indication of future segments and joints is a slight
cleavage or groove (a), forming at the apex of the bud. This
groove marks the first development of the claw; 1.e., the dactyl-
opodite (dc), and propodite (pr).
Fifth Day (Lobster No. 3, Fig. IV). At this stage of develop-
ment the outlines of the dactylopodite (dc), and propodite (pr) are
becoming more definite, as indicated by the constrictions for the
606 Victor E. Emmel
first (1) and second (2) joints, (counting from the distal segment).
The groove (a) for the two jaws of the claw has grown deeper.
Sixth Day (Lobster No. 4, Fig. V). The anlage of all the
segments of the appendage have appeared; 7. e., the dactyl-
opodite (dc), propodite (pr), carpopodite (cp), meropodite (me),
and ischiopodite (7s). “The dactylopodite is now more completely
differentiated from the propodite.
It is interesting to note here that the direction of differentiation
is from the distal portion backward toward the proximal region
of the bud, a method of differentiation characteristic of regener-
ation in many animal forms (see Zeleny, ’06; Child ’o6, p. 410).
Seventh and Eighth Days (Lobsters Nos. 5 and 6, Figs. VI and
VIL) The four joints and five segments are clearly defined and
the whole bud is now assuming a forward curvature.
The important fact for our present purpose in the six stages
just described, is the position or spatial relations of the two
terminal segments (7. ¢., the dactylopodite and propodite) with
reference to the basal segment of the limb.
It should be observed that each figure represents a nearly
constant point of view—the postero-lateral aspect shown in Fig. I.
The plane of the paper thus presents a common plane of reference
and comparison in each case. Let this plane be called the
“reference plane.”’
Now if in Figs. III and IV, we compare the position of the two
segments of the claw (dc and pr), it may be seen that the plane
of cleavage for the two jaws of the claw, or groove (a) is practi-
cally at right angles to our “reference plane.” In other words,
if we compare the “biting plane” of the claw at this early period
with the normal position of the body, the claw will open almost
vertically upward.
The same is practically true for Figs. Vand VI. In Fig. VII,
however, the plane of cleavage (a) is no longer vertical to the
“reference plane.’”’ ‘This is due to the fact that a slight twisting
or torsion of the terminal segments has begun. ‘The dactylopo-
dite (dc) and propodite (pr), and possibly the carpopodite (cp),
are gradually turning in such a way as to throw the dactylopcdite
farther inward toward the median line of the body; so that at
Regeneration of the Cheliped of the Lobster 607
this stage, the “biting plane” of the claw is inclined away from
the “reference plane” at an angle of about 20°. The later
phases of this torsion appear in the further development.
Twelfth Day (Lobster No.7, Fig. VIII). At this period of the
regenerating cheliped the lobster is rapidly approaching the molt.
The segments and joints are now almost completely developed.
The regenerating limb is assuming a rounded, plump appearance,
as the result of the large growth of tissue compressed within the
epithelial membrane. ‘The significant fact here is the position of
the claw. ‘The torsion has progressed to such a degree that the
plane passing through the dactylopodite (dc) and propodite (pr)
or “biting plane,” is now inclined at an angle of about 45° to its
original position.
Thirteenth Day (Lobster No. 8, Fig. EX). ‘This phase of the
development marks the culmination of both the regenerative
process and the torsion of the chela. At this stage the lobster
has moulted its old shell, and Fig. 1X, represents the completely
regenerated limb with its joints and segments expanded to their
normal shape.
The complete torsion of the chela is now readily perceived.
Instead of being parallel, as in Figs. III and IV, the “biting
plane” of the dactylopodite (dc) and propodite (pr) is at right
angles to the “reference plane.” Thus it is clearly evident,
that beginning with the earliest phase of the regenerating cheliped,
with the claw nearly vertical in position, a torsion of the terminal
segments has gradually taken place during the development, so
that the dactylopodite and propodite have rotated through an angle
of g0°, and attained the horizontal position of the normal claw.
c. Relative Size of the two Segments of the Claw (Plate I)
In the earliest period at which the differentiation of the claw
is apparent in the regenerating bud (Fig. III), the partially
developed propodite (pr’) of the claw is relatively smaller than
the opposing dactylopodite (dc), a relation which 1s just opposite
to that which holds in the adult structure.
On the fifth day (Fig. IV) there is less difference in size, although
608 Victor E. Emmel
the dactylopodite (dc) is still clearly larger and farther developed
than the propodite (pr’) element of the claw. By the sixth day,
however (Fig. V), this relative inequality has disappeared and
the two segments are practically equal. ;
The seventh day (Fig. V1) marks the transition to a relation in
which the propodite jaw (pr’) is slightly more developed, and by
the eighth day (Fig. VII) it clearly forms the larger segment of
the claw. During the remaining development, the propodite and
dactylopodite gradually acquire the proportions of the normally
developed chela (Figs. VII] and IX). ‘Thus, according to these
observations on the regenerating chela, it is evident that in the
course of its differentiation the claw develops from an early stage
in which the propodite portion of the claw 1s proportionately
smaller, to a stage in which it is proportionately larger than the
opposing dactylopodite.
d. Transitional Characters in the Regenerated Crushing Claw
(Plate IT)
In the lobster the chelipeds are differentiated into two types of
claws, the “toothed” or “nipping” claw, and the “‘crushing” claw.
When the “crushing” cheliped has been amputated and another
cheliped regenerated from the breaking plane, the claw of the
latter frequently displays such a close resemblance to a “nipping”’
claw that it is not easy to distinguish the regenerated crusher from
a normal “nipping” claw.
Figs. XXI, XXII and XXIII represent the normal and regener-
ated chelipeds of a seventh(?) stage lobster. ‘The normal left
“nipping’ and the right “crushing” claws are shown in Figs.
"At this stage the two types of claws are clearly differentiated, although it should be observed that the
older the animal the more highly developed the ‘‘crusher” becomes. This specimen was one of a
number of lobsters used in an experiment upon the reversal of the chele, and in this individual the ‘‘ crusher”
had been removed later than the “‘nipper”’ in order, if possible, to give an advantage to the regener-
ating “‘nipper.”” It might be added that although the regenerated chelipeds frequently appeared so
much alike that it was necessary to wait until another moult before it could be determined whether a
crusher had been developed or not, still no conclusive evidence was obtained that ‘‘reversal’’ ever
occurs in either young or old lobsters, a result similar to that of Przibram’s (’02) on the European
lobster.
Regeneration of the Cheliped of the Lobster 609
XXI and XXII, respectively, and the regenerated right “crusher”
in Fig. XXIII.
Both chelipeds were autotomously removed, the left on July 30
and the right August 4. ‘Iwenty-seven days later, the lobster
moulted and measured26.5mm._ Both chelipeds had regenerated,
but in this individual as well as in many of the other lobsters used
in the experiment indicatedin the footnote, the claws looked very
much alike. ‘The regenerated left chela (“nipper’’) had all the
characteristics of the original “nipper;” the right chela, on the
contrary, was unlike the original “crusher” and showed characters
transitional between the original “nipping” and “crushing”
claws.
Ina morphological comparison of the regenerated right “crush-
ing”’ claw with the original “nipping” and “crushing” claws the
following contrasts may be observed (see Plate IJ)
OriGINAL Ricut Craw | REGENERATED RiGut CLAw
(Fig. XXII) (Fig. XXIII)
| Oricrnat Lert CLaw
(Fig. XX1)
Hormof claw... ..6.- |Proportionately elon- | Proportionately thicker | Proportionately elongated
gated and slender and more massive and slender
Ratio of breadth to
6 ay : : :
length of claw....... ee Se 26 SS EaRs = .34 HEED .27
1omm g.8 mm. 9-3 mm.
Dactylopodite ......... Slender and nearly | Distinctly crooked and | Almost straight and com-
straight stubby paratively slender
|
Dentition ............ Pointed cutting teeth, | Characteristic broad | Cutting teeth predominate.
grouped in periodic |
sequence (p,p) with
formula 1:3:5:7*
Number of spines (sp)
on inner border of
LOO emer ereyeeieters 5
Tactile hairs on inner |
and outer border of |
Glawsyere rors 2 | Very numerous
crushing tubercle + (t.t.
etc.), with only a rudi-
ment of periodic se-
quence (f’)
wa
Sparse { ' |
Sequence of 1:3:5:7 well
marked (p)
Quite numerous
|
Dominant morphologi-
“Nipping” type
cal characters
“Crushing” type |
Transitional between
“nipping” and ‘‘crush-
ing” types
*According to Stahr’s schema. (Stahr, °98, p. 459)
{Formed by a fusion of periodic teeth.
(Herrick, ’o5)
tin an older lobster, tactile hairs on the crushing claw almost entirely disappear.
610 Victor E. Emmel
From this comparison, it is evident that the claw on the regen-
erated right cheliped differs from the original “crusher” and
closely resembles the “nipping” claw, in general form and
proportions, the character and arrangement of teeth, and even in
the number of spines and tactile hairs. The conclusion seems
clear that the cheliped, which regenerates after the removal of the
“crusher,” is a type transitional between the original “nipping”
and “crushing” claws.
In regard to the universal character of this rule it as already
been intimated that the transitional type may not always be pres-
ent in the regenerated “crusher,”’ especially in the older lobsters.
This may be partially due to the conditions under which the limb
was removed; 7.¢., if it is removed early in the interval between
two moults, the regenerating bud will have a longer time to develop,
and may consequently be farther advanced in its differentiation
toward the original type before the next moult occurs.
II. COMPARISON WITH THE NORMAL DEVELOPMENT OF THE
CHELIPED
a. Torsion in the Larval Stages (Plate IT)
The torsion of the limb in regeneration which has just been
described, was discovered before I had seen Herrick’s (05a)
work on normal torsion. I have since gone over the larval
development of the lobster on some carefully preserved material
which Mr. P. B. Hadley kindly gave me, and have verified
Herrick’s work and made drawings to facilitate comparison with
the regenerating appendages.
In the first larval stage; 7.e., just after hatching, the claw of the
cheliped, as well as the claws of the first and second thoracic
legs, opens in a vertical plane with a slight inclination outward.
A dorsal view (Fig. XVIII) shows the dactylopodite (dc) in a
position vertically above the propodite (pr).
In the second larval stage a change in position begins, and in
the third stage (Fig. XIX) the dactylopodite (dc) and propodite
(pr) have turned over and inward, so that the claw now opens
at anangle of about 45° to its former plane. ‘The rotation of the
Regeneration of the Cheliped of the Lobster 611
cheliped is completed at the fourth stage (Fig. XX), the dactyl-
opodite (dc) and propedite (pr) are at right angles to their original
position and the claw now opens inward on the nearly horizontal
plane characteristic of the normal limb. Atthe same time it may be
observed that the claws of the first (1’) and second (1°) thoracic
legs have retained their original vertical position. ‘Thus “the
position of the great forceps has been reversed by rotation through
go°, in consequence of which their inner or anterior faces have
become their under sides” (Herrick, p. 130).
A direct comparison of the rotation of the regenerating cheliped
with the torsion in the larval stages may be made by means of
Figs. XV, XVI and XVII. ‘These figures represent the dorsal
view of the regenerating lobsters, Nos. 3, 7 and 8, respectively
(see p. 605). “The dactylopodite and propoditeon the fifth day stage
of regeneration (Fig. XV) lie in a plane vertical and outward
similar to the position for the normal claw in the first larval stage
(Fig. XVIII). On the twelfth day (Fig. XVI) the two segments of
the claw (dc and pr) lie ina plane at 45° from their former position
and correspond closely to the third larval stage type (Fig. XIX).
On the thirteenth day the regenerated claws of the moulted lobster
open inward on the horizontal plane shown in Fig. XVII.
Thus in both the regenerative and the ontogenetic development,
the “great claw” of the cheliped rotates through an angle of go°
to the horizontal plane of the normal claw.
b. Relative Size of the Dactylopodite and the Opposing Propodite
in Normal Development (Plate 1)
Having observed that in the regenerating claw the propodite
part of the claw which was at first smaller, gradually surpassed
in size the opposing dactylopodite, the question naturally arose
whether this, too, might be a feature of the ontogenetic develop-
ment. If so, it was evident that it must be sought in the larval
stages, for at the fourth stage the propodite, part of the claw,
is already slightly larger than the dactylopodite.
The larval specimens upon examination showed just such a
morphological transition in the development of the claw. In
the first larval stage the propodite part of the claw (Fig. X, pr’)
612 Victor E. Emmel
is nearly a third smaller than the opposing dactylopodite (dc).
In the second stage (Fig. XI) the propodite Jaw (pr’) is only
slightly the smaller, while in the third larval stage (Fig. XII) the
two jaws of the claw are practically equal in size. At the fourth
stage (Fig. XX, Plate II) the propodite jaw (pr’) is larger than the
opposing dactylopodite, and the claw is now assuming character-
istic normal proportions. Thus early in the development of the
cheliped, the dactylopodite is the larger of the two jaws ofthe
claw. Then by a gradual transformation the propodite jaw
becomes equal, and finally inthe adult structure is much the larger
of the two segments of the claw.
Nor, indeed, is this an unexpected course of ontogeny. For
the comparative morphology of the crustacean appendages
certainly indicates that the cheliped is merely a highly modified
leg appendage. In the lobster a transitional series, almost equal
to a demonstration, exists between the terminal segments of the
thoracic legs and the claw of the cheliped. ‘The third and fourth
legs have no claws (Fig. XIII), but in the first and second thoracic
legs (Fig. XIV) an elongated process (pr’) has grown out from the
distal part of the propodite, thus forming a Jaw to meet the oppos-
ing dactylopodite (dc). In the cheliped this process becomes the
propodite segment of the highly developed claw. ‘That this
propodite part of the claw is a true process which has grown out
from the second segment independently of the joint for the
dactylopodite, is clearly shown by the well marked groove or
shoulder (g, Figs. XIV, X, XI,) between the propodite jaw (pr’)
and the dactylopodite in the early stages of the developing chela.
From this series it 1s evident that the “great” claw of the cheliped
has been evolved by a distal elongation of the propodite process
or “claw-like” projection, against which the terminal segment
“bites,” thus forming a claw with greater facilities for grasping
prey.
c. Development of the “Crushing” Claw
The development of the external morphological features of
the chelipeds has been carefully described by Herrick (’05), and
my observations do not add anything of an original character.
Regeneration of the Cheliped of the Lobster 613
But a brief description seems necessary here in order to complete
the comparison between the regenerative and normal method of
developing this appendage.
In the adult lobster two distinct types of claws are found, the
more primitive “toothed” or “nipping” claw, and the larger
and phylogenetically younger (according to Stahr, ’98; and Przi-
bram, ’or) “crushing” claw. But in the larval stages and up to
the fifth moult, on the contrary, both claws are “similar” and of
the “toothed” type. At about the sixth stage (Hadley, ’05) the
“crushing” claw begins to differentiate. “The claw becomes
wider, broad tubercle-like teeth develop, and the tactile hair of
the toothed type (Plate II, Fig. XXI) gradually disappear in succes-
sive moults. “Thus the adult ‘‘crushing”’ claw comes to be char-
acterized by the almost entire absence of tactile hairs, and the
presence of the broad crushing teeth; by a dactylopodite relatively
smaller, and by being on the whole larger and more massive than
the “toothed” or “nipping” type.
‘These facts in the ontogenetic development serve to show the
relation between“ nipping” and “crushing” claws, and give signifi-
cance to the fact that the regenerating crusher for a long period
has a form intermediate between these two types.
III. RESUME
Three characteristic phenomena may be observed in the
regeneration of the cheliped, each of which resembles corre-
sponding stages of the ontogenetic development.
(a) In the very early stages of differentiation the cleavage of
the claw first appears at the apex of the regenerating bud insuch
a manner that the dactylopodite is vertically above the propodite.
The claw would, therefore, open upward and outward. In the
later course of development a marked transition in_ position
takes place. The terminal segments gradually rotate over and
inward, so that when the moult occurs, a torsion of about go° has
been produced, and the claw now opens inward on the naturally
characteristic horizontal plane.
(b) Contemporaneously with this torsion, a transition in the
form of the claw also occurs. At an early stage the dactylopodite
614 Victor E. Emmel
is larger than the opposing segment of the claw. Later the pro-
podite part of the claw rapidly develops, and becomes equal and
finally larger than the dactylopodite. By the time of moulting
the terminal segments have practically assumed their normal
proportions.
(c) During the regeneration of the “crushing” cheliped it
was observed that after the first moult, the regenerated “crushing”
claw could not always be recognized as such, but is of a type
intermediate between the “nipping” and “crushing” claws.
The ontogenetic development of the cheliped to which this
series of changes correspond is as follows:
(a) At the first larval stage the claw of the cheliped together
with the claws of the first and second chelate legs open upward
and outward, the dactylopodite being in a position vertically
above the propodite. The chele legs retain their original
vertical position, but in the cheliped a gradual change occurs
during the second and third larval stage, so that at the fourth
moult the claw has rotated through go° and assumed a horizontal
position. |
(b) In the first larval stage the propodite jaw of the claw is
much smaller than the opposing dactylopodite, but in the second
and third stages it increases in size and ultimately becomes pro-
portionately much the larger of the two segments of the claw.
(c) In the first four larval stages both claws are similar in
type. At about the sixth moult one claw begins to differentiate
toward a “crushing” claw. During ensuing moults this claw
passes through transitional stages and is finally completely
transformed into a normal “crushing”’ claw.
In a word, the torsion and other transitional morphological
changes in the regeneration of the cheliped are parallel to similar
phenomena in the ontogentic development of this appendage.
IV. DISCUSSION
In view of the fact that there is such a close similarity or paral-
lelism between the regenerative and ontological processes just
described, the question arises whether this parallelism has any
phylogenetic significance.
Regeneration of the Cheliped of the Lobster 615
The general question of the repetition of phylogenetic and
ontogenetic processes in regeneration is still open. Such facts
as the regeneration of an mecca type of claw in the shrimp
(Miller, *80); the reproduction of a walking leg in place of a
maxilliped in Portenus (Przibram, ’o1); the formation of a lens
from the iris in Urodeles (Wolff, ’94); the regeneration of a five,
instead of four-fingered hand in the Axolotl (Barfurth, ’94); have
led to a diversity of interpretation. Weismann (’02), on the
basis of his “determinant” theory, speaking of the differences
between the regenerated and the original structure, holds that
“there remains nothing for it but the assumption that the regen-
eration determinants have remained at a phyletic lower level,
while the determinants which direct embryogenesis have varied
and either developed farther or retrogressed”’ (p. 28).
On the other hand Herbst, Driesch, Morgan, and others, do not
see a dependence of regenerative processes upon fundamental
biogenetic laws, but rather hold that a similarity in development
is due to a similarity in conditions; “that the mistake is not in
stating that the two processes are sometimes similar or even
identical, but in stating the matter as though the regenerative
process repeats the embryonic method of development,” and
“that it may be entirely misleading to infer that ancestral charac-
ters have reappeared” (Morgan, ’O1, pp. 213, 214).
Quite recently, Schultz (’05) has reafhrmed the atavistic signif-
cance of the process in a striking case of regeneration in the claws
of five species of Russian crayfish: A. fluvitalis, A. pachypur, A.
colchicus, A. kessleriand A. leptodactylus. The regenerated claws
in each species were not only unlike the normal type of claw in each
case, but, moreover, the remarkable fact was observed that they all
resembled more or less closely a single type of claw, namely, the
type characteristic of A. leptodactylus. Schultz’s atavistic inter-
pretation of the case is supported by the recent work of Skorikows,
whose study of the genetic relationship among the crayfish from
the standpoint of geographical distribution, etc., makes A. leptodac-
tylus var. colchica the parent form of all Russian crayfish. Schultz
accordingly maintains that “we see here a dependence of regenera-
tion upon fundamental biogenetic laws, a dependence which
616 Victor E. Emmel
Herbst, Delage, Driesch, and others, can vainly deny or slight,
as “explaining nothing”’ (p. 46).
In regard to this controverted question, the present observa-
tions on the lobster seems to favor a recapitulation theory in
regeneration. Since it is difficult adequately to account for the
similarity between the ontogenetic and regenerative processes on
the basis of a “similarity of conditions” in the organism. ‘The
ontogenetic torsion and proportional development of the claw
take place in the larval stages of this crustacean. At this period
the organic conditions must be widely different from those under
which the same phenomena occur in the regenerative process
of the adult lobster, for during the larval stages the organism
undergoes such an extensive metamorphosis in both function
ang eae (Hadley, ’05) that the fourth moult of the young animal
‘ BLESS the most surprising leap in the whole history of develop-
ment”’ (Herrick, ’05). Even the differentiation of the claws into
the “nipping” and “crushing” types, which occurs after the larval
stages, marks the change from a “symmetrical” to an “asym-
metrical” “equilibrium” in these appendages, and 1s correlated
with the establisment of the “bottom-living”’ habits of the lobster
(see Przibram, ’05, p. 238; Herrick, ’95, p. 180).
‘This parallelism between the regenerative and normal develop-
ment, therefore, seems to be more adequately interpreted by the
conclusion that im the regenerative process of the cheliped we
meet with a recapitulation of characteristic phases of its ontogeny;
phases which, if investigators are at all correct in interpreting the
origin of the crustacean cheliped through a modification of the
thoracic leg appendages, are themselves in turn the recapitula-
tion of a phylogenetic development.
Anatomical Laboratory
Brown University, Providence, R. I.
May 23, 1906
Regeneration of the Cheliped of the Lobster 617
LITERATURE CITED
BarFutH, D., ’94—Die experimentelle regeneration tiberschiissiger Gliedmassen-
teile bei den Amphibien. Archiv. f. Entw.-Mech., i, pp. 91-116,
1894.
Cuitp, C. M., ’06.—Contributions toward a Theory of Regulation. I. Archiv.
f. Entw-Mech., Bd. 20, pp. 380-426, 1906.
Emmet, V. E. ’05.—The Regeneration of Lost Parts in the Lobster. Report of
Rhode Island Commission of Inland Fisheries, pp. 81-117, 1905.
Sp. paper, No. 20.
’06.—The Relation of Regeneration to the Moulting Process of the
Lobster. Report of Rhode Island Commission of Inland Fisheries,
pp- 258-313, 1906. Sp. paper, No 27.
Haptey, P. B., ’05.—Changes in Form and Color in Successive Stages of the
American Lobster. ‘Report of Rhode Island Commission of
Inland Fisheries, pp. 44-80, 1905. Sp. paper, No. 19.
Herrick, F. H., ’95:—The American Lobster. Bull. U. S. Fish Commission,
pp- 1-252, 1895.
’05.—The “Great Forceps” of the American Lobster. Science, n. s., vol.
xxi, Pp. 375, 1905.
’o§ a.—Torsion of the Crustacean Limb. Biol. Bull., vol. ix, No. 2.
pp- 130-137, 1905.
Moreau, ’o1.—Regeneration. New York, r1got.
o2—Regeneration of the Appendages of the Hermit Crab and Crayfish.
Anat. Anz., Bd. 20, pp., 598-605, 1902.
Mutter, F., *80.—Haeckel’s Biogenetisches Grundgesetz bei der neubildung ver-
lorener Glieder. Kosmos, viii, 1880-81.
PrzipraM, H., ’o1.—Experimentelle Studien tiber Regeneration. Archiv. f. Entw-
Mech., Bd. xi, pp. 320-345. 1901.
’o2.—Experimentelle Studien tiber Regeneration. Archiv. f. Entw.-
Mech., Bd. xii, pp. 507-527, 1902.
’05.—Die “‘Heterochelie” bei decapoden Crustacean. Archiv. f. Entw.-
Mech., Bd. xix, pp.181-247, 1905.
ScHuttz, E., ’o1—Ueber das Verhiltnis der Regeneration zur Embryonalent-
wicklung und Knospung. Biol. Centralblatt, Bd. xxi, pp. 360-
368, 1902.
’05.—Ueber atavistische Regeneration bei Flusskrebsen. Archiv. f.
Entw—Mech., Bd. xx, pp. 38-47, 1905.
Sraur, ’98.—Neue Beitrage zur Morphologie der Hummerschere mit physio-
logischen und phylogenetischen Bemerkungen. Jena. Zeitschr.
fiir Naturwissenschaft. Bd. xxxil, pp. 387-482, 1898.
618 Victor E. Emmel
STEELE, M. I., ’04.—Regeneration of Crayfish Appendages. Univ. of Missouri
Studies, vol. 11, No. 4, 1904. ;
WEIsMANN, A., ’02.—The Theory of Evolution. London, 1904.
Witson, E. B., ’94. The Embryological Criterion of Homology. Biol. Lectures,
Marine Biol. Laboratory, Woods Hole, 1894.
"03.—Notes on the Reversal of Asymmetry in the Regeneration of the
Chelz in Alpheus Heterochelis. Biol. Bull., vol. iv, pp. 197-214,
1902-03.
Wo rr, G., ’94.—Entwickelungphysiologische Studien. I. Die Regeneration der
Urodelenlinse. Archiv. f. Entw.-Mech., i, pp. 380-390, 1894-95.
ZELENY, C., ’05.—Compensatory Regulation. Journ. of Exp. Zool., vol. i,
pp. I-102.
’06.—The Direction of Differentiation in a Regenerating Appendage.
Science, n. s., vol. xxill, p. 527, 1906.
EXPLANATION OF PLATES
(All figures are from camera drawings)
a—Cleavage or groove between the two jaws of the claw.
bs—Basipodite.
c—Cheliped.
cp—Carpopodite.
dc—Dactylopodite.
ex—Exopodite. :
g—Groove or projection between the dactylopodite and the claw-process of (pr’) the propodite in
early development.
h—Tactile hairs. .
is—Ischiopodite.
’—First thoracic leg.
’—Second thoracic leg.
me—Meropodite.
p—Group of teeth in periodic sequence.
p’ —Indications of a former sequence in the teeth of the ‘‘crushing” claw.
pr—Propodite.
pr’ —Propodite-part of claw opposing the corresponding jaw or dactylopodite.
sp—Spines on lateral edge of propodite.
t—Tubercle-like teeth one crushing claw. 1, 2, 3 and 4, respectively, the first, second, third
and fourth joints of the cheliped.
Prate I
I-IX—Successive stages in the regeneration of the right cheliped of fifth stage lobsters.
I—Side view of Lobster No. 3 (see table, p. 605). Gives the postero-lateral view of the
regenerating cheliped (reg) typical for Figs. II-IX. 4.5
II—Stump of cheliped one day after autotomy, before any regeneration is apparent. bk
‘‘breaking-plane”’ or surface of the basipodite, at the center of which the regenerating
bud will appear. (Lobster, No 1) 12.8%
I1J—Regeneration, 3.5 days. (Lobster, No 2) 12.8X
IV—Regeneration, 5 days. (Lobster, No3) 12.8%
V—Regeneration, 6 days. (Lobster, No 4) 12,8
ViI—Regeneration, 7 days. (Lobster, No 5) 12.8%
VII —Regeneration, 8 days. (Lobster, No 6) 12.8X
VilI—Regeneration, 12 days. (Lobster, No 7) 12.8%
X1IX—Regeneration, 13.5 days. Completely regenerated cheliped, just after lobster had molted
to sixth stage. (Lobster No 8) 9X
X-XII—Show relative size of the two segments of the claw of the right cheliped in the larval
stages (posterior view). 12,8
X—First larval stage.
XI—Second larval stage.
XII—Third larval stage.
XIII-XIV—Terminal segments of the third leg (XIII), and the claw of the first leg (XIV) of a
first stage larval lobster (12.8% ). These figures, together with Figs. X, XI and XII,
evidently indicate successive phases in the phylogeny of the ‘“‘great forceps’ of the
cheliped.
Oe ie
REGENERATION OF THE CHELIPED OF THE LOBSTER
Victor E, EMMeL
PATE KTV
Tue JourNat or ExpERIMENTAL ZOOLOGY, VOL. III, NO. 4
PLATE I
Prate IT
XV-XVII—Torsion in the regeneration of the right cheliped. Dorsal view of fifth stage lobsters,
showing different phases of the regenerating limb.
XV—Regeneration, 5 days, dactylopodite (dc) outward and above the propodite (pr?).
(Lobster No3) 4.5
XVI—Regeneration, 12 days, “‘biting plane” of claw turned inward at angle of about 45° to
its original position. (Lobster No7) 4.5
XVII—Cheliped completely regenerated, 13.5 days. Lobster had just moulted and the complete
torsion of the cheliped permits the claw to open inward on a nearly horizontal
plane. (Lobster No 8) 4.5
XVIII-XX—Torsion of the right cheliped in normal development.
XVITI—First larval stage; claw of cheliped (c) opens upward and outward. 9X
XIX—Third larval stage; claw opens inward at an angle of about 45° to its original position. 9X
XX—Fourth stage; claw opens inward on nearly horizontal plane. 4.5
XXI-XXIII—Transitional characters in the regeneration of a ‘‘crushing” claw. Lobster in seventh
(?) stage. (Dorsal view)
XXI—Original left “‘nipping” claw. 4.5
XXII—Original right “‘crushing” claw. 4.5
XXTII—Regenerated right ‘‘crushing” claw. 4.5%
REGENERATION OF THE CHELIPED OF THE LOBSTER PLATE II
Victor E. EMMEL
XV XVI
XXI XXII XXIII
Tue JourNAL oF ExperiMENTAL ZOOLOGY, VOL. III, NO. 4
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From the Physiological Laboratory of the University of Kansas
THE INFLUENCES OF GASES AND TEMPERATURE
ONE THE CARDIAC AND -RESPIRATORY> MOVE-
MENTS IN THE GRASSHOPPER
BY
EULALIA V2 WALLING
The object of the experiments recorded in the following pages
was to ascertain the influences of certain gases upon the cardiac and
respiratory movements of the grasshopper, both in the uninjured
animal and when the heart and respiratory centers were isolated
from all or part of the body.
The principal parts of the respiratory organs are the paired
spiracles, with their air sacs, and the tracheal tubes, that ramify
from these to all parts of the body. ‘The mechanical respiratory
movements consist of an expiratory and an inspiratory phase.
During the latter, the active contraction of the abdominal muscles
increases the breathing space by enlarging the antero-posterior
and dorso-ventral diameters of the abdomen. In consequence of
the lowered pressure therein produced, air passes into the tracheal
tubes. During expiration the muscles relax and air enters the
air sacs.
The heart extends as a delicate tube longitudinally above the
intestine, directly beneath the chitin, along the whole length of the
abdomen. When entirely exposed to view it is seen to pulsate
rhythmically throughout its whole extent. By a system of valves
it is divided into a series of eight chambers, that communicate
through slits or ostia with a surrounding pericardial sinus.
Through the aorta, which arises at the anterior end of the heart,
and the aortic branches, blood is conveyed to all parts of the body,
along the paths of the tracheal tubes.
It was demonstrated by Ewing" that the respiratory movements
persist not only in decapitated animals but also in the isolated
abdominal segments, in which are contained the ganglia of the ven-
1Ewing,H.Z. Kansas University Science Bulletin, 1904, Vol. ii, p. 305.
Tue JourNat or ExperIMENTAL ZOOLOGY, VOL. 11, NO. 4.
822 Eulalia V. Walling
tral nerve cord, that are the centers for the reflex and respiratory
movements of the special segments to which they belong. I found
that the heart also will continue its rhythmical contractions for
hours, when left attached to a narrow strip of chitin along its mid-
dorsal line and entirely removed from the body; moreover, that
segments cut from the heart will continue their pulsations for long
periods of time, especially if kept moist with Ringer’s solution.
Whether there are dorsal ganglia as described by Carlson? for
Limulus, is a question that I am now investigating. Certain it 1s,
that electrical stimulation on the dorsal side accelerates, and burn-
ing this region with a very fine needle destroys the action of the
special segment that was injured.
More than a thousand grasshoppers of different species were
employed in this investigation. Besides normal, decapitated and
deviscerated specimens, abdominal segments and isolated heart
pieces were employed. In the deviscerated animals, the viscera
and air sacs were removed, thus leaving the heart, ventral nerve
cord and respiratory muscles intact. ‘The experiments were
repeated many times and the data for each set of experiments
represent average results.
I take this opportunity to express my thanks for valuable sug-
gestions and help to Professor Hyde, under whose direction this
work was conducted.
Vitality Varies with Conditions.—When kept under the most
favorable conditions; that is, when placed in a wire cage containing
sodand growing grass, the animals live from seventeen to twenty-
five days, according to the species; they live on an average three
days, and certain species five days, under the same conditions
when decapitated or without food. Respiration and heart action
last about thirty-three hours in deviscerated animals and ab-
dominal segments. When the isolated heart is kept moist in a
moist chamber, or with Ringer’s solution, it pulsates two days, but
it becomes dry and stops beating i in nine hours when exposed to
the air. Post-mortem examinations showed, as a rule, that heart
action outlasts that of respiration.
‘Carlson, A. J. | American Journal of Physiology, 1904, vol. xii, p. 67.
Cardiac and Respiratory Movements in the Grasshopper 623
Effect of Vitiated Air.—In each of these experiments, a single
grasshopper was placed in a hermetically sealed tube ten inches
long and one-half inch in diameter. One grasshopper was con-
fined with each isolated heart, to furnish the vitiated air.
The results from many experiments were, that normal animals
continued to breathe forty-two hours; those of one species (Arphia)
lived four days. Often when placed in fresh air, the hearts con-
tinued their movements nine hours after the breathing had ceased,
and frequently both cardiac and respiratory activities were resumed
in fresh air, after they had stopped for many hours in vitiated air.
The decapitated animal and the isolated heart not protected from
drying, lived as long as in fresh air when not kept moist and often
when the isolated heart was moistened with Ringer’s solution,
after removal from the vitiated air its activity would return and
continue ten hours longer. It was interesting to learn that both
the respiration and heart action could be resumed after they had
ceased for many hours.
Ether.—One end of a glass tube containing the grasshoppers was
placed under water and the other end was joined to a bottle
partly filled with commercial ether. ‘Through this system air was
forced under constant pressure. Both normal and decapitated
animals stopped breathing in this atmosphere of ether in from
five to ten minutes, indicating that the brain exercised no influence
under these conditions. ‘Three hours’ exposure to the ether did
not inhibit the heart action, but after four hours the ether had
injured the tissue so that neither respiration nor heart action
returned when the animals were placed in fresh air. Isolated hearts
ceased beating in one minute in the ether and when exposed longer
than forty minutes, were past recovery in fresh air. Inthe uninjured
animal the ether reaches the heart indirectly during respiration.
When that ceases, it is fed with venous blood, as in the previous
and following experiments, with the addition, however, of some
ether in solution.
Carbon Dioxide-—The carbon dioxide, which was purified by
passing through solutions of sodium carbonate, alkaline and acid
potassium permanganate and alkaline pyrogallic acid, constantly
flowed through the glass tube in which the grasshoppers were
624 Eulalia V. Walling
confined. All connections were tested and made perfectly air
tight. ‘The exit end of the tube dipped under oil.
Observations from many experiments showed that, although
the respiratory movements in the normal animal ceased in from
twenty to sixty seconds, the heart action continued about six hours
longer. Moreover, if the grasshoppers were subjected fifteen hours
to the gas, the respiration would return, provided the animals were
then removed to fresh air; and what is more remarkable, the heart
action, but not the respiration, will be resumed in fresh air after
an exposure to the carbon dioxide for forty-eight hours. In some
cases only a few minutes, in others several hours, and ina few
instances, it was necessary to expose them to fresh air for twenty-
four hours before the heart resumed its rhythm. Isolated hearts
directly exposed to the gas unlike those in the uninjured body cease
their activity in from twenty to sixty seconds and will, like those in
the body, resume their rhythm in fresh air after an exposure of
forty-eight hours to the carbon dioxide gas. ‘The gas inhibits the
respiratory movements and those of the isolated heart in exactly
the same time. ‘The results indicate that the tissue was not
injured by long exposure to the gas, but probably its oxidative or
metabolic action was inhibited.
Carbon Monoxide.—Carbon monoxide was obtained by heat-
ing a mixture of sulphuric and oxalic acids, and purified by pass-
ing through the same solutions that were employed to purify
the carbon dioxide. ‘The gas passed under a slight pressure con-
stantly through the glass tube containing the grasshoppers, into
a vessel containing a solution of cuprous chloride. Grasshoppers
exposed to this gas behave much the same as do those in car-
bon dioxide. Respiration stops in the same time in both gases,
but animals may recover respiratory movements after an exposure
of thirty hours to carbon monoxide, while they cannot recover if
they remain in carbon dioxide more than fifteen hours. On the
other hand, the heart action in the normal animal and in the iso-
lated hearts, directly exposed to carbon monoxide for forty-eight
hours, will not recover in fresh air, indicating that carbon monoxide
is more damaging to cardiac activity than 1s carbon dioxide.
H ydrogen.—Hydrogen obtained by the interaction of HCl on
Cardiac and Respiratory Movements 1n the Grasshopper 625
zinc, was passed through solutions of sodium carbonate, sodium
hydroxide, acid and alkaline potassium permanganate and alkaline
pyrogallol and further through sulphuric acid when dry hydrogen
wasneeded. Under slight pressure the hydrogen passed constantly
through the glass tube containing the grasshoppers. ‘The open
end of the tube was drawn out into a long point that dipped under
oil. The connections were all sealed, tested, and made perfectly
air tight.
A study of the data obtained from many experiments on the
influence of hydrogen upon the respiratory and heart movements,
reveals interesting results. In some of the experiments the cardiac
and respiratory activity continued five days in an atmosphere of
dry hydrogen. It will be remembered that this 1s as long as these
functions persisted in the grasshoppers kept in fresh air without
food. Theaverage duration of activity, however, 1s not as great in
hydrogen as it is in air, because the animals are often in a comatose
condition. ‘That is, they are very restless and active for a few
minutes, then they become quiet and respiration seems to stop
for from five minutes to two hours. Occasionally an appendage
is moved and then they revive, become active and move about in the
tube, so that periods of activity and lethargy alternate until they die.
Isolated hearts directly exposed to the hydrogen, behave in some-
what the same manner. The first effect is an acceleration of
the heart beat. For example, the heart may contract sixty times
a minute before it is put into the hydrogen, but one hundred times
per minute after one minute exposure, eighty-six per minute after
five minutes, only thirty times per minute after ten minutes, and
in about twenty minutes all contractions have ceased, only to
begin again in from two to four hours. They then continue to beat
in the dry hydrogen for about twenty-seven hours or in moist
hydrogen twenty-three hours. It was interesting to note, that
the hearts exposed to the hydrogen did not become dry although
they were not moistened with Ringer’s solution.
It would appear from these experiments, that the absence of
oxygen is not so injurious to the heart and nerve tissue in the grass-
hopper as might have been inferred from the experimental results
on higher forms.
626 Eulalia T. Walling
Oxygen.—The gas from a cylinder of oxygen was purified by
washing through solutions of sodium carbonate, sodium hydrox-
ide, acid and alkaline potassium permanganate and distilled water,
respectively. ’o obtain dry oxygen, the gas was further washed
through sulphuric acid. As in the other experiments, the gas passed
constantly under slight pressure through the glass tubes contain-
ing the grasshoppers, and the free end of the tube dipped under
oil.
An inspection of the many results secured from the influence of
oxygen upon the cardiac and respiratory movements, disclosed the
fact that these functions persist longer in animals kept in that gas
than when living in fresh air without food. In some normal grass-
hoppers, these functions continued eighty-nine hours, while in fresh
air, the same species lived but seventy-two hours. ‘There was not so
much difference in the effects of dry and moist oxygen as there was
between the dry and moist hydrogen.
Isolated hearts, moistened with Ringer’s solution when first
placed in oxygen, continued to pulsate actively for about thirty
hours. ‘They could not be kept moist as could those which were
placed in moist chambers in fresh air, and which contracted on an
average forty-eight hours; therefore, a direct comparison between
these and those subjected to oxygen cannot be made.
While the grasshoppers are inactive, breathing faintly and peri-
odically in hydrogen, they are active, moving about in the tube
frequently, in an atmosphere of oxygen, but at times do not breathe
at all, being in what appears to be an apneeic state. Possibly if
they had had food, they would have lived much longer than did
those in air.
High Temperature.—In these experiments the grasshoppers -
and isolated hearts moistened with Ringer’s solution were observed
through the glass door of a digester in which the temperature could
be carefully regulated and observed.
In one series of experiments, the temperature was gradually
raised during three hours from 25° C. to 68° C. It was a note-
worthy fact that as the temperature was raised, the first effect upon
the isolated heart was to retard its contractions for a few minutes.
A heart that contracted rhythmically sixty times a minute at 25° C.,
Cardiac and Respiratory Movements in the Grasshopper 627
contracted only forty-eight times a minute at 30° C. and then the
action increased rapidly, so that at 48° C., it was beating at more
than twice its normal rate. In Coloptena femur rubrum the pul-
sations were increased to about two hundred per minute. As the
temperature was raised beyond 48° C., the contractions became
slower and stopped entirely in temperatures between 62° C. and
68°C. On an average the respiratory rate increased from 48
per minute gradually to 66 per minute at 41° C., then the rate de-
creased to 60 at 43° C. It then increased to 112 at 51° C., from
there on decreased and ceased at 57° C.
In another set of experiments, the isolated heart was removed
froma temperature of 25° C., in which the contractions were on an
average fifty-six per minute, and subjected directly to a tempera-
ture of 57°C. Here the heart’s action was instantly increased to
one hundred and fifty contractions per minute. During the next
hour the temperature was gradually raised from 57° C. to 68°C.,
with the result that the rhythmical contractions decreased and
ceased entirely at 68° C., but when the heart was then placed
in a temperature of 25° C., its action was again resumed.
In a third set of experiments, normal grasshoppers, abdominal
segments and isolated hearts were subjected to a temperature of
14° C., that was gradually raised during four hours to 63° C., and
then lowered again to 14° C., or room temperature. Ina tempera-
ture of 14° C., there were, in the normal insect, on an average forty
respiratory contractions per minute and in the abdominal seg-
ments twenty-five contractions per minute. In-that time the 1so-
lated heart contracted about thirty-six times. As the temperature
was raised from 14° C. to 54° C., the respiratory movements in the
normal animal, and in the abdominal segments increased from 40
to 110 and 50 contractions per minute, respectively, then decreased
and ceased at 59° C., while the isolated heart increased its action
from 36 beats in a temperature of 14° C. to 160 beats in a tempera-
ture of 48° C., then gradually slowed, and stopped entirely at
62° C., one-half hour after the respiratory movements had ceased.
The specimens were then subjected to a gradually but rather rap-
idly falling temperature, but the cardiac and respiratory activities
were not again resumed.
628 Eulalia V. Walling
Low Temperatures.—When the effect of a gradually lowered
temperature was to be studied, the grasshoppers were placed with
a thermometer in a test tube around which the temperature was
gradually lowered, first by placing the tube with its contents near
a large dish containing a mixture of chopped 1 ice and salt, and, as
the temperature oa lower in the tube, it was gradually sub-
merged in the salt and ice mixture, until a temperature of —17° C.
was obtained. For studying the influence of still lower tempera-
tures the tubes were placed in a liquid air chamber, where a
temperature of —100° C. was obtained.
The following are the results of many experiments upon normal
and decapitated grasshoppers and upon abdominal segments and
isolated hearts.
The respiratory and heart action becomes slower and fainter as
the temperature falls from 20° C. to 5° C. At this temperature, the
animals are usually inactive and breathe faintly only five or six
times a minute, if at all, while the isolated heart ceases to contract.
The respiratory movements in the normal animal, however, may
not stop until the temperature falls to o° C. Normal animals and
isolated hearts kept in a temperature of —10° C. for about one hour
will recover their activity if the temperature is very gradually
raised to that of the room. Several nymphs, young immature
grasshoppers, were kept for one-half hour in a temperature of —30°
C., then the temperature surrounding them was gradually raised
during the following eighteen hours to that of the room. Respira-
tion did not return but the heart action did. Probably those that
were subjected to —30° C., or even —100° C. would have recovered
if the temperature had been more gradually raised during several
days to that of the room.
SUMMARY
Normal grasshoppers can live under most favorable conditions
in the laboratory twenty-five days, and under the same conditions
but without food three to five days; whereas the isolated heart beats
nine hours if unprotected, but if kept moist with Ringer’s solution
it will beat for two days.
Cardiac and Respiratory Movements in the Grasshopper 629
‘ In vitiated air in sealed tubes the normal grasshopper lives
two days, but the heart beats as long as it does in fresh air only
more slowly and at irregular intervals.
In carbon dioxide the heart and respiratory movements cease
within a minute and may remain inactive as long as two days,
and then when the specimens are placed in fresh air for about two
hours the movements are again resumed. Carbon dioxide may
act upon the cardiac cells and nerve centers as a reaction product,
inhibiting enzymes or metabolic activity in the cells. Carbon
monoxide is slightly more toxic than carbon dioxide in its effect
upon the heart and respiratory centers.
Hydrogen has a most remarkable effect upon the respiratory
and cardiac action. For the first few hours it has an inhibitory
influence upon the heart and respiratory movements both in the
normal and isolated states. After the comatose condition has
lasted about four hours, the respiratory and cardiac actions revive,
and in some cases the movements continue for four days. The
contractions are conspicuous in consisting of long intervals of cessa-
tion alternating with shorter ones of activity. In moist hydrogen
the activities cease sooner than in dry hydrogen. If the energy
of the nerve, heart and muscle cells depends upon oxidative pro-
cesses, then the necessary oxygen for the four days activity must
have been supplied by an amount stored in some manner in the
cells. .
In normal grasshoppers and isolated hearts the contractions of
the heart continued in some cases longer in oxygen than in air.
It was observed, however, that periods of inactivity alternated with
periods of strong respiratory contractions.
It is interesting to note that the cardiac cells can contract in a
temperature as high as 66°C., and cease at about 0° C. More-
over, that they can withstand for at least half an hour, a tempera-
ture of —30°C., and probably if the thawing was carried on very
slowly, they could withstand a temperature of —100° c.
Whether the heart tissue in the isolated heart is capable of con-
traction independent of the influence of intrinsic nerve cells, I
have as yet not been able fully to determine, but hope soon to
obtain definite proofs in regard to this question.
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