BIOLOGICAL BULLETIN

OF THE

fIDarine Biological laboratory

WOODS HOLL, MASS.

Editorial Staff.

E. G. CON KLIN The University of Pennsylvania.
JACQUES LOEB The University of California.
T. H. MORGAN Bryn Mawr College.

W M. WHEELER American Museum of Natura/
History.

C. O. WHITMAN The University of Chicago.
E. B. WILSON Columbia University.

FRANK R. LILLIE The University of Chicago,

VOLUME V

WOODS HOLL, MASS.
JUNE, 1903, TO NOVEMBER, 1903.

BIOLOGICAL BULLETIN

OF THE

flDarine Biological Xaboratcr\>

WOODS HOLL, MASS.

Editorial Staff,

E. G. CONKLIN The University of Pennsylvania.
JACQUES LOEB The University of California.
T. H. MORGAN Bryn Mawr College.
W. M. WHEELER The University of Texas.
C. O. WHITMAN The University of Chicago.
E. B. WILSON Columbia University.

FRANK R. LILLIE The University of Chicago

VOLUME VI.

WOODS HOLL, MASS.
DECEMBER, 1903, TO MAY, 1904.

PRESS of

HE NEW ERA PRINTING COMPANV
LANCASTER, PA.

CONTENTS OF VOL VI.

No. i. DECEMBER, 1903

PAGE.

C. M. CHILD : Form Regulation in Cerianthits, II. The Effect

of Position, Size and Other Factors upon Regeneration, II.

Discussion of Results i

CHARLES ZELENY : A Study of the Rate of Regeneration of the

Arms in tJie Brittle-star, Ophioglypha lacertosa 12

D. B. CASTEEL AND E. F. PHILLIPS : Comparative Variability of

Drones and Workers of the Honey Bee 1 8

HENRY H. LANE : The Ovarian Structures of the Viviparous Blind

Fishes, Luc if it ga and Stygicola 3^

No. 2. JANUARY, 1904

C. M. CHILD : Form Regulation in Cerianthus, III. The Initia-

v
tion of Regeneration 55

E. A. ANDREWS: An Aberrant Limb in a Cray-fish 75

ROBERT M. YERKES : The Reaction-time of Gonionemus murbachii

to Electric and Photic Stimuli 84

No. 3. FEBRUARY, 1904

E. G . SPAULDING : The Special Physics of Segmentation as shown
by the Synthesis, from the Standpoint of Universally Valid
Dynamic Principles, of all the Artificial Parthenogenetic

Methods 97

N. YATSU : Experiments on the Development of Egg Fragments in

Cerebratulus 123

THOS. H. MONTGOMERY, JR.: Some Observations and Considera-
tions upon the Maturation Phenomena of the Germ Cells... 137

No. 4. MARCH, 1904

T. H. MORGAN: Notes on Regeneration 159

E. H. HARPER: Notes on Regulation in Sty/aria lacustris 173

CARL HARTMANN : Variability in the Number of Teeth on the
C/aws of Spiders, showing their Unreliability for Systematic

Description 19*

E. P. LYON : A Biological Examination of Distilled Water 198

iii

IV CONTENTS

PAGE.

No. 5. APRIL, 1904

HARRY BEAL TORREY : On the Habits and Reactions of Sagartia

davisi 203

FRANK E. LUTZ : Variation in Bees 217

ALBERT M. REESE: The Sexual Elements of the Giant Salamander,

Crypto 1) ranch us allegheniensis 220 v

E. G. SPAULDING : The Rhythm of Immunity and Susceptibility of
Fertilized Sea-urchin Eggs to Ether, to HCl, and to Some
Salts 224

GEORGE T. HARGITT : Budding Tentacles of Gonionemus 241

No. 6. MAY, 1904

WILLIAM MORTON WHEELER: A Crustacean-eating Ant (Lepto-

genys elongata) Buckley 251

WM. S. MARSHALL: The Marching of the Larva of the Maia

Moth, Hemileiica maia 260

C. M. CHILD: Form- Regulation in Cerianthiis, IV. 7 he Role

of Water-Pressure in Regeneration 266

HELEN DEAN KING : Notes on Regeneration in Tubularia crocea.. 287

Research Seminar of the Marine Biological

Laboratory for the Year ipoj 307

Vol. VI. December, /<?oj. No. i

BIOLOGICAL BULLETIN.

FORM REGULATION IN CERIANTHUS, II.

THE EFFECT OF POSITION, SIZE AND OTHER FACTORS UPON
REGENERATION. ( Continued. )

c. M. CHILD.

II. DISCUSSION OF RESULTS.
Tlic Factor of Position.

In the experiments described above, which were selected from
a large number, the effect of position on regeneration has ap-
peared in every case. Considering first the effect of position on
the rapidity of regeneration, we 'find that in each series the
rapidity of regeneration is dependent on the position which the
piece occupied in the parent body, a decrease in the rapidity of
regeneration occurring with increasing distance from the oral
end. This relation holds for the aboral ends of pieces as well
as for the oral ends, though the regenerative phenomena at the
aboral ends are in most cases less sharply defined than at the
oral ends. Further analysis is possible in such series as 54 and
55. The oral ends of the two sets of pieces 54*4 and $$A are
situated at approximately the same level of the body, but the
aboral ends of the pieces 54/4 are much nearer the aboral end of
the parent body than those of 55^4. The data given above for
this series show very clearly that the rapidity of regeneration at
the oral ends of both sets of pieces was equal, except in the final
stages, while at the aboral ends it was much greater in the shorter
pieces, i. e., those in which the aboral cut surface was near the
oral end of the parent body. From these experiments we may
conclude that the two ends of a piece regenerate independently of
each other, at least to a certain extent, the result depending in
each case on the level which a particular cut surface occupied in
the parent body.

Near the aboral end the regenerative capacity is reduced nearly

2 C. M. CHILD.

or quite to zero, cut surfaces at this level showing no regeneration
beyond the union of ectoderm and entoderm over the cut mus-
cular layer.

The influence of temperature in determining the level at which
the regenerative power is reduced to zero will be discussed in
another section. As regards the amount of regeneration, the
effect of position is similar to its effect on rapidity. Not only is
there a decrease in the rapidity of regeneration toward the aboral
ends, but the total amount of oral or aboral regeneration also de-
creases in the same manner. In certain cases, as in Series 54,
55 and 35, the difference in size of pieces may counteract the
effect of position in later stages of regeneration, so that short
pieces show slightly less oral regeneration than long pieces with
oral ends at the same level (Series 54^ and 55/2), or the amount
of oral regeneration on long aboral pieces may equal that on
much shorter pieces from a region nearer the oral end (Series 35,
A and B). But aside from exceptions of this kind the rapidity
and the amount of regeneration depend upon the level in the
parent body from which the cut surface is taken, both being
greatest at the oral end and decreasing aborally.

The existence of a difference in regenerative power at different
levels of the body being established, the question as to its nature
and significance next requires consideration. I believe that it may
be due in part to a difference in the general reactive capacity of
the tissues in the different regions, i. e., to a decrease in the re-
active power of the tissues with increasing distance from the oral
end. That such a difference does exist is indicated by various
facts in the normal anatomy and physiology of the animal.
We must consider first the anatomical features which bear upon
this point. There is a marked decrease in the thickness of the
body-wall and especially of the muscular layer, toward the aboral
end ; the number of mesenteries also decreases toward the aboral
end, until only a single pair remains ; all new mesenteries appear
first at the oral end and extend gradually aborally, thus indicating
that growth in circumference begins orally. The physiological
differences of different regions which have been noted are : the
greater sensitiveness of the oral region to tactile and other stim-
uli, and the greater contractility of the muscles of this region
when stimulated.

FORM REGULATION IN CERIANTHUS. 3

Since I am convinced that the power of regeneration is due,
not to any special regulatory mechanism, but to those properties
of organized matter which cause "normal" or "typical" growth
and differentiation, I believe that the differences in regenerative
power and the differences in normal anatomical and physiological
reactive capacity must all depend, at least in some degree, on the
general reactive capacity of the tissues.

The differences in reactive capacity in different regions may be
due to differences in the intensity of metabolic change, or they
may depend upon other conditions. It is of interest to note in
this connection that the differentiation and concentration of ner-
vous tissue is greatest about the oral end in the actinians. It is
at least not impossible that this fact is correlated in some manner
with those above mentioned. For the present, however, the pos-
sibility of such a correlation is merely suggested as the data are
still insufficient for positive conclusions.

There is also a possibility that the difference in the rapidity
and amount of regeneration at different levels may be due in part
to differences in internal pressure resulting from circulatory cur-
rents in the enteron. Discussion of this phase of the problem is,
however, reserved for a future time.

The Factor of Size.

My observations along this line are to a large extent merely
incidental, since, owing to the relatively slight influence of this
factor, its effects were not clearly recognized until it was too late
to complete the series of experiments necessary for further study.
Certain conclusions may, however, be drawn from my work and
these are briefly mentioned here.

It is evident from Series 54 and 55 that great differences in size
of pieces do not affect the rapidity of regeneration in earlier
stages. The pieces 54^ and 55^4, although widely different in
size, regenerate orally with equal rapidity except in the final
stages. Aborally the smaller piece regenerates more rapidly than
the larger because its aboral end lies in a region of greater cellu-
lar activity. As regards the total amount of regeneration at both
ends the small piece exceeds the laige piece, because of its posi-
tion in the parent body. The only effect of size was noted

4 C. M. CHILD.

at the final stage of the experiment when it was seen that
the smaller pieces were falling behind the larger pieces with re-
spect to size of the regenerated structures. In Series 35 the
possible effect of size upon the final result was noted, but here
also there was no visible effect during the earlier stages.

The results afforded by these two series are confirmed by a
large number of experiments. In no case, when pieces were
above a certain minimal size, could any effect of size upon rapid-
ity of regeneration be observed during the earlier stages. In the
later stages it was found that regeneration became less rapid and
ceased in the smaller pieces earlier than in the larger. Even in
this respect differences were slight in pieces of widely different
size.

The amount of regeneration is then not proportional to the
size of the piece. As regards both oral and aboral cut surfaces
smaller pieces show a relatively much greater amount of regen-
eration than larger pieces ending at the same level. In a piece
one tenth the length of the body the tentacles regenerate at first
with the same rapidity as in a piece nine times as long. Only
after the tentacles are well formed and several millimeters in
length does a difference appear. In many pieces so short that
they appeared to consist only of the regenerated disc and ten-
tacles these organs were of the same size up to a late stage as in
pieces many times as large.

As regards the nature of the effect of size in regeneration in
CeriantJms and in many other forms a few points which have
suggested themselves to me may be discussed in the hope of aid-
ing in the analysis of this phase of the problem. It may be
stated as a general rule that as regeneration advances the stim-
ulus to regeneration diminishes. We may suppose that a certain
amount of material or energy is necessary for the production of
regenerated structures of a certain size. This energy is derived
from the substance of the body in cases like the present where
no food is supplied. The smaller pieces possess of course a
smaller amount of material or energy available for regeneration.
This is sufficient, however, for the earlier stages, but as the
amount diminishes it is probably given up less readily by the
other tissues. The well-known regulative power of animal tis-

FORM REGULATION IN CERIANTHUS. 5

sues during starvation supports this view. Now as regeneration
advances it is clear that in the smaller pieces the point where the
stimulus to regeneration is no longer sufficient to cause the
transfer of material or energy from the other tissues, would be
reached earlier than in larger where the total supply of available
material is much greater. Consequently regeneration in the
smaller piece is retarded earlier than in the larger, even though
the stimulus may be as great. The small piece is not neces-
sarily exhausted, for if a new cut is made it may regenerate
again, but the reserve supplies are held so closely by the other
tissues that the decreasing regenerative stimulus is insufficient to
render them available. A new cut surface means an increased
stimulus, and under these conditions material which was not
available during the later stages of the preceding regeneration
may now be made available by the increased stimulus, though in
this second regeneration the supply will run out sooner than
before.

In the larger pieces of Ccriant/nis it is probable that the stim-
ulus to regeneration ceases, or reaches the level of normal rep-
arative stimuli, before a shortage of material occurs. A con-
venient designation for the condition of the smaller pieces is
" relative exhaustion." They are not absolutely exhausted, since
they may regenerate further under sufficiently powerful stimulus ;
they are, however, exhausted so far as the stimulus to which
they are subjected is concerned. There is nothing new in the
idea of relative exhaustion ; it is a well-known phenomenon in
biology and its role in regulative phenomena is undoubtedly im-
portant.

It has been impossible to determine with any degree of exact-
ness the minimal size of pieces in which regeneration is possible.
In small pieces the inrolling of the cut margins becomes so irregu-
lar that the piece often takes a form in which normal regenera-
tion is impossible simply because of the relative position of parts.
Moreover, since the regenerative power is different at different
levels the minimal size of pieces differs according to the region of
the body from which they are taken. Short pieces in the
cesophageal region are not available for the aboral cut margin
of the oesophagus unites with the aboral cut margin of the body-

6 C. M. CHILD.

wall, thus producing a condition in which the oesophagus opens
aborally as well as orally to the exterior and the enteron is com-
pletely closed. As will be shown at another time, regeneration
in such pieces is always slight.

The smallest pieces in which normal regeneration has been ob-
served were about one twentieth of the body-length and from
the region just posterior to the oesophagus. When the regenera-
tion was complete these pieces appeared to consist of little but
tentacles and disc, which were only slightly smaller than in
pieces many times as large. My observations prove that typical
regeneration is possible in very small portions of the body, pro-
vided they represent complete transverse or oblique sections of
the body ; pieces in which the transverse continuity is inter-
rupted roll up in such manner that typical regeneration can
never occur.

The Effect of Temperature on Regeneration.

No special experiments in relation to temperature were per-
formed, but my work upon Cerianthus extended from September,
1902, to February, 1903. In September the temperature of the
water was very high ; during October, as the weather became
cooler, it fell gradually and continued to do so as long as ob-
served, i. e.,, until February. A comparison of similar series of
pieces begun at different times during this period affords an inter-
esting illustration of the effect of temperature. The various
series for which data are given in this paper will serve for this
purpose.]

Series 22 was begun September 24; Series 35, October 20;
Series 45, November 7 ; Series 54, 55 and 56, December 15.
If the series be considered in this order a considerable decrease in
the rapidity of regeneration is noticeable. In Series 22, Septem-
ber 24, the marginal tentacles first appeared in the pieces A four
days after section. In Series 54, 55, 56, December 15, the mar-
ginal tentacles in the pieces A from about the same level of the
body as Series 22A appeared eleven days after section. Between
these two extremes lie the other series, and in every case the
later in the season the series was begun the less rapid the regen-
eration. It is not necessary to go over the data in detail to

FORM REGULATION IN CERIANTHUS. 7

illustrate this point. In general it may be stated that the rapidity
of regeneration in winter during the earlier stages is only about
one third of the rapidity in summer.

The amount of regeneration as well as the rapidity also differs
with the season. During September and October regenerated
tentacles attained a length about twice as great as in similar pieces
during January and February.

The length of time during which increase of size in regenerated
structures, e. g., tentacles, continues does not differ widely at high
and low temperatures, and since regeneration is less rapid it is
evident that the total amount must decrease with the temperature.

The most interesting point in connection with the effect of
temperature on regeneration is the increase in size of the region
at the aboral end in which regeneration does not occur. During
the earlier months of my work the only pieces which failed to
regenerate were the aboral tips comprising about one eighth of
the body-length. Later, as illustrated in Series 45, an aboral
piece about one fifth of the body-length failed to regenerate. In
December, January and February pieces comprising more than
the aboral third failed to regenerate or showed only slight traces
of regeneration, as in Series $6B. Thus the portion of the body
incapable of regeneration increases as temperature decreases.

In general, the effect of temperature upon regeneration in
Ccriantlms is what might be expected, since the activities of liv-
ing substance in general increase and decrease with the tempera-
ture. At high temperatures it is only the extreme aboral end
which cannot regenerate typically. From this " inactive " region
regenerative power increases toward the oral end. As the tem-
perature falls the limit of " inactivity " must advance toward the
oral end, and more and more of the body be included in it.

The Number of Regenerated Tentacles.

It was determined that the number of tentacles regenerated is
always less than the original number, and furthermore, that it
decreases as the distance between the oral end of the piece and
the oral end of the body increases.

It is difficult to determine with certainty the number of ten-
tacles in living specimens of C. solitarius, but in fixed material

8 C. M. CHILD.

they can readily be counted. A considerable amount of material
fixed for this purpose was lost by accident when too late to re-
place it, so that exact data cannot be given at present. The
decrease in the number of tentacles with increasing distance from
the oral end is, however, always noticeable even on cursory ex-
amination of the pieces.

Another spieces was also employed for this purpose, viz : the
small, whitish, undetermined species mentioned in the preceding
paper. In this form the tentacles are much less numerous than
in C. solitarius, and can usually be counted without difficulty in
the living specimen. This species differs somewhat from C. soli-
tarins in the extent of regenerative capacity, but the course of
regeneration is the same, and since the structure is in general
similar. to that of the other members of the family, the results
obtained from this species may be accepted as typical. In all
cases the regenerated tentacles were counted only after they had
attained a length of several millimeters ; time being thus afforded
for the establishment of the small tentacles in the growing region,
which sometimes regenerate somewhat later than the others.
Only marginal tentacles were counted since the labial tentacles
are fewer in number than the intermesenterial chambers and re-
generate less regularly than the marginal tentacles. The follow-
ing series are given as examples :

Series 2, October 7. Original number of marginal tentacles
2 1 . After removal of disc and tentacles the

D

B

E

body was cut into five pieces, A, B, C, D, E
(Fig. 8). Number of marginal tentacles re-
generated A, O, died ; B, 15 ; C, 14 ; D, 13 ;
E, o, did not regenerate.

Scries 5, October ig. Original number of
marginal tentacles 19. After removal of disc

and tentacles body cut into five pieces, A. B.

FIG. 9.
C, D, E (Fig. 9). Number of marginal ten-

FIG. 8. tacles regenerated : A, o, died ; B, 17 ; C, I 5 ; D,

13 ; E, o, did not regenerate.

These two series are sufficient to show that the number of ten-
tacles regenerated depends on position and has no relation to size
of the piece. In both series the piece B is only half the size of
C, yet it regenerates a larger number of tentacles.

FORM REGULATION IN CERIANTHUS. 9

These relations hold for all cases recorded, and, since the mar-
ginal tentacles correspond to the intermesenterial chambers, they
are exactly what might be expected. The number of marginal
tentacles which regenerate on a given piece is determined by the
number of intermesenterial chambers which it possesses, or the
number which it acquires by regeneration of mesenteries. The
arrangement of mesenteries in Ceriantlins was described in the
preceding paper. All mesenteries appear first at the oral end
and extend gradually in the aboral direction. In general, the
younger a mesentery the' less its length. Consequently as we
pass aborally the number of mesenteries actually present de-
creases. After section at any level where regeneration is possible
a powerful stimulus to growth exists ; the mesenteries which
extend into the piece remain and become united with the new
oesophagus and others regenerate. It appears that the power
to regenerate a given mesentery extends for a certain distance
aboral to the end of that mesentery and since the mesenteries
terminate at all levels we may now conclude that the " determi-
nation " of the body- wall for the regeneration of mesenteries shows
a similar arrangement. Thus the youngest, shortest mesenteries
can be regenerated only within a very short distance from the
oral end, the next older and longer within a somewhat greater
distance, and so on. The experimental results confirm and es-
tablish this conclusion in every case. The addition of new
material to the body of CeriantJius in producing increase in cir-
cumference and number of tentacles and mesenteries may be
conceived as a wedge of material which enters the body-wall at
the oral end and continually forces its way aborally, thus sepa-
rating the older parts of the body more and more widely. This
conception of the method of growth in the transverse direction
agrees with the view expressed above that the general reactive
capacity of the tissues is greatest at the oral end. The stimulus
to the formation of new mesenteries always becomes effective first
at the oral end and from here gradually extends aborally until it
is insufficient under ordinary conditions to give rise to a mesen-
tery. Even at this level, however, a mesentery may be formed
if the stimulus increases in intensity, or if the tissue becomes
more susceptible to it. The resulting conditions give rise to rapid

IO C. M. CHILD.

growth and we may suppose that formative stimuli which were
insufficient to produce visible results so long as the piece
formed an integral part of the parent-body, either become more
powerful after artificial section or else that the new embryonic
tissue becomes more susceptible.

A comparative account of the factors of position, size and tem-
perature in C. solitarius and the other species studied is rendered
unnecessary by the close agreement between all four species.
Similar results were obtained in all cases. The only points worth
noting here are slight differences in the degree of regenerative
power. Ceriantlius nicinbranaccus regenerates less rapidly than
the other species. In the small whitish undetermined species the
aboral region in which typical regeneration does not occur is
relatively much longer than in C. solitaries at the same tempera-
tures. During October only about the aboral fifth or sixth of
the body is incapable of regeneration or regenerates incompletely
in C. solitarius, while in the other species a cut surface only a
short distance aboral to the middle of the body is either incapable
of regeneration or regenerates incompletely. This species is much
less common than C. solitaries and was only rarely obtained ; no
opportunity offered of determining its regenerative power during
the colder months. Presumably, however, regeneration would
have failed to occur except in the region near the oral end.

One important result of this study may be summed up in the
statement that the rapidity and the amount of regeneration in
pieces from the body of Ccriantlms depend primarily on the pre-
vious relations of pieces to the parent-body, i. t\, on the position
and consequent functional condition. This fact is of importance
as indicating that the phenomena of regeneration are due not to
any special regulatory mechanism which bring about return to a
" normal" or "typical " form, but to the same properties which
cause growth and differentiation under normal conditions.

SUMMARY.

i. The power of regeneration from cut surfaces in Ccriantlms
is greatest at the oral end and decreases aborally, becoming null
a certain distance from the aboral end. This decrease of regen-

FORM REGULATION IN CERIANTHUS. I I

erative power is shown by differences both in rapidity and in
amount of regeneration.

2. The size of the piece does not influence the rapidity or the
amount of regeneration, except in the latest stages. Since the
regenerative power is different at different levels, the minimal size
of a piece capable of regeneration also differs at different levels,
but inversely as the regenerative power.

3. Rapidity and amount of regeneration increase and decrease
with the temperature.

4. The assumption of a special regulatory mechanism is not
necessary for the explanation of form regulation in Cerianthus.

HULL ZOOLOGICAL LABORATORY,

UiNIVERSlTY OF CHICAGO, July, 1903.

A STUDY OF THE RATE OF REGENERATION OF

THE ARMS IN THE BRITTLE-STAR, OPHIO-

GLYPHA LACERTOSA.

CHARLES ZELENY.

Two interesting internal factors regulating the rate of regenera-
tion of the arms in the brittle-star, Ophioglypha lacertosa, were
discovered in the course of a study of the problem at the Naples
Zoological Station during the past winter (190203). As some
months must elapse before a full discussion of the results can be
published it has been thought advisable to give the general data
in a preliminary paper. This seems especially desirable at the
present time because of the high interest taken in the experi-
mental evidences of a far-reaching correlation between the parts
of the individual in both animals and plants.

The experiments to be described give data which shoiv that tJic
rate of regeneration of the an/is varies on the one handivith the
size of the animal and on the other with the number of removed
arms. The first-mentioned correlation gives a maximum rate of
regeneration for the medium sized individuals with a pronounced
decrease for the smaller as well as for the larger ones. The second
correlation, with one exception to be mentioned, gives us an increase
in the rate of regeneration of an arm as we pass from the cases
with a smaller to those with a greater number of removed arms.
The series with all five arms missing is excepted in the statement
because the animals in this lot in every instance died or showed
evidences of decay before the completion of the experiment.

Method. Forty-five perfect specimens were divided into five
equal groups of nine each, care being taken to distribute them in
such a way as to make the sets approximately equivalent as re-
gards size of individuals. The operation consisted in the removal
of one or more arms by a transverse cut at the disk level. In
the first series one arm was removed, in the second two contiguous
arms, in the third three contiguous arms, in the fourth four, and
in the fifth five arms. The animals were kept in ten " battery "
jars, two for each series, and were not fed during the whole period

12

REGENERATION OF ARMS IN BRITTLE-STAR. 13

of the experiment. Measurements of the lengths of the regener-
ating arms were taken 22, 33 and 46 days after the operation.
As stated above the specimens in the series where all five arms
were removed did not retain their vitality for a sufficient length
of time to allow of comparison with the others and they will
therefore be excluded from the following tabulation.

As the rate of regeneration varies with the size of the animal
as well as with the number of removed arms the proper relations
can best be represented by means of curves. The figure accom-
panying this paper gives the average of Series I. and II. (those
with respectively one and two arms removed) in one curve and
of Series III. and IV. (those with respectively three and four arms
removed) in the other. The same arrangement is followed for
each of the three measurements taken respectively 22 days, 33
days and 46 days after the operation. This brings out the de-
sired relations more clearly than would have been possible if all
the individual data had been included. In the figure the abscissae
give the size of the animal as represented by the disk diameter
in millimeters. The ordinates give the length of the regener-
ating arm or arms, also in millimeters. In the series where more
than one arm was operated on the regeneration length as given
is an average of all the regenerating arms of the individual. As
the individual disk diameters are not exactly equivalent in the
different series it was found convenient in taking the averages for
the combination curves to use arbitrarily disk diameters equal to
whole millimeters as the points for comparison. The curves in
the figure are therefore constructed on this basis. The unbroken
line in each case gives the average of Series I. and II. (one and
two arms removed) and the broken line of Series III. and IV.
(three and four arms removed).

Statement of Data and Discussion. The curves show very
distinctly the correlation between the rate of regeneration and
the size of the animal on the one hand and the number of re-
moved arms on the other.

I. Taking up first the size correlation we find that, starting
with the smaller individuals, as we advance toward the larger
ones there is a general increase up to a maximum at a diameter
of i 2 to 15 mm. This is most striking in the two later meas-

CHARLES ZELENY.

10

i .

12

13 14 15 = 16 17 18 19

6

I 5

rt

T3

D 4-

S 3

m

i-n

ffi-I

i-n

urn
i-n

I-E

i-n

7 9 10 ii 12 13 14 15 16 17 18 19

Disk diameters in millimeters >

FIG. i. The abscissae, represent the disk diameters in millimeters and the ordinates
the regenerated arm lengths, also in millimeters. The unbroken line gives the aver-
age of Series I. -II. (the specimens with one and two arms removed). The broken
line gives the average of Series III. -IV. (the specimens with three and four arms re-
moved). The uppermost curves show the conditions 22 days, the middle curves 33
days, and the lowermost curves 46 days after the operation.

^^HS REGENERATION OF ARMS IN BRITTLE-STAR. I 5

urements taken 33 days and 46 days after the operation. Thus in
the 3 3 -day measurement for Series I. and II. the regenerated
length increases from 1.07 mm. for a disk diameter of 7 mm. to
a maximum of 2.37 mm. for a disk diameter of 14 mm. and then
goes down to .21 mm. for a iQ-mm. diameter. Also for the
Series III. and IV. at the same time the length increases from
2.04 mm. at a diameter of 7 mm. to a maximum of 3.45 mm. at a
12-mm. diameter and down again to 1.36 mm. at a diameter of
1 8 mm. The medium sized individuals tints have the maximum
rate of regeneration.

2. More striking still is the very constant difference between
the average of Series I. and II. as compared with Series III. and
IV. This shows a very decided advantage in favor of the ani-
mals with the greater number of removed arms. The difference
is evident in the upper curves of the figure from measurements
taken 22 days after the operation but becomes more striking in
the 33-day and 46-day curves. For example, in the 33-day
curve for a 1 2-mm. diameter (the diameter at which we have
the maximum rate of regeneration of Series III. and IV.) we get
a regenerated length of 2.08 mm. for Series I. -II. and of 3.45
mm. for Series III. -IV., an advantage of 1.37 mm. or 66 per
cent, in favor of the latter. Likewise at a diameter of 14 mm.
(where the Series I.-II. has its maximum regeneration) we get
2.37 mm. for Series I. II. and 2.77 mm. for Series III. -IV., an
advantage of .4mm. or 17 per cent, in favor of Series III. IV. In a
similar manner in the curves obtained from the 46-day measure-
ments we get at a 12 -mm. disk diameter a regenerated length of
2.46 mm. for Series I.-II. and 5.42 mm. for Series III. -IV.,
and at a 15 -mm. diameter 3.14 mm. for Series I.-II. and 3.72
mm. for Series 1 1 1. -IV. which represents an advantage for the
group with the greater number of removed arms of respectively
2.96 mm. (= 1 20 per cent.) and .58 mm. ( = 18 per cent.) for
the two points named.

We must therefore conclude that when more than one arm is
removed the regenerative energy as expressed in the replacement
of the lost arms is greatly increased. Not only is the total re-
generative energy greater in this case but the energy expressed
in each arm is greater than the total energy when only one is
removed.

I 6 CHARLES ZELENY.

Expressing this in mathematical form, if E l represents the re-
generative energy exhibited in the replacement of the lost arm
when only one is removed, assuming that increase in length is a
measure of such energy, and E n represents the energy exhibited
in regeneration when more than one arm is removed, # being the
number of absent arms, then not only is E > E, but also E ln~>E,

J n~^ \ n' -^ 1

or E n >nE r Therefore when we remove n arms we increase the
total regenerative energy by more than n times the amount ex-
hibited when only one is removed. The force of this statement
is made especially strong when we consider that throughout the
experiments the animals received no food supply whatever.

Expressing the relation in still another way, let us take a
brittle-star with arms A, B, C, D and E, in which a r b v c v d^ and
e l represent the respective lengths these arms will attain after a
definite period of regeneration, supposing that one alone is cut off
in each case. Now let us suppose instead that the first four are
cut off, then after this same period of time we get for the regener-
ated lengths a 4 > a v b 4 > b lt c 4 > c v d > d r Now in the first case
mentioned we cannot assume that the stimulus of removal and
the resultant reaction of regeneration are purely local and con-
cern only the tissues in the immediate vicinity of the cut surface
for we then get into difficulty as soon as we try to explain the
cases where four arms are simultaneously removed. Here we
find we must add a considerable quantity (/- 4 ) to each of the ori-
ginal single regeneration lengths, e. g., <? 4 = ^ -f- ;- 4 . Then a 4 -f b
4- c 4 -(- d 4 = a l + b l -f c\ + d l + R 4 where R 4 (= -V 4 ) represents
the total response of the organism as a whole which must be
added to the local effects of the operation stimulus. If, on the
other hand, we consider the influence of the organism as a whole
on the regeneration of its arms as one of retardation, we must take
the values a 4 , b, c 4 and d 4 as representing most nearly the origi-
nal local stimulus effect. Then without changing the values of
r 4 or R A we may rearrange the formulse, making a l = a 4 r 4 ,
etc., and ^ + b^ -f c l -f </, = ^ 4 + /> 4 -f c 4 + d 4 R 4 .

But whether we consider the influence of the organism as a
whole to be one of acceleration or one of retardation we must
recognize in either case that the regeneration rate is not a matter
which involves only the local conditions at the wounded surface

REGENERATION OF ARMS IN BRITTLE-STAR. I /

as determined by the direct action of the operation. It seems, on
the other hand, to be bound up with intricate reactions affecting
the whole character of the activities and organization of the ani-
mal. A more direct application of the above statements to the
special theories of regeneration would be out of place at the
present time.

We may sum up my results on the rate of regeneration of the
arms of the brittle-star, Ophioglypha lacertosa^ as follows :

1. There is a definite relation between the size (/. c., age (?) )
of the animal and the rate of regeneration of its arms. The maxi-
mum rate is exhibited by individuals of medium size (with a disk
diameter of 12 to 15 mm.). Both the smaller and the larger ones
give a diminishing rate as we go away from this point.

2. The greater the number of removed arms (excepting the
case where all are removed) the greater is the rate of regenera-
tion of each arm.

HULL ZOOLOGICAL LABORATORY,
THE UNIVERSITY OF CHICAGO,
October 12, 1903.

COMPARATIVE VARIABILITY OF DRONES AND
WORKERS OF THE HONEY BEE. 1

D. B. CASTEEL AND E. F. PHILLIPS.

INTRODUCTION.

According to the theory of germinal variation it would be
concluded that the workers of the honey bee, Apis uuilifica,
being produced from fertilized eggs, would show more variation
than would the drones which come from parthenogenetic eggs.
This variation would be manifested by coloration and by relative
size of parts, and it might be expected that a series of measure-
ments made on like parts of drones and workers would show a
smaller degree of variability for drones than for workers. To test
this fact a series of measurements have been made and the results
tabulated.

Owing to the difficulty of measuring the extent of coloration
on the segments of the abdomen this could not well be used for
this work, and so a series of measurements on the wings were
chosen although coloration is practically the only difference
usually observed between the varieties of Apis mcllifica.

The wings are also desirable for other reasons. They are of
classificatory importance in systematic work, do not shrink when
preserved in alcohol and are easily examined with a microscope
by simply clipping off the wing and mounting in alcohol on a
slide. They also give more accurate results since the extent of
coloration would vary according to the retraction of the seg-
ments of the abdomen in preserving and it would be practically
impossible to get the individuals normally extended in all cases.

The reason for taking up this work was rather indirect and
should perhaps be stated since the results throw some light on
a widely separated line of work. Perez 2 (1878) took an Italian
queen fertilized by a French black drone, and after some time
examined 300 drones from this queen. As the queen was pure

1 Contribution from the Zoological Laboratory of the University of Pennsylvania.

2 Perez, J., " Memoire sur la ponte de 1'abeille reine et la theorie de Dzierzon.
Ann. Sci. Nat., 6 Ser., Zool., T. 7, 1878.

18

COMPARATIVE VARIABILITY IN THE HONEY BEE. 1 9

Italian her drones would also be pure Italian, since they are pro-
duced from parthenogenetic eggs, and the fact that she was
mated with a black drone should make no difference. He found,
however, that 149 of the drones did show some markings which
he thought indicated hybridism, and from these observations
rejected the theory of Dzierzon. His results were criticised
severely and all manner of arguments were used against them,
atavism, impurity of the queen and other reasons being given in
explanation. Weighing the arguments of Perez and those pre-
sented in opposition to them, however, would lead one to believe
that Perez had the best of the argument. If then we accept the
theory of Dzierzon, and it is well established, we must account
for the results of Perez.

An examination of a large number of hives has shown us that
the coloration of the drones cannot be used as a test of their
purity, and that, therefore, Perez' work is inaccurate, since he
used this test as the basis of his argument. Drones from an
Italian queen fertilized by an Italian drone show gradations in
amount of coloration of the segments of the abdomen which
would easily lead one to conclude that some of them were not
pure, provided the evidence for their purity was not so strong ;
while at the same time the workers from the same queen show a
uniformity of marking which is very striking. In the face of
these facts it is evident that extent of coloration could not be
used as a basis for investigation in relation to parthenogenetic
development in the case of the bee. It might also be added that
the fact of the irregularity of coloration of the drones is well
known to most bee-keepers, and a number of these men have
stated to us that they do not consider the coloration of the drones
as in any sense a test of purity.

This little investigation led to the conclusion that possibly the
drones showed more variation in other ways than did the work-
ers, and to test this the measurements here recorded were made.

We wish to express our appreciation to Mr. E. L. Pratt, of
Swarthmore, Pa., for material furnished, and especially to Mr. E.
R. Root, of Medina, Ohio, for material and for many courtesies
shown during investigations carried on by one of us in his apiary.

2O D. B. CASTEEL AND E. F. PHILLIPS.

MEASUREMENTS.

For the measurements taken in this work we have chosen
veins and cells which in the bee differ from the typical hymen-
opterous wing and which are to a certain extent typical of the
bee in their direction and extent. In this choice we have fol-
lowed the discussion of the venation of the Hymenoptera of
Corrlstock and Needham. 1 The measurements were : (i) Length
of vein radius (7?) ; (2) diagonal length of cell radius-four (7? 4 ) ;
(3) length of vein media-two (^/,) ; (4) length of medial cross-
vein (;) ; (5) ratio between m and M 2 , and (6) number of hooks
or hamuli on the hind wing. An attempt was made to
measure the angles formed by the union of veins radius-four (7? 4 )
and radius-sector (Rs\ and veins radius-five (7? 5 ) and radius-
sector (RsJ, but on account of the difficulty of getting the exact
angle at which the veins branch these measurements were dis-
continued through fear of inaccuracy. In all cases right wings

c R

FIG. i.

were measured. The measurements were made from camera-
lucida sketches, Leitz ocular 2, objective 3 with lower lens re-
moved and sketch made at table level, this giving a -magnification
of forty-two diameters. In all cases this magnification is retained
in the tables.

The choice of the veins and cells measured perhaps needs
some explanation since each one has certain peculiarities in direc-
tion or extent. It should be stated, however, that we do not
think it makes much difference what veins are chosen for a com-
parison of variations in this case since all our observations show

^omstock, J. H., and Needham, J. G., " The Wings of Insects," Chap. III.
(continued), IX., "The Venation of the Wings of Hymenoptera." Amer. Nat.,
Vol. 32, pp. 413-424, 12 figs., 1898.

COMPARATIVE VARIABILITY IN THE HONEY BEE. 21

about the same degrees of variability of the two sexes. Any
other veins or cells would no doubt show like variations.

Length of Vein R. In the typical hymenopterous wing the
media (M) branches from the vein R + J/ at a 1 point nearer the
base of the wing than in Apis. The length of the vein R + M
would therefore be desirable for measurement, but from the diffi-
culty of getting exact measurements this was discarded, and in its
place we took the measurement from the point where M branches
off from R + M to the point where R divides into R l and Rs or
the length of R, which is therefore shorter than in the typical
hymenopterous wing.

Diagonal Length Cell R 4 . In the typical hymenopterous
wing veins R 4 and R 5 are nearly at right angles to the vein from
which they branch, while in the bee, R 4 is bent out to about
135 and R 5 to 160. This makes the cell R 4 considerably longer,
and the diagonal length varies according as the angle R 4 Rs
varies. The measurements were made from the proximal side
of R 5 Rs to the anterior angle of R 4 J/,.

Length of Veins M, and in. In the bee's wing there is a bend-
ing in of the veins M 4 and M s toward the base of the wing with a
corresponding lengthening and shifting of vein m. This vein
gives a convenient measurement for the relative length of wing
since it varies almost directly as the length increases. J/ 9 was
chosen because it is correlated in its length with ;//, and forms a
convenient relative measurement for the breadth of the wing.

Ratio between m and M. r As stated above, the lengths of m
and M. y are correlated in their variation, so in order to test the
relative variabilities of the two veins in drones and workers, we
computed the ratios between the two M z : m \ : i : x ; x in
every case being carried to two decimal places. From these
computations it was found that the variation of m is in inverse
proportion to that of J/ 9 as will be shown later.

Number of Hooks on Hind Wing. This count was taken to
see whether the hind wing varied as did the fore wing, and the
number of hooks served as a conservative test.

Besides these measurements and calculations we looked for
cases of abnormal wings in which the subcostal (Sc], radius-
two (R. 2 ] and cubital-two (Cu.^) veins might be present, these

22 D. B. CASTEEL AND E. F. PHILLIPS.

being absent normally in the bee, and also for all other cases of
abnormalities in the venation. In none of the wings observed
were S<~, R 2 or Cn. 2 present. The other abnormalities will be

discussed later.

THE CHOICE OF MATERIAL.

The individuals used were not all taken from the same hive,
since observations show that all colonies do not vary to the same
extent, at least in coloration. For this reason it appeared best
to use individuals from different hives and different strains in
order to get a more correct idea of the natural variations. In all
cases individuals were selected at random. The material used

was as follows :

DRONES.

I. Fifty individuals from Medina, Ohio, May 16, 1903.
Hybrids, Italian and black.

II. One hundred individuals from Medina, Ohio, May 23,
1903. Italians (?) from a peculiar strain bred by F. A. Hooper,
Jamaica, very light in color.

III. One hundred individuals from same hive as I. May 25,
1903.

IV. One hundred individuals from Medina, Ohio.

V. Fifty individuals from Medina, Ohio, May 9, 1903.
Italians.

VI. One hundred individuals from Swarthmore, Pa., August
20, 1903. Italians from a peculiar strain bred by E. L. Pratt,
the queen having an entirely yellow abdomen.

WORKERS.

I. Fifty individuals from same hive as Drones V., May 9, 1903.

II. Three hundred and fifty individuals from Philadelphia, Pa.,
August 10, 1903. Italians.

III. One hundred individuals from Philadelphia, Pa., May 15,
1902. Hybrids, Italian and black.

LENGTH OF VEIN R.

The measurement of the length of this vein was found to be
somewhat difficult owing to the hairs covering the angles and to
the difficulty of getting the exact middle of the curve at the place
where R { and ^separate. However, with considerable care and

COMPARATIVE VARIABILITY IN THE HONEY BEE.

the reexamination of cases showing the greatest variation we
think the figures are nearly correct. Since the drones varied over
5 mm. on an average more than the workers and any error in
measurement could scarcely be more than I or 2 mm. we feel
justified in concluding that in this case the drones vary consider-
ably more than the workers. The greatest variations were in
Lot III. of the workers, where the variation was from 32 mm. to
42 mm., and in Lot VI. of the drones, where the variation was
from 35 mm. to 48 mm. Lots I. of the workers and V. of the
drones taken from the same hive at the same time show a range
of variability in the ratio 8: I I.

TABLE I.

VEIN R.

Drones.

Lot.

3

3 1

3 2

33

34

35

36

37

38

39

40

41

42

43

44

45

4 6

47

4 8

Av.

I.

I

I

2

2

3

12

6

12

i

5

3

2

40.16

II.

I

I

I

7

8

22

19

21

ii

6

3

35-99

III.

2

3

I

2

5

13

13

12

10

14

12

3

6

3

I 41.42

IV.

2

2

H

16

23

15

13

8

5

i

i

34.38

V.

I

3

3

5

II

8

7

5

3

3

i

36.92

VI.

3

9

ii

18

H

10

14

8

8

3

i

I

40.42

3

3

16

26

37

49

46

5

42

55

40

37

26

27

23

8

6

4

2

Workers.

I.

2

i

3

4

ii 17

9

3

3746

II.

4

13

21

65

78

70

7i

17

9

2

38. 4 2

III.

I

I

5

ii

10

26

19

12

10

2

3

37-40

I

3

10

27

35

102

114

91

84

J 9

12

2

DIAGONAL MEASUREMENT OF CELL R.

This measurement being taken in one of the most variable
regions of the wing shows a remarkable difference in the range
of variability of the two sexes. The measurement of this distance
was quite easy since the limits are well marked and easily distin-
quishable and in no case do we think there was room for doubt
in the length to more than i mm. This fact taken with the
length makes these measurements a very good test of the relative
variability. In this region of the wing a very large part of the
abnormalities were found, so that we conclude that this portion
shows more variation than any other ; but in spite of this tendency

D. B. CASTEEL AND E. F. PHILLIPS.

to vary, the workers' wings were quite constant. The greatest
range of variation in workers was found to be 13 mm., while the
least range in drones was 19 mm. The fact that the average
length of the cell in drones was between 96 mm. and 102 mm.,
while the average length for workers was about 75 mm., will
however account for part of this greater range since, with a given
range of variability, the greater the length, the less the actual
variation. This, however, does not account for the extreme differ-
ence which we find and in this case again, as in the first set of
measurements, we find a much greater variability in drones than

in workers.

TABLE II.

DIAGONAL CELL J? t .

Drones.

Lot.

75'

So 1

87'

89

90

9 1

92

93

94

95

96

97

98

99

IOO

I.

i

I

2

2

2

2

3

4

6

II.

i

2

6

10

9

6

10

6

s

9

13

III.

I

I

i

3

6

7

IV.

7

6

5

8

5

13

13

ii

8

5

7

V.

i

2

i

3

i

5

4

VI.

I

I

I

i

i

2

12

II

4

6

12

13

I

I

i

2

9

13

18

19

IS

39

34

28

27

40

5

Lot

101

102

103

104

i5

106

107

108

109

no

in

112

"3 1

"5

Average.

I.

2

2

3

3

4

I

5

3

3

I

101.68

II.

9

4

i

i

96.49

III.

7

8

9

9

it

10

5

8

4

6

i

I

I

J

104. 14

IV.

2

4

3

i

i

i

96.15

V.

3

6

6

8

4

2

3

I

lOI.gS

VI.

II

6

8

2

3

2

I

i

I

98.77

34

30

29

24

23

13

14

16

8

7

I

2

I

I

1 No individuals found in columns omitted.

Workers.

Lot.

69

70

7 1

72

73

74

75

76

77

78

79

So

81

82

Average.

I.

I

I

3

6

6

12

7

3

6

3

I

I

76.36

II.

I

10

18

20

42

37

7i

62

38

24

16

4

4

3

75.08

III.

I

i

3

5

8

19

20

12

12

16

2

i

76.24

2

12

22

29

53

57

97

85

56

39

33

7

5

3

LENGTH OF VEIN M z .

This is not a specialized vein, and while it has for its anterior

boundary the edge of the cell R v it shows comparatively little

abnormality. A few cases of extra veins thrown in in the

region of this vein will be discussed later, but it is located out-

COMPARATIVE VARIABILITY IN THE HONEY BEE.

side the portion of greatest abnormality of the wing. The prin-
cipal reason for the measurement of this vein was to get a ratio
with the vein ;//, but as the actual measurements add to the evi-
dence, it seems advisable to give the table. The greatest range
of variability in workers was 9 mm., the least for drones 1 1 mm.,
or, if we drop the two quite abnormal cases in Lot V., 9 mm.
Taking into consideration the relative lengths of the average veins,
34 5 mm. and 45 mm., this makes the greatest variation in workers
about equal to the least variation in drones, while the greatest
range in drones is over twice that of the greatest range in workers.

TABLE III.

VEIN M

Drones.

Lot.

32

33

34

35

36

37

38

39

40

41

42

43

44

I.

3

2

II.

6

8

12

6

21

16

17

III.

I

IV.

I

3

3

15

9

V.

2

4

VI.

I

I

2

i

2

I

6

II

13

II

28

33

33

Lot.

45

46

47

48

49

So

5 1

S*

S3

54

55

56

Average.

I.

2

8

2

,5

15

8

2

2

I

48.82

II.

6

5

2

I

42.^6

III.

3

6

7

17

is

2.S

ii

8

I

3

2

I

49-43

IV.

IS

13

IS

14

6

3

2

I

45-72

V.

8

S

6

10

ii

2

I

I

46.96

VI.

2

3

7

II

14

13

IO

15

13

2

3

48.64

36

32

45

55

5i

58

32

27

16

6

5

i

Workers.

32

33

34

35

36

37

3S

39

40

I.

7

12

19

9

I

2

33-82

II.

8

40

64

134

59

31

10

3

i

34.83

III.

4

IO

42

22

10

I I

I

34.6i

19

62

125

165

70

44

II

3

i

LENGTH OF CROSS- VEIN in.

This vein shows no abnormalities and may therefore be con-
sidered as entirely outside the region so well marked about the
cells R and R 5 , which shows so much abnormal variation. If it
be argued that, since the more anterior veins tend to be abnormal,

26

D. B. CASTEEL AND E. F. PHILLIPS.

they are not therefore good tests of the comparative variability,
then, since vein m is entirely outside this area, this measurement
would serve as an answer to that argument and is in itself a
sufficient test. As in the case of vein M 2 , the measurements of
this vein were made principally for the sake of getting a ratio of
variation between two veins which meet at nearly a right angle,
but as the drones show greater variation than the workers, al-
though not to so great a degree as in some other cases, and as
this is a constant vein, we add the table. The greatest range in
workers is 16 mm., the least in drones 15 mm.; the average for
workers almost 13 mm., the average for drones 20 mm.; the
greatest for drones 23 mm. The average lengths in the two cases
(72 mm. and 97 mm.) reduces this difference about one third, so
that the least variable drones are about as constant as the least
variable workers, but the average range for drones still remains
considerably greater than for workers.

TABLE IV.

VEIN m.

Drones.

Lot.

78

79

80

81

82

83

84

85

86

87

88

89

9 o

9 1

92

93 94

95

96

I.

I

I

I

3

3

I

6

II.

i

4

3

7

12

8

17

8

III.

2

2

IV.

I

i

4

I

2

3

2

ii

II

6

16

5

12

7

8

5

3

V.

2

2

3

4

2

4

VI.

I

I

i

2

2

5

5

6

10

12

14

13

I

i

I

4

2

3

3

2

13

H

8

27

13

28

35

35

41

36

Lot. | 97

98

99

too

101

IO2

103

104

105

106

107

108

109

no

in

112

"3

114

"5

Avg.

I.

I

4

2

7

5

2

3

3

3

4

IOO.O4

II.

14

8

5

6

3

2

2

95-65

III.

4

9

S

16

4

12

8

ii

10

4

2

4

2

2

I

i

I

102.43

IV.

2

88.84

V.

9

13

i

3

2

3

i

i

97.08

VI.

6

7

7

5

2

i

94-63

34

43

20

37

16

20

H

IS

13

8

2

4

2

2

I

i

I

Workers.

63

64

65

66

67

68

69

70

7 1

72

73

74

75

76

77

78

Avg.

I.

I

2

2

4

6

13

7

8

4

2

I

72.30

II.

I

2

3

IS

12

69

35

73

42

43

40

I I

3

i

72.00

III.

i

2

1

10

16

18

27

13

8

2

2

72.43

I

1 2

5

I.I

IS

83

57

104

76

64

5^

15

6

i

COMPARATIVE VARIABILITY IN THE HONEY BEE.

RATIO BETWEEN M, AND ;;/.

Z

To get this ratio the length of the vein m was divided by the
length of the vein M 2 and the division carried to two decimal
points giving a ratio in the form M^.in: : i : x. To get the com-
parative variability of the two sexes the individuals were tabulated
according to the values of x and in this tabulation the values of
x to two decimal points were used, but owing to the length of
such a table we have combined these values and give them ac-
cording to x carried to one decimal point. This is advisable also
for another reason ; owing to the relative smallness of the num-
bers in the first part of the proportion certain columns remained
empty since, for example, no combination of figures between 32
and 57 and between 63 and 1 15 can give a ratio of i : 1.99. In
order then to get a series of numbers which represents what is
no doubt more nearly the true scale of ratios, the first table has
been reduced to a table with x carried to but one decimal point.
In tabulating these ratios the individuals were not grouped ac-
cording to lots as in the other cases since we found practically
little difference between the lots in any one sex. The average
ratio for drones is 1 : 2.06 and for workers i : 2.08 so that no ac-
count need here be taken of the difference in averages since it is
so slight. The range of the values of x for workers is from 1.70
to 2.34 or .64 ; that for drones from 1.68 to 2.90, or, omitting one
wing with a very abnormal ratio, from 1.68 to 2.57 or .89 show-
ing .25 greater range in drones than in workers. It might also
be said, since our long table cannot be used here, that this
greater range is not caused by a few abnormal cases but that the
variations are shown by a gradually decreasing number of indi-
viduals in each value of x in both drones and workers, barring,
of course, certain values of x which as above mentioned are im-
possible with the figures worked upon.

TABLE V.

RATIO BETWEEN M AND m.

Value of j-.

i-7

1.8

1.9

2

2.1

2.2

2-3

,4

-.3

2.6

Drones.

13

51

81

126

8 9

70

36

19

12

3

Workers.

I

II

53

119

217

Si

17

I

28

D. B. CASTEEL AND E. F. PHILLIPS.

The working out of these ratios brought to light another point
which explains this greater range in the values of x in the propor-
tion. It was found that a certain inverse ratio exists between the
lengths of ;;/ and M 2 so that the area of the cell istM 2 bounded
distally and posteriorly by these veins remains more nearly con-
stant for wings of the same area than do the bounding veins.
This does not mean that the area of the cell istM 2 does not vary,
for a little calculation shows that it does vary considerably. The
ratios of the wings for each length of M were gathered together
and an average taken of the ratios in each case, with the result
that we found a relatively constantly decreasing ratio with the
increase in the length of M 2 . The range of these ratios for drones
was from 1.80 to 2.57, omitting again the one abnormal wing
mentioned above, and for workers from 1.77 to 2.21, a difference
in this case of .33 in the ranges of the two sexes. This would
indicate seventy-five per cent, more range of variation for the
drones than for the workers and this difference is directly corre-
lated with the great difference in the size of M 2 found to exist.

TABLE VI.

RATIOS FOR LENGTHS OF VEIN M .

mm.

32

33

34

35

36

37

38

39

40

4i

42

43

44

Drones.
Workers.

2.21

2.19

2.05
2.13

2.06

2.01

1-93

2.57
1.90

2.43

1.86

2-35

1-77

2.25

2.28

2.16

2.14

mm.

45

46

47

48

49

5

51

52

53

54

55

56

A

r.

Drones.
Workers.

2.09

2.O6

2.O2

2.03

1.98

2.OO

1.91

1.88

1.83

1. 80

1.79

1. 80

2.1
2.

D6
38

HOOKS ON THE HIND WING.

The number of hamuli or hooks on the hind wing which are
used to fasten the two wings together during flight were used as a
means of testing the variability of this wing. The examination of
1,000 wings has shown that this is not the most variable feature of
this wing, but that far more variation occurs in the breadth of the
wing and in the angles of the veins which form the cross sup-
ports. As the area of the hind wing increases the increase takes
place principally by a widening of the wing although by no
means entirely by that method. The drones especially show

COMPARATIVE VARIABILITY IN THE HONEY BEE.

2 9

this widened wing, in some cases the width equalling the length/
It may safely be stated, although no measurements were made
on this point, that the area of the back wing is more variable than
that of the fore wing. Since the number of hooks is correlated
with the length of the wing they do not, therefore, show as much
variation as would be found on the other parts of the hind wing.
The least range of variation in number of hooks in workers was
over seven points, the greatest, ten ; the least for drones, nine,
the greatest eighteen, or omitting one very abnormal wing with
but twelve hooks, twelve. The relative amounts of variation are

FIG. 2.

shown far more clearly here by examining the numbers of indi-
viduals in each case which have the same number of hooks. For
the workers the greatest number is 139 individuals which have
21 hooks while for drones the greatest number is 98 with 22
hooks, the average number for drones being a little higher than
for workers. In workers the descent in numbers of individuals
from 21 is far more sudden than that for drones, which is really

TABLE VII.
HOOKS ON HIND WING.

Drones.

12

16

r ?

18

19

20

21

22

23

24

25

26

27

28

29

Avg.

I.

4

4

8

II

9

4

6

i

i

2

21. s6

II.

2

I

12

21

23

2O

17

2

2

20.12

III.

I

7

12

15

28

14

14

6

2

I

22.C9

IV.

4

10

17

22

27

12

2

5

i

20-33

V.

4

2

II

IO

9

7

i

4

I

i

21-54

VI.

I

3

3

7

6

23

23

19

6

6

I

i

22.42

I

2

5

34

54

83

89

98

52

47

18

10

4

i

i

Workers.

I.

6

9

12

ii

6

5

I

21.42

II.

4

13

42

74

94

61

45

ii

4

2

21. 08

III.

2

ii

18

18

33

8

6

4

20-37

6

24

66

101

139

80

57

20

5

2

3O D. B. CASTEEL AND E. F. PHILLIPS.

the true test of the relative variability far more than is the range
of variation.

So far we have discussed the range of variability in each case,
but a far more important test of the comparative variability is the
relative centralization of individuals about the average point in
the table. If, for example, the drones showed exactly the same
range as did the workers, the fact that in every one of the tables
the workers show a greater number of individuals with the average
dimensions, together with a rapidly decreasing number in each
direction, while the drones show a more nearly equal number of
individuals over several dimensions on each side of the average
point, would lead one to conclude that the drones show far more
variability than do the workers. It is not necessary to go over
all the tables to point out this fact, but even a hurried examina-

FIG. 3.

tion will show the facts as above stated. If, for example, the
tables were put in the form of curves of variation the workers
would show a longer and narrower curve in every case than the
drones, and were these curves expressed in the form of mathe-
matical formulae the fact would be brought out still more strongly
that the males in this case show more variation than do the
abortive females.

It may be well here to offer some explanation as to why curves
were not used in this work, since that is the usual method for
work on variation. It was not the purpose of this investigation
to work out the law of variation for the two sexes, but merely to
show which sex did vary to the greater extent. This purpose
has been fulfilled and is shown in the tables. In order to get a
true curve of comparative variability it would be necessary to

COMPARATIVE VARIABILITY IN THE HONEY BEE. 31

measure many times the number of individuals which were used
and to extend the observations over a far greater range of varie-
ties. No curve made with 500 individuals would express the
true law of variation, nor would ten times that number be suffi-
cient, and since the formulation of the law of variation for par-
thenogenetic and fertilized forms for this particular kind of par-
thenogenesis, arrenotoky, is too important a matter to be based
on an inaccurate mathematical formula, it seems better to us to
state simply the fact that greater variation does occur in the males
and leave the formulation of the law to be worked out with a far
greater range of observations and measurements. And then, too,
it is by no means certain that this variation follows any fixed law.
If the variation were caused entirely by germinal variation, or by
any other one factor, then it might be assumed that the law of
this variation could be stated in the form of a mathematical for-
mula, but as will be shown later, it appears probable that a large
part of the greater variability of the drones is due to chance and
is therefore not in accordance with any law. It may be argued
that variation according to chance is but a way of stating our
ignorance of the true law, but if there is a law for this variation it
is certainly very obscure, and the working out of this law would
require an extremely large number of measurements taken from
individuals, each one with its life history known together with a
high degree of mathematical ability in its formulation.

ABNORMAL WINGS.

As noted previously, in all wings examined, record was made
of wings having veins which are not typically found in the bee.
Fig. 4 shows, in dotted lines, where these abnormalities occur
most frequently. It is very difficult to record these irregularities
in any kind of a table, since the irregular veins vary widely in ex-
tent and do not arise at exactly the same place in many cases.
An attempt was made to classify these according to the veins from
which they branch, their extent and direction. Manifestly any
tabulation must be considered as merely a matter of convenience
in examination. In this we have recorded cases where a vein
bends (b in table) from its true course, showing but a tendency
toward abnormality, as well as the well-marked cases. The ex-

D. B. CASTEEL AND E. F. PHILLIPS.

tent of the abnormality is expressed roughly in the terms, very
small (vs), small (s), almost complete (ac) and complete (c).
The letters used to designate these abnormalities are in no way
connected with the naming of the normal veins, but are chosen
merely as a convenient means of marking the irregularities. It
will be understood that it is impossible to draw at all times the
same line between, for example, the terms small and very small,

FIG. 4.

and the table is of no value except to give an idea of the number
of cases of abnormalities.

MALES.

a. 82 b, 32 vs, 109 s, I ac, 2 irregular.

b. 7 s, I double s, 4 ac, 6 c.

c. 3 vs, 18 s, 2 ac, 13 c, 2 irregular.

d. lib, 27 s, I double s, 7 ac, 2 c.

e. 9 s, I ac, 5 c.

f, g. 8 irregular at anterior end.
h. 5 s, 2 c, I very irregular.
i. none.
Other irregularities, 14.

FEMALES.
2 b.

2 b.

I b, 3 TS, 4 s.

3 b, 6 vs, 9 s.
I b, I vs, 3 s.

2 irregular at anterior end.

none.

2 lost veins ; I almost lost.

none.

Deducting the cases where more than one irregularity occurs
on one wing we have 271 irregular drone wings and 37 worker
wings. Leaving out of consideration those cases in which merely
a bend is recorded there are 206 irregular drone wings and 30
irregular worker wings or almost seven times as many for drones
as for workers.

The figure and these tabulations make clear what has been
said previously about an area of irregularity in the wing. This
region is rather well defined and no irregularities in venation were
seen outside it.

A point which may well be recorded is that none of these veins

COMPARATIVE VARIABILITY IN THE HONEY BEE. 33

could be considered as in any sense mutations since there are in
all cases varying degrees of abnormality indicating a gradual
variation in the directions indicated.

No attempt has been made to correlate definitely these ab-
normalities with any lengths of the neighboring veins, but in a
general way it can be said that the largest wings of each sex are
the most abnormal, and this fact explains to some extent why
the drone wings are so much more abnormal than those of the
workers. From this it would appear that the cause of these
abnormalities was the need of extra strengthening of the cells
as they became larger, and that the irregularities are the result
of extra growth energy, which has a chance to show itself when
room is allowed for its manifestation.

SOME EXPLANATIONS OF THESE RESULTS.

With theseYacts before us it seems desirable to find, if possible,
some explanations for these peculiar results. As stated in the
introduction the theory of germinal variation would lead one to
believe that the parthenogenetic individuals of the bee would
show less variation than those from fertilized eggs and if there
were no complicating factors it may be supposed that this would
be true, but there are certain factors which modify this result so
that the ratio of variation is exactly reversed.

In the first place that kind of parthenogenesis in which males
only are produced (arrenotoky), is not such a specialized form of
agamic reproduction as are those kinds of parthenogenesis in
which females or both males and females are produced. When
females only result from parthenogenetic eggs (thelytoky], a
crossing of two lines of heredity seldom occurs and amphimixis
can bring about little variation. In the case of the Aphids and
Daphnids we have a similar condition except that once in a year
or once in some life cycle males also are produced parthenoge-
netically and then there is an opportunity for the blending of hered-
itary traits through fertilization. To this last form of partheno-
genesis the name amphoterotoky is applied. It is thus seen that
in arrenotoky this mixing of hereditary traits is not dispensed
with to such an extent as in the cases of thelytoky and ampho-
terotoky. On the other hand, the production of females par-

34 D. B. CASTEEL AND E. F. PHILLIPS.

thenogenetically is of far more use to a species where gamic
reproduction is unnecessary, since fertilization is not necessary to
give a stimulus to the egg so that it may develop, and what the
species loses in lack of cross fertilization is more than made up
in the advantage it has through its parthenogenetic power.
Since then arrenotoky is the least specialized form of partheno-
genesis, and since a crossing does occur at every second genera-
tion in species with this power, it follows that according to the
theory of germinal variation we would find more variation in
arrenotoky than in either thelytoky or amphoterotoky. In fact
the decrease in variability would not be very great, since in
every case but one half the crossing is dispensed with.

Variation in parthenogenetic forms has been observed previ-
ously. Weismann l found that the parthenogenetic ostracod,
Cyprus reptans, showed variation, and Warren 2 found consider-
able variation in Daphnids. Both of these cases fall under
amphoterotoky, so that on a priori grounds we would expect
still more variation in arrentoky. It is also held by some that
males tend to vary more than females, and perhaps this tendency
has something to do with what we find in the case examined.
Davenport and Bullard, 3 found 2^4 per cent, more variation in
males than in females in swine. Darwin 4 gives a considerable
number of cases showing the same tendency, and others have
observed similar facts. On the other hand there are cases in
the Odonata (Gouiphus and Macrothemis, Calvert 5 ) and in the
Lepidoptera (Thy re us abbotii, Field 6 ), in which the females are
more variable than the males so, that we must not assume too
much on this ground.

This still leaves considerable variation in drones to be accounted

1 Weismann, A., "The Germ-plasm," 1893.

2 Warren, E., "An Observation on Inheritance in Parthenogenesis," P. R. Soc.
Lond., Vol. LXV., pp. 154-8, 1899.

3 Davenport, C. B., and Bullard, C., " Studies in Morphogenesis," VI. "A con-
tribution to the quantitative study of correlated variation and the comparative varia-
bility of the sexes." Proc. Am. Soc., Vol. 32, pp. 85-97, 1897.

4 Darwin, Charles, "The Descent of Man." London, 1871.

5 Calvert, P. P., "The Odonate Genus Macrothemis and Its Allies," Proc. Boston
Soc. Nat. Hist., Vol. 28, pp. 301-332, 1898. "On Gomphus fraternus, externns and
crassus (Order Odonata)," Entomol. A r eios, March, 1901.

6 Field, W. L. W., " A Contribution to the Study of Individual Variation in the
Wings of the Lepidoptera," Proc. Am. Ac. Sc , Vol. 38, pp. 389-396, 1898.

COMPARATIVE VARIABILITY IN THE HONEY BEE. 35

for and the following facts seem to us to help in this. The
workers in a hive are hatched from cells one fifth of an inch in
width and the size of these cells is remarkably uniform. On the
other hand the drones hatch from cells which are generally one
fourth of an inch in width, but often hatch in worker cells and from
cells of all intermediate sizes. In the making of the comb under
natural conditions there are a great many irregular cells formed
which are transition cells between the worker and drone cells,
and from these, if used for brood at all, drones are produced. It
is true that sometimes a worker is produced in a drone cell, but
this is very rare, provided there are any worker cells in the
hive. Drone pupae, on the contrary, are frequently seen in
worker cells and are very noticeable on account of the exception-
ally high arched cap which the workers put on when the larva
is sealed up. This then gives to drones a greater amount of
variation in the room provided for their growth while in the
plastic state.

A bee larva will grow until it fills the cell in which it is placed
and the young bee which emerges will be the size of the cell
from which it came, within certain limits. This is shown in the
production of queens by the modern methods of queen rearing
used in apiculture. A young worker larva, less than one day
old, is lifted from its cell and put into a cell cup of queen size.
The workers complete this cup and form a queen cell and the
larva in this cell grows to a much larger size than would be
possible if it had remained in its original cell. Once in a while
the bees will attempt to make a queen from a drone larva and
while, of course, this is a failure yet the result is a very large
drone. Generally, however, the drone under these conditions
dies before reaching the imago stage. These facts show that the
growth of an individual is limited by the size of the cell and also
undoubtedly by the amount of food received during the unsealed
larval stage. Then it follows that since the cells from which

o

drones hatch vary from one fifth of an inch to over one fourth of
an inch in width, while those from which workers hatch are
quite uniform, that the variation in size will be^ considerably
greater for drones than for workers.

This supposition is further strengthened by some of the facts

36 D. B. CASTEEL AND E. F. PHILLIPS.

brought out by measuring the wings. Referring again to the
table of the ratios varying according to the length of vein M 2 we
find that the vein m varies inversely as the length of M v The
length of vein /// represents the ratio of the length of the wing
while vein M 2 represents the ratio of the width. Probably most
of the drones which have the shortest vein M 2 are those hatched
from the smallest cells and the wings could not increase in width
and therefore to meet the needs of the animal in making the
necessary area of wing for flight the vein ;;/ must be lengthened.
On the other hand, those drones hatched from the largest cells
would be allowed greater room for the development of vein M 2
and vein ;// need not be so long.

Another fact which seems to indicate this is that those drone
pupae which are developing in worker cells are covered over by
a very high cap, making the length of their cell much greater
than that of the ordinary drone cell. The drones which hatch
from these cells are long and narrow when compared with those
from drone cells proper.

The drones in Lot II. were taken from a hive in which there
were no drone cells except possibly a very few in the corners of
the frame or near the top bar of the frame since all the combs
were made on what beekeepers call foundation and the cells were
uniformly of worker size. These drones show the least variation
since they were all hatched under the same conditions. The
drones of Lots I. and III. were hatched in old irregular combs
and the tables show considerably greater variability.

The greatest number of abnormalities were found on the largest
drone wings and the throwing in of extra veins is probably caused
by the necessity for greater strengthening of the wings. Just
how these extra veins arose is not easy to explain. They may
be sports, or reversions to an ancestral type, or the result of extra
growth-energy or caused by the splitting of normal veins so that
it is rather difficult to say just what factors bring about this extra
amount of variation. While we speak of these as abnormal veins
it must be noted that we do not know whether they are really
abnormal or whether they are but the manifestations of a tendency
possessed by all bees but which can develop only under certain
conditions, just as the ovaries of the workers can develop only
when extra room and food are provided.

^H COMPARATIVE VARIABILITY IN THE HONEY BEE. 37

If then germinal variation will not explain all these variations
and if we accept the explanation offered as a partial and possible
statement of the cause, then it would appear that the mere chance
as to which cell happens to be the receptacle of a drone egg
determines its variation. While it is probable that even this
" chance " is according to fixed law, the fact remains that in any
event this law is beyond the possibility of formulation from any
observations except those extending over far more individuals
than those here used. On this account we consider ourselves
justified in our tabulation of results rather than in the plotting
of curves and expression in mathematical formulae, since that
would be undesirable except with far more measurements and
with material gathered under conditions better controlled. Our
tables show the variation as it actually exists in a state of nature
and the real laws can be worked out only from observations from
control experiments and this it is hoped will be possible in the
near future.

We do not wish to be considered as advocating the inadequacy
of the theory of germinal variation to explain variation, since
we have no means of knowing whether these variations can be
inherited but simply wish to express the facts as we find them,
and leave the explanation of the bearing of germinal variation on
this problem for future investigation.

October I, 1903.

EXPLANATIONS OF FIGURES.

FIG. I. Fore wing of honey bee, normal. Cells and veins are named according
to Comstock and Needham.

FIG. 2. Hind wing of honey bee, normal.

FIG. 3. Typical hymenopterous wing according to Comstock and Needham.

FIG. 4. Part of fore wing of honey bee showing (in dotted lines) where acces-
sory veins were seen to occur in the wings examined. Lettering purely arbitrary as
explained in text.

THE OVARIAN STRUCTURES OF THE VIVIPAROUS
BLIND FISHES, LUCIFUGA AND STYGICOLA. 1

HENRY H. LANE, A.M.

I. INTRODUCTORY.

During the spring of 1902, Dr. C. H. Eigenmann collected a
number of specimens of blind fishes in the caves of western Cuba,
within a radius of I 30 kilometers of Havana. The fishes belong
to the two distinct but closely related genera, Liicifuga and
Stygicola. It has been my good fortune to have the oppor-
tunity of studying the reproductive organs more particularly,
the ovarian structures --of these blind fishes, with special ref-
erence to their method of reproduction. It was discovered
upon examination of the specimens that they are viviparous, 2
a fact long known in regard to some of their deep-sea rela-
tives. Owing to the lateness of the season when they were col-
lected, unfortunately but one female was pregnant. This one
measured only 65 mm. in length and contained four foetuses
borrowing a term to designate the post-larval stages of the young
until birth 18-20 mm. long. These foetuses were in an ad-
vanced stage of development, very probably being within a few
days, or possibly hours, of birth, since a number of young only
25 mm. long were caught in the water. No other prenatal speci-
mens having been secured, it has been impossible to study the
early stages of development. My attention has been particularly
directed to the ovarian structures of the mature females secured.
A few of the young specimens, evidently taken not long after
birth, were also examined.

I wish here to express my deep sense of obligation to Dr. C.
H. Eigenmann for his assistance and criticism in the preparation
of this paper.

1 Contributions from the Zoological Laboratory of Indiana University, No. 58.

2 Eigenmann, '03, p. 236, pi. 21.

33

OVERIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 39

II. SYSTEMATIC POSITION OF LUCIFUGA AND STYGICOLA.

Poey 1 ('60) described these fishes in 1860, giving them the
generic name of Lucifuga, recognizing them, however, as two
species, dentatus and subterraneus. Later Gill, '63. separated
dentatits from the other and created the genus Stygicola fortit.
The two genera are different in that Stygicola has teeth on the
palatines, where Liicifitga has none, and the teeth in the jaws of
the former are larger than those of the latter. There is also a
very noticeable difference in the depth of the head at the nape, in
adult individuals. The two species or genera are however so
nearly alike that it is only after a prolonged comparison that the
above technical differences were made out.

The several species of blind cave fishes, found in Indiana, Illi-
nois, Kentucky and Missouri, are not related to the Cuban
species. The latter are descended from marine forms which have
worked their way through underground channels into the Cuban
caves. Related genera that still live in the ocean about Cuba
are Brotula 2 and Ogilbia? in moderate depths and Bassosetus*
and Apkyonus 5 in deep water.

All of these genera belong to the family Brotulidae, a deep-sea
group comprising about forty-five genera and one hundred
species, living mostly in the tropical seas of both hemispheres.
The two genera under consideration in this paper are the only
ones found in fresh water. Jordan and Evermann (op. cit., Pt.
III., p. 2498) observe very properly that " these fishes are closely
related to the Zoarcidae. In spite of various external resem-
blances to the Gadidae their affinities are rather with the blen-
nioid forms than with the latter."

III. HISTORICAL.

Numerous contributions to our knowledge of viviparity in
fishes have been made from the time of Cuvier to the present.

1 Lucifuga, Poey, " Memorias," II., 95, 1860 (su&ferraneas) ; Lucifuga subter-
rannts, Poey, "Memorias," II., 96, 1860; Luiijuga dentatus, Poey, "Memorias,"
II., 102, 1860; Stygicola dcntata, Gill, Proc. Ac. Nat. Sci. Phil., 252, 1863.

2 Brotula (vid. Bull. 47, U. S. Nat. Mus. , Jordan & Evermann, "fishes of
North America," Pt. III., p. 2500).

3 Ogilbia {vid. idem, Pt. III., pp. 2502, 2503).

4 Bassozetns, Gill (viJ. idem, Pt. III., p. 2507).

5 Aphyoniis, Giinther (yiJ. idem, Pt. III., p. 2525).

4O HENRY H. LANE.

Among the most important ones are the following, most of which
I have consulted in connection with my own investigation :

Cuvierand Valenciennes, in their " Histoire Naturelle des Pois-
sons," I., Paris, 1828, have a short general account of viviparity
in. fishes, and mention is made of it frequently throughout their
work in the description of such fishes as bring forth living young.
Much of their work has been superseded by the more accurate
observations of later investigators.

Rathke, in 1833, published his " Bildungs- und Entwickel-
ungsgeschichte des Blennius viviparus odes des Schleimfisches."
This was long the best paper on the subject.

In 1844 (Ann. des Sc. Nat., t. I., 3d series, p. 313) Duvernoy
published a paper on Pcecilia snrinamensis, which is frequently
referred to by more recent writers, but which I have not had the
opportunity of consulting.

Tn 1846, Cuvier and Valenciennes described the genus Ana-
blcps, one species of which, A. gronovii, formed the subject matter
of an important paper by Jeffries Wyman, in the Boston Journal
of Natural History, Vol. VI., No. IV., p. 432, 1857. I shall
refer to this article more at length below.

In 1853, Louis Agassiz (Am. Jour, of Science, XVI., 2d series,
Nov., 1853) described a new family of fishes from California
the Embiotocidas, which embraces the genus Cymatogaster. The
only species of this genus, C. aggregates, was studied in detail
by Dr. Eigenmann, and the results published in the Bulletin of
the U. S. Fish Commission, Vol. 12, p. 401, 1892 (1894).

Another very important paper, " On the Development of Vi-
viparous Osseous Fishes and the Atlantic Salmon," by John A.
Ryder (Proc. U. S. Nat. Mus., 1885, pp. 128-162, Pis. VI.-XII.)
will be noticed frequently below.

In 1887, Dr. Franz Stuhlmann made a detailed study of
Zoarccs viviparus, Cuv., the results of which he published under
the title, " Zur Kenntnis des Ovariums der Aalmutter (Zoarces
vivipariis, Cuv.)." Frequent references to this volume will be
made below.

IV. VIVIPARITY IN GENERAL.

Cuvier and Valenciennes (loc. cit.} give a general account of
viviparity in fishes so far as known at that time ; but since their

OVARIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 4!

statements are now either matters of common knowledge or else
not in accordance with the facts as revealed by later investiga-
tions, I shall not speak of their work further.

Wyman (op. cit.~) classifies viviparous fishes into two groups
"according to the position occupied by the embryo during the
period of growth. In the first group may be arranged those
fishes in which the ovum leaves the ovary in an undeveloped state,
and in which the process of eolntion (sic] is not commenced until
it readies the lower portion of the oviduct. The fishes which this
group comprises are nearly all, if not all, Plagiostomes. The
best known are Spinax, Carcharias, Mustcllus, Galeus, and
Torpedo. ... II. In the second group those fishes are comprised
in which the gestation is wholly or in part ovarian, the last stages
only of the process usually occurring in the oviduct. Among
the genera included in this division are Sihirns, Blcnnius, Ana-
bleps, Ptfcilia and Embiotoca. In all of these genera impregna-
tion takes place in the ovary, and, as seems probable, while the
ovum is still invested with its original envelopes."

Wyman found that each of the foetuses in A. gronovii is envel-
oped in a separate sac of vascular tissue, much too large for the
foetus enclosed, the extra space being filled up with an albuminous
fluid. He seems to regard these foetal sacs simply as extensions
of those within which the ova were suspended.

Eigenmann, considering only teleosts, found two types of
viviparity (op. cit., p. 404) ; he says :

" At least two types of viviparity may be distinguished in
fishes ; first, those in which the yolk furnishes all the intra-
ovarian food ; and second, those in which the greater part of the
food is furnished by the ovary.

" In the first type the number of young is not less than in re-
lated oviparous forms, while the number of young in the second
is always greatly reduced. . . . The size and development of
the young in this class (type I.) of fishes at the time of birth is
of course much less than in the second class of viviparous
fishes."

As will be seen, Lucifuga and Stygicola belong to the second
type of both Wyman and Eigenmann. The number of young is
small and they are born in quite a mature condition.

42 HENRY H. LANE.

V. GROSS ANATOMY.

For the sake of clearness the following terms will be used in
the sense here given :

Oviduct the single duct leading from the ovary to the uro-
genital pore. Ovisac the forward continuation of the oviduct
which covers the ovary. Ovary the structure containing the
eggs. Stroma the supporting tissues of the ovary itself.

The term ovary is also used in a general way to include the
ovisac and the ovarian structures proper. The context in every
case will determine what is meant.

In Liicifitga and Stygicola the ovary is enclosed between two
layers of peritoneum above the posterior portion of the alimentary
canal. It may extend so far forward as to lie in part even beside
the stomach.

The ovary has a bilateral arrangement. Externally it is a
Y-shaped, bifurcated, subcylindrical organ, whose greatest diam-
eter is immediately posterior to the point at which the division
begins (Fig. i). The two horns lie on the right and left sides
respectively and may enclose between them the posterior portion
of the stomach. Interiorly the stem of the Y is divided by a
median partition with which the ovarian structures proper are
associated and which extends to near the oviduct, though here
only the portion attached to the ventral wall is found (Fig. 2).
From the tips of the ovarian horns slender though comparatively
strong threads of connective tissue, inclosing blood vessels, run for-
ward and fasten to the peritoneal walls, thus very securely hold-
ing the ovary in position. Dorsally, the ovary is attached to the
peritoneal lining of the body cavity by the mesovarium ; ventrally,
there is a corresponding attachment, the mesorectum. The ovi-
duct, which opens externally at the urogenital pore, increases
gradually in size as it approaches the ovary and finally merges
into the ovisac, or outer wall of the ovary.

A somewhat immature specimen shows finely those structures
connected with the support of the ovary. In it one sees that
each horn is supported by its own fold or lamina of peritoneum ;
that these two laminae become united at or near*the point of di-
vision of the horns and are continued posteriorly as a single
though thicker mesovarium supporting the body of the ovary

OVARIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 43

and the oviduct. Below there is no sign of a mesorectum in the
region of the ovarian horns, except for a short distance near the
base of one of them, but posterior to them there is such a mem-
brane, inclosing several blood vessels, and itself somewhat thicker
than the mesovarium above. It is also clear from an examina-
tion of this ovary that, as will be noted more particularly below,
the egg-bearing tissue, the ovary proper, forms a thick median
partition in the ovisac.

The external appearance of the ovary agrees very closely with
Eigenmann's description (op. cit., p. 418) of that of Cy matog aster :

" The ovary is' a spindle-shaped bag, divided anteriorly into
two arms which indicate the bilateral origin of the present struc-
ture. One of these arms, the left, is usually smaller than the
other. . . . The ovaries of the two sides have evidently been
united from behind forward, so that externally only the two an-
terior horns show the bilateral structure, and one of these horns
seems to be in process of phylogenetic resorption."

While there is frequently a difference in the size of the two
horns in Lu'cifnga and Stygicola, there is no uniformity in this
matter. There seems to be no evidence that the right or left
portion of the ovary is "in process of phylogenetic resorption."
Ryder found that in Gainlnisia patrnelis " the ovary is a simple
unpaired organ, the greater part of which lies on the right side
of the body-cavity below the air bladder. . . ."

The size of the ovary varies, of course, with the age and size
of the female, as well as with the state of development of the ova
or embryos contained in it. One female of the genus Litcifiiga,
which had a length of 65 mm. and which contained four foetuses
nearly ready to be born, had an ovary with a length --measur-
ing to the extremity of the longer horn --of 16 mm., and a di-
ameter of 8 by 9 mm. As the foetuses were 1 8 to 20 mm. long,
it was not surprising to find that their tails were bent over. An-
other female of the same genus, 83 mm. long, had evidently
given birth to young only a short time before her capture and
had an ovary 12 mm. in length.

The point of division into the two horns is usually about five
twelfths the distance from the anterior tip of the ovary to its pos-
terior end. As already stated, the two horns rarely show equal

44

HENRY H. LANE.

PlATE '

Fit 3

OVARIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 45

development, there being always a more or less marked differ-
ence in size.

Between the ovisac and the ovary proper there is a lumen of
varying size. When there are larvae present, the ovisac and the
oviduct are extremely thin and so stretched, especially near the
close of gestation, that their cellular structure cannot be made
out with any satisfaction (Fig. 5). Shortly after birth of the
young they contract and assume the form and appearance found
in the ovaries of mature but non-pregnant females (Fig. 3). The
wall of the ovisac is then quite thick, and the lumen very small.
The histological structure of the ovisac will be described below.

In non-pregnant ovaries, the stroma is a mass, which, in-
ternally, has a bilateral arrangement and occupies most of the
space within the ovisac (Figs. 2, 3). It is, in general shape,
fusiform with its largest diameter just posterior to the division
of the ovisac into the two horns down both of which it is con-
tinued along their median surfaces, forming the prongs of a Y.
In the middle of the ovary the stroma forms a median parti-
tion ; somewhat posteriorly this partition is cut across (Fig. 3),
and still further back only the ventral part remains (Fig. 2).
It has many lobes which are usually somewhat pointed and
comparatively large and distinct, the indentations sometimes leav-
ing merely a "neck" of tissue to support them. The whole
stroma at this time is fully distended by the large amount of lymph
contained in the sinuses described below. Where the ova are well
advanced they can be seen by the unaided eye in the form of
opaque dots. When the ova are surrounded by follicles, they
lie some distance below the surface of the stroma and there is
a tubular indentation of the epithelial covering of the latter down
to the follicle (Fig. 7, /?). In a circular space over the egg the
epithelium is apparently continuous with the follicle. It is only
on very close inspection that the independence of the follicle can
be made out. It is then found to be of only a single cell in thick-
ness beneath the epithelial indentation. A similar position of the
epithelium was noted by Stuhlmann over the ova in the ovary
of Zoarccs and was called " Delle " by him.

The pregnant ovary is quite different in appearance from that
of a non -pregnant female. A cross-section of the former shows

46 HENRY H. LANE.

it to be an almost bilaterally symmetrical organ, but without any
folds or pockets in which the embryos are contained as is the
case in Cymatogaster 1 and numerous other species of Embiot-
ocidae. During the intra-ovarian development of the embryos,
or rather the development of the foetuses within the oviduct
of Liicifjiga, the ovarian structure proper or stroma which forms
the thick median partition in non-pregnant ovaries, becomes
gradually reduced and compressed into a narrow wall (Fig. 5).
The stroma is much thickened both dorsally and ventrally near
the oviduct (Fig. 4), where the partition is incomplete, but ante-
riorly its greatest thickness is near the median plane (Fig. 5). The
arrangement of the stroma in each horn of the ovary is as in non-
pregnant ovaries (Fig. 6, ov.st.}.

The single oviduct runs from the caudal end of the ovary
proper to open at the urogenital pore. In pregnant females it is
widely distended for some distance when the foetuses are well
advanced, but in the non-pregnant females it is a rather cylin-
drical, thick-walled, muscular tube with numerous folds or laminae
on its inner surface, covered with a layer of columnar epithelial
cells, 12 fj. in depth. It is not materially different, except as to
dimensions, from the ovisac described above. Stuhlmann 2 sim-
ilarly found the oviduct of Zoarces to be a tube composed of the
same cell-layers as the ovary, with the exception of the " ger-
minal " and follicular epithelia.

VI. HlSTOLOGICAL PART.

/. The Walls of the Ovary or Ovisac.

The following system will be used to facilitate cross references
to the descriptions of the various ovaries. Each ovary will be
referred to by a letter, A, B, C, etc., the meanings of which are
as follows :

A represents a female of the genus Stygicola with a length of
95 mm.

B represents a female of the same genus, but with a length of
128 mm.

C represents a female of the genus Lucifuga, length 87 mm.

1 Eigenmann, op. cit., p. 418.

2 Op. cit., p. 10.

OVARIUM STRUCTURES OF VIVIPAROUS BLIND FISHES. 4/

D and E represent females of the same genus with a length of
83 and 65 mm. respectively. E was pregnant; the others were
non-pregnant.

An examination of the ovisac and oviduct reveals quite a range
of variation, depending in the main upon the condition of preg-
nancy or upon the length of time that had elapsed since the close
of that period.

In the ovary of D, which had not been pregnant at all, or at
least for so long a time that the ovarian structures had regained
their normal form, the wall of the oviduct and the ovisac is from
100 to 150 // in thickness at different places. Structurally, the
ovisac consists of at least four cell-layers. The outer, a sinuated,
peritoneal layer, immediatly beneath which there is a thicker layer
of longitudinal muscle fibers ; below this there is another some-
what thicker transverse band of muscle fibers ; on the inner sur-
face there is an epithelial layer containing numerous capillaries.
This will be described in detail below. The nuclei of the longi-
tudinal band are rod-like in appearance ; the nuclei of the second
muscle layer appear more nearly round, being evidently the cross-
sections of nuclei of the same form as those in the longitudinal
band. The innermost layer of epithelial cells has nuclei oval or
round in shape, while the peritoneal layer shows few nuclei, but
those which do appear are rod-shaped in section. Quite numer-
ous capillaries are found between the cells of these several layers
and in some places there are large blood vessels.

In the ovary A from a female which had evidently given birth
to young but a short time previous to her capture, the ovisac
measures only 15 to 20 fi in thickness. Structurally it con-
sists of four or five thin cell-layers, between which there are
anastomosing capillaries. The outermost layer consists of peri-
toneum, the cells of which are very much elongated and com-
pressed. The muscle fibers beneath are mostly transverse and
of the non-striated type. The inner layer is epithelial and is
also much compressed. The nuclei of the muscle-fibers are long,
narrow, rod-like structures which stain deeply, as would be ex-
pected, with haematoxylin : the nuclei of the epithelium are oval
in form. The condition of this ovary does not permit me to go
into greater detail.

PLATE II

OVARIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 49

In the ovary of the pregnant female E, whose young were
almost ready for birth, the ovisac is thinner than in the case just
described. The different cell-layers can scarcely be distinguished,
though where the cells themselves are visible, the nuclei in section
have the rod-like form mentioned above. The capillaries have
mostly disappeared, apparently being closed by the crowding to-
gether and stretching of the cellular structure ; but in places one
comparatively large vessel appears, containing two or more rows
of corpuscles, side by side. At this place the wall is enlarged
somewhat to accommodate the vessel.

In the ovary of C, which contained ova quite well advanced,
the ovisac is very similar to that described for D. But in some
portions of this ovary the muscle layer is restricted almost
entirely to longitudinal fibers, the transverse layer being much
reduced. Capillaries penetrate freely through these muscle
layers in all directions ; those in the lining epithelium are larger
than those in the other specimens already described.

In the ovary of B the ovisac is similar to that in C, but the peri-
toneal covering is not so distinct ; there is the same arrangement
of muscle-fibers - - the outer longitudinal and the inner transverse,
the latter being much the deeper. The innermost epithelial layer
is composed of " pavement " cells with quite large distinct nuclei.
Numerous capillaries are found in this inner lining.

Compare in this connection Eigenmann's description of the
ovarian wall in Cymatogaster (op. cit., p. 418) :

" The ovarian walls are composed, first, of the thin peritoneal
membrane ; second, of a layer of longitudinal muscle fibers ;
third, of a layer of circular muscle fibers, inside of which there is,
in places, a layer of longitudinal fibers ; fourth, of a very thin layer
of cells with flattened, deeply stainable nuclei ; fifth, of a layer of
epithelium. This layer is derived from the peritoneum."

Stuhlmann found the ovarian wall in Zoarces to have a toler-
ably deep, non-striated, muscle layer, the fibers of which were
closely packed together next to the peritoneal covering, but
toward the lumen they were split apart by numerous sinuses
containing blood vessels. The oviduct was similarly composed,
except that there were few if any clefts between the fibers and
there were fewer blood vessels.

5O HENRY H. LANE.

The unique feature in the ovisac as well as in the epithelial
Covering of the stroma of Lucifuga and Stygicola is the presence
of capillaries in the lining epithelium (Fig. 8). So far as could
be determined this condition has never been observed in the ovary
of any other form. So numerous are these capillaries that they
attract attention at the first glance. The epithelium itself is often
reduced to extreme thinness, sometimes serving merely as a mem-
brane to contain the blood.

VII. THE OVARY.

The ovarian structure itself is highly vascular and much lobed.
There is a tendency in some instances for these lobes to be ar-
ranged in a bilaterally symmetrical pattern, when seen in cross-
section, though this is not equally evident in all ovaries or even
in all parts of the same ovary. The ovarian structures of the
different specimens examined, while presenting numerous points
in common, are yet characteristically different in every case.

The ovisac of A had but recently contained young, to judge
from its extreme thinness ; the stroma was so large that it gave
promise of containing embryos. Instead of that condition, how-
ever, it was found that the large size was due to the mass of
stroma which is composed in part of highly vascular tissue.
Numerous blood vessels penetrate the stroma in all directions
while around the ova themselves there is a network of capillaries.
The greater portion of the stroma is split up into numerous
sinuses, many of which are larger than any of its blood vessels.
These are closely similar in appearance to the " lymph-spaces ):
described by Stuhlmann (op. cit., p. 19) for Zoarccs and no doubt
serve the same purpose (Fig. 7, /.5.).

The ova of A are few in number, less than ten over 60 /J. in
diameter appearing in any cross-section. Five or six ova are of
quite large size, visible even to the naked eye, and measuring
from 300 to 800 11 in diameter. They have a large amount,
proportionately of yolk-substance. The smaller ova are about
50 to 60 IJL in diameter, and are of the usual appearance of
ova of that size. The cells of the stroma in this ovary are very
irregular in shape, indistinct in outline, and usually of inconsid-
erable size. The nuclei are round, oval, or elongated, appar-

OVARIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 5 I

ently influenced as to form and shape by the cell-body. The
entire surface of the stroma is covered by a layer of epithelium,
with a depth of 10 to I 5 ij.. The nuclei of these epithelial cells
appear quite distinct, are of a comparatively large size, and are
round or oval in shape.

By far the largest amount of space in this epithelial layer is
given up to the numerous capillaries contained in it. They are so
numerous that, in cross-section, they appear as a row of large per-
forations, there being no more than a scant cell thickness between
them. The average diameter of these capillaries is less than
eight micra, in many instances being only five micra. This con-
dition is comparable to that described above for the ovisac and is
also unique (Fig. 8).

The ovarian stroma of E, which contained the mature foetuses,
has been squeezed and crowded into a median position by the
young (Fig. 5). The cellular structure resembles that of the
ovary just described, except for such variations as would be
caused by its closely packed condition. The capillaries of the
epithelial layer, covering its surface, are not so numerous as in the
ovary of A, but are of larger size. The larger blood vessels are
more nearly cylindrical in form and have their walls more thick-
ened than have those in the first ovary. The lymph-spaces in
this ovary are compressed by the foetuses and temporarily elimi-
nated.

Quite different in appearence from either of the two just de-
scribed, though somewhat intermediate between them in some
respects, and more advanced than the first in others, is the ovary
of D. In this the ovarian stroma has not so many nor such large
lymph-sinuses as A, but on the contrary has more nearly the ap-
pearance of that in E, from which it differs conspicuously, how-
ever, in not showing a "crowded" appearance, and in having quite
numerous ova of various sizes, though none of the latter are so
large as those of A, and in many cases are grouped together in
" nests " in a way largely unknown in A. The blood vessels are
comparatively numerous, large and quite thick walled. The
capillaries in the epithelial covering of the ovary so conspicu-
ous is that of A are so few in this case as to be visible only
when carefully searched for. The cellular structure near and

52 HENRY H. LANE.

next to the surface is dense and without important sinuses. The
outlines of the cells are very indistinct, but the nuclei are alto-
gether similar to those of the ovaries previously described.

The ovary of C approaches more nearly to the condition of A
than has any of the others ; but it differs very characteristically,
since in many places it contains " nests " of ova much more con-
spicuous than any seen in A, while the largest ova in this speci-
men are larger than those in A. The ovarian structure itself,
while evidently of the same character as that of A, does not con-
tain quite so many lymph-spaces, and the walls of the sinuses are
somewhat thicker and denser.

The ovary of B is almost exactly in the same stage as that of
A. It differs from C in that the egg-nests have given place to
single ova of considerable size and greater development than
most of those in the latter.

It will be noted that the " nests " of ova are conspicuous in
Lucifuga, though inconspicuous or lacking in the specimens of
Stygicola examined. Whether this is a constant distinction can
only be determined by the examination of more material than I
have in hand.

VIII. BLOOD SUPPLY TO THE OVARY.

A small artery, with a diameter, in different ovaries, of 20 to
75 fjt, enters each horn of the ovary and runs back near the
inner surface of the horn. In the main portion or body of the
ovary, the two arteries occupy parallel courses near the center,
separated by perhaps one third the diameter of the ovary. Since
none of the specimens at hand were injected, the course of these
arteries could not be traced except in a general way. But it is
plain that they extend posteriorly in a tortuous course through
the ovary and give off numerous branches, which find their way
to or toward the surface, where they form the capillaries so dis-
tinctly visible in some of the ovaries in the epithelial covering.
The blood from the epithelial capillaries of the anterior half of
the ovary is collected by veinlets, frequently quite large and dis-
tinct in the vicinity of the larger ova, which join to form larger
veins that pour their contents into the chief vein of the ovary at
the " horseshoe bend" (infra). This largest vein has two branches

OVARIAN STRUCTURES OF VIVIPAROUS BLIND FISHES. 53

(one going out by either horn) which are united near the point
of division of the ovarian horns, forming a single " horseshoe"-
shaped vessel. The veinlets which return the blood from the
posterior part of the ovary collect into one vessel which joins the
right horn of the "horseshoe" at a considerable distance in
front of the fork of the ovary, after running above and parallel
to the portion with which it unites, for the distance, in one speci-
men at least, of nearly 2 mm.

It quite frequently occurs that red-blood corpuscles are pres-
ent in the ovarian sinuses of Stygicola and Lucifitga, though their
presence may be due to accident. As indicated elsewhere, these
sinuses are very probably filled with a plasma or lymph.

BIBLIOGRAPHY.
Agassiz

'53 (Embiotoca.) Am. Journ. ofSci., Vol. XVI., 2d ser., Nov., 1853.

Cuvier et Valenciennes.

'28 Histoire Naturelle des Poissons. Paris, 1828.

Duvernoy.

'44 Observations pour servir a la connoissance du developpement de la Poecile de
Surinam (Poecilia surinamensis). Ann. des Sc. Nat. Zoolog., ser. III., T.
I., 1844.

Eigenmann.

'92 Cymatogaster aggregatus Gibbons ; A Contribution to the Ontogeny of Vi-
viparous Fishes. Bull. U. S. Fish Commission, Vol. 12, p. 401, 1892

(1894).

Eigenmann.

'97 Sex-Differentiation in the Viviparous Teleost Cymatogaster. Archiv f. Ent-
wickelungsmechanik der Organism, Bd. IV. Leipzig, 1897.

Eigenmann.

'03 The Freshwater Fishes of Western Cuba. Bull. U. S. Fish Commission,

for 1902 (1903), pp. 213-236 (3 pis.).
Gill.

'63 Stygicola. Proc. Ac. Nat. Sci., Phila., 1863, 252.

Girard.

'58 Explorations and Surveys for a Railroad from the Mississippi River to the
Pacific Ocean, IV., Fishes, 1858. (Embiotoca.)

Jordan & Evermann.

'98 Bull. 47, U. S. Nat. Mus., Fishes of North America. 1898, Pt. III.

Ludwig.

'74 Uber die Eibildung im Tierreich. Arb. Aus dem zoolog-zootom. Inst.

Wiirzburg, Bd. I., 1874.
Poey.

'60 Memorias, II., Havana, 1860.

54 HENRY H. LANE.

Rathke.

'33 Abhandlungen zur Bildungs- und Entwickelungsgeschichte des Menschen
und der Tiere, II. Leipzig, 1833, I., Abth. : Bildungs- und Entwickelungs-
geschichte des Blennius viviparus oder des Schleimfisches. 61 pp.
Ryder.

'85 "On the Development of Viviparous Osseous Fishes, etc." Proc. U. S.
Nat. Mus, 1885, pp. 128-162, Pis. VI. -XI.

Stuhlmann.

'87 "Zur Kenntnis des Ovariums der Aalmutter (Zoarces viviparus, Cuv. ).
Tab. IV. (1887).

Wyman.

'57 Observations on the Development of Aiiableps gionovii. Bost. Journ. of
Nat. Hist, Vol. VI., No. IV., 1857.

The paper by William Wallace, B.Sc., "Observations on Ovarian Ova and Folli-
cles in certain Teleostean and Elasmobranch Fishes," Quart. Jonrn. Micr. Sei.,
XLVII, (July, 1903), pp. 161-213 (3 pis.), was not seen by me until after my paper
was in the Editor's hands.

EXPLANATION OF PLATES.

FIG. I. External ventral view of the ovary of Stygicola. Portions of the peri-
toneal covering are visible along the sides.

FIG. 2. Cross-section of ovary near the beginning of the oviduct proper. Two
large ova at the sides.

FIG. 3. Cross-section of non-pregnant ovary with stroma in two lobes one dorsal,
the other ventral.

FlG. 4. Cross-section of pregnant ovary. The section is made through a region
corresponding to that of Fig. 3.

FIG. 5. Cross-section of pregnant ovary through the middle portion. The ovisac
collapsed when the foetuses were removed.

FlG. 6. Cross-section of pregnant ovary through the horns.

FIG. 7. A portion of a cross-section of a non-pregnant ovary, showing a part of a
large ovum (0} surrounded by its follicle (fl.c.} ; the epithelial covering of the stroma
dips down, forming a tube to the ovum (D). Bausch and Lornb one sixth objective ;
2-in. ocular; tube length, 160 mm.

FIG. 8. Portion of the epithelial covering of a non-pregnant ovary showing the
capillaries (r/j. ). Bausch and Lomb one twelfth objective ; i-in. ocular.
a. anterior. mr. mesorectum.

cps. capillaries. o. ovum.

d. dorsal. ovs. ovisac.

D. the"Delle." ov.sf. ovarian stroma.

fl.c. follicular cells. /. posterior.

l.s. lymph sinus. v. ventral.

mov. mesovarium.

All drawings by the author; outlines made with Abbe camera; details put in free-
hand but with the closest possible regard to accuracy.

Vol. VI. January, /poy. No. 2

BIOLOGICAL BULLETIN.

FORM-REGULATION IN CERIANTHUS, III.
THE INITIATION OF REGENERATION.

C. M. CIIfLD.

In the first paper of this series ('03^) the typical course of re-
generation in a cylindrical piece was described ; in the second
paper ('03$) some of the factors influencing the process of regener-
ation as a whole were discussed ; these papers have served to clear
the ground for a detailed analytical study of the process of re-
generation in Cerianthus in its various manifestations. In this
and following papers of the series various phases of this subject
will be considered.

CHANGES IN FORM CONSEQUENT UPON SECTION.

The reduction in size of the opening at the end of a cut piece
by the bending inward of the cut margins was described in its
simplest form in the first paper of this series. A somewhat more
general consideration of this peculiar process is necessary before
proceeding to the discussion of other points.

Early in the course of my experiments upon Ccriantlnis it was
noted that in nearly every case, however the pieces might be cut,
the body-wall became rolled or folded in such a manner that the
opening into the enteric cavity resulting from the cut \vas much
reduced in size or was closed by approximation or contact between
different parts of the body-wall. The usual result of the infold-
ing is the complete removal of the entodermal surfaces from con-
tact with the external water, /. c\, the piece rolls up or closes in
such mariner that the entoderm is on the inside. For conve-
nience we may designate inrolling about a transverse axis as
transverse inrolling, and inrolling about a longitudinal axis as
longitudinal inrolling.

At first glance this process appears much like an adaptive re-
action. In some cases it is almost as if the animal or part were

55

50 C. M. CHILD.

consciously closing the artificial openings. In the following
paragraphs the principal forms of inrolling in the cut pieces are
described.

The case of the closure of the ends of a cylindrical piece which
was described in the first paper is the simplest of all. Collapse
occurs with the escape of the water from the enteron and within

FIG. 2.

FIG. 6.

FIG. i.

FIG. 4.

FIG. 8.

FIG. i.

FIG. 7.

FIG. 9.

a few moments the cut ends begin to bend inward and finally
close the openings except for the small slits between the folds.
The diagrams, Figs. I, 1 2 and 3, illustrate this case, Fig. I repre-
senting the cylindrical piece at the time of section, Fig. 2 the
longitudinal section of the ends after the bending in of the cut
margins, and Fig. 3 a transverse section, indicating the flattening

1 The diagrams representing the inrolling are much less highly magnified than pre-
ceding figures.

FORM REGULATION IN CERIANTHUS. 57

of the piece as it lies on a flat surface. In pieces of this kind
complete collapse and contact of the body-walls is prevented by
the large mass of mesenteries and mesenterial filaments which
occupy the enteron. These are not represented in the figures,
but they fill the whole enteron after collapse. Any solid mass
in the enteron would of course have the same effect.

A piece cut from the extreme aboral end of the body (Fig. 4)
differs in certain respects from the piece just described. Figs. 5
and 6 show the changes in a piece of this kind. Here the cut
end becomes rolled inward to a much greater extent than in the
previous case so that the enteron is nearly filled by the inrolled
portion and the cut surface is so situated that closure by growth
of new tissue from this surface is impossible. The reason for the
greater degree of rolling in this piece as compared with the longer
piece is undoubtedly to be found in the absence of mesenteries,
except a single pair, in the aboral region. Since the enteric
cavity is not filled with a mass of mesenterial filaments as in a
region further orally the inrolling continues until the entire cavity
is practically obliterated by the inrolled parts.

If a cut be made in one side of the body or a piece removed as
in Fig. 7, the cut edges roll inward as in other cases, but in addi-
tion to this the body becomes bent at the level of the cut, so that
here also the inrolled edges are brought into contact (Figs. 8
and 9).

The widest departures from the typical form are found, how-
ever, in those pieces which were cut longitudinally as well as
transversely. In these the results differ to some extent accord-
ing to the shape and relations of the pieces. Fig. 10 represents
a cylindrical piece split longitudinally on one side. One form
after collapse and inrolling of cut margins is shown in Fig. n.
Fig. 12 represents a transverse section and Fig. 13 a longitudinal
section of one end. In Fig. 14 another form of closure is rep-
resented ; here the ends fold over to a greater extent so that the
opening is entirely on one side of the piece. Fig. I 5 represents
a transverse section of this piece and Fig. 16 a longitudinal sec-
tion in the plane indicated by the vertical line in Fig. 14.

In most cases, however, the right and left longitudinal cut
edges do not roll inward with equal rapidity and the result is

C. M. CHILD.

that the piece rolls up spirally on its longitudinal axis. Such a
case is shown in Fig. 17 ; here the inner and outer coils of the
spiral are on the same level at the outer end of the piece. Fig.
19 shows a spirally coiled piece in which the inner coils are
higher than the outer. Figs. 18 and 20 represent longitudinal
sections of one end of these pieces and Fig. 2 i a transverse sec-
tion.

The Figs. 1 121 represent only the chief types resulting from
pieces like Fig. 10. All possible intermediate forms and modifi-

FIG. 13.

FIG. 15.

FIG. j i .

FIG.

14-

FIG. 16.

FIG. 10.

cations of these different types occur, the differences depending
on various conditions, but chiefly on the relative rapidity of the
inrolling in the different directions.

Semi-cylindrical pieces or longitudinal strips may roll either
longitudinally or transversely. The greater the length and the
less the breadth of the strip the more likely it is to roll trans-
versely. Figs. 2224 show a strip and two forms of trans-
verse rolling which it may undergo. The longitudinal strips

FORM REGULATION IN CERIANTHUS.

59

often roll longitudinally soon after section, but by gradual inroll-
ing of the ends finally become rolled transversely.

Loeb ('91) has suggested that the cut edges roll inward be-
cause the inner layers of the body- wall are stretched to a
greater degree than the outer layers ; this view assumes that
all layers are more or less similar in elasticity and therefore that
the layer that is most stretched will undergo the greatest con-
traction when the tension ceases. It is difficult to understand
why one layer of the body should be more stretched than
another, since all have been subjected to the same conditions,
viz., the tension resulting from the fluid pressure on the walls of
the enteron.

FIG. 17.

FIG. 1 8.

FIG. 19.

FIG. 20.

FIG. 21.

FIG. 22.

FIG. 23.

FIG. 24.

I am inclined to believe that this remarkable capacity for roll-
ing which the pieces exhibit is due primarily to a difference in
elasticity between the different layers of the body-wall, though it
may be increased or modified by other factors. The succession
of layers in the body-wall is as follows : ectoderm', longitudinal
muscles, mesogloea, entoderm. The mesoglceal layer is fibrillar
in appearance and, while not as thick as the muscular layer, is
well developed.

Judging from the fact that this layer is not folded or wrinkled,
even in strongly contracted animals, the inference that it possesses

60 C. M. CHILD.

a considerable degree of elasticity appears justifiable. Under
normal conditions the body-wall is subjected to tension. By sec-
tion of the body at any point the internal pressure is removed
and collapse occurs ; the body-wall is no longer under tension
and contraction of the elastic layer begins. If the ectoderm and
muscles are to a large extent passive in this elastic contraction
the result will be not simply a reduction in surface area, but an
inrolling of the body-wall, since the mesoglcea is situated near
its inner surface.

The fact that the region near the cut surface is always more
strongly rolled than other parts may perhaps be the result of the
direct injury to the tissues in this region, causing contraction,
but here as elsewhere the contraction must be greater in the
inner portions of the body-wall than in the outer, otherwise inroll-
ing could not occur. More probably, however, the greater
degree of inrolling near the cut surface is largely, if not wholly
due to the fact that the physical obstacles to the inrolling offered
by resistance of other tissues, etc., are much less near a free end
or cut surface than elsewhere and the effect of elasticity is there-
fore greater. It appears probable from the preceding consider-
ations that the mesoglcea plays the chief part in the inrolling
about the cut surface as well as in regions distant from it.

Objection to this view may, however, be made on the ground
that a tonic muscular contraction resulting from the injury is not
only a possible but a much more probable cause of the inrolling.
A brief consideration of the facts is sufficient to show that the
inrolling cannot be explained as the result of muscular contraction.

This is evident first from the fact that it occurs in all directions,
longitudinally and obliquely as well as transversely. If it were
the result of muscular contraction we should expect it to occur
only transversely since the body-wall contains only longitudinal
muscles. It is difficult to understand how the contraction of
longitudinal muscles could account for the inrolling of a longi-
tudinal cut margin, since the muscle fibers are parallel to the
cut. Moreover, there is no apparent reason why the inner por-
tions of the muscular layer should contract more strongly than
the outer, since all must be equally affected by the injury. And
finally, observation renders it very evident that the inrolling is

FORM REGULATION IN CERIANTHUS. 6 1

not due to muscular contraction, for although strong contraction
occurs at the time of section the muscles relax within a few
moments and before inrolling begins. Moreover, the muscular
contraction consequent upon stimulation of pieces after inrolling
has occurred causes in most cases a more or less complete un-
rolling, provided the inrolling was in the transverse direction but
does not affect inrolling in the longitudinal direction. In the
light of these facts it is difficult to escape the conclusion that the
inrolling is caused by some part of the body-wall axial to the
muscular layer, viz., either entoderm or mesogloea. The delicate
cellular layer of entoderm cannot be supposed to possess any
such elasticity ; there remains therefore only the mesoglcea.

It now remains to consider whether the different forms of in-
rolling described and figured above are all explicable on the
basis of elasticity of the mesogloea. For this purpose we may
regard the tension as resolved into longitudinal and transverse
components.

As regards the inrolling of cylindrical pieces with transverse
cut margins (cf. No. I of these studies, '03^, also Figs. I and 2
of the present paper), it is easy to see that it can proceed only
a certain distance. Since the elastic tension is present in all
parts of the cylindrical piece reduction in size may occur, but the
cut ends are the only regions where marked change of form can
take place. These are bent inward until the more prominent
folds come into contact and the size of the opening is reduced.
Beyond this the inrolling cannot go since contact between dif-
ferent parts of the margin and the radial folds into which the
contracting margin is thrown both oppose further change. The
appearance of the radial folds requires a word of explanation.
Their presence would seem at first glance to indicate that elastic
tension exists only in the longitudinal direction. A brief con-
sideration will show, however, that this is not the case. After
escape of the water from the enteron and collapse of the body,
reduction of the circumference occurs throughout the whole
piece, undoubtedly in consequence of the elasticity of the body-
wall. There is, however, no physical ground for greater con-
traction in the transverse direction at the ends than elsewhere
since there is no break in the transverse continuity of the body-

62 C. M. CHILD.

wall. The longitudinal component must cause inrolling at the cut
end until either the local tension due to the formation of radial
folds in consequence of the in rolling or the mutual contact of
appressed portions of the inrolled wall opposes a resistance equal
to the elastic tension. It is probable that in the oral half of the
body, the mass of mesenteries and mesenterial filaments also
oppose more or less resistance to the inrolling margins.

The flattening of the piece which often occurs (Fig. 3) is simply
the result of gravity. If the collapsed piece lies on one side
during several hours the weight of the body-wall is sufficient to
bring about the flattening to a greater or less extent. In con-
sequence of the flattening the openings at the ends are frequently
elongated in the plane of flattening and slit-like, the inrolling
occurring chiefly on the two margins of the slit.

In pieces from the extreme aboral end the inrolling at the cut
margin may proceed much further (Figs. 4-6). Here the body-
wall and especially the muscular layer is much thinner and must
offer much less resistance to the elastic tension. Moreover, the
enteron is practically empty in this part of the body. In con-
sequence of these conditions the inrolling may proceed so far
that portions of the margin are directed orally (Fig. 6).

The closure of a lateral cut by bending of the whole piece
(Figs. 79) especially resembles a definite adaptive reaction, but
can be explained as the result of elastic contraction. A cylindri-
cal piece such as Fig. I does not become bent or curved so long
as elastic tension on opposing sides of the body is equal. If in
any way the tension on one side be reduced in effectiveness the
body must bend toward that side. A transverse cut through the
body-wall on one side, or the removal of a piece as in Fig. 7
interrupts the continuity of the body-wall. The longitudinal
component of the elastic tension acts on the parts above and
below the cut and causes contraction and inrolling of their edges.
But by removal of a piece of the body-wall an open space is left
and the longitudinal component of tension on the opposite side
of the body causes bending of the piece so that the concave sur-
face is on the side of the cut. The larger the piece cut out from
the one side the greater will be the bending since it will continue
until contact between the cut margins affords a resistance equal
to the opposing tension.

FORM REGULATION IN CERIANTHUS. 63

Figs. 10-21 require little explanation. Here transverse con-
tinuity is interrupted by a longitudinal cut on one side. The
form of the piece after inrolling is at least in large part a matter
of chance, being dependent upon the relative rapidity with which
the different margins roll inward.

In Fig. 1 1 the inrolling at the ends has been less than in
Fig. 14 and the resulting form is different. In Figs. 1721 the
spiral form is due simply to the fact that one longitudinal cut
margin rolled inward somewhat more rapidly than the other.
An oblique spiral results from more rapid inrolling of the longi-
tudinal margin near one end. It is clear that various conditions
such as the degree of contraction of the muscles of a certain part
of the body, the resistance offered by the mesenteries, the posi-
tion of the piece in the aquarium, etc., may constitute conditions
affecting the result.

The frequent rolling about a transverse axis of longitudinal
strips cut from the body is clearly the result of the predominance
of the longitudinal component of tension. It is interesting to note
that this transverse rolling occurs only when the muscles are
fully relaxed. If the piece be stimulated sufficiently to cause
strong muscular contraction more or less complete unrolling
often occurs. Pieces of this sort frequently roll about a longi-
tudinal axis after cutting while the muscles are more or less con-
tracted and then as the muscles relax after a longer or shorter
time begin to roll transversely and continue until completely
rolled up in a single or double spiral.

In cases of spiral or transverse inrolling (Figs. 17, 19, 23, 24)
there is little resemblance to an adaptive reaction. As will
appear, typical regeneration is impossible in these cases. Since
it is scarcely to be supposed that in pieces of a certain form the
reaction is adaptive in nature while in pieces of other forms it is
due merely to elasticity it is preferable at least to attempt to
analyze the apparently adaptive reaction. In the present case I
think the analysis has demonstrated that the various methods of
inrolling are all explicable on the basis of elastic contraction of
the mesoglcea. The apparently adaptive character of the inroll-
ing in cylindrical pieces where it results in more or less perfect
closure of the ends is due to the particular physical conditions

64 C. M. CHILD.

present in such pieces. The inrolling of pieces after section is
not then a definite reaction adapted to close the wound, except
in so far as we may regard the presence of an elastic layer in the
body-wall as an adaptation.

After the inrolling is completed gradual reduction in the size of
the whole piece continues until the artificial openings are closed
by new tissue or otherwise and the water pressure is again estab-
lished in the enteron. This reduction in size can scarcely be due
to the loss of tissue in the absence of food, for that is much less
rapid. The piece appears to contract continuously after collapse
and closure and if the closure and distention with water is pre-
vented in any way, becomes much reduced within a few days.
Frequently new wrinkles or folds appear as the contraction pro-
gresses, indicating that it is not due to actual loss of material
but to some other cause.

There can be no doubt that this reduction in size of collapsed
pieces is simply a continued reduction in the surface area of the
tissues resulting from mechanical conditions. It is due at least
in part to the elasticity of the body-wall (or especially of the
mesoglcea). This being effective in all directions must cause
gradual reduction in size of the whole after the inrolling of the
margins is completed, unless it is counterbalanced in some way,
which is not usually the case. This quality of the body-wall is
remarkable ; pieces kept under conditions where distention with
water is impossible often contract to half the size after section
and collapse. If they are then permitted to close and become
distended with water they may again attain in two to three days
almost the original size, provided the period during which they
remained contracted was not too long. The longer the period of
collapse the slower and less complete is the return to the
original size. These facts indicate that in the absence of the ten-
sion due to internal water-pressure the tissues gradually rearrange
themselves in accordance with the altered physical conditions.
There is no return to the " normal " form unless mechanical con-
ditions once more become normal.

In his study of Ccriantlius, Loeb ('91) has attempted to explain
the collapse of tentacles and other phenomena by loss of turgor in
the cells. As I shall show in a later paper, this explanation is

FORM REGULATION IN CERIANTHUS. 65

wholly incorrect. The question as to whether osmotic phenom-
ena play a part in the changes above described requires, however,
a moment's consideration. As regards the inrolling after section
and the reduction in size of the collapsed pieces there is certainly
no reason for supposing that it is due to changed osmotic condi-
tions. It is difficult to understand how, in a form like CcriantJius
section of the body-wall at one level should cause changes in
turgor in the cells of the whole piece or of those at a distant
region unless we suppose that special stimuli producing these
changes arise from the region of the cut. If this be the case
then the change is not primarily osmotic but reactive. More-
over, the phenomena are so obviously due to elasticity that the
search for any other explanation is clearly unnecessary.

THE ROLE OF THE SLIME SECRETION IN THE CLOSURE OF THE
ENTERIC CAVITY AFTER SECTION.

As has been shown, the inrolling of the margins of the piece
under ordinary conditions approximates the various parts of the
cut surface, and thus reduces the size of the opening. The radi-
ating wrinkles and folds into which the inrolling portions are
thrown and the frequent protrusion of parts of the mesenteries
through the opening render the closure by contact imperfect.
There are always slits and angles between the various parts,
through which the enteric cavity is in communication with the
exterior.

In spite of this fact I have often found pieces distended with
water, before any closure of the ends by new tissue has occurred.
A series of experiments in which the body-wall was sectioned
transversely at some level and the oral portion, still bearing ten-
tacles and disc intact, was used, will serve to illustrate this point.
In every case collapse of the tentacles and body occurred imme-
diately after section, owing to the escape of water from the en-
teron, but very frequently the whole oral piece including the ten-
tacles was again distended with water in less than an hour.

O

Examination of the aboral cut end in such cases showed that
inrolling and approximation of the margins had occurred, but
frequently distinct spaces between the wrinkles could be observed
opening into the enteron. If the end were spread open with

66

C. M. CHILD.

needles collapse occurred at once, but was followed by renewed
extension in a short time. Pieces with tentacles and disc intact
show these changes much better than others, since the phenom-
ena of distention and collapse are especially conspicuous in the
tentacles. Moreover, in these pieces the presence of the mouth
permits much more rapid entrance of water than is possible in
pieces with oral end removed, since in these latter there is no
apparatus for forcing the water into the enteron. The rapid
distention of pieces under the conditions described is made pos-
sible only by the ectodermal slime secretion which under normal
conditions forms the tube.

The manipulation incidental to section of the body and the
stimulus of the cut itself cause a rapid secretion of this slime
during the operation and for some time after. The secretion is
tenacious even when first formed and clings closely to the body.

After inrolling has occurred at a cut surface only ectoderm is
visible from without and where different parts of the inrolled
margins are in contact the contact is usually in part ectodermal.
The slime is secreted over this inrolled portion and forms a
tenacious coating which closes all the crevices between the in-
rolled portions of the body-wall. Thus, so far as the escape o
water in appreciable quantities is concerned, the cut end may be
closed within a short time after section, almost as soon, in fact,
as the inrolling is completed.

If the piece is left undisturbed the slime accumulates and the
closure becomes more and more complete until finally the thin
membrane of new tissue constitutes the definitive closure. If at
any time before the definitive closure the slime be carefully
removed with needles without causing violent contraction or
changes in form of the piece, collapse will occur at once, showing
clearly that the slime alone prevented the escape of the water.

THE GROWTH OF NEW TISSUE FROM THE CUT SURFACE.

The closure of the ends by new tissue was briefly described in
the first paper of the present series, but the conditions which
determine it were not discussed.

As described, the course of the process at both oral and aboral
ends in typical regeneration is as follows : First the appearance of

FORM REGULATION IN CERIANTHUS. 6/

a thin membrane of new tissue between those regions of the cut
surface which are sufficiently approximated ; the growth of this
membrane until the whole opening is closed ; the increase in
size of the area of new tissue as the piece becomes distended.

During the course of my experiments it was found that certain
definite conditions are necessary for this growth of new tissue.

Mention was made of the fact that the new tissue appears first
in the folds and wrinkles where two cut surfaces are most closely
in contact and that from these regions it extends until closure is
complete. In pieces rolled spirally (Figs. 17, 19, 24) or in any
such manner that the cut surfaces are not brought into contact
no appreciable growth of new tissue occurs ; the cut edges heal,
but may remain without further change for months.

Thus, in these spirally or transversely rolled pieces typical
regeneration of new tissue from the cut surface does not occur.
Moreover, this is true of all cases in which there is no approxi-
mation or contact of two cut surfaces or parts of a cut surface.
Never is a thin membrane of new tissue found growing out from
a cut surface and without other connections. When present it
always connects two cut surfaces or the two sides of a fold where
different regions of the cut surface have been approximated.

This is a point of considerable importance ; indicating as it does
that there is nothing in the cut surface itself which initiates re-
generation, the necessary condition being found rather in the
relations of different cut surfaces or their parts. Never do we
find regeneration of the body-wall occurring in the manner
represented in the diagram, Fig. 25, as a continuation with free
margin of the old tissue. New tissue arising from cut surfaces
always appears between two cut surfaces which are in contact or
closely approximated as in Fig. 2. These surfaces become
united by new tissue which then increases in amount under cer-
tain conditions, thus forming a thin membrane connecting the two
parts of the cut surface. In the ordinary closure of the end of a
cylindrical piece the new tissue first appears, as has been noted,
in the folds and wrinkles where parts of the cut surface are
closely approximated (Figs. 68, '03^), but from this it spreads
rapidly until the whole space is covered and the end closed
(Fig. 9, '03).

68 C. M. CHILD.

The process is briefly as follows : After exposure of a cut sur-
face some slight proliferation occurs which results in healing
unless another cut surface be so near that the cells arising from

o

both are in contact ; if this is the case then organic union be-
tween the two cut surfaces is rapidly established. In the closure
of the ends of cylindrical pieces this process is usually completed
in a few days, but in certain other cases it may proceed much
more slowly. For example, in pieces which are split down one
side (Fig. 10) and in which both of the longitudinal cut margins
roll inward as in Fig. 11, the process of closure often requires
two months or more for completion. In such cases the longitu-
dinal cut margins usually roll inward so far that they are not in
contact. At one or both of the ends, however, the closure may
occur in nearly the typical manner. From the end the new
tissue begins to grow along the longitudinal cut, and as it grows
actually draws the cut edges together to a certain extent. The
process may be compared for the sake of illustration to that of
sewing up the longitudinal slit in the piece from one or both ends.
If we take, for example, a case where two cut surfaces are in con-
tact at one point and diverge at an acute angle from this point,
we find that the growth of new tissue always begins at the point
of contact. From this point growth and the formation of a thin
membrane continue for a certain distance along the diverging
cut surfaces, the extent of the membrane depending in a given
species on the angle of divergence of the surfaces. This thin
membrane is itself somewhat elastic and so tends to approximate
the cut surfaces in greater or less degree unless opposed by other
conditions. The approximation of the surfaces renders possible
a further extension of the membrane between them, and so the
process continues unless at some point the cut surfaces are so
situated that the elasticity of the new tissues is insufficient to bring
them into contact, or near enough to permit the extension of the
thin membrane between them. In such a case the process of
closure must cease, as often occurs. That this is actually what
occurs I have convinced myself by repeated examination of speci-
mens cut in such manner that at least some parts of the cut sur-
faces were not in contact while others were. The diagrammatic
Figs. 2630 will serve to illustrate the process. Fig. 26 shows

FORM REGULATION IN CERIANTHUS.

6 9

a cylindrical piece slit down one side and represented as cut
across obliquely to show the separation of the cut surfaces ;
the cut surfaces have rolled inward and the oblique section at the
lower end of the figure shows that the inrolling along the longi-
tudinal cut is so great that the cut surfaces are not in contact.
The growth of new tissue and closure begins at the oral end (it
may begin at the aboral end also) where the cut surfaces are
much more closely approximated and the numerous folds afford

FIG. 25.

FIG. 27.

FIG. 30.

FIG. 28.

FIG. 26.

FIG. 29.

FIG. 3 r.

various points of contact. From this region it gradually extends
along the longitudinal cut, drawing the cut surfaces together as it
proceeds, as is shown by the two transverse sections, Figs. 28
and 29, Fig. 28 from a level where union has already occurred
(the upper transverse line in Fig. 27) and Fig. 29 from a level
just beyond where the cut surfaces have united (the level of the
lower transverse line in Fig. 27). In Fig. 28 the margins have
been drawn together and united, in Fig. 29 they are not yet in
contact but are nearer together than at the level of the oblique
section at the lower end of Fig. 27. In Fig. 30 the closure is

7<3 C. M. CHILD.

finally complete. In these figures only one end has been shown ;
usually, however, closure proceeds from both ends, and the mid-
dle portions are the last to become united.

It is evident that in a spirally or transversely rolled piece this
process can never occur, for even if it begins in some fold or local
approximation of the cut surfaces it cannot continue, because
approximation of the cut surfaces to a degree sufficient to permit
their union by new tissue is impossible as long as the piece
remains spirally or transversely rolled.

Description of all possible cases of closure of pieces cannot of
course be attempted. Results depend so largely on chance, that
every piece affords, it might almost be said, a different solution of
the problem. The examination of hundreds of pieces has, how-
ever, convinced me that the essential features of the process are
those described above.

The cut surfaces appear to remain capable of giving rise to new
tissue for an indefinite period. Often closure of pieces slit open
longitudinally is completed only two or three months after sec-
tion ; yet during all this time the cut surface retains the power
of producing new tissue under proper conditions, though it has
no power to produce anything more than the proliferation con-
nected with healing, provided it is not in contact with another
surface. Union is as complete and perfect, though perhaps not
as rapid, when it occurs two months or more after section as
when it occurs within a few days.

When we compare C. solitariits and C. membranaceus we find
that in the latter species the thin membranous growth of new tis-
sue if once begun between cut edges in contact, may continue
until it forms a connecting membrane between widely separated
cut surfaces ; in C. solitaries , on the other hand, the membrane
is incapable of bridging over spaces so wide ; the new tissue
ceases to extend long before a point is reached where the cut
surfaces are so widely separated. This difference is so marked
that it raises the question as to whether there is a fundamental
difference in the conditions and method of growth of new tissue in
the two species. Figs. 6-9 of paper I. ('03^) and Fig. 31 of
the present paper (an aboral end) illustrate the closure of open-
ings in C. membranaceus. In Fig. 31 the new tissue which is

FORM REGULATION IN CERIANTHUS. /I

growing over the opening is in two parts which are advancing to
meet near the middle. The concave free margin of both portions
is noticeable. The dotted lines represent various stages in the
growth of the new tissue. It is evident that it appeared first in the
angles where two cut surfaces were in contact or very closely ap-
proximated. In C. solitarius closure by new tissue of only very
much smaller pieces is possible. I believe the explanation of this
difference is to be found in the different quality of the thin mem-
brane of new tissue in the two cases. In C. meuibranaceus it is
much thicker, more resistant, and less easily ruptured than in C. soli-
tarius. The new tissue arises at a region where the cut surfaces are
close together and may extend from this to regions wHere they are
more widely separated. As was shown above, it exerts a certain
degree of tension on the parts connected by it. As the distance
between the cut surfaces increases a point may finally be reached
where the tension is equal to the cohesive power of the tissue ele-
ments. Beyond this the new tissue cannot extend. In C. solitarius
this limit is attained with a slight separation of the cut surfaces,
while in C. membranaceus the new tissue is capable of resisting
much greater tension and so of extending over wider spaces.

The membrane extending between the two cut surfaces may be
compared with a fluid film bounded by lines diverging at an acute
angle. The film extends a certain distance from the apex of the
angle, this distance being determined with a given fluid by the
size of the angle. The free margin of the film is always concave
toward the opening of the angle. So long as the relation between
cohesion and adhesion remains the same and the angle does not
change the film can never extend beyond a certain point, since
the surface-tension will cause rupture. As the angle and surface-
tension decrease or as the adhesion increases the film will spread.
If the*arms of the angle are sufficiently pliable or capable of
movement they may be drawn together by the surface-tension of
the fluid, and thus permit further extension of the film.

In Cerianthus the thin membrane of new tissue which may be
compared to the fluid film, arises at the apex of the angle, /. e.,
where the two cut surfaces are in contact. The membrane ex-
tends along the diverging surfaces to a certain point. Its free
margin is always concave (Fig. 31, also Figs. 68 ; '03^). The

/2 C. M. CHILD.

distance to which the membrane spreads between the surfaces
depends upon its composition and the degree of divergence
of the surfaces, just as in the case of the fluid film. The
thicker membrane with greater resistance will grow farther just
as the film with less surface-tension will spread farther, other
things being equal. Even the elasticity of the newly formed
membrane is paralleled by the tension to which the fluid film is
subjected. In both cases the margins of the space may be
approximated by this tension and thus permit further spreading of
the connecting film or membrane.

The illustration of the fluid film has been employed primarily
as an analogy. It is not to be supposed that the thin membrane
growing between two cut surfaces behaves in all respects like a
fluid film extending across an angle. Yet the close parallelism
between the two series of phenomena must raise the question as
to whether after all the growth of new tissue from a cut surface in
Cerianthus may not be, at least to a large extent if not entirely,
determined by the laws which govern the behavior of fluids. The
following facts point toward this conclusion : except so far as heal-
ing of a cut surface is concerned new tissue arises from a cut sur-
face only when it is in contact with another, the thin membrane
of new tissue which is under tension spreads between diverging
cut surfaces to a certain point, beyond which no growth occurs,
unless the surfaces are brought nearer together ; the point where
growth ceases differs in different species, depending on the quality
of the membrane ; the cut surfaces may themselves be approxi-
mated by the tension of the membrane and so further growth of
the membrane made possible ; the free margin of the membrane
is always concave in the direction of growth, /. c., the margins of
the membrane extend further than its middle region. In all of
these respects the thin membrane and the fluid film behave simi-
larly. The conclusion is at least probable that the similarity in
behavior is due to the fact that similar conditions are present. I
think it probable, therefore, that the appearance and growth of
new tissue from the cut surfaces of the body of CcriantJnts is
governed, at least to a large extent, by the laws of capillarity.
Qf course the cellular structure of the tissue may complicate con-
ditions, and the thickening and structural differentiation which

FORM REGULATION IN CER1ANTHUS. 73

occur in the membrane after its formation bring into play other
factors. These need not be considered here, however. Certainly
the phenomena are far from being adaptive or teleological in any
sense although the closure of the cut might appear at first glance
to be an adaptation. It is difficult at present to see how they
can be due to anything except simple physical conditions, though
it is possible that increased knowledge may afford another explan-
ation. Provisionally then we may regard the delicate thin mem-
brane which appears in the angles between cut surfaces as pos-
sessing some of the properties of a fluid and as subject, at least in
large degree, to the laws of capillarity.

Whether these suggestions are correct or not, the two facts
above mentioned are of great importance, viz., that regeneration
of new tissue from cut surfaces occurs only when two surfaces
are in contact, and that the new tissue cannot extend indefinitely
between diverging cut surfaces but ceases at a certain point de-
termined by the angle of divergence of the two surfaces and the
(physical) quality of the membrane, /. e., is different in different spe-
cies. The only possible inference from these facts is that all con-
ditions for regeneration are not given in the living tissues them-
selves, nor in these plus the normal environment as a whole, but
that the formation of new tissue from a cut 'surface is probably
dependent upon certain simple physical conditions similar to
those which govern the existence of a liquid film between two
diverging boundaries.

Healing of the cut surface does not require these conditions ;
for this the necessary conditions, which are very probably also
primarily due to capillarity, are established by the cut itself. The
same conditions are not, however, adequate for the formation of
a membrane of new tissue from the cut surface.

In this case then the conditions for new growth and closure
of a wound are to be found, not in the absence of a certain part,
nor in the presence of a special stimulus at the cut surface, but
in simple, external, physical relations of parts. Discussion of the
bearing of these facts may be postponed to another time. Atten-
tion may be called, however, to the difficulty of reconciling these
facts with the neo-vitalistic theories of life and especially with that
of Driesch which is based upon the phenomena of form-regula-

74 C. M. CHILD.

tion and has adopted a modification of the Aristotelian idea of an
entelechy as the basis of organic form.

SUMMARY.

1. The inrolling of the margins and the closure of openings
by contact of the inrolled margins is the result of the elasticity of
the body-wall. This elasticity must be greater in the inner por-
tions than in the outer portions, in order to produce the results
observed. The facts indicate that the mesogloea plays the most
important part in this elastic contraction.

2. Openings between folds of the inrolled body-wall may be
stopped by the ectodermal slime secretion. This method of clo-
sure often occurs in pieces before the formation of new tissue
and permits the existence of considerable water-pressure in the
enteron.

3. Contact or close approximation between two cut surfaces
or parts of a cut surface is a necessary condition of the growth
of new tissue from these surfaces. A single exposed cut surface
may heal over but no further growth occurs from it.

4. The new tissue having arisen at a point of contact between
two cut surfaces is capable of extending in the form of a thin
membrane for a certain distance between diverging cut surfaces ;
the distance to which it extends is determined in a given species
by the angle of divergence of the cut surfaces, and in different
species by the thickness and quality of the membrane.

5. The new tissue rising between two cut surfaces behaves in
certain respects as if subject to the laws of capillarity.

HULL ZOOLOGICAL LABORATORY,

UNIVERSITY OF CHICAGO, September, 1903.

BIBLIOGRAPHY.
Child, C. M.

'033 Form- Regulation in Cerianthus, I. The Typical Course of Regeneration.

Biol. Bull., Vol. V., No. 5, 1903.

'O3b Form- Regulation in Cerianthus, II. The Influence of Position, Size and
other Factors upon Regeneration. Biol. Bull., Vol. V., No. 6; Vol. VI.,
No. I, 1903.

Loeb, J.

'91 Untersuchungen zur physiologischen Morphologic, I. Heteromorphose.
Wiirzburg, 1891.

AN ABERRANT LIMB IN A CRAY-FISH.

E. A. ANDREWS.

A striking aberration in the form of a third, left-walking leg
of a female Cambarus Bartoni found in class dissection in Febru-
ary, 1892, seems of enough interest to warrant its being put on
record.

A view of the anterior face of the limb (Fig. i) shows a mark-
edly forceps-like structure in addition to the usual forceps at the
end of the limb, so that there are four instead of the usual two
terminal points.

The added structure is, however, not a true forceps with one
movable finger, but a movable piece with two immobile prongs

FIG. I. Camera sketch of anterior face of left third leg of C. Bartoni. Genita

opening indicated in black.

that otherwise resemble the index and pollex of a forceps. This
is evident in the enlarged view, Fig. 2.

The real forceps in this limb is nearly normal, but on compar-
ing it with an anterior view of the third walking leg of a normal
C. Bartoni (Fig. 3), of about the same size, we may note some
differences. Thus, in place of the straight-lined articulation of
dactyl and propodite, we find the propodite presenting a hollowed,
socket-like face where the dactyl articulates. Again, while the
dactyl and the index are both normal in form, the dactyl is not
a straight continuation of the propodite, but bends down at a
noticeable angle, thus increasing the wide divergence of the
double set of tips of this limb.

The propodite departs from the usual form in being wider dis-
tally where it bears, as it were, a large protuberance that is

75

7 6

E. A. ANDREWS.

truncated to articulate with the unusual pronged structure or
second claw-like ending of the limb. There is also an abnor-
mality in the propodite, indicated in Fig. 2, and suggesting
some former injury ; it is a slight indentation upon the middle of
the anterior face.

The movements possible to the dactyl in the normal, alcoholic
specimen (Fig. 3) are a swinging of 4.11 mm. in one plane to

FlG. 2. Anterior view of terminal part of Fig. I. Camera. Zeiss 2, a.

bring about direct apposition of the tips of the forceps, with no
overlapping and also a very slight lateral movement.

In the aberrant limb the dactyl is so set that apposition is im-
perfect ; the dactyl passes the tip of the index by about .5 mm.
while the entire swing is the same as above, 4. 1 1 mm. The
actual gape of the forceps is restricted to that same amount, .5
mm. All movement in this forceps is strictly in one plane.

Next considering the monstrous, pronged structure we find
that, starting from the position shown in Fig. 2, there is a possible

AN ABERRANT LIMB IX A CRAY-FISH. 77

movement of 2.5 mm. in the plane of the real forceps, one half
of this being towards the forceps and one half away from it.
There is also mobility at right angles to the above plane, in a
general antero-posterior direction. Anteriorly this is 1.5 mm.
and posteriorly about .7 mm., a total swing of 2.2 mm. On
moving the abnormal structure and the normal dactyl as far as
possible toward one another they came just into contact. The
angle of divergence between the index and the most remote part
of the abnormal structure is greater than the extreme opening of
a normal claw.

The form of the pronged structure is remarkable for its sym-
metry : the two prongs (Fig. 2) differ but slightly in shape and
in size. But the one standing nearer to the claw is slightly

FIG. 3. Camera sketch of an anterior view of a terminal part of the third left
leg of a normal C. Bctrtoni.

thicker and less sharply pointed and it also lacks the clear, per-
forated horn-like tip present upon the other prongs as upon
normal claws. That this tip was lost by wear or by accident
seems evident from the rough surface ending the prong and from
the fact that staining liquids easily enter at this point.

Since the anterior and posterior faces of the prongs are not
alike one could not put either prong into a space of the shape of
the other prong ; the two prongs are symmetrical about a plane
between them ; they are mirror images of one another, except for
the above noted difference in form. This symmetry extends to
such details as the distribution of clusters of bristles or hairs and
to the arrangement and number of the serrations along the op-
posed faces of the prongs.

This latter detail deserves special description. These serrations
are like those along the opposed faces of the dactyl and the index
and they add greatly to the impression that the pronged structure

78 E. A. ANDREWS.

is in some sort an imitation claw. On both dactyl and index is a
long series of transverse plates closely crowded together and
freely projecting to give the serrated appearance noticeable under
a low power. Each plate is itself serrated near its tip but these
fine serrations are seen only with a higher power. As these
plates stand nearer to the posterior than to the anterior face of
the claw, they are more readily seen from a posterior view. Each
plate, like these in Fig. 4, stands obliquely transverse and is
shaped like a scalene triangle with bluntly rounded apex. It is
just below this apex that the outer and distal edge bears a series
of sharp teeth. This fine serration is on the edge that faces pos-
teriorly as well as distally and the plates overlap one another so
that the teeth could not be seen from an anterior view, such as
Fig. 2, were it not that the plates are so transparent that the
teeth can be seen through the next overlapping plate. Each
plate has a central canal that passes from the epidermis through
the length of the plate and ends at the surface in the blunt apex :
it passes by the serrations without any connection with them.
Morphologically these plates seem to be flattened setae or hairs.
In this claw there are 61 plates on the dactyl and 67 on the index ;
three or four are broken. As seen in Fig. 2 the series of plates
is longer on the index where the proximal six or seven plates are
opposed by a bare space upon the dactyl. With this exception
the plates of the index and dactyl correspond, each plate having
its duplicate in the opposite series.

In the pronged structure there are two series of serrations show-
ing this same symmetry ; some of each series are represented in
Fig. 4.

To save space the two series are drawn as if close together
while in reality the rigid prongs always held the two series far
apart (Fig. 2). The plate marked T is about the thirty-fifth one
from the tip of the prong that has a perfect terminal spine. The
edges with teeth are those nearer to the tip and also those farthest
away from the serrations of the opposite series. 1 The edge turned
toward the plate of the opposite series is smooth and free. The
third edge of the triangular plate is the line of attachment and is

1 The use of these serrated plates may be to aid in cleaning the animal rather than
to aid in preparing food by acting against the opposed series ; they may be like the
combs on the legs of certain birds.

AN ABERRANT LIMB IN A CRAY-FISH. 79

i

somewhat anterior to the free tip of the plate. The terminal
plates are smaller than the others and have a smaller number
of teeth. This number may be as high as twelve toward the
middle of the series. In all these respects the plates of the mon-
strous growth agree with those of the real claw. No difference
was found between single plates of the dactyl and the index, nor
between plates of the two prongs nor between plates of the nor-
mal and abnormal growths.

The pronged structure, however, differs from the natural claw
both in number of plates and in their arrangement at the angle.
While the normal claw has 61 and 67 plates the pronged struc-

FIG. 4. Camera sketch of posterior view of serrated plates from the abnormal pronged

structure. 2-D.

ture has 53 on the prong with a broken tip and 54 on the other.
At the angle (Fig. 2) the plates have the arrangement shown in
Fig. 5 : the terminal plates are crowded together and the two
series interfere at the angle. Plate 52 of the imperfect prong
steps out of rank and stands partly in between plates 53 and 54
of the series on the perfect prong, which is indicated by the letter
T on the fifty-second plate. The angle thus has plates of both
series carried into it till they fuse into one curved line. More-
over, these plates at the angle are not the same as the terminal
plates of the normal claw, nor do they agree in number of teeth
with plates at that distance from the tip of the normal claw.
They are evidently special terminal plates in their own series but
not directly comparable with the normal terminal plates. The
prongs are shorter than the index and the dactyl and have not
room for a full number of plates. Where they have a free edge

So

E. A. ANDREWS.

it is set with plates at the same rate as on an equal length of claw
edge. If the common base of the prongs were to be split for
about two fifths of its length and the prongs so lengthened they
could bear about seventy plates each and the prongs would be
much more like the normal claw ; still it would be necessary to
transform the present terminal plates at the angle into plates of
the right character in the new series and to make new terminals
at the new proximal ends.

But little was made out regarding the internal anatomy of this
abnormal limb either from preparations cleared and stained or
from sections ; but it was evident that the muscles in the propo-

FIG. 5- Camera sketch, 2-D, of a posterior view of the plates at the angle between

the two prongs.

dite were not arranged as in a normal propodite. At the distal
end there were two muscle masses that seemed to connect with
opposite edges of the articular end of the pronged structure.
These would probably move this structure up and down in the
plane of the claw. The usual muscles of abduction and adduc-
tion seemed to be developed, but attached to the dactyl in an
abnormal way in connection with the above extra muscles and
with abnormal widening of the distal part of the propodite.

The gist of the above description is that this abnormality is a
case in which the propodite is to some extent double and bears a

AN ABERRANT LIMB IN A CRAY-FISH. 8 I

normal claw as well as a pronged structure that simulates a claw
even in details and was probably movable after the manner of a
dactyl. This pronged structure is remarkable for its symmetry.

Comparing this with other described cases we find in the first
place that it is unusual in being upon a walking leg. Of the
thirty-one cases of abnormal appendages quoted by Bateson, 1 two
are of antennae, four are of non-chelate legs, and all the rest of
chelae except one, which is of a chelate walking leg.

Of the eleven additional cases given by Herrick 2 only two are
of walking legs. However, this relative infrequency of described
abnormalities in walking legs may be due, in part, to the greater
ease with which other cases are collected or noted.

In the second place, it is unusual in being a monstrosity of the
propodite. Bateson found the greater number of cases of repe-
tition of parts, in the Crustacea, are repetitions of extra dactyls
upon a normal dactylopodite (some fifty cases), and that next in
frequency are the cases of extra index upon a normal index (some
fifteen cases).

In the third place it seems to fall into none of the four cate-
gories established by Bateson, but rather to be like the excep-
tions, of which he found only two.

It has, however, resemblance to the case 815 of Bateson and
more to the case shown in Fig. 195 of Herrick and still more to
the case shown in Fig. 2 of Faxon, 3 which was described as
follows : " This leg is provided with two chelae. One of them
has the ordinary form and structure, but is bent at a strong angle
with the long axis of the leg. The second appears to have been
budded off from an amputated surface of the propodite. It con-
sists of two fingers which have the form of the normal dactyl us
and index, but neither is articulated with the other at the base.
The two fingers together seem to be morphologically equivalent
to a single segment, and represent a two-branched supernumerary
dactyl us."

Though the pronged structure we have described is markedly
like a claw in its symmetry yet any tentative attempt to interpret

1 Bateson, " Materials for the Study of Variation," 1894.

2 Herrick, "The American Lobster," Bull. U. S. F. C., 1895.

'Faxon, "On Some Crustacean Deformities," Bull. M. C. Z., VIII., 1880.

82 E. A. ANDREWS.

it morphologically would seem to meet with more difficulty in
assuming it to represent a claw than in assuming it to represent
two fused dactyls or a branched dactyl. Were it a claw with
fused articulation of dactyl and index we would have a limb so
doubled distally as to have an extra segment and a lack of coin-
cidence between the two series of segments. A propodite would
spring from propodite instead of from a carpodite ; and if we
bear in mind the partial double appearance of the propodite and
regard it as a fusion we would have a carpodite and a propodite
springing from a carpodite, and so on.

Bateson's thorough study led to the conclusion that almost all
cases could be interpreted as repetitions of claws in which there
was more or less suppression of index or of carpus. The
pronged structure would then be regarded as two party fused
dactyls placed face to face and we would expect to find some
representative of the two indices. On the line of imagined fusion
there is a slight eversion of membrane where the pronged struc-
ture articulates with the propodite, but there is no reason for
regarding this as of any morphological significance.

In the case described by Faxon, as quoted above, Bateson
thought he had found a representative of the required indices in
a small protuberance shown in Faxon's Fig. 2 ; this however
was an error for I am informed by Faxon that " the artist un-
fortunately represented a protuberance which does not exist."

There are thus two cases in which pronged structures have
nothing with them to countenance the idea that they represent
double dactyls with even traces of double indices. Moreover, it
will be seen from the above Fig. 2 that the prong nearer to
the dactyl is not a mirror image of that dactyl but that it rep-
resents the index and likewise the other prong is not a mirror
image of the index but represents the dactyl ; this is true since
all have their serrations nearer to the posterior face than to the
anterior face. There is thus a departure from Bateson's rule of
symmetry and we have to deal with a very unusual abnormality
that is not interpretable in the same way as most of those hitherto
known.

But any morphological interpretation seems somewhat prema-
ture and unsatisfactory in the lack of more knowledge of the

AN ABERRANT LIMB IN A CRAY-FISH. 83

mode of formation of such structures. The appearance of the
limb suggests a new growth following some injury in which the
material for claw making was partly severed and displaced.
This might happen, we can suppose, not only in the egg and in
the young, but in the adult, especially at the periods of shedding
when the interior of the claw is soft and the blood peculiar.
That limbs may regenerate from a peripheral wound was shown
by Herrick for the tips of the claws and by Morgan l for large
parts of the limb. Possibly then such a monstrosity as this
might arise in regeneration following an injury to the propodite.
An attempt to get experimental evidence resulted in failure,
but this is what would be expected from the rarity of such mon-
strosities and from the difficulties in keeping the material long
enough. In that attempt 103 mature Canibanis affinis were oper-
ated on in February. In each a deep cut was made in the
carpodite of each chelate walking leg at a point corresponding to
the pronged structure in this abnormal limb. In ten days many
had healed, some could again use the dactyl and some had
dropped the parts peripheral to the cut. Subsequently a piece
was removed where the cut had been made in order to prevent
such rapid healing. The breeding season then came on and
after some months all the specimens had died without shedding
and no new formations were found.

1 Morgan, " Regeneration of the Appendages of the Hermit Crab and Crayfish,"
Anat. Anz., XX., 1902.

THE REACTION-TIME OF GONIONEMUS MURBACHII
TO ELECTRIC AND PHOTIC STIMULI. 1

ROBERT M. VERKES.
CONTENTS.

PAGE.

Problems and Methods 84

Reaction-Time to Electric Stimuli 85

Reaction-Time to Photic Stimuli 86

Relation of Reaction-Time to Region Stimulated 88

Reaction-Time of Tentacles 89

Relation of Quality of Stimulus to Time of Reaction 91

Absolute and Relative Variability 92

PROBLEMS AND METHODS.

The reaction-time method as applied to the study of the func-
tioning of the nervous system has already given us certain im-
portant facts in human neuro- physiology, and it promises much
more valuable results when its application to representatives of
the various animal phyla makes a comparative survey of the time
relations of neural processes possible. The value of reaction-
time studies lies chiefly in the knowledge which they give us of
the biological significance of the nervous system. " Certainly
they are not important as giving us knowledge of the time of per-
ception, cognition or association, except in so far as we discover
the relations of these processes, and the conditions which are most
favorable for them. To determine how this or that factor in the
environment influences the activities of the nervous system, and
in what way system may be adjusted to system or part process
to whole is the task of the reaction-time investigator."

For the reaction-time measurements which furnish the ma-
terial of this paper chronoscopic methods were employed. All
reactions to light were measured by means of a stop watch read-
able to tenths of a second, but for the reactions to electric stimuli,
which were very much quicker, it was necessary to make use of

1 From the Marine Biological Laboratory, Woods Holl, Mass.

2 Yerkes, Robert Mearns : "The Instincts, Habits and Reactions of the Frog."
Harvard Psychological Studies, Vol. I., 1903, p. 509 {Psychological Review Mono-
graph Supplement, Vol. IV.).

84

REACTION-TIME OF GONIONEMUS MURBACHII. 85

an instrument readable to hundredths or thousandths of a second.
For this purpose a Hipp chronoscope, readable to thousandths,
was placed in circuit with the stimulus electrodes and the reac-
tion-key. The electrodes were connected in such a way that the
chronoscope circuit was made, and the record thereby started, the
instant the stimulus circuit was completed. The motor reaction
of the medusa in response to the electric shock served to break
the chronoscope circuit, thus stopping the record. The experi-
menter was then able to read from the chronoscope dials the
time which intervened between stimulus and reaction (reaction-
time). Cattell's falling screen served as a regulator for the
chronoscope. 1

The reaction-key used in these measurements of the time of
reaction to electric stimuli consisted of a frame for the support
of an easily sliding rod, one end of which carried a cork disk and
the other a platinum point by which the circuit was completed.
The movement of the medusa against the disk when a stimulus was
given, caused the rod to slip upward, thus breaking the chrono-
scope circuit.

REACTION-TIME TO ELECTRIC STIMULI.

Gonionemus reacts to an electric current, indirectly applied, in
from one to five seconds, according to the strength of the stimu-
lus, and the position of the electrodes. The following averages
indicate the facts. Since it was not possible to get more than
four or five satisfactory reactions in series with any one animal,
the averages, unless otherwise marked, are for five reactions.

I. Reactions to a 4-Mesco-cell current, with electrodes on
opposite sides of the bell, not in contact with the organism.
M. 1.023 sec. ; M.V. 0.168 sec. ; R.V. 2 16.0.

II. Same, with 2-cell current. M. 1.489 sec.; M. V. 0.199
sec. ; R. V. 13.4.

III. Reactions to a 4-cell current, with electrodes 5 mm. apart
in contact with the margin of the bell. M. 0.605 sec. ; M. V.
0.128 sec. ; R. V. 21.

Repetition of the 4-cell stimulus at intervals of a minute causes

1 For fuller description of the chronoscopic method used see Harvard Psycholog-
ical Studies, Vol. I., pp. 601-605.

2 R.V. = (M.V. X I0 ) / M - = Relative Variability.

86

ROBERT M. YERKES.

a rapid lengthening of the time of reaction. Thus : first reaction,
.506 sec. ; second, 1.003 ; third, 3.607.

As compared with the reactions of the small medusa Gonione-
j/n/s, those of the jelly-fish Cyanca arctica are slow. Some indi-
vidual reaction-times of a single individual (Cyaned) to a four-cell
current, with electrodes in contact with opposite points on the
margin follow.

Reaction.

Reaction-Time.

Deviation from Mean.

I

.026 sec.

.504 sec.

2

.987

457

3

.3 2 4

.206

4

.636

.106

5

.629

.099

6

.760

.230

7

.2OO

330

8

1.328

.202

9

I.SOO

.270

10

1.610

.080

Mean.

1.530 Mean variation. .248

Relative Variability 16.2.

REACTION-TIME TO PHOTIC STIMULI.

The reaction-time of Gonionemus to increase in light intensity,
as I have stated in another paper, 1 varies with the strength of the
stimulus, temperature, condition of the organism, etc., from one
to thirty seconds. To daylight the organism usually responds in
about seven seconds ; to sunlight the reaction is at first much
quicker, but it rapidly lengthens as the organism is exposed to
the influence of the intense light. The relation of time of reac-
tion to intensity is indicated by the following averages : Weak
daylight, 9.4 sec. ; daylight, 7.0 sec. ; sunlight, 5.5 sec.

Moreover, the reaction-time varies with the size, sex, and pig-
mentation of the individual, as well as with such external condi-
tions as temperature, density, and chemical constitution of the
medium. Increase in temperature gradually shortens the time
from about 8-9 sec. at 19 C., to 2-3 sec. at 32 C. Decrease
in temperature lengthens the time, until reactions fail entirely at
about 10-12 C.

1 Yerkes, Robert M., with the assistance of James B. Ayer, jr.: "A Study of the
Reactions and Reaction-Time of the Medusa Gonionema miirbackii to Photic
stimuli." Amer.Journ. Physiol., Vol. 9, 1903, pp. 279-307.

REACTfON-TIME OF GONIONEMUS MURBACHII.

Between these reaction-times of the Coelenterata and those of
most vertebrates, as well as of many invertebrates, there is a strik-
ing difference in rapidity. Whereas, the jelty-fish and medusa re-
spond to an electric stimulus in from one to four seconds, the fish
or frog responds in a fraction of a second, usually not more than
one fourth, and sometimes one tenth. Observe the reaction of
the fiddler crab to a shadow, and note how quick it is in com-
parison with the reaction of Gonionemus to the same change in
illumination. Is this difference in reaction-time due to a differ-
ence in sensitiveness (/. r., is the latency period of stimulation
longer) ; is there a difference in the rate of impulse transmission,
of central nerve processes, or of muscle contraction ? Such ques-
tions should be answered by means of reaction-time investigations.
The rate of impulse transmission (presumably nerve transmission)
is much slower in the medusa Gonionevnis than in the vertebrates
and most invertebrates thus far studied. Furthermore, it differs
for different regions of the medusa ; the margin and the radial

Frog

Medusa.

Electric
Stim.
Intensity.

M.

M. V.

R. V.

Light
Intensity.

M.

M. V.

R. V.

I

2
4

.301 sec.
.231 "
.103 "

.085 sec.

-034 "
.012 "

28.2

14-7
ii. 6.

Weak daylight.
Daylight.
Sunlight.

9.4 sec.
7.0 "

5-5 "

3.16 sec.
2.39 "
1. 60 "

33-6

34-1
29.0

canal regions transmit impulses much more rapidly than do the
inter-radial regions. The exumbrellar layer of tissue, so far as
I have been able to determine, does not transmit impulses at all. *
The frog reacts to such an electric stimulus as was applied to
Gonionevius in . i 50 .200 sec. In comparison the medusa's reac-
tion-time is very long ; but it differs in yet another respect it
is far more variable. The reaction -times and variabilities of the
reactions of frogs to three intensities of electric stimulation as
determined in an experimental investigation - are here given for
comparison with the results given by Goiiioncinus to three inten-
sities of light.

1 Yerkes, Robert M. : "A Contribution to the Physiology of the Nervous System of
the Medusa Gonionema Murbachii. Part II. The Physiology of the Central Nervous
System,' 'Amer. Jour. Physiol., Vol. 7, 1902, p. 193.

2 Harvard Psychological Studies, Vol. I., 1903, pp. 616-618.

88 ROBERT M. YERKES.

This table shows that the relative as well as the absolute vari-
ability is higher for the medusa than for the frog. In general it
is true that variability increases with increase in the time of reac-
tion. Stimuli or .intensities of stimulation which give extremely
short reaction-times may be expected to give low indices of vari-
ability ; similarly animals which are slow in reacting exhibit high
degrees of variability. The reflex reaction is absolutely and rela-
tively the least variable among the common types of action ; the
instinctive reaction is much more variable, and most variable of
all in time of execution as also in form, is the voluntary reaction
so-called

RELATION OF REACTION-TIME TO REGION STIMULATED.

As might be expected the reaction -time of Gonioneinits varies
with the region stimulated. When the electrodes are placed in
contact with the margin at the bases of the radial canals the re-
action is noticeably quicker than when the inter-radial regions or
other portions of the bell are stimulated. The average reaction-
time to a four-cell current applied to the inter-radial portions of
the margin is .605 second ; for the radial canal regions it is .507
second. It is not necessary, however, to make measurements to
thousandths or even hundredths of a second to exhibit this fact ;
stimulating different regions and simply watching the responses
will make clear the differences in reaction-time. Again the time
of reaction to light varies according as the light falls upon the
subumbrellar or the exumbrellar surfaces. It is much shorter
when the subumbrella is exposed to the light (3.4 seconds as
compared with 17.4 seconds for the other position). 1

It is not at all likely that the differences in reaction-time here
noted for electric and photic stimuli are due to the same condi-
tions. The quicker reaction to stimulation of the radial canal
regions is doubtless due to the higher transmission rate of the
differentiated nerve tracts along the radial canals. Stimulation
of any other portion of the bell causes reaction less quickly sim-
ply because the tissues transmit impulses less rapidly, since they
possess less highly specialized nerve tracts. In case of the
quicker reaction to light when the medusa is resting with the

1 Anier. Jour. Physiol,, Vol. 9, 1903, p. 301.

REACTION-TIME OF GONIONEMUS MURBACHII. 89

subumbrellar, instead of the exumbrellar, surface toward the light,
a difference in sensitiveness is apparently the cause of the dif-
ference in reaction-time. Certain organs which are especially
sensitive to light are found on the subumbrellar surface of the
margin, and it is when they are most fully exposed to the action
of light that the organism responds most promptly to the stim-
ulus. The rate of impulse transmission is probably the same no
matter which surface is stimulated by light, but the latency period
of stimulation is far greater for stimulation of the exumbrella.

These experimentally demonstrable facts clearly prove that the
nervous system of the medusa Gonionemus consists of cells which
possess irritability and conductivity in high degrees. And they
further show that rapidity of reaction is directly dependent upon
specially differentiated paths of conduction.

REACTION-TIME OF TENTACLES.

A study of the reaction-time of the various parts of Gonionemus
(tentacles, manubrium, margin, bell), when normally functioning,
and when isolated by operation, throws interesting light upon
certain problems in the physiology of the nervous system.

Cutting off the tentacles close to the bell causes Gonionemus a
severe shock. If only one tentacle is cut off the usual response
is a contraction of the bell, which may occasionally lead to a
swimming bout. The severity of the shock, or as we would
commonly say, the strength of the stimulus which Gonionemus
receives from tentacle excision, evidently varies directly with the
size of the organ. Small tentacles frequently may be cut with-
out causing any visible reaction except slight contractions of the
adjacent tentacles; large tentacles cause one or more contractions,
and in general the larger the organ the greater the number and
force of the contractions. Excision of the primary tentacles,
those at the bases of the radial canals, apparently causes the
most severe shock, for when one of them is suddenly clipped off
the animal frequently swims about rapidly for a considerable
length of time. In every way its reaction is more vigorous than
those caused by excision of smaller tentacles or of large tentacles
in other positions.

It would appear from this that the radial canal tentacle is of

ROBERT M. YERKES.

special significance in the life of the medusa. And in support of
this belief it is worth noting that they are almost always held dif-
ferently from the others. When the majority of the tentacles of a
"bell up" (exumbrellar surface uppermost) individual are resting
on the bottom of the vessel, the primary tentacles are usually held
slightly higher in the water than the others. They are used for
attachment and for food seizing sooner than the others. These
facts point toward either a specialization or a modification in func-
tion which is of interest because of its bearing upon certain
neuro-anatomical facts which have been presented by Miss Hyde. 1
She finds well-defined cell-fiber tracts along the radial canals.
This being the case we should expect the radial canal tentacles to
have a more important and direct influence upon the reactions of
the organism than have the other tentacles.

When the medusa is stimulated to motion by light the tenta-
cles contract from . I .2 second before the bell. At times tentacle
reactions occur in the absence of a general bell contraction.
As determined with a stop-watch the reaction-time of the normal

I. REACTION-TIME OF NORMAL TENTACLES TO DAYLIGHT.

Tentacle.

M.

M. V.

R. V.

No. i

2.2 sec.

0.22 sec.

IO.O

" 2
" 3

3-4 "
3.6 "

0.62 "
0.46 "

18.2

12.6

General averages.

3-i-

0.43 +

13.6

II. REACTION-TIME OF TENTACLES OF EXCISED MARGIN TO DAYLIGHT.

Tentacle.

M.

M. V.

R. V.

No. I

" 2

" 3

2.5 sec.
2.7 "
2.9 "

o.i 6 sec.
1. 06 "
0.79 "

6.4

39-2
27.2

General averages.

2.4-

0.67 "

24-3

II. REACTION-TIME OF EXCISED TENTACLES TO DAYLIGHT. (AVERAGE OF

FIRST THREE REACTIONS.)

Tentacle.

M.

M. V.

R. V.

No. i

" 2

" 3

5.3 sec.
4.2 "
4-4 "

2 30 sec.
1.92 "

1-33 "

43-4
45-2
30.2

General averages.

4-5-

1.85 "

39-6

1 Hyde, Ida H.: " The Nervous System of Gonionema Murbachii," BIOLOGICAL
BULLETIN, Vol. IV., 1902, pp. 40-45.

REACTION-TIME OF GONIONEMUS MURBACHII. 9!

tentacle to the increase in light intensity caused by suddenly
uncovering a dish containing the medusa is from two to five
seconds. Reaction-time averages for three conditions of the
tentacle are given in table on page 90.

A fact significant in this connection is that the excised tentacle
rapidly loses its power to react to photic stimuli. To the first
four or five repetitions of a stimulus it usually reacts quickly,
then the time of reaction, as is shown in the series herewith pre-
sented, rapidly increases until reaction fails entirely.

SERIES OF REACTIONS OF AN EXCISED TENTACLE TO DAYLIGHT.

Reaction I 4.6

2 2.5

3 8.7

4 15-6

5 35-o

6 No reaction except to

7 , mechanical stimulation.

The reaction-time of the normal tentacle, 3.1 seconds, is con-
siderably shorter, as would be expected, than that of the bell.
Its variability is low. The reactions of the tentacles of excised
margins are slightly quicker, 2.4, according to the results pre-
sented, than are those of the normal animal, but they are also
more variable. The quickness of these reactions may possibly be
due to a temporary increase in the irritability of the margin caused
by the operation. Finally, the reactions of excised tentacles
are much longer, 4.5, and more variable than are those of either
the normal animal or the excised margin. This may mean that
the tentacle contraction in response to light is. normally initiated
by stimulation of the margin, or that the ability of the organ to
react is lessened by its separation from the bell. At any rate there
is a marked difference here indicated in the time of reaction of
isolated and normally attached organs, a difference which may
possibly be an indication of a function of the central nervous
system or of the special organs of light stimulation which are in
all probability situated in the margin of the bell.

RELATION OF QUALITY OF STIMULUS TO TIME OF REACTION.

The motor reaction of Gonionemus to increase in light is much

slower than that to other forms of stimuli. This is due in part

92 ROBERT M. YERKES.

to difference in strength of stimulus, but it is of interest to en-
quire whether the quality of the stimulus is not of importance.
We may ask, for example, whether the reaction-time to the thres-
hold stimulus of all modes of stimulation is the same. If it is
not, quality of stimulus is evidently significant. Wundt l presents
the following figures in support of his statement that the reaction-
time to the threshold intensity of all modes of stimulation is the
same.

Threshold Stimulus.

Mean.

Mean Variation.

Sound.
Light.
Touch.

337 sec.
33 1

-327

0.50 sec.

0-57
0.32

The results which I have gotten with frogs in working with
electric and tactual stimuli cause me to question the appli-
cability of this statement to the reactions of all organisms. It
seems highly probable that the just perceptible stimulus reac-
tion-time is by no means the same for different qualities of
stimulus. Those modifications of the vital processes which make
survival possible appear even in the responses to minimal stimuli.
In one case the just perceptible stimulus may cause nothing
more than a slight local change in circulation, excretion, muscu-
lar action, in another it may produce, just because of the particu-
lar significance of the stimulus for the life of the organism, a
violent and sudden motor reaction. 2

ABSOLUTE AND RELATION VARIABILITY.

As already pointed out 3 it is generally useless to compare re-
action-times with respect to variability unless the reaction-time
value as well as the absolute variability is considered. If, for
example, to an electric stimulus Gonionemus reacts in 2.0 seconds,
with a variability of 0.5 sec. ; and to a photic stimulus in 6.0
seconds, with a variability of 1.5 sec., it is not correct to say that
the reaction-time to light is three times as variable as that to
electricity. As a matter of fact the two variabilities as such are

1 Wundt, Wm. : " Grundziige der physiologischen Psychologic," Fiinfte Auflage,
Leipzig, 1903, Dritte Band, S. 428-429.

2 Harvard Psychological Studies, Vol. I., 1903, p. 625.

3 Amer. Jour. Phvsiol., Vol. 9, 1903, p. 291.

REACTION-TIME OF GONIONEMUS MURBACHII. 93

not equal, but when we consider the reaction-times we find that
the ratio of variability to reaction-time is in each case 1:4. Al-
though the absolute variability is in one case three times as great
as in the other, it is 25 per cent, of the average reaction-time in
both instances.

Heretofore I have expressed relative variability as a ratio
(M.V. : M.), or as a percentage value of the mean (M.V. = x per
cent, of M., in which case x = R.V., the relative variability.)

Obviously it is always important in comparative reaction-time
work to know the relative variability of reactions ; in fact it is
often quite impossible to make significant comparisons of results
until this value is found. For this reason I have given in every
table of this paper the percentage value of the mean variation in
terms of the mean. This value I have called the relative vari-

*

ability (R. V.). It is obtainable by the formula recently used
by Myers, ' v.c. = m.v. x loo/av. In this formula, which as
inspection shows gives the ratio (in per cent.) of m.v. to av., v.c.
is a value called by Myers the variation-coefficient, and av. is the
mean (M.). Supposing a reaction-time of .180 second to have
an absolute variability of .020 second, then by the formula
(.020 x lOO/.iSo) the variation-coefficient (Myers), or what I
prefer to call the relative variability (R. V.), is I i.i + . If we
chose this value might be written ii.i -(-per cent., thus indi-
cating that the absolute variability (M. V.) is ii.i + P er cent, of
the average reaction -time (M.).

Since Pearson 2 in this statistical work has made use of a quan-
tity which he calls the "coefficient of variability," and which is
obtained by the formula

C. V. = - - x 100,

it seems unwise to use the term variation -coefficient, suggested
by Myers, for this new quantity in reaction-time work. As the
value which we obtain by Myers' formula is in reality the per-

1 Myers, Chas. S.: "Reports of the Cambridge Anthropological Expedition to
Torres Straits." Vol. II., "Physiology and Psychology," Part II., 1903, p. 212.

2 Pearson, Karl : " Mathematical Contributions to the Theory of Evolution, III.,
Regression, Heredity and Panmixia," Phil. Trans. Roy. Soc. London, Vol. 187, A,
pp. 253-318.

94 ROBERT M. YERKES.

centage value of the mean variation in terms of the mean, I see
no reason why we should not call it the relative variability, in
contrast with the absolute variability. Thus the confusion with
Pearson's quantity which will inevitably result from the use of
variation-coefficient can be avoided.

Myers' formula gives us precisely what we need for the direct
comparison of reaction-times, with respect to their variableness,
either to different stimuli or of different organisms. Strange to
say most investigators of the time relajtions of neural processes
have paid little or no attention to the variability of their results ;
none, so far as I know, have ever determined the relative varia-
bility throughout their work. It may be objected that those who
have use for the relative variability can find it for themselves since
the reaction-time and its mean variation are usually given. But
the value is far too important to be left half-way determined ; in
fact it is even more useful in most cases than the mean variation.
Every one who has had experience in dealing with reaction-time
results will admit that the reaction-time to a particular stimulus
has different meanings according to its variability, and that it is
never possible to compare reaction-times without considering this
value. It is clear then that no reaction-time statistics sJiould be
published without determinations of the relative variabilities.

Conventionally we compare human reaction-times to visual,
tactual and auditory stimuli without noticing their variabilities
or the strength of the stimulus employed. Jastrow : in a table of
results, collected from the papers of many investigators, which is
intended to indicate the differences in time of reaction for the dif-
ferent senses gives these averages : Visual reaction-time .185 sec-
ond ; tactile, .148 ; auditory, .139. Not even the mean variability
is given in connection with the averages. Since reaction-time
varies with the strength of the stimulus it is possible by varying
the stimulus-intensity to get any one of the above reaction-times
with any of the qualities of stimulus named. This being true,
how are we to make valuable comparisons of reaction-times to
different kinds of stimuli ?

As before stated the threshold intensities of all modes of stimu-
lation may be regarded as directly comparable. No matter what

1 Jastrow, Joseph : "The Time Relations of Mental Phenomena," New York, 1890,
p. II.

REACTION-TIME OF GONIONEMUS MURBACHII. 95

the form of the stimulus, the threshold gives the longest and
most variable reaction-time which can be obtained by the use of
that particular quality of stimulus. Now, as the intensity of the
stimulus is increased the variability decreases. Why may we not
choose equality in relative or in absolute variability as a basis of
comparison ? If it should be found and I am now gathering data
for the settlement of the point that the relative variability is the
same for the threshold reaction-time to all qualities of stimuli,
equality in relative variability would be the most satisfactory
basis ; if, on the other hand, absolute variability is a constant
quantity at the threshold, it should be used in preference.

NOTE. Reasons have recently appeared for returning to the
original spelling of the name of the medusa. Gonioncnnts there-
fore is used instead of Gonionenia.

Vol. VI. February, 1904.. No.

BIOLOGICAL BULLETIN.

THE SPECIAL PHYSICS OF SEGMENTATION AS
SHOWN BY THE SYNTHESIS, FROM THE STAND-
POINT OF UNIVERSALLY VALID DYNAMIC
PRINCIPLES, OF ALL THE ARTIFICIAL
PARTHENOGENETIC METHODS.

E. G. SPAULDING.

The genesis of this paper is a twofold one. In the first place
the careful perusal of the literature which has appeared in very
recent years on the matter of the obtaining of artificial partheno-
genesis in various forms by a number of methods, and which in-
cludes also varying theories of the process of segmentation as in-
terpretations of these data, this perusal readily convinces one that
such a consistent and far-reaching synthetic view of the nature of
segmentation as known data would seem to warrant one in try-
ing to obtain is quite lacking. In fact no attempt seems to have
been made to show that all the methods employed must result
in bringing about one and the same series of physical events in
the cell preceding and during segmentation, to which the process
resulting from normal fertilization is no exception. That vital
(?) phenomena can be reduced to a purely physical basis will
doubtless be disputed as long as any details connected therewith
remain unstudied or in any way ambiguous. The absence of
such a complete reduction is in itself, however, no disproof of
the correctness of the view as a theoretical standpoint, and suc-
cess in it will at least always remain a scientific ideal. 1 A clear
and detailed demonstration that the effectiveness of the various
artificial parthenogenetic methods can be explained if it is held
that one series of physical events always occurs in the process of
segmentation would seem therefore to go a considerable way in

1 The position that this standpoint is logically necessary for biology as a science is
discussed in the author's article, " The Contrary and the Contradictory in Biology;
a Study of Vitalism," in The Monist, July, 1903.

97

98 E. G. SPAULDING.

the attainment of that ideal. To attempt to do this states ac-
cordingly the purpose of this paper.

The second genetic element was the desire to get such a uni-
tary view as at least a preliminary to and if possible a justifica-
tion of the attempt to initiate segmentation by new methods,
viz., by the application of the electrical current to the unfertilized
eggs of the starfish, and although these experiments were unsuc-
cessful, the theory, although based on the experimental work of
others, is offered for what it may be worth as an endeavor to
gain an end, the value of which in itself will not be denied.

i. EXPERIMENTAL DATA.

A brief recapitulation of the results already obtained by arti-
ficial parthenogenetic methods may, as a preliminary to subse-
quent discussion, be pardoned.

In the starfish egg parthenogenesis may be produced by : (i)
the use of HC1 ; l (2) increasing the osmotic pressure of the
surrounding medium ; 2 (3) by lowering the temperature ; 3 (4) by
mechanical agitation. 4 By the first method it is held that the
"parthenogenesis of Asterias eggs is to be produced by means of
specific (hydrogen) ions," at least this is the interpretation of the fact
that 100 c.c. of sea water plus 3-5 c.c. N/io HC1 acting for
from 3 to 20 minutes on the eggs, which are then removed, brings
about the desired result. 5 In the case of the second method, 6
although the results are stated somewhat ambiguously, the maxi-
mum number of parthenogenetic eggs seems to have been se-
cured by using 15 c.c. of 2*4 N KG + 85 c.c. of sea water at
about 23 C. for 15 minutes, then transferring. As for the third,
" e gg s f Asterias may be made to develop parthenogenetically
by exposing them for a definite length of time to a temperature
of i -/ C., in sea water, and then raising the temperature."
As an interpretation of this, we find it stated that "the produc-

1 Loeb, Fischer u. Neilson, Archiv fiir die geschichlliche Physiologie, Bd. 87, 1901.
2 Greeley, A. W., BIOLOGICAL BULLETIN, IV., 3, Feb., 1903, says that Neilson
found this method successful.

3 Greeley, A. W., Am. Jour, of Physiologv, VI., 1902, p. 296.

4 Mathews, A. P., Am. Jour, of Physiology, VI., II.

5 Loeb, Fischer u. Neilson, loc. cit.

6 Greeley, loc. cit.

PHYSICS OF SEGMENTATION. 99

tion of artificial development by lowering the temperature is
brought about by an extraction of water from the protoplasm,
just as if the eggs had been placed in a solution of higher os-
motic pressure than that of the sea water," l though no explana-
tion of the reason for this is offered. A suggestion as to this is
however made by Mathews in his comments on the fourth
method, that " the getting of parthenogenesis by agitation may
be due to a dissolution of the nuclear membrane, since the cen-
trosome originates close to the nucleus, or it may cause the eggs
to lose water like the cells of sensitive plants. The loss of water
could be caused only by lowering the osmotic pressure in the
cell, and this by decreasing the number of molecules in the cell ;
and this in turn by synthetic processes."

In other forms artificial parthenogenesis may be obtained by
similar or slightly different methods ; c. g., in Arbacia by osmotic
pressure, 50 c.c. :2 ^- TV MgG 2 or NaCl + 50 c.c. sea water, 3 and
at least a segmentation by lack of oxygen, by heat, or by ex-
posure to alcohol, chloroform, or ether; 4 in Chcetopterus likewise
by the use of KG, KNO 3 , K 2 SO 4 (2^4 N+ 100 c.c. sea water),
NaCl, MgCl 2 , CaCl 2 and sugar, 5 in Amphitntns by Ca(NO 3 ) 5
(2 c.c. N -\- 99 c.c. sea water) ;'' in Nereis by osmotic pressure,
(20 c.c. 2^/2 N KG + 80 c.c. sea water, 30 minutes), 6 in
Podarke obscura by use of the same solution. 7 As theories and
interpretations of the results obtained by these factual methods,
we find in addition to those already cited the following, which are
quoted in abstract :

" All that the spermatozoon needs to carry into the egg for
the process of fertilization are ions, Mg, K, HO or others, to
supplement the lack of the one or counteract the effects of the
other class of ions in the sea water, or both. The ions and not
the nucleins in the spermatozoon are essential to the process of

1 Greeley, loc. cit.
2 Mathews, loc. cit.

3 Loeb, J., Am. Jon?: of Physiology, Vol. III., Nos. III. and IX. and Vol. IV.,
IV.

4 Mathews, A. P., Am. Jour, of Physiology, IV., VII.
5 Loeb, J., Am. Jour, of Physiology, IV., IX.
6 Fischer, M., Am. Jour, of Physiology, VII., III.
7 Treadwell, BIOLOGICAL BULLETIN, III., 5.

IOO E. G. SPAULDING.

fertilization ; l or the spermatozoon may carry enzymes." " Either
of these two causes affects the most important qualities of life
phenomena, /. c., causes the proteids (i) to change their state, or
(2) to take up or lose water." 2 Further details as to these two
possible events are not given, however, but it is quite evident that
the two may be coincident, so that the latter change may take
place in any case.

To summarize systematically, a cell division can be caused
in various forms by one or more of the following classes of stim-
uli : (a) mechanical ; (//>) heat (or cold); (c) osmotic ; (V) chem-
ical, or, if one will, ionic ; the third for the reason that either of
the electrolytes MgCl, or NaCl, or the non-electrolyte sugar,
maybe used ior Arbacia ;* therefore no specific chemical effect is
to be accepted here. The fourth, a distinctly chemical effect, is
evident, for HC1 is effective for Asterias eggs and KC1 is not ;
so also only the Ca ion for Amphitritus. Here then it is the
kation that is considered to cause the segmentation, but that a
fundamental chemical effect different to an ionic, i. c., electrical

/

charge effect is present is shown by the fact that, keeping the
osmotic pressure and the number of charges on the kation the
same, but changing the ions, the effect is different. This is con-
firmed by the comparison of the action of KC1 and NaCl on
muscle. 4 A specific chemical effect is therefore not done away
with even if the difference in effect is reduced to a difference in
the path of the charge moving around the atom. For the cause
of this latter difference must in turn be a fundamental difference
in the atoms themselves. The same kind of proof of an irredu-
cible and ultimate chemical difference is found in the results of
Lillie's work on the effect of Na, K, Ca and Mg salts on Aren-
icola and Polygordius, and of Mat hews on the different stimu-
lating effects on the nerve of NaCl, NaBr, Nal and NaFl. This
fundamental chemical difference is related to the difference in so-
lution tension, as Mathew's work this past summer has shown.
However, not alone the stimuli, the external agents initiating

, J. , Am. Jour, of Physiology, III., III.
2 Loeb, J., Am. Jour, of Physiology, III., IX.
3Loeb, Am. Jour, of Physiology, IV., IV. and III., IX.
4 Loeb, Am. Jour, of Physiology, III., VIII.

PHYSICS OF SEGMENTATION. IOI

segmentation are to be put into the above classes, but also, with
the addition of the class, surface energy, which is of special im-
portance here, the phenomena taking place within the cell itself,
preceding and during cleavage. We accordingly consider the
cell to be a physico-chemical object, whatever else it may be, and
subject therefore to general physical principles. The special na-
ture of the physical processes that occur in it is to be demon-
strated by showing that the effectiveness of the physical methods
used for causing segmentation implies that, or at least can be ex-
plained, if by each method only one and the same series of events
is made to take place. The bringing together of results in this
way exemplifies what we have termed synthesis, and the internal
agreement with which it is identical makes for the probable cor-
rectness of our theory.

2. GENERAL PHYSICAL PRINCIPLES TO BE OBSERVED IN THE
INTERPRETATION OF THESE DAT A. 1

If the energies both within and without the cell belong to the
classes named, we must in our endeavor to get at the meaning of
the data at hand be guided strictly by the most general funda-
mental chemical and physical principles valid for those. These
principles, some of which are of course well known, may be
stated as follows :

I. The " first law " of energetics, that of the conservation of
energy. This is considered to have an experimental basis in the
fact that, e. g., a weight of one kilogram falling 424 mecers raises
the temperature of one kilogram of water I C. as indicated on
an arbitrarily selected scale. This is interpreted to mean that
the kinetic energy of the falling body is quantitatively equal to
the heat energy gained in the rise in temperature. However this
cannot be strictly proven, for the two energies are qualitatively
different, and have no common factor. It would therefore be

1 The principles as stated are to be found in no one author, but are with their criticism
the result of the study of the works of Planck, Mach, Ostwald, Helm, Wald, Riecke,
and others; some of these are as follows: Planck, " Prin. d. Erh. d. Energie " ;
Ostwald, " Vorlesungen iiber Naturphilosophie," " Allgemeine Chemie," and other
writings; Helm, " Die Energetik nach ihrer gesch. Entw. " ; Rankine, Philos. Alag.,
1867 (4); Mach, " History of Mechanics," " Warmelehre," "Pop. Lect," "An-
alyse der Empf" ; Riecke, " Lehrbuch d. Physik."

IO2 E. G. SPAULDING.

quite as logical, though not as practical to interpret the two as
quantitatively different. To interpret as equal is therefore to base
the law of conservation on an assumption not proven, yet not
disproven. 1

In the second place the law is based upon the impossibility of
a system's continuing to do work unless energy is received from
without. In this the assumption is implicitly contained that work
cannot be created ex niliilo. Were it possible, however, for a
system to receive from without the work (energy) which it itself
does, a perpetual motion would be possible.

II. The second law prevents this. In every " Ausgleichung"
or transformation (Umgleichung) of energy, some heat is pro-
duced, only part of which at best is again available, for the reason
that it tends to " dissipate " ; it cannot pass from the body of the
lower to that of the higher potential (temperature) but only con-
versely. The entropy of the universe is accordingly said to
increase. This characteristic of heat energy is a special case of
a law (the second) valid for all the energies. Its meaning is that
all events have a definite direction.

According to the first law then if one form of energy disappears
another form or forms held to be quantitatively equal to it must
appear. Energy may therefore be defined as that which in
changing conserves itself. Implicit in both the first and second
laws is the definition of it as that which does work, but that there
are objections to this is evident from the facts stated in the
second, that some energy in the form of heat with no difference
of potential (7~) cannot do work.

III. The factors of energy. Already present though unre-
cognized in the early development of mechanics, but made ex-
plicit first in thermodynamics, and later extended to all forms of
energy is the view that each is made up of the product of two
factors, a potential or intensity, on the one hand, and an extensity
or capacity factor, on the other. In heat energy these factors
are respectively temperature and entropy Qj T (specific heat), in
kinetic energy f^and MV, in volume energy (gases and solutions)

1 This procedure illustrates the necessary dogmatism of all science, and the superi-
ority of a pragmatic to a logical justification, a subject which will be treated at length
in another paper.

PHYSICS OF SEGMENTATION. 103

pressure and volume, in surface energy surface tension and sur-
face, in chemical energy chemical intensity (avidity) and mass.

IV. The law of events or action. Eveiy event, i. e., the going
over of energy from one body to another or the transformation into
another form is conditioned (<?) not by the absolute quantity of
energy involved, but by the potential factors which ($) must be
opposed to each other in direction and be of different value, i. e., a
potential difference must exist ; (c) this potential difference must
not be compensated by a third potential, i. c., must be uncompen-
sated. Unless these three conditions are fulfilled a system re-
mains in its state of equilibrium ; if they are given between a
system and its environment work is done either on it or by it,
the amount of which depends upon the product of the total ex-
tensity into the potential difference. If such conditions are held
to exist wholly within a system so that change occurs in it, this
is equivalent to dividing the system into environment and smaller
system, the limitation of which in every case is arbitrary though
for practical purposes necessary.

V. The direction of the energy transfer is always from the
higher to the lower potential, the one falling as much as the
other rises until equilibrium is reached. By this event however
a new potential difference between a second and a third energy
form may be created, and with the getting of equilibrium by this
a series of events is formed. Assuming the potential of a second
energy to have increased or to be continuously increasing, the re-
sult is that in the " Ausgleichung " between this second and a
third within the system, the second may be of the same intensity
at the end as at the beginning of this event, while the third
shows a rise in potential. This kind of event during which one
potential is kept constant is called isocyclic. In comparison to
the third the second potential presents here a relative fall. Con-
versely those events in which the extensity factor remains con-
stant and the intensity alone changes are called adiabatic.
Both kinds of changes can be brought about by manipulation of
a system which is isolated with the exception of the manipula-
tion. In natural events, however, there is always a change of
potential as well as of extensity. The process of getting equi-
librium is quite consistent with an absolute rise in two potentials

IO4 E. G. SPAULDING.

within a system in relation. to a fall in a third potential outside of
the system ; at the same time the extensity factors of the first
two may diminish. Under these circumstances work is done on
the system. Conversely, two potentials within a system may
get equilibrium and, at the same time, both decrease absolutely
in relation to a third external, which rises. The system then
does work. Both of its extensity factors may however increase
in this process, but this does not necessarily mean a gain in
energy, for energy equals potential x extensity.

Concrete instances of these possible cases, which have been
selected as bearing directly on our special problem, we shall find
in the physical events making up the process of segmentation
and cleavage.

3. SURFACE TENSION AND OSMOTIC PRESSURE.

In the instance of the normal progress of the event of segmen-
tation, i. e., with a cleavage of both nucleus and cytoplasm, no
matter whether this is the result of natural or artificial fertiliza-
tion, it is an undeniable fact that, coincident with and as a culmi-
nation of all the processes taking place in the cell there is a de-
crease in surface tension in at least certain parts of the surface.
This follows necessarily from the change in the radius of curva-
ture of the approximately spherical form of the egg to the
increased radii of certain parts of the surface of the constricted
form. For surface tension, the potential of surface energy, varies
inversely with the radius of curvature, and is greatest in the
spherical form. From this it follows that in cleavage there is in
any case an average decrease in surface tension accompanying
the redistribution of this. The very fact that these changes
occur may be advanced as a proof that in protoplasm we are
dealing with either a solution or a fluid. Accordingly the sur-
face energy is to be considered as due to or identical with the
"attracting forces " (cohesion) of the fluid particles. This redis-
tribution of surface tension may be correlated with and is doubt-
less confirmed by observed protoplasmic streamings. 1

It would not, however, occur of itself; it must have an ultimate
cause, either within or without the system (the egg), and in either

1 Cf. Butschli, "Protoplasm."

PHYSICS OF SEGMENTATION. 1 05

case is possible only if it is either the immediate result or cause of a
change in the potential of that energy which is opposed to the
surface tension, viz., osmotic, /. e., the " repelling " forces of the
substance in solution. 1 The change in surface tension is one
part of the " Ausgleichung " of the potential difference existing
between it and the potential opposing it, viz., osmotic pressure.
The necessary coexistence of these two kinds of energy in a
solution and the characteristics of each bring it about however
that when one potential decreases the other does also, although
there may be a relative fall of one and rise of the other, thus
making an isocyclic event possible. For just as the surface
tension decreases with an increase in surface and conversely, so
also is a decrease in osmotic pressure accompanied normally by
an increase in volume (and therefore of surface) and conversely.
It is evident then that in segmentation with its change of form
and redistribution of surface tension we are always dealing im-
mediately with the interrelations of two energy forms, surface and
osmotic, and mediately with any causes which may act on them,
whatever these may be. If we succeed in showing that the
various physical agents used for producing artificial partheno-
genesis can be effective only by in any case causing changes in
either one or both of these energies, then we shall have obtained
that unification of evidence which is our purpose.

4. FACTORS CONDITIONING VARIATIONS AND CHANGES IN

THESE ENERGIES.

The existence of an uncompensated potential difference within
the egg can be accounted for in two ways. It may be either
the result of changes already going on in the egg, e. g., the
becoming active of preferments 2 and so the formation of new
chemical compounds, processes of maturation, or those leading
to " natural death," 3 but this is the same as saying that such a
difference is already there and that it leads to others ; or the

1 In reply to the possible objection that colloidal solutions have no osmotic pres-
sure there is experimental evidence that they have this and are diffusible, thus showing
that there is no essential difference between these and other solutions. Cf. Hober,
" Physikalische Chemie der Zelle u. Gewebe," s. 43, et seq.

2 Cf. Hofmeister, " Chemische Organization der Zelle."

3 J. Loeb, BIOL. BULL., Nov., 1902, " Maturation, Death, etc., in Asterias."

IO6 E. G. SPAULDING.

result of action between the egg and its environment, in which
case the egg may either have done work, as, e.g., in a medium
of less osmotic pressure, or work may have been done upon it
by means of mechanical agitation, heating, abstraction of water,
or fertilization by a spermatozoon. Any of these possible events
must take place in complete agreement with the general conditions
above outlined, and will in every case of cleavage ultimately
condition a change in the relation of two kinds of energy, surface
and osmotic.

That this is true is shown by consideration in detail of the
characteristics of these two energies. Surface energy is due to
the mutual attraction of the molecules of a fluid, which here forms
one part, viz., the solvent, of protoplasm. The molecules at a
certain distance from the surface are each free to adjust their
mean position under the influence of the surrounding molecules,
the mean position being that in which each is acted upon equally
on all sides, with the result that the mutual attraction is not
rendered manifest. At the surface, however, if this be free, or
relatively free, as is shown by experiment (/. e., if a chemical dif-
ference between medium and egg exists) the molecules are vir-
tually acted upon only by those lying internal to them. The
result is a system of forces manifesting their action throughout
the fluid and at right angles to the surface, /. t\, radially and
tending to reduce the surface to the least possible area, i. e., the
spherical form. The surface in contact with a chemically different
medium so that mixture does not take place, acts like or is in
fact a membrane or film.

The factors by which variations in the attraction of these
particles for each other are determined are : (i) Their chemical
nature, and consequently (2) the density of the fluid, (3) the
temperature, a rise in which decreases the tension, (4) the pres-
ence of electrical charges.

Osmotic (volume) energy is identical with the mutually re-
pelling forces of molecules in a solvent, and follows the law of
gases. Its two factors are accordingly volume and pressure, and
in a natural event as the former increases the latter decreases.
The conditions upon which variations in this depend are accord-
ingly (i) for equal weights of dissolved substance, and equal

PHYSICS OF SEGMENTATION. I O/

volumes, chemical constitution, (2) nature of solvent, (3) elec-
trolytic dissociation, (4) heat. Changes may be brought by
chemical interaction, enzymes, heat, mechanical agitation, or
amount of solvent present.

In the egg, which is a system of coexisting energies, it is
then directly with the attracting and repelling "forces," indirectly
with anything conditioning them, that our problem is concerned.
To be sure the molecules of both solvent and dissolved sub-
stance, the solute, must be admitted each respectively both to at-
tract and to repel. In the former, however, the attracting forces
predominate as is shown by the slight effect of temperature ; the
repelling may therefore be ignored. In the latter the converse
is the case, for the same reason.

The form and surface of the egg at any time is accordingly de-
termined directly by these two energies, and indirectly by any of
the above enumerated conditions modifying them, for any uncom-
pensated potential differences which may rise through the action
of either internal or external causes must be equilibrated with a
resultant change in surface and perhaps in volume.

The valid objection may here be offered, however, that a uni-
form or average decrease in surface tension does not account for
the change in form accompanying segmentation. This can doubt-
less be met by showing that, at the same time that a decrease
takes place, a rearrangement also results because of the exist-
ence in the egg of localized chemical differences, i. e., the egg
is organized. That these exist is shown : (i) experimentally by
the different staining reactions of, e. g., the nucleic and cyto-
plasmic cells to acid and basic dyes ; * and (2) by observed mor-
phological differences and changes. Granted this chemical or-
ganization, localized changes in it and so of the osmotic pressure
necessarily present in such a solution can be brought about either
normally by the entrance of a spermatozoon, whether its action
be enzymatic or chemical, or abnormally by ions, temperature,
agitation, or abstraction of water. Differences in osmotic pres-
sure thus arising may remain localized either because of mem-
branes within the egg, /. e., of further chemical differences, or by
reason of the relatively slow diffusibility of colloidal particles.

1 Cf. the researches of Picton and Linder.

IOS E. G. SPAULDING.

The chemical changes in the solute to which they are due may
in turn be accompanied by electrolytic phenomena, and so by an
attraction of unlike and repulsion of like charges, so that with
all of these factors taken together reasonable grounds are fur-
nished for the understanding of the presence of irregularities in
the cleavage form. The fact of this constricted form and of the
possibility of its explanation on the above basis must be con-
stantly kept in mind in the subsequent considerations. If then
the final event in the series of changes leading up to segmenta-
tion is that ending with a change of form and average decrease
and rearrangement of surface tension, after which a " resting
stage" of relative equilibrium exists for at least some time, then
this series of events must have resulted from or be in part iden-
tical with a disturbance of equilibrium within the cell before the
event of cleavage. The various theoretically possible ways in
which this disturbance can be brought about must therefore be
presented systematically and in detail.

5. POSSIBLE WAYS OF CREATING A DIFFERENCE BETWEEN THE
TWO POTENTIALS CONCERNED, PRESSURE AND TENSION.

There is, first, the possibility that the surface tension may
be greater than the osmotic pressure of the moment can with-
stand, /'. e., that a potential difference in the direction of surface
tension osmotic pressure has been formed. This of course is
equalized with the establishment of equilibrium, but therewith
both extensity factors, surface and volume must have decreased
and the cell have lost water, while both intensities, tension and
pressure, however, have presented an absolute increase. A po-
tential difference in this direction might theoretically be caused in
two ways, either (i) by increasing the surface tension or (2) by
decreasing the osmotic pressure, in each case keeping the other
potential constant. Conversely, if the osmotic pressure is first
increased, e. ., by an analytic chemical process resulting from
oxidation, or if the surface tension is first decreased by, e. g.,
electrical charges, then a potential difference in the direction of
osmotic pressure --surface tension will exist. This being un-
compensated, the resulting process of equilibrating is identical
with the taking place of an increase in both extensity factors and

PHYSICS OF SEGMENTATION. 1 09

a decrease in the intensities ; but in the equilibrating of poten-
tials the decrease in the tension is less than in the pressure, for
the former is already lower ; it therefore presents a relative rise.
This scheme offers a systematic mode of procedure for taking
up the examination of the various methods of producing segmen-
tation and development. However, since we are concerned only
with the bringing about of an average decrease in surface ten-
sion, we shall use only the second class of possibilities.

A. Factors Decreasing Surface Tension Directly.

One theoretically possible way of decreasing the tension di-
rectly is by means of either a rise in temperature or by bringing
to the surface (the poles of the egg) like electrical charges.

The attracting forces, it has been seen, may be regarded as due
exclusively to the solvent, which for protoplasm, we have every
reason to believe, is only water ; consequently any direct decrease
in surface tension by changing the solvent chemically seems to be
excluded for practical reasons. But a rise in temperature lessens
the attraction potential directly at the same time that it increases
the osmotic pressure ; therewith is equilibrium done away with,
work being done on the egg from without. The pressure being
too great for the tension, the volume must increase, with absorp-
tion of water, until equilibrium is established. The extent of sur-
face therewith increases, but the potential, surface tension, decreases
absolutely, although, relative to the pressure opposing it, it rises
and the egg thus in turn does mechanical work by displacing the
surrounding medium. The direction of these events is thus de-
termined by the difference in potential.

With this theoretically possible course of events agrees exactly
the observed swelling and liquefaction of cells when heated, 1 as
well as the starting of development in Arbacia by a rise in tempera-
ture, observed by Mathews. 2 This constitutes the first example
of factual methods agreeing with our synthetic point of view, and
indicates that liquefaction consists either in increasing the pres-
sure by analytic processes, or in absorption of water, or in
both.

1 Greeley, A. W., BIOLOGICAL BULLETIN, IV., 3, 1903; V., i, 1903.

2 Ainer. Jour. Physiology, IV., VII.

IIO E. G. SPAULDING.

Such liquefaction phenomena do not of course in every case
mean, nor are they the whole of cell division, neither do they in any
case account for this except by also taking into consideration the
chemical organization previously referred to. But that such a
liquefaction takes place during segmentation is evidenced also by
(l) the greater susceptibility of the fertilized egg at that time to
ether, HC1, KG, etc., results obtained by the author in work
on Arbacia, and an account of which will appear in a subse-
quent paper, and (2) by the results obtained by Lyon this sum-
mer, which indicated a rhythm in the use of oxygen and the
giving off of CO 9 by the egg. The last is at the maximum
during segmentation, which accordingly points to a greater
splitting of molecules at that time and a consequent increase in
osmotic pressure and absorption of water.

Another theoretically possible direct cause of a decreased ten-
sion would be the presence at the surface of the egg of like
electrical charges. But as this concerns our own experimental
work \VG postpone its consideration until further on.

A. fourth possible method here would be that of mechanical
agitation, by means of which the attracting forces of the mem-
brane of the cell would be lessened ; for in a physical experiment,
at least, the form is charged, the average tension therefore de-
creased by mechanical work done from without. By analogy
this may hold good of the egg, and to it corresponds the method
of agitation used on starfish eggs. Its efficacy may be further ex-
plained if it is recalled that agitation may also result in a molecular
splitting and consequent increase in pressure. As this com-
pletes the possible cases under this class of methods we may
pass to the consideration of the other class.

B. Factors Directly Affecting the Osmotic Pressure.
We are here concerned with the establishment of a potential
difference in the direction of osmotic pressure surface tension
by directly raising the former. Two classes of theoretically
possible methods for doing this are to be considered, by bring-
ing about (i) analytic chemical changes in the solute and (2)
physical changes of pressure.

PHYSICS OF SEGMENTATION. Ill

I . Chemical Changes in Colloidal Particles by (a) Chemical Methods.

Other conditions such as the amount of solvent being assumed
as constant, the first class of changes can in theory be caused by
any agent which increases the number of molecules or of parti-
cles, i. c., which causes a splitting up of these within the egg.
For, as is well known, in a given volume the pressure varies
directly with the number of molecules. Under these circum-
stances, in order that subsequently there may be equilibrium
between pressure and tension, both potentials must decrease, the
latter however presenting a relative rise ; at the same time both
the volume and surface increase, water is absorbed, and the
medium outside the cell is really of smaller volume and greater
pressure than before.

This series of events agrees with that which we have seen must
take place in segmentation, viz., an average decrease of tension.
Confirmatory of the correctness of our theory and quite agreeing
with it are a number of experimental methods for obtaining arti-
ficial parthenogenesis when the direct effect can be only chemical
or ionic or both, but in any case not purely electronic. Thus in
Asterias eggs HC1 is effective, but not KC1, from which a specific
H ion effect is inferred. * So also are Ca ions alone effective for
Amphitritus? 1 (The effect of the hydroxyl ion, or of O, can
have only the same direct chemical effect primarily ; by these
conditions segmentation is retarded or exhibited, thus indicating
that for segmentation analytic processes are essential ; for, e. g.,
oxygen in the presence of ferments means a splitting up of mole-
cules.) The accepting of a specific chemical effect of these ions is
not invalidated by the admission of an " electronic" effect also,
but is necessitated by just such data as the above, which are the
results of an application of the " method of difference." Just
how this chemical effect is caused, whether directly by action on
the protoplasm, or indirectly by first making certain " prefer-
ments " in the egg active cannot be definitely stated, but this
deficiency does not in itself alter the correctness of the above
view. The effectiveness of another practical method is thus found

1 Loeb, Fischer and Neilson, Archiv fur geschichtliche Physiologic, Bd. 87, 1901 ;
previously cited.

2 Fischer, loc. cit.

112 E. G. SPAULDING.

to be explainable upon the basis of our theory and the usefulness
of the attempt to synthesize therewith demonstrated.

From exactly this same standpoint also can the effect of an
entering spermatozoon be explained, whether its immediate action
be chemical or enzymatic, and thus the physical processes of
normal fertilization and segmentation made clear, at least in part.

Clicinical Changes by ($) Physical Means.

Theoretically possible physical means for producing this split-
ting of particles or of molecules are heat, and mechanical agita-
tion ; the former, as a rise in temperature, may be supposed to
have this direct effect in agreement with well-known phenomena
in pure chemistry, or, as a preliminary to this, to first make cer-
tain enzymes in the egg active ; or it may be considered to have
a purely physical effect, for since PV '= RT it directly increases
the pressure and decreases the tension. All of these three effects
may and probably do coexist. Agreeing with these theoretical
possibilities is the practical one of starting development in Arbacia
by the action of heat (rise in 7").

In the same way can also the practical method of causing par-
thenogenesis, e. g., in Asterias eggs by mechanical agitation, 1 be
explained, i. e., in analogy to facts in chemistry. For example, in
the " diazo-compounds " mechanical shock starts analytic proc-
esses. Such compounds are accordingly said to be " metastable."
At the same time that the pressure is increased by such analytic
processes the potential difference thus formed may be further
augmented by a decrease in the tension also caused by agitation,
as pointed out above.

The effectiveness of this entire class of methods, both physical
and chemical, for causing analytic changes in the solute may be
explained as follows : Work is first done on the cell in strict ac-
cordance with the general physical principles stated. As a re-
sult of the analytic chemical processes taking place, the pressure
is at first increased, possibly also the tension decreased at the
same time. If the potential difference thus created is uncompen-
sated by a third, /. c., if the pressure of the surrounding medium
has been kept constant, c. g., by retransferring the eggs to sea

1 Mathews, Am. Journal of Physiology, VI., 2, 1901.

PHYSICS OF SEGMENTATION. 113

water in the HC\-Asterias method, /. c., if another potential dif-
ference exists than in the cell, the latter must in turn do work ;
its two potentials accordingly decrease, the extensity factors in-
crease, water is absorbed, and as a result of new localized chemi-
cal and osmotic differences in the egg plus accompanying elec-
trical changes, the constricted form of cleavage appears.

(Semipermeable Membranes.')

In the above presentation the assumption is implicitly made
that the surface of the cell acts as a semipermeable membrane,
so that, first, only specific ions can enter, and second, others,
those within the cell cannot make their exit, while water can pass
either way. Only on these conditions can osmotic pressure be
truly exerted.

The question accordingly arises, if this possibility is done away
with if the so-called membrane is due to, or identical with, only
the surface tension of a fluid in which colloidal particles are in
solution. The answer to this on the basis of the evidence at
hand and to accept the position generally taken is negative, but
again brings us to the matter of chemical organization. Rhum-
bler 1 has shown experimentally that particles in the surface are
not freely displaceable, nor do protoplasmic streamings to the
outside take place ; this can be accounted for only by assuming
a foam structure and a state of solution. This means that the
apparent membrane is a result of the forces of surface tension.
Furthermore, with very probable reasons for the surface being
chemically different to the interior as it is to that of the surround-
ing medium, and with the known fact of the difficult diffusibility
of colloidal particles, it is quite intelligible that at the same time
that a displacement to the interior is rendered difficult the specific
chemical nature of the surface film should prevent the passage
through it of some ions while allowing that of others, quite
analogous to the artificial semipermeable membranes of Pfeffer.
The same explanation would hold good for any other apparent
membranes, like that of the nucleus, within the cell whose mor-
phological differentiation can be established. 2 All these factors,

1 Rhumbler, " Aggregatzustand u. Physikalische Besonderheiten des Zellinhaltes,"
Zeitschrift fur allg. P/iysiologie, I., 3, '02.

2 Cf. Hober, loc. fit., p. 47, for confirmatory evidence.

114 E. G. SPAULDING.

viz., local chemical differences, membranes within the cell, diffi-
cult diffusibility of colloidal particles, splitting of molecules,
localized processes, resulting electrical changes, make the con-
stricted form of the egg at cleavage with its rearrangement and
average decrease of tension not only intelligible, but a priori
probable.

//. Means for Changing the Osmotic Pressure Physically.
Purely physical changes in the repelling forces of the cell are
possible, and the theoretical methods to be inferred from these
are confirmed by a number of practical methods for producing
artificial parthenogenesis.

() Hypertonic Solutions.

The first of these both theoretically and also practically pos-
sible methods is the direct raising of the pressure within the cell
by first surrounding it with a hypertonic solution and then sub-
sequently transferring it to the sea water whereupon segmenta-
tion takes place. This method is used for Arbacia, Astcrias,
Cluctoptenis, Nereis and Podarkc, and is perhaps the most impor-
tant of the artificial parthenogenetic means. Its effectiveness
may be explained as follows : In a hypertonic solution there is
at first an uncompensated potential difference between the pres-
sure within and without the egg, which is equalized by the pass-
ing of water through the membrane of the egg to the medium.
The relative number of molecules of solute within the egg is
accordingly increased. At the same time with a decrease in the
egg's radius of curvature accompanying the decrease in size the
surface tension has been increasing to compensate the increased
internal pressure, though this compensation never quite takes
place as long as water continues to be withdrawn, but theoretic-
ally is always somewhat behind. The egg thus receives energy
from without. With the transferral of the egg to sea water,
which is of lower pressure than it is, a potential difference in the
opposite direction next exists which is uncompensated by the
tension owing to the permeability of the membrane to water ;
accordingly an event, viz., absorption of water, takes place until
the pressure both within and without is the same. In this proc-

PHYSICS OF SEGMENTATION. I I 5

ess, however, both the pressure and the tension of the egg de-
crease, as our theory demands, while the volume and surface in-
crease until equilibrium is attained between the two energies
within and the pressure without. But therewith new potential
differences between these and other energies, c. g., chemical, may
have been created, by which the series is continued up through
the various stages of development.

However, in this method it is not directly evident why, instead
of segmentation taking place, simply the former size of the egg
is not recovered when it is returned to sea water. To account
for the constriction actually occurring it is again necessary to
make the assumption which is nevertheless theoretically justifi-
able in analogy to well-known phenomena in chemistry, that in
connection with localized chemical differences some of the col-
loidal particles are naturally in a " metastable " condition, which
is done away with and chemical changes started by the with-
drawal of water, and which therefore cannot subsequently be
regained. 1 It may also well be in analogy to known instances
elsewhere that so-called preferments become active only on the
condition of a certain degree of concentration being present and
that, becoming active with the concentration here present, they
initiate chemical changes which finally cause the constriction of
the cleavage form.

Action of Temperature Changes.

The second of the class of methods we are here considering,
viz., the physical, has to do with variations in temperature. Two
possibilities therefore exist, a raising and a lowering of the tem-
perature of the medium and so of the egg.

A raising of the temperature directly increases the pressure,
but it may also produce, as has been seen, a molecular splitting
either directly or through the mediate action of preferments,
and, as before stated, decrease surface tension. Any one or all
three of the effects taken together are essential with the forma-
tion of a potential difference in the required direction ; the start-
ing of development in Arbacia eggs by heating may be ex-
plained in this way.

1 Cf. Ostwald, Vorlesungen liber Naturphilosphie, s. 271 et 353, et seq.

Il6 E. G. SPAULDING.

Greeley ! found however that artificial parthenogenesis could
be produced in Asterias eggs by exposing the eggs to a tempera-
ture of i 7 C. and then allowing this to rise, keeping them in
the same sea water all the time. This can be explained as follows :

A lowering of the temperature directly reduces the osmotic pres-
sure of both medium and egg, yet increases the surface tension.
A potential difference within the egg in the direction of tension-
pressure is thus created, the equalization of which necessitates a
loss of water. This view is confirmed by the spherical form as-
sumed and the losing of water by many species as a result of
exposure to low temperatures. 2 The loss of water as a result of
the increased contracting forces cannot continue indefinitely for
the reason that, by virtue of the increased osmotic pressure
caused by it, the egg itself would tend to absorb water. Con-
sequently the three processes must be considered to occur until
there is equilibrium in the entire "system" of egg-medium ; like-
wise they would continue to take place, though in opposite direc-
tions, when the temperature was subsequently raised; i. i\, the
pressure and tension would then both simultaneously decrease
and the egg absorb water. Consequently this method would
agree with our theoretical demand, that the event of cleavage is
always identical with a decrease in surface tension.

(c) Electrical CJianges.

The third possible way of bringing about changes in the egg
which are in themselves physical (electrical), but perhaps due
directly to chemical causes as we have indicated, is by the use
of electrolytic methods. These changes may have to do with
both pressure and tension at the same time and we have referred
to them previously as perhaps to. be necessarily considered as
present in any case in order to account for the constricted
form of cleavage. For it can be shown that every method that
we have analyzed may, at the same time that it results in the
changes in chemical composition, pressure and tension, have
also accompanying these a change in the electrical condition of
the egg.

1 Am. Journal of Physiology, ^ 7 I., 1902, p. 296.

2 Greeley, Am. Journal of Physiology, 1901, VI., p. 122; BIOLOGICAL BUL-
LETIN, III., p. 165 and V., p. 42.

PHYSICS OF SEGMENTATION. I IJ

Lillie * in two recent papers has emphasized the importance
and essentiality of these electrical phenomena for segmentation,
but, inasmuch as he ignores to a certain extent the consideration
of the factors we have emphasized, and because we believe it
can be shown that his own view is incomplete without this, it may
be allowed us to quote quite extensively and in abstract.

Lillie in his first paper finds that " the tendency of colloidal
particles to collect at the electrodes indicates that they carry a
surface charge, either positive or negative." Precipitation can
be caused by ions bearing charges of opposite sign to those
of the particles, liquefaction by those of like sign. " The
researches of Picton and Under, and Hardy indicate that the
nucleo-proteids especially the chromatin of dividing cells and of
spermatozoa are pronouncedly acid and therefore electronega-
tive ; cytoplasmic proteids are conversely basic and positive.
Accordingly a difference of electrical potential exists between
cytoplasm and chromatin in the cell, which difference is greatest
at the time of mitosis, when the chromatin is most strongly acid.
This potential difference may constitute the primary and deter-
mining condition of mitosis."

In confirmation of the correctness of this view he finds that
"in all cases the appearance of the cytoplasmic radiations and
the formation of the mitotic figure are accompanied by a passage
of the nuclear chromatin into a phase rich in nucleic acid.
Evidently the two parallel series of changes are intimately con-
nected. Furthermore, the marked resemblance between the rays
of the mitotic figure and the electric and magnetic lines of force
are additional indications that the process is essentially electrical
in nature. The position of the chromosomes during mitosis in-
dicates a mutually repellent action similar to that of similarly
charged bodies. The same action takes place in the chromatin
filament whereby it assumes a coiled or spiral form."

In his second paper his purpose is to show the necessity of
these electrical conditions for segmentation. Evidence for this is
that " cytoplasmic cleavage in fertilized Astcrias and Arbacia
eggs is prevented in solutions of non-electrolytes, although the

1 R. S. Lillie, American Journal of Physiology, VIII., IV., Jan. I, 1903, and
BIOLOGICAL BULLETIN, IV., 4, March, 1903.

IlS E. G. SPAULDING.

nuclear division continues. In these solutions the electrolytes
present in the egg must diffuse outward. Cleavage therefore
depends on the presence of these in the cytoplasm. Likewise a
strong tendency to fusion shows itself in blastomeres transferred
to these solutions during early cleavage stages ; therefore further
cleavage also depends on the presence of electrolytes in the cyto-
plasm. This action is due to the ions into which they dis-
sociate. ' '

If all this is admitted it seems to us quite necessary to admit
or infer therefrom that the presence of electrolytes in the eggs
means also the presence of osmotic pressure; one cannot exist
without the other ; consequently their dissociation is identical
with an increase in pressure. This would be in perfect agree-
ment with our own theory, but is quite ignored by Lillie. He
does, however, recognize with us the necessity of accepting a de-
crease and rearrangement of surface tension at cleavage, which he
accounts for by a difference of electrical potential between the egg
and medium. For " Lippman and Helmholtz have shown that
the surface tension is greatest when the electrical potential at the
boundary is zero, and decreases as the latter increases, for like
electrical charges at the surface oppose the tension and diminish
it. From this is evident the importance of electrolytes in seg-
mentation ; for the production of a potential difference between
the egg and the medium separated by a semipermeable membrane
is accompanied only by a migration of ions. Therefore, there are
ions within the egg originally, and a potential difference implies
that ions of like sign are respectively at the surface and in the
interior. This state of affairs is found at cleavage. Observation
of the direction of the fibrils in the egg during mitosis agrees
with this view, /. c., they correspond with the lines of force.'
How is this difference of potential between the surface and the
interior established ? The answer is that " during segmentation
especially is the chromatin markedly acid, the cytoplasm basic."
This means in our opinion that there is chemical organization.
" Agreeing with the results of Nernst and Olsen, the chromatin
represents a charged body by the action of whose negative
charges the anions are repelled toward the periphery, or the
poles, and the kations are attracted to the nucleus." A curren

PHYSICS OF SEGMENTATION. IIQ

passes therefore in the direction of the gradient from periphery to
center. Such inductive phenomena resulting from the increased
acidity of the nucleus during mitosis must in turn be due, we find,
to the preceding chemical changes resulting perhaps from the
action of ferments. Accordingly, it results that "the center of
the astral radiations is the region of highest positive potential, the
surface of negative, and hence the decrease in the surface tension
by the like charges present."

But a uniform decrease in tension is no change in form ; to
bring this about the tension must be unequal. To explain this,
Lillie says, "There are indications, e. g., the elongation of the
spindle axis, that the primary lowering of surface tension by the
above agencies is at the two sides of the egg opposite the astral
centers. From the position of the astral centers during meta-
phase and telophase it is to be expected that the surface negative
charges are densest near regions adjoining the long axis of the
egg and that there surface tension is lowered to the greatest de-
gree. The effect in these regions will increase as the daughter
groups of chromosomes approach the poles, since the inductive
action increases as the distance decreases. The surface tension
at the regions adjoining the astral centers must therefore de-
crease as the daughter groups approach the surface, /. c., the
difference between the surface tension at the poles and at the
equatorial region progressively increases. Eventually the egg
is surrounded by an equatorial surface zone possessing higher
tension and acting like a constricting band ; a cleavage furrow
follows."

" From the fact that not all of these events takes place when
the egg is placed in solutions of non-electrolytes, it is clear that
cleavage depends on the presence of ions, and that in fertilization
the spermatozoon carries either these necessary electrolytes into
the egg or ferments which initiate their formation and action."

That the acceptance of the events either identical with or simi-
lar to those so ingeniously outlined by Lillie is necessary in
order to complete the theory of segmentation advanced in our
own paper, may be admitted. But that the two are not contra-
dictory to but rather must supplement each other seems quite as
necessary to admit. The constricted form of segmentation must

1 2O E. G. SPAULDING.

be accounted for, but if this is done by electrolytic theories, then
the existence of osmotic pressure cannot be denied. Lillie, how-
ever, neglects its consideration and in this respect his vievv is
incomplete. If the necessity for segmentation of the presence
of electrolytes in the egg is proven by such data as Lillie has
advanced then osmotic phenomena must be also present, and
that they play an important part cannot be denied in the face
also of the evidence from artificial parthenogenetic methods.
Rather, conversely, starting with the known fact of the effective-
ness of, e. g-., the osmotic pressure methods, the possibility of
these leading to the electrical changes should be shown. The
objection may be made that, although present, osmotic pressure
may nevertheless be left out of consideration for the reason that
it is of low intensity. This objection does not hold good, we
think ; for, given a semipermeable membrane and the tendency
of surface tension to reach its maximum, the latter will do this
until balanced by at least as high a pressure as in the normal
surrounding medium, sea water, of the marine forms we are con-
sidering.

It therefore seems possible and even necessary to unite the
two views ; to take the position that the electrical phenomena
described by Lillie cannot be done away with but, being essen-
tial, they can nevertheless, as we have experimental evidence to
show, be brought about in a number of different ways, such as
amount of water present, action of heat, agitation, etc.; and that
they cooperate with the factors we have emphasized in making
up the physical and chemical events of cleavage processes. This
possible perfect agreement of Lillie's theory with our own, into
which all the factual methods have been shown to fit, adds,
it seems to us, one more confirmatory element to the synthesis
which it has been our purpose to attain.

The electrolytic methods which have been referred to as the
third class which bring about physical changes may therefore be
explained from the standpoint of this more complete view. As
our factual result we have artificial parthenogenesis produced in
Asterias eggs by the action of TV/io HC1 solution (2-5 c.c. + 100
sea water) for about one hour, with a subsequent transferral to
sea water. Dissociation takes place in the dilute solution used.

PHYSICS OF SEGMENTATION. I 2 I

The negative results obtained with KCl indicate that it is the H
and not the Cl ion that is effective. Two views of the way in
which this ion acts are conceivable. First, an interpretation we
have referred to previously, it may be that only the H ions penetrate
the membrane, either because of i/s specific chemical nature or
because of their greater diffusion velocity ; or, second, that, with
no penetration occurring, the cytoplasmic membrane simply at-
tracts the H ions to the surface.

Both views however present difficulties if the H ions are con-
sidered to have a purely inductive (physical) action. The first
view in its very genesis, because, firstly, unless the surface is
negative all the time it is hard to understand why the positive
ions should even be attracted to it, much less penetrate it ; if, on
the contrary, it is at first positive they would not reach it, being
repelled, unless their velocity of diffusion overcame this repul-
sion ; but secondly, because if penetration does occur it is difficult
to understand how at least many of the ions can reach the
nucleus from which point possibly to, in turn, induce negative
charges at the surface ; for cytoplasm and H ions are like-
charged ; these would, however, tend to cause a repulsion of
cytoplasmic particles and therefore an increase in pressure. The
second view meets with objections because, with no penetration
occurring, it is difficult to understand also here why, firstly, if the
surface is not yet negative before mitosis, either H or any other
kations should be attracted, or, secondly, if the surface is nega-
tive at all times and the cytoplasm positive, so that the surround-
fhg H ions may induce further negative surface charges, why
the H and not other kations as well should be effective. That
they are might seem to be explained by the comparative diffusion
velocity of H, K and Cl ions (H is 313, Cl 65.9, K 63.8) ' from
which it might be assumed that the K ions are not effective be-
cause with them the Cl ions while in the case of HC1 the H ions
have the greater comparative velocity. But this explanation
would not hold good for explaining positive results with Ca(NO. j )
on A nipJi itritns- for the diffusion velocity of Ca, is 62, that of
NO 3 6s. 3

1 Hober, loc. cit., p. 7 2 -

2 Fischer, Am. Join: of Physiology, VII., III.
3 Hober, loc. cit., p. 191.

122 E. G. SPAULDING.

Between the difficulties of the two views it seems necessary
to conclude that certain ions penetrate the membrane because of
their and its specific chemical nature and of their greater com-
parative velocity and notwithstanding the possible repulsion, and
that there follows a specific chemical effect on the chemically
organized cell contents accompanied by those electrical and
osmotic phenomena above considered. That there is a chemical
effect is indicated also by the different results obtained in deter-
mining the rhythm of immunity of fertilized Arbacia eggs to
ether, HC1, KC1, etc., an account of which will appear in a later
paper. Furthermore, from this standpoint there should be in
theory no effect resulting from the use of either the anodal or
kathodal end of the current on the eggs of, e. g., Aster ias, and
the negative results of such experiments carried on by the author
this summer are confirmatory of this view.

CONCLUSIONS AND SUMMARY.

Under the experimentally justified assumption that the organ-
ism is a peculiar complex of energies, so that general physical
principles are therefore valid for it, it is found that the effective-
ness of both normal and artificial fertilization methods can be
explained from one standpoint, viz., firstly, that the necessary
condition for the event of cleavage, is the creation, previous to
it, of an uncompensated potential difference between osmotic
pressure and surface tension by increasing in a chemically or-
ganized egg either absolutely or relatively the pressure or by
decreasing the surface tension ; secondly, that the event of
cleaving is itself identical with the equilibrating and compensat-
ing of this difference, which necessiates an average decrease in
both the potentials, osmotic pressure, and surface tension ; and
thirdly, that there is an accompanying unequal distribution of
electrical charges at the surface and at the center in such a way
that constriction results therefrom.

This constitutes the synthesis which we purposed, and is
offered only as an attempt, that, although in itself justifiable, pre-
sents much that is incomplete and tentative.

COLLEGE OF THE CITY OF NEW YORK,

DEPARTMENT OF PHILOSOPHY, December 22, 1903.

EXPERIMENTS ON THE DEVELOPMENT OF EGG
FRAGMENTS IN CEREBRATULUS. 1

N. YATSU.

The question, "To what extent can the principle of germinal
localization be applied to the unsegmented egg, and how far, on
the other hand, may the specification of the embryonic regions be
considered a progressive process that falls under the category of
epigenetic phenomena?" (Wilson, 1903'") led me in the summer
of 1903 to carry out a series of removal-experiments on the eggs
of Cerebratulus lacteus, during the later part of my stay at the
Tufts College Marine Laboratory at Harpswell, Me. I wished
to determine whether the cytoplasmic localization is progressively
established, and especially to ascertain the conditions existing in
the egg just prior to the first cleavage. To this end I have
examined the development of egg fragments, obtained by cutting,
at four successive periods between discharge of the egg and the
first cleavage, namely, (i) before the dissolution of the germinal
vesicle, (2) at the metaphase of the first polar mitosis, (3) at the
period of conjugation of the egg- and sperm-nuclei and (4) after
the constriction of the first cleavage appeared. The result shows
that the percentage of abnormal larvae steadily increases as the
egg approaches the two-cell stage. When I appreciated the im-
portance of comparing with these results the development ot
isolated blastomeres of the two-cell stage, the breeding season was
nearly over, and I was only able to make a few experiments,
which are not numerous enough to give a satisfactory basis of
comparison. I hope, however, to carry on more complete ex-
periments at the first opportunity.

A word about cutting the eggs. A drop of water containing
several eggs was spread on a slide, and some of the water was

1 For the preparation of the present paper my best thanks are due to Professor E.
B. Wilson for his kindly suggestions and criticisms. I am also indebted to Professor
J. S. Kingsley for kindness shown me at the Harpswell Laboratory.

2 E. B. Wilson, "Experiments on Cleavage and Localization in the Nemertine
Egg," Arch. f. Ent-w/n., Bd. 16, Heft 3, p. 440.

123

124* N. YATSU.

removed by a pipette. The egg, thus flattened a little, was cut
with a fine scalpel. It sometimes proved better, in order to
spread the water evenly, to use a little albumen fixative, rubbed
on the slide, and thoroughly washed off with a brush. In doing
so, the quantity of glycerine left on the slide was infinitesimal, so
that it did not affect at all the development of the eggs. My
experiments consisted in cutting off a portion of cytoplasm from
unsegmented eggs at the four periods already mentioned, in the first
two cases fertilizing the fragments, and rearing the resulting em-
bryos up to the pilidium. For the sake of uniformity I have
confined my work to the nucleated fragments. Cleavage was
studied up to the eight-cell stage. Among the pilidia thus ob-
tained, I found many abnormal ones, and they were compared
with the normal larvae. Every pilidium was drawn with a
camera between 48 and 58 hours after fertilization. During
drawing they were kept still by sucking out some of the water
from under the cover-glass, which was supported by a piece of
thread. Thus they could barely move without distortion of their
shape. It should here be noted that defective larvse remain
always defective ; moreover, the defective parts become more and
more prominent as the larvae grow. No size regulation takes
place among the pilidia ; the smaller the original egg pieces, the

smaller the pilidia.

SERIES A.

Development of Fragments obtained before the Disappearance of tlic

Germinal Vesicle.

A portion of cytoplasm was cut off from the egg immediately
after its release and while the germinal vesicle was intact, and
the nucleated piece was fertilized. Care was taken not to injure
the germinal vesicle, and most of the operations were done upon
eggs in which it was eccentrically situated, so as to cut off as
much cytoplasm as possible. Since the germinal vesicle, as a
rule, lies nearer to the animal pole, it may be inferred that most
of my sections were performed in the vegetative hemisphere,
although I did not record the exact plane in this series.

Thirty-five egg-fragments were able to develop up to the
pilidium stage, the rest having either died or been rejected on
account of polyspermy. The result may be tabulated as follows :

DEVELOPMENT OF EGG FRAGMENTS IN CEREBRATULUS. 125

Perfect pilidia 30 { ^""^ } ........... 5-7 per cent.

Defective pilidia 5 ............................................ 14.3 per cent.

It is striking that comparatively few larvae turned out to be
defective. All of these are shown in Fig. I, C-G, 1 a glance at
which shows that they are certainly abnormal, yet their defect is
not so great as those obtained at later periods (Series B and C).

FIG. i.

i C represents a larva which would be perfect, if there were an
apical organ. \D is another defective one with three apical
organs, two tufts having been about to fuse ; in other respects it is
perfect. A pilidium represented in \E is much more abnormal
than the preceding two. It has two apical organs and a pair of
ciliated lappets of smaller size. The shape of the gut also is not
normal. Another pilidium (i/ 7 ) resembles the one with three
apical organs just described (i-D), but differs from it in having
three apical organs widely separated from one another --two on
the left side, one on the right. In this larva the left ciliated lobe
is indented. Lastly we have a very defective pilidium (i G),
which, like i C, has no apical organ. While the ciliated lobes

1 All the figures throughout the paper have been drawn by the camera ; 84
diameters magnification.

126 N. YATSU.

are comparatively normal, the gut is quite abnormal, the stomach
being larger than the oesophagus, and the position of the mouth
deviating from the normal. Nevertheless each part is complete,
except the apical organ. It should not be overlooked that the
apical organ is abnormal in every one of five defective pilidia,
while both the gut and ciliated lobes are fairly unaffected by the
operation. From this it is probable that the basis of the apical
organ is vaguely foreshadowed as early as the stage at which
the crerminal vesicle is still intact.

o

It is highly important to note that the formation of a pilidium
does not depend on the size of the piece, since one fourth of the
perfect larvae are decidedly smaller than the rest. The larva, lA,
is one of the larger group, and iB one of the smaller. Both are
perfect in every respect, but one is a little larger than one half of
the other. From the fact that, in spite of cutting the eggs at
random, a large percentage of perfect larvae were produced, that
the perfect ones vary greatly in size, and that the defects are not
so considerable as at a later period, the most natural interpreta-
tion one can draw would be that, antecedent to the dissolution of
the germinal vesicle, the egg cytoplasm still shows little or no
definite specification of the germ regions.

SERIES B.

Development of Fragments obtained at the MetapJiase of the first

Polar Mitosis.

The eggs were cut when the mitotic figure of the first matura-
tion division was completed ; this can readily be seen as a clear
space at the animal pole. The nucleated fragment was fertilized.
Sixty-five eggs thus operated were able to develop into larvae.
Owing to a change of consistency of either the gelatinous egg
envelope or the egg itself, I found it more difficult to operate at
this stage than at any other. The result was :

Perfect pilidia 34 52.3 per cent.

Defective pilidia 31 47.7 per cent.

Notwithstanding the difficulty of the operation, it was compara-
tively easy to cut off a considerable part of cytoplasm from the
egg of this stage on account of the peripheral position of the

DEVELOPMENT OF EGG FRAGMENTS IN CEREBRATULUS. I 27

mitotic figure. Consequently in some cases the fragments were
quite small. In this series all the defective larvae were more
abnormal than in the preceding. The abnormalities may be
classified as follows :

Defect in apical JLarvee with supernumerary apical organ 4

' ' no apical organ I o

" ciliated lobes indented or not well developed 16

organ. (

Defect in ciliated J

lobes.

Defect in gut.

I

only one ciliated lobe II

no gut 6

no oesophagus 5

no stomach I

stomach cutoff from oesophagus 2

gut not well developed 7

14

27

r 21

FlG. 2.

The disturbance of the apical organ (2,B and 26") is of special
interest, indicating that its basis covers quite a large area over the
animal pole, since in this series it was impossible to cut off cyto-
plasm from the top of the egg. While in Series A the defective
part was chiefly restricted to the apical organ, we find that in the
stage under consideration the abnormalities extend to the ciliated
lobes and gut also. As will be seen from the above table, a
large number of the defective pilidia have abnormal ciliated
lobes ; in some, one of the lobes is very short (2^?), while in oth-
ers the lobes are indented (2/T), reminding us of Pilidiinn braii-
chiatinn. Quite commonly one lobe is entirely wanting (2 A, 2C
and 2/7). The defect in the gut is remarkable. In a few larvae

128 N. YUTSU.

there is no gut at all, these being not far from the " Dauerblas-
tulae " (26"); in other cases the gut is represented by a shallow
depression (2 A"). In some defective larvae either the stomach or
the oesophagus may be wanting (28, 2/T). In a few cases the
stomach is cut off from the oesophagus (2E).

It is noteworthy that there occur a few defective larvae which
are as large as the perfect ones (2A). And among the perfect
pilidia only three were smaller than the others.

From the increased number of defective larvae in this series, it
may be inferred that, since the fading of the germinal vesicle, the
regional specification has advanced. But the occurrence of per-
fect pilidia equal in number to the defective ones cannot be over-
looked. It is possible that the production of the perfect larvse
is because the injury was too small to have materially affected
the organ-bases, or because the plane of section was such as not
to disturb their normal proportions. The number obtained is,
however, too great to lend much probability to either of these
suggestions. A more likely interpretation is that the egg still
possesses a considerable power of regulation.

SERIES C.

Development of Fragments obtained at t/ie Period of Conjugation

of the Egg- and Sperm-nuclei.

The eggs were fertilized and a portion of cytoplasm was cut off
a little after the second polar body was extruded, /. e., at about
the stage at which the egg and sperm nuclei
came to fuse. In this series the polar bodies
gave a very good landmark for orientation of the
egg. The accompanying cut shows the direc-
tion of section plane (Fig. 3). The eggs which
were able to develop up to pilidium were not as
FlG numerous as in Series B, but they gave a fairly con-

clusive result, as shown in the table on p. 102.
Most of the pilidia of this series are defective, as tabulated.
Not only that, the defective parts correspond in a general way
to the region from which the cytoplasm was cut. 1 It is very

1 The apparent contradictory result as in the case of Nos. 17 and 23 is probably
due to the volume of the cytoplasm cut oft".

DEVELOPMENT OF EGG FRAGMENTS IN CEREBRATULUS. 1 2Q

No. of Eggs.

Section Plane.

Condition.

Remarks.

I

AB

D(efective)

Ciliated lobes short.

2

4 (

D

No apical organ ; ciliated lobes asym-

metrical.

3

C(

D

One of ciliated lobes wanting.

Gut not well developed.

4

it

D

Apical organ on the right side.

5

t t

P(erfect)

6

CD

P

7

t

P

8

<

P

9

(

P

I0 1

t

D

Two apical organs. Gut double.

ii

EF

D

Ciliated lobes defective.

12

t

D

One of ciliated lobes wanting.

13

t

D

One of ciliated lobes defective.

/I4

(

D

Gut defective.

w

(

D

No apical organ ; one ciliated lobe want-

ing ; gut comparatively large.

f 15

D

One of ciliated lobes wanting.

w

( C

D

One of ciliated lobes wanting.

16

GH

P

17

D

One of ciliated lobes wanting.

18

( <

D

Neither of ciliated lobes developed, but

the margin ciliated.

19

c

D

One of ciliated lobes wanting.

20

D

Ciliated lobes defective.

21

C

D

Ciliated lobes defective.

22

t

D

One of ciliated lobes wanting.

C

D

No apical organ ; only one lobe present ;

/ 2 3 2

gut imperfect.

\23'

(C

D

No apical organ ; only one lobe present ;

gut imperfect.

Summary :

Perfect pilidia 6 24 %

Defective pilidia 20 76 %

important to observe that most of the perfect larvae were pro-
duced from the egg cut along CD. The basis of the apical
organ must, therefore, have existed below the line CD. This
fact, and the frequent occurrence of a defective apical organ in
series B taken together, it may safely be concluded that the basis
of this organ takes the form of a broad ring a little above the
equator. But how it finally takes an apical position I am at a
loss to imagine. Although Professor Wilson's experiments 3 were
not directed to this problem, his results seem to tally well with

1 In this egg the nuclei conjugated in the vegetative hemisphere ; the section
plane passed nearly through the equator.

2 In this egg the sperm-nucleus must have been present in the piece which I
thought enucleated.

3 Wilson, /. f., pp. 432, 433 (Fig. 10), 436.

130 N. YATSU.

my conclusion. He obtained through the section near the ani-
mal pole along op (his Fig. 3, B, on p. 240) the animal larva
without any apical organ, the vegetative one in this case having
been provided with the multiple apical organs (his Fig. 10, E, on
p. 433). On the other hand, in another case, he found that both
the animal and vegetative larvae had the normal apical organ

(his Fig. 10, A and B] when he cut along the plane near the
equator (kl of his Fig. 3, B]. In this case it may be inferred that
the section plane bisected the basis of the apical organ. He
gives still another instance of the animal larva with the apical
organ (his Fig. 10, C) obtained when he cut below the equator
along ;;//;/ (his Fig. 3, -5).

The pilidium No. 10 (4-D) is a very important one. It was
produced from an egg from which about one third of the animal
hemisphere was cut off. This was about as large as a perfect
larva if seen from the side, but it was extremely compressed
laterally. It has not only two apical organs but two guts. I
suspect that it might have been disturbed by some unknown
cause at the two-cell stage because, as a rule, half-larvae obtained
by isolation of blastomeres are larger than one half of the normal
larvae. In another pilidium (No. 4) I found the apical organ
shifted from the normal position (4^). In spite of this, it swam
with the apical pole directed forward like a normal pilidium.

DEVELOPMENT OF EGG FRAGMENTS IN CEREBRATULUS. I 3 1

In three cases I got two larvae from one fertilized egg. There
are three possibilities to explain this : first, when the operation is
done before the fusion of the egg- and sperm-nuclei ; second,
after the first cleavage mitosis came to the telophase ; third, when
the egg is doubly fertilized and the segmentation nucleus is cut
apart from the sperm-nucleus. Since my operations were done
not so late as the second case, the result may be due either to the
first or the third cause. Whatever the cause may be, the com-
parison of the resulting larvae is very interesting. ^.A and ^.A'
(Nos. 14 and 14') show a most instructive pair of pilidia. The
larger of the two is almost normal, except that the gut is very
defective, while the smaller one has a comparatively large gut.
Professor Wilson found a similar pair of larvae by cutting the
blastulas (cf. his Fig. 1 1, A and B}. Another pair (^B and ^B' ,
Nos. 15 and 15') are also important; both of them are normal,
but one ciliated lobe is wanting in each. In still another pair,
4.C and 46"' (Nos. 23 and 23'), both are devoid of the apical
organ and have a very defective gut. Either of them is barely
more than the lappet.

Now let us see how the basis of the ciliated lobes and gut are
disposed in the egg of this stage. Most of the defective larvae
have abnormalities in the ciliated lobes in some way or other.
We can distinguish two kinds of defect ; in one only one ciliated
lobe has been developed, the other being entirely suppressed, so
that the mouth can be seen from the side (^.A' , 4.8' , ^C and
4ZT). The other kind of defect is shown in ^.F. In this case
both the ciliated lobes are present, but they are very short and
almost straight. This difference may be ascribed to the fact that
the basis of the ciliated lobes is more or less bilaterally situated
in the vegetative hemisphere near the equator. As for the basis
of the gut I know very little, but it is certain that it lies near the
vegative pole (cf. ^A and

SERIES D.

Development of Fragments obtained before the Completion of the

first Cleavage.

The eggs were cut between the period of the appearance of the
first cleavage furrow and the completion of the division. This

132

N. YATSU.

experiment was begun at the end of my stay at Harpsvvell, and
I can give here only four cases.

(i) The egg was cut along AB ($A) and a perfect pilidium
was produced which is represented in 5 C. It is important to ob-
serve that, in spite of cutting off the cytoplasm from the animal
pole, the apical organ has developed undisturbed.

(2 and 3) The eggs were cut along CD (5/4). From each
blastomere arose a pilidium with one ciliated lobe and no apical
organ. The cases are too few to draw any conclusion, but the
importance of the cytoplasmic bridge connecting the blastomeres
and of constriction of the first cleavage upon the arrangement of
organ bases, is not to be overlooked.

(4) The egg was cut along EF ($A). From the left half a
perfect but dwarf pilidium resulted. This is a case worth de-
scribing, because the egg neither rounded up, as is usually the
case, nor divided into two unequal halves, but soon after the cut
surface was closed, the right half gradually increased in size ac-
companied by the decrease of the left half, and thus the egg was
divided into two equal blastomeres.

SERIES E.

Development of the Blastomeres Isolated at the Two-cell Stage.

The blastomeres of eight eggs were separated at the two-cell
stage when the cytoplasmic bridge had disappeared and the blas-
tomeres had assumed a spherical form. From every blastomere a
normal pilidium was developed. Some were about half the nor-
mal size, while some, for reasons that I cannot explain, were very

DEVELOPMENT OF EGG FRAGMENTS IN CEREBRATULUS. 133

much smaller. It is remarkable that the development of the iso-
lated blastomeres differs very widely from that of fragments of the
period preceding. From a comparison with Series D it seems
likely that the result may be different according to the period of
cutting /. e., before or after the disappearance of the cyto-
plasmic bridge (cf. Series D, eggs B and C) and the pressing to-
gether of the blastomeres. In any event, further careful exami-
nation of the egg of this stage with reference to the question of
egg specification is very desirable.

CONCLUSION.

From the above series of experiments we have seen that, if the
cutting-off of a portion of cytoplasm is done before maturation, it
only slightly affects the normal development ; but if the opera-
tion is done at the first maturation stage the formation of the
organs of the pilidia is considerably affected, and still more if it
is done at the time of fusion of the germ-nuclei. We cannot,
therefore, escape the conclusion that there must take place some
progressive changes in the general make-up of the egg during
the period extending from the time of dissolution of the germinal
vesicle to the fusion of the germ nuclei. This period falls into
two subdivisions, the first extending from the fading of the
germinal vesicle to the metaphase of the first polar mitosis, and
the second from the growing period of the sperm-nucleus to its
conjugation with the egg-nucleus. The importance of these two
periods has been correctly perceived by Delage (1901), Fischel
(1903) and recently by Professor Wilson (1903). It is hardly
necessary to state here that, although these two periods can
be artificially separated by delaying fertilization, yet, under natural
conditions, there is no pause between them, as the spermatozoon
enters the egg before its maturation.

Let us next see what visible changes take place in the egg
cytoplasm as the result of the above two acts, i. e., the dis-
appearance of the germinal vesicle and the entrance of the
spermatozoon in the matured cytoplasm. As soon as the ger-
minal vesicle breaks up, the nuclear fluids flow out and may
diffuse through the egg. At this time currents may be formed
carrying the egg cytoplasm from one spot to another. Although

134 N - YATSU.

in the Cerebratulus egg we have so far no direct evidence to sup-
port the occurrence of the last phenomenon, yet my observations
on sections clearly show that a segregation of egg material does
actually takes place at this period, the yolk accumulating in the
lower hemisphere, while the clear and more finely granular
protoplasm collects especially at the top of the egg, where,
in the iron hematoxylin-Congo-red preparations, it stands out
beautifully stained blue in contrast with the red yolk. After the
fading of the germinal vesicle the eggs not only become fecun-
dable, but also acquire much more power of forming cytasters
than they had before. It is hardly necessary to state that in
many forms remarkable changes take place in the egg at or
after the entrance of the spermatozoon. It may further be
pointed out, however, that the part played by the spermatozoon
in causing rearrangements of the egg-substance is of a subsidiary
character, as is shown in the case of parthenogenesis.

To the question as to what degree of localization exists in the
cytoplasm of the Cerebratulus egg before the fading of the germi-
nal vesicle, I am not in a position to answer, and it is almost
impossible to find direct evidence. But the results of my experi-
ments harmonize with Boveri's view that there is at first only a
simple promorphological condition such as polarity and bilater-
ality, which may give a basis, so to speak, for a more definite
grouping of material arising at the time of the flowing-out of the
nuclear fluids. Otherwise it is impossible to understand the
sudden increase in the proportion of abnormal embryos arising
from the fragments obtained subsequent to the fading of the
germinal vesicle. In the eggs of some forms (c. g. Myzostoma)
the segregation of material has in a measure taken place long
before maturation. In such cases the horizontal section of the
egg before the disappearance of the germinal vesicle will, I think,
produce a defective embryo. My observations on the later
periods render it probable that at this time there must be a certain
number of predetermined regions more or less firmly fixed,
though this is not so clearly shown as in the ctenophore egg. It
is extremely desirable to carry out careful studies with respect
to the egg specification prior to the first cleavage in other forms.

So far as I could ascertain (up to the eight-cell stage), the cleav-

DEVELOPMENT OF EGG FRAGMENTS IN CEREBRATULUS. 135

age of egg fragments was perfectly normal in Series A, B and C.
It is especially noteworthy that the nucleated pieces of the fertil-
ized eggs cleave exactly like a normal egg. Whatever amount
of cytoplasm be cut off, the nucleated piece always divides at first
into two and then into four equal blastomeres. Since these frag-
ments so often gave rise to defective embryos, it is probable, not
only that the two and four blastomeres differ from each other,
but also that they do not correspond with those in the normal
case. The same is true in the eight-cell stage, and so on. From
this it seems further probable that cleavage of an egg takes its
normal course irrespective of the localization of embryonic
regions within. The cleavage pattern is stamped on the cyto-
plasm ; but the end-result is governed by a different set of factors.
The cleavage factors seem, therefore, to differ in this case from
the morphogenic ones. " Though there is often a close and con-
stant connection in the normal development between the process
of cleavage and that of localization and differentiation, this con-
nection is not a necessary relation" (Wilson, I9O3 1 ).

It is rather striking that, if the operation is done before the
completion of the first cleavage, most of the egg fragments give
rise to defective larvae, while all of the isolated blastomeres at
the two-cell stage develop into perfect pilidia (though the result
is not conclusive, since my cases^were too few). It is easy to
conceive, however, that natural cleavage comes into operation in a
quite different way from the artificial section, and it is probable
that by the natural cleavage all the organ bases are equally dis-
tributed into two blastomeres which would be very unlikely after

an artificial section.

SUMMARY.

1. Before the germinal vesicle fades, there is no evidence of
definite specification in the egg regions.

2. Dissolution of the germinal vesicle initiates the establish-
ment of the germinal localization.

3. In the period between the entrance of the spermatozoon
and the fusion of the germ nuclei, the localization becomes more
definite.

4. The basis of the apical organ is not at the animal pole, but

1 Wilson, /. c. , p. 439.

136 N. YATSU.

somewhere above the equator as a broad zone. The bases of
the ciliated lobes and the gut lie mainly in the vegetative
hemisphere.

5. The cleavage (up to eight-cell stage) is normal in an egg
fragment obtained from the unsegmented egg, whatever be the
amount of cytoplasm cut off, or at whatever period. It is probable
that the cleavage factors do not here necessarily coincide with the
morphogenic ones.

ZOOLOGICAL LABORATORY, COLUMBIA UNIVERSITY,-
October 29, 1903.

SOME OBSERVATIONS AND CONSIDERATIONS

UPON THE MATURATION PHENOMENA

OF THE GERM CELLS. 1

THOS. H. MONTGOMERY, JR.

In a series of studies on the spermatogenesis of the Hemiptera,
of Peripatus 3 and of the Amphibia, 4 I have endeavored to prove
the following points :

1. That the chromosomes retain their individuality from gen-
eration to generation to that extent that a chromosome of one
generation is not a new formation, however much its chemical
substance has became changed by metabolism, but represents at
least a part of a particular chromosome of the preceding genera-
tion.

2. That the first maturation mitosis in the species studied re-
sults in the separation from each other of entire univalent chromo-
somes, and hence is a transverse or reduction division ; while the
second maturation mitosis results in the longitudinal splitting of
univalent chromosomes, and therefore is an equational division.

3. That the so-called "reduction in number" of the chromo-
somes is effected before the maturation divisions, by a pairing
(conjugation) in the early growth period (synapsis stage) of the
spermatocytes, of univalent chromosomes of like volume, each
such composite chromosome then being bivalent with relation to
the number in the spermatogonia.

4. That this conjugation of univalent chromosomes in the
synapsis stage, is a conjugation of paternal with maternal chromo-

1 Contributions from the Zoological Laboratory of the University of Texas, No. 54.

2 " The Spermatogenesis in Pentatoma up to the Formation of the Spermatic!,"
Zoolo<r. Jahrit., 12, 1898 ; " Chromatin Reduction in the Hemiptera : a Correction,"
Zoolog. Aitz., 22. 1899 ; "A Study of the Chromosomes of the Germ Cells of Meta-
zoa," Trans. Amer. Phil. Soc., 20, 1901; "Further Studies on the Chromosomes
of the Hemiptera heteroptera," Proc. Acad. Nat. Sci., Philadelphia, 1901.

3 "The Spermatogenesis of Peripatus (Peripatopsis) balfouri up to the Formation
of the Spermatid," Zoolo^. Jahrb., 14, 1900.

4 " The Heterotypic Maturation Mitosis in Amphibia and its General Significance,"
BIOL. BULL., 4, 1903.

137

138 T. H. MONTGOMERY, JR.

somes ; and hence that the first maturation mitosis separates
paternal from maternal chromosomes.

The third and fourth of these conclusions were new ; the
second has been a matter of much controversy, while the first
has a considerable number of cytologists in its support. If
these results can be generally established they will give a co-
herent basis for understanding the part played by the chromo-
somes in heredity, taken in conjunction with the important con-
clusion of Van Beneden [ that the male pronucleus has a number
of chromosomes equal to that in the female pronucleus ; and of
Henking 2 and O. Hertwig 3 that the maturation phenomena cor-
respond very closely in the ovogenesis and spermatogenesis of
the same species.

It is not my purpose here to go over the whole controversy
and discuss in full the opposing views, for that has been done in
my preceding papers, but rather to draw attention to a few of
the more important results, and to add some new observations.

i. THE FIRST MATURATION MITOSIS IN AMPHIBIA.
Janssens and Dumez 4 have very recently reexamined the
spermatogenesis of Amphibia, particularly with regard to my
results on Plcthodon and Desmognathus, and decide that my
position is both untenable and unproved : that the heterotypic
mitosis is an equational division, and not a separation of entire
univalent chromosomes as I had maintained. They state (p.
433) : " On est tout etonne de ne trouver dans le texte de Thos.
H. Montgomery aucun argument pour cette these." They do
not mention my strongest argument at all, namely that the space
enclosed by the heterotypic chromosome is a space separating
two whole univalent chromosomes, and not a longitudinal split
between two halves of a single chromosome, because this space
is largest in the earliest stages, and not, as one would expect if it

1 " Recherches sur la maturation de Foeuf et la fecondation," Arch. Biol., 5,
1883.

2 " Ueber Spermatogenesis und deren Beziehungen zur Eientwicklung bei Pyrrho-
coris apterus M.," Zeit. iviss. Zoo/., 51, 1890.

3 " Vergleich cler Ei- und Samenbildung bei Nematoden," Arch, inikr. Aitat,
36, i8qo.

4 " L' Element nucleinien pendant les cineses de maturation des spermatocytes
chez Batrachoseps attenuatus et Pletodon cinereus," La Cellule, 20, 1903.

MATURATION PHENOMENA OF GERM CELLS. 139

were a longitudinal split, smallest at those periods. In other
words, I showed that at the earliest stage when the chromosomes
can be distinguished, this space is largest, while the true longi-
tudinal split is found in the axis of each arm of a heterotypic
chromosome. They add : " L'auteur continue et, sans en donner
la moindre preuve cette fois, que les deux brandies de r anse s'en-
ronlent r une antoitr dc I'anfi-e pour constituer les dyades enrou-
lees, qui, d'apres tous les auteurs, se trouvent dans les spermato-
cytes I avant la mise au fuseau des chromosomes." It is only
necessary to rejoin that the comparison of the chromosomes as
shown in my consecutive figures is sufficient proof. But when
they say that my figures " sont fort schematisees," I simply an-
swer that is not true, and that all were made with great care with
the use of a camera lucida.

Janssens and Dumez do, however, bring up one good criticism.
They note quite correctly that in the spermatogonia the chromo-
somes are of unequal lengths, while the two halves of a bivalent
heterotypic chromosome are always of equal length. And they
reason that if my view were correct that one of the heterotypic
chromosomes is formed by the pairing of two univalent chromo-
somes, that I would have to demonstrate how two such con-
jugating chromosomes are always of the same length. For they
argue that if the heterotypic chromosome were formed by a lon-
gitudinal splitting, such a splitting would fully explain the length
equality of the two arms of the chromosome.

This is a good criticism, but I find it answered by a study of
the relative volumes of the chromosomes in the equatorial plate
stage of the spermatogonia. In every case where the pole view
shows all the chromosomes lying in one plane, we can determine
by carefully drawing them that there are just 12 pairs of chro-
mosomes present, the two of each pair being of equal volume and
frequently of similar form. One will find many cases where
the pole view of a chromosome plate does not show this dis-
tinctly, but that is only when the chromosomes are irregularly
arranged, and when their long axes do not lie in the plane of
the equator.

A series of figures demonstrate this. Fig. I is the only case
where all 24 chromosomes were seen in their entirety on pole

I4O T. H. MONTGOMERY, JR.

view ; in Figs. 25 only such chromosomes were drawn as could
be sent in their whole length ; and in none of the figures were
any chromosomes omitted that could be seen in their entirety.
Two chromosomes of a corresponding volume are marked with
the same letter, one in capitals and other in lower case, as, e. g.,
A and a. All these cases were drawn, after numerous prelimi-
nary sketches, as accurately as possible with the camera lucida,
and then those which seemed to correspond were lettered alike.
Thus in Fig. I, the large chromosomes A and a are alike, but
differ in volume from all others ; B and b are markedly alike,
C and c, and all appear clearly paired. Sometimes, as in the case
of G, g, //, h, the two of a pair appear dissimilar in the drawings,
but this is simply because the curvature of one lies in a different
plane from that of the other. Thus in Fig. 3 are shown also
all 24 chromosomes, but the two marked x and y were seen so
obliquely that it could not be determined whether they were
alike. Fig. 6 shows a lateral view of a spermatogonic spindle,
showing also a similar pairing of some of the chromosomes ; and
Fig. 7 shows the same phenomenon in an oblique pole view of a
portion of one plate of daughter chromosomes of an early sper-
matogonic anaphase.

An examination of these Figs. 1-7, shows that the chromo-
somes are paired according to their volumes, that the two of a
pair are very frequently of the same form ; and that in most
of the cases the two of a pair lie in the spindle close together.
The only interpretation for this last condition is that correspond-
ing chromosomes must have been arranged close together in the
continuous chromatin spirem of the prophase, so that in the
spirem A would be next to a, B to b, and so on. But on a
study of nuclei in the late prophase (loose spirem) stage, I was
not able to determine this positively, for until the chromosomes
have shortened to their definitive forms, they are so irregular
and twisted that it is practically impossible to determine their
exact lengths on sections ; probably crush preparations (such as
those employed by Sutton) could be used with advantage here.
At least there is no doubt of a continuous linin spirem in the
spermatogonic prophase and of the arrangement of the chromo-
somes in a chain along this thread. And it is probable that

MATURATION PHENOMENA OF GERM CELLS. 14!

similar chromosomes are contiguous in this spirem, since in the
metakinesis like chromosomes usually lie near each other ; and,
as Fig. 7 shows, they retain their contiguity even in the daughter
cells.

Therefore we find, what Janssens and Dumez insisted that I
should demonstrate, twelve pairs of chromosomes of the same
length in the equatorial plate of the spermatogonia.

Now for the proof that such chromosomes unite into pairs in
the spermatocytes, an explanation which, according to the Belgian
cytologists, " est absolument fantastique et demanderait a etre
rigoureusement demontree." On none of my preparations were
there stages between the early anaphase of the last spermatogonic
mitosis and the synapsis stage of the growth period. In the
synapsis stage in Desmognathus the long and slender chromosomes
are so intricately coiled together that it is impossible in sections
to determine their exact relations. The cell has a distinct
polarity, the nucleus at one end, at the opposite the greatest mass
of cytoplasm containing the idiozome body. When the chromo-
somes begin to separate sufficiently for their boundaries to be
distinguished, each appears as a loop or U with its ends at that
part of the nucleus nearest the idiozome body. This is shown in
Fig. 8, where only five of the loops are drawn in their entirety.
On a transverse section of such a stage (Fig. 9) one finds 24
cross-cut portions of loop ; every two of these portions corre-
spond to the two arms of one of the U-shaped loop of Fig. 8.
Therefore there are U-shaped chromosomes, with a particular
arrangement, to the number of 12 ; hence they must be bivalent
with regard to the 24 chromosomes of the spermatogonia. At this
early stage (Fig. 8) there is no sign that the space enclosed by
such a loop has arisen by a longitudinal splitting of a chromosome,
for in fact the characteristic shape of the loops is the same now
as at later stages (Figs. 10, 11). Now this was the main basis
of my argument before an argument that Janssens and Dumez
ignore : that if this were a longitudinal split, we should find its
commencement in such an early stage.

The true longitudinal split commences in the stage of Fig.
8, is very prominent in that of Figs. 9, 10 ; this is a clear split-
ting of each chromatin granule of the arms of each bivalent

142 T. H. MONTGOMERY, JR.

chromosome, but the width of this split never becomes wider
than that shown in Fig. 1 1 . Janssens and Dumez, as all the
workers on amphibian spermatogenesis before them, have entirely
overlooked this split of each arm of a bivalent chromosome.
With material stained in iron haematoxyline, and sufficiently
destained, this split, though narrow, is perfectly distinct.

There is a point brought up by Janssens and Dumez to which
attention must be drawn. They maintain (i) that there is no
regular occurrence of a band of linin (that marked k in the figures
of my previous paper), joining the two univalent arms composing
a U, and placed at the bend of the U ; and (2) that the linin
spirem appears continuous and not broken into as many segments
as there are U-shaped loops. A reexamination shows me that
they are right in regard to there being here a continuous linin
spirem. And this is exactly what I gave especial study to
proving to be the case in the corresponding stages of Pcripatns.
But I must maintain against these writers, that in Plethodon and
Desmognathus, as in Peripatus, there is at no stage in the sper-
matocytes a continuous chromatin spirem. Sometimes one arm
of a U-shaped chromosome seems to be continuous with the end
of an arm of another, as in Fig. 1 1 (and Fig. 5 of my preceding
paper). But this is unusual, and generally one finds, as in Figs.
8, 10 and 11, that the ends of the U-shaped chromatin loops are
connected with the ends of other loops only by linin. Hence
the boundaries of the bivalent chromosomes are perfectly distin-
guishable in most cases. As to the first point, I admit that the
U-shaped loop, which I regard as composed of two univalent
chromosomes attached at the bend of the U, does not show in all
cases a band of linin at its bend ; but it does nevertheless in many
cases. However, on this point I did not place great insistence,
as Janssens and Dumez maintain.

The U-shaped bivalent chromosomes of Fig. 1 1 shorten and
condense into forms such as shown in Fig. 12. By the conden-
sation of the chromatin the longitudinal split becomes hidden.
This is not a remarkable phenomenon, as maintained by the
Belgian writers ; I and others have described it for frequent
cases in arthropods. It reappears in the anaphase of the first
maturation mitosis as a split along which the chromosomes

MATURATION PHENOMENA OF GERM CELLS. 143

divide in the second mitosis. Finally the definitive shape is
reached, as shown in Figs. 13 and 14. One peculiarity has often
been described in heterotypic chromosomes : as they are placed
in the spindle (Fig. 13), very frequently at the middle of each is
one thickening, and this is more frequent than two thickenings.
By comparison of a chromosome, such as that of Fig. 14, with
earlier conditions, such as those of Fig. 12, it becomes evident
that such a thickening corresponds to the separated ends of the
U-shaped loop, which have finally came into close juxtaposition.

When we consider these points, we find two important facts :
(i) that the twenty-four chromosomes are regularly paired in the
spermatogonia, and that there the two of a pair lie close together ;
and (2) that there is no evidence that the space enclosed by any
one of the twelve heterotypic chromosomes has been formed by a
longitudinal splitting. In the spermatocytes there are twelve
loops of the shape of a U or V. There is a longitudinal splitting,
but along the long axis of each loop. The simple explanation
of these facts is that in the spermatocytes, in the synapsis stage,
there takes place the close conjugation of every two such chro-
mosomes as were found in the spermatogonia ; that two together
constitute a U-shaped loop ; and that therefore the first matu-
ration division results in separating entire univalent chro-
mosomes.

The difference of opinion between Janssens and Dumez and
myself is more one of interpretation than of observation, though
they did not notice the pairing of the chromosomes in the sper-
matogonia, nor yet the true longitudinal split. They frankly ad-
mit that by their interpretation the reduction in number of the
chromosomes remains a mystery. They give no explanation of
why there should be regularly disposed U-shaped loops. In as-
suming that a heterotypic chromosome has been formed by a
longitudinal splitting, instead of by a junction of two univalent
chromosomes, they contend for a kind of splitting very wide at
the middle of the chromosome, but narrow at its ends ; yet no
such longitudinal splitting is known in any other case, and its
difference is brought out sharply by comparison with the un-
doubted longitudinal splitting in the chromosomes of the later
prophases of the spermatogonia. These heterotypic chromosomes

144 T - H - MONTGOMERY, JR.

are different in form from others, just because they represent
pairs of univalent chromosomes. If they arose by simple longi-
tudinal splitting, why should they differ so in form from the
chromosomes of the spermatogonia, or of the spermatocytes of
the second order ?

And here my thanks are due to Janssens and Dumez for this
critique of my interpretation, because it induced me to study
anew the amphibian spermatogenesis, and this reexamination
brought out the fact, strong in support of my position, that the
chromosomes are regularly paired in the spermatogonia.

2. THE INDIVIDUALITY OF THE CHROMOSOMES.

By the idea of the maintenance of the chromosomal individu-
ality we do not mean that a chromosome remains chemically
unchanged from generation to generation (for after every mitosis
a daughter chromosome grows to the size of a mother chromo-
some before it divides in the next mitosis), but that, despite great
metabolic and structural changes, a chromosome of any generation
is the descendant of a particular chromosome of the preceding
generation, and is not a new formation. A chromosome of one
generation represents a chromosome of a preceding, just as much
as a cell of one generation represents a particular cell of a pre-
ceding generation. This idea was first propounded by Rabl, 1 and
has steadily gained in support. The workers on ovogenesis
have, for the most part, taken the position that it is not proved ;
but the reason there is simply the great duration of the growth
period of the ovocytes, during a part of which chromosomal
boundaries are not distinguishable. The students of spermato-
genesis, on the other hand, are fairly unanimous in support of
the view.

It is very important that this idea should be firmly established,
and certain considerations would show it to be so. There is first
the fact that from generation to generation the number of chromo-
somes remains the same, from the stage of the fertilized egg to
that of the ovocyte or spermatocyte of the first order. Even the
form of the chromosomes is maintained through these generations,
as shown in the case of the cleavage of Ascaris. In the sperma-

1 " Ueber Zelltheilung," Morfliol. Jahib., 5, 1885.

MATURATION PHENOMENA OF GERM CELLS. 145

togenesis of some Hemiptera there is no rest stage in the growth
period of the spermatocytes, so that the chromosomes can be fol-
lowed from the spermatogonia to the spermatids. In Peripatus
this is but a short rest stage, and during it the boundaries of the
chromosomes can be readily distinguished.

On the experimental side excellent evidence has been brought
in support of this view, particularly by the study of abnormalities,
by Boveri, l Zur Strassen, 2 Morgan, 3 and Herla. 4

Evidence fully as strong as that from experimentaal study has
been obtained by observations upon certain modified chromo-
somes of insects, which are :

3. THE HETEROCHROMOSOMES.

I offer this name to include those peculiarly modified chromo-
somes to which have been given the names " accessory chromo-
somes " by McClung, 5 " small chromosomes " by Paulmier and
"chromatin nuceoli " by myself. They have been described for
the Hemiptera by Henking (/. c.), Paulmier, and myself; for the
Orthoptera by Wilcox, 7 McClung, Sutton, 8 cle Sinety ; 9 and for
the spider by Miss Wallace. 10

1 "Zellen-Studien," Zoo/. Ja/irb., 1888 ; " Befruchtung," Ergcbn. Anat. Entw.,
1891 ; " Ueber die Befruchtungs- und Entwickelungsfahigkeit kernloser Seeigel-
Eier," Arch. Enfwickmeck., 2, 1895 ; " Mehrpolige Mitosen als Mittel zur Ana-
lyse des Zellkerns," Verh. Phys. Ges. Wilrzburg, 35, 1902.

2 " Ueber die Riesenbildung bei Ascaris-Eiern," Arch. Entwickmech., 7, 1898.

3 " The Fertilization of Non-nucleated Fragments of Echinoderm Eggs," Arch.
Entwickmech., 2, 1895.

4 "Etude des Variations de la Mitose Chez 1'Ascaride Megalocephale," Arch.
Bio/., 13, 1893.

5 " A Peculiar Nuclear Element in the Male Reproductive Cells of Insects,"
Zool. Bull., 1899; "The Spermatocyte Divisions of the Acrididre," Bull. Univ.
Kansas, 1900 ; " Notes on the Accessory Chromosome," Anat. Anz., 20, 1901 ;
" The Accessory Chromosome Sex Determinant ? " BIOL. BULL., 3, 1902 ; " The
Spermatocyte Divisions of the Locustidse," Kansas Univ. Sci. Bull., 1, 1902.

" The Spermatogenesis of Anasa tristis," Journ. JMorph., 15, 1899.

7 " Spermatogenesis of Caloptenus femur-rubrum and Cicada tibicen," Bull. Mus.
Coinp. Zool. Harvard, 27, 1895.

8 " The Spermatogonial Divisions of Brachystola magna," Kansas Univ. Quar-
terly, 9, 1900 ; "On the Morphology of the Chromosome Group in Brachystola
magna," BlOL. BULL., 4, 1902.

9 " Recherches sur la Biologic et 1' Anatomic des Phasmes," La Cellule, 19, 1901.
10 " The Accessory Chromosome in the Spider," Anat. Anz.,\%, 1900.

146 T. H. MONTGOMERY, JR.

These are chromosomes which preserve to great extent their
compact form during the whole growth period of the spermato-
cytes, and during the rest stages of the spermatogonia, and re-
tain throughout this whole period the deep staining characteristic
of the other chromosomes only during the height of mitosis.
Thanks to this peculiarity they can be followed with extreme
certainty from generation to generation, even during rest stages ;
and so are splendid evidence for the thesis of the individuality
of the chromosomes.

Now there are two kinds of these. In the Orthoptera there is
an unpaired one in the spermatogonia, larger than the other
chromosomes ; in the Hemiptera they are paired in the spermato-
gonia, and usually smaller than the other chromosomes. Other-
wise in their behavior they are very similar in these two groups
of insects. To include both these kinds the name " heterochro-
mosomes," as expressing a difference from the other chromosomes,
can be advantageously applied; and this would include (i)
the "accessory chromosomes" (unpaired in the spermatogonia),
and (2) "the chromatin nucleoli " or "small chromosomes"
(paired in the spermatogonia). McClung regards them as sex
determinants ; I have considered them to be chromosomes that
are in the process of disappearance, in the evolution of a higher
to a lower chromosomal number.

Now these can be followed from generation to generation with-
out in the Hemiptera undergoing those profound changes which
characterize the other chromosomes after a mitosis. In the Figs.
15-23 they are the chromosomes marked N, n ; Figs. 16 and
17 show them in the spermatogonic and first spermatocytic mi-
toses of Anasa; Figs. 18, 19 for the same stages in Corizits ;
Figs. 20, 21 for Trichpcpla ; and Figs. 22, 23 for the spermato-
gonic monaster and late prophase of the first maturation respec-
tively of Protcnor. In all these cases they can be recognized in
mitosis by their much smaller size.

Recently I have found them to occur in the same number and
form in the ovogonia ; Fig. 15 shows a pole view of the chro-
mosomal plate in the ovogonium of Anasa ; Fig. 16 a similar view
of the spermatogonium of the same species, and the chromosomes
marked N and n are found to correspond exactly.

MATURATION PHENOMENA OF GERM CELLS. 147

Now for Protenor I found that in the spermatogonic chromo-
somal plate there are always exactly thirteen elements (Fig. 22) ;
the two smallest of these are the heterochromosomes marked
N, n they are paired, and in the following synapsis stage conju-
gate to form the smallest bivalent chromosome N, n of Fig. 23 ;
these two chromosomes are then quite similar to the hetero-
chromosomes (chromatin nucleoli) of the other Hemiptera. But
there is a large element in the spermatogonium (X, Fig. 22),
unpaired there, and which does not conjugate with any other
chromosome during the synapsis stage, but remains unpaired in
the spermatocyte (X, Fig. 23). This element I called the
" chromosome .r." Now, as McClung has also pointed out, this
chromosome behaves exactly as does an accessory chromosome
in the Orthoptera, being unpaired in the spermatogonia, and not
conjugating with any other chromosome during the synapsis
stage. Therefore in Protenor occur both kinds of heterochromo-
somes, the small paired ones, A 7 and ;/, and the large unpaired
one, X. N and n are "chromatin nucleoli" according to my
terminology, while X is an "accessory chromosome"; thus
both kinds of heterochromosomes occur in the same cell, and
their likenesses and differences were fully discribed by me for this
species. Both are recognizable through the whole growth period
of the spermatocytes by their compact form and deep staining ;
but only the small pair, N and n, can be recognized in the rest
stage of the spermatogonia. In three other Hemiptera, Alydits,
Hannostcs and CEdancala I showed that in the spermatogonia
occurred also an uneven number (thirteen) of chromosomes ; but
in these the odd chromosome does not maintain its compact form
during the growth period of the spermatocyte, and so is not
recognizable there ; but in the first maturation mitosis it is
immediately recognizable as the only chromosome that has not
conjugated with another to form a bivalent one. Now such a
chromosome of A/ydits, Hannostcs and CEdancala agrees with
the chromosome X of Protenor in not pairing with another during
the synapsis stage ; but differs from it in behaving like the other
chromosomes after a mitosis, i. e., in losing its compact structure
and strong affinity for chromatin stains.

Why should a heterochromosome be sometimes unpaired in the

148 T. H. MONTGOMERY, JR.

spermatogonia, sometimes paired ? When they are unpaired
they are larger than the other chromosomes. This might imply
that such an unpaired heterochromosome really represents two in
close union, /'. c., is already bivalent in the spermatogonium.
And this I think is a very probable explanation, in view, first, of
the behavior of the unpaired chromosomes during the growth
period of the spermatocyte in Protcnor (fully described in my
paper, "The Germ Cells of the Metazoa") ; and, second, of the
fact that such a chromosome sometimes shows a distinct con-
striction at its middle (shown for Haruwstes in my paper,
" Further Studies on the Chromosomes," etc.).

It is hoped that these considerations, in endeavoring to show
the likenesses and differences of the two kinds of heterochromo-
somes, will bring more uniformity in the interpretation of these
modified chromosomes, and that such chromosomes should always
be taken into account in any discussion of the idea of the indi-
viduality of the chromosomes.

4. THE CONJUGATION OF THE CHROMOSOMES IN THE SYNAPSIS
STAGE, AND ITS RELATION TO THE REDUCTION DIVISION.

It is now determined for a considerable number of cases that
in the early portion of the growth period of both ovocytes and
spermatocytes there takes place the process known as the " re-
duction in number" of the chromosomes. Thus if there are
twenty-four chromosomes in the spermatogonium, twelve are
found in the maturation period before the first mitosis. This
fact was first established by Boveri (" Zellen-Studien," /. c.) and
by Brauer. 1 Really the name applied is a misnomer, for there is
no loss of chromosomes, no true " reduction " in this number,
but it is a conjugation of the chromosomes. Riickert 2 sought to
explain it by stating that in the prophase of the first maturation
division the chromatin spirem breaks into only half the normal
number of segments. This, however, is inadequate as an ex-
planation, for I showed in the " Spermatogenesis of Peripatus "
that in the prophases of the first maturation mitosis there is a

1 " Zur Kenntniss der Spermatogenese von Ascaris megalocephala,".4;r/z. J///J-;-.
Anat., 42, 1893.

2 " Zur Eireifung bei Copepoden," Anat. Heftz, 4, 1894.

MATURATION PHENOMENA OF GERM CELLS. 149

continuous /////;/ spirem, which probably does not break into
segments until the metakinesis of the first maturation mitosis, but
no continuous chromatin spirem. Hence it is not a question of
chromosomes which were already contiguous remaining con-
tiguous, but of chromosomes which were first separated (except
for their linin connections) conjugating to form pairs during the
synapsis stage. The criterion of the synapsis stage is such a
pairing; and the term "conjugation" of the chromosomes rep-
resents the facts much better than the term " reduction in
number."

The bivalent chromosomes so formed by conjugation, in the
Hemiptera, Peripalns and Amphibia, become so placed in the
equator of the spindle of the first maturation mitosis, that entire
univalent chromosomes become separated. This is a true reduc-
tion division in the sense of Weismann. Each spermatocyte
of the second order thus receives whole univalent chromosomes
in one half the normal (somatic) number. While the majority
of writers still hold that no such reduction division occurs, the
idea being abhorrent to them, there are still a number who have
furnished an array of facts that can be interpreted only as speak-
ing for such an occurrence ; thus Riickert, Hacker, Vom Rath,
Korschelt, Henking, Paulmier, McClung, Sutton, Nichols, Grif-
fin, Van Winiwarter, Lillie, Schockaert. But the arraying of
names on the one side against those on the other is no argument
in itself, and we may pass to the discussion of certain facts which
harmonize completely with the occurrence of a reduction division,
and remain unexplainable on any other basis.

First, it may be recalled that there is a divergence of opinion
as to which of the two maturation mitoses is the reduction divi-
sion, some holding that it is the first and others, the second.
There is no good reason, save the probability that there would
be expected uniformity in such important processes, that this
division should always be in the first mitosis, or always in the
second ; for it. is quite possible that there is a difference in this
regard in different objects. In the discussion which follows we
will assume it to be the first maturation mitosis since there
occurs the reduction division in the objects specially studied
by me.

I5O T. H. MONTGOMERY, JR.

Now I reached the conclusion ("A Study of the Chromo-
somes," etc.) that in the synapsis stage there is effected a
conjugation of paternal with maternal chromosomes ; under
"paternal " understanding those derived from the spermatozoon,
and under "maternal," those from the ovotid. The arguments
for this were stated as follows :

1. In Ascaris megaloccpliala itnivalcns there is the normal num-
ber of two chromosomes. The ovotid and spermatid have each
only one. In the fertilized egg there is one derived from the sper-
matid, one from the ovotid ; therefore the bivalent chomosome
found in the maturation period of the spermatocyte or ovocyte
must have been formed by the conjugation of a paternal with a
maternal chromosome.

2. In the spermatogenesis of the Hemiptera there are usually
two small heterochromosomes in the spermatogonia. These unite
to form a bivalent one in the spermatocyte. They become
separated from each other in the first maturation mitosis so that
no spermatid receives more than one. Evidently then in the
fertilized ovum since only one comes from the spermatid, the
other must come from the ovitid. Therefore in the conjugation
of the two in the synapsis, it is a conjugation of a paternal one
with a maternal one. That was reasoned out without any
knowledge of such chromosomes of the ovogenesis. Now I
add Fig. 15, showing among the chromosomes of an.ovogenic
monaster stage the two small elements N and ;/, which are
heterochromosomes of the same number and size as those found
in the spermatogonium (Fig. 16, N, ;/). Therefore, there must
be a conjugation in the ovogenetic synapsis stage, as well as
in the spermatogenetic, of a paternal heterochromosome with a
maternal one.

3. That besides the heterochromosomes, whenever there is
recognizable in the spermatogonic chromosomal plate a pair of
chromosomes notably different from the others in volume, there
is always found in the first maturation mitosis a particular bivalent
chromosome notably different in volume from the other ones,
and so evidently formed in the synapsis by the conjugation of
the two peculiar univalent ones of the spermatogonium. This
bivalent chromosome is so placed in the first maturation spindle

MATURATION PHENOMENA OF GERM CELLS. 151

that its two univalent elements pass to opposite cells, so the sper-
spermatid has nevermore than one of them. One of those in the
spermatogonium must accordingly have come from the spermatid
and one from the ovotid, which combined to give rise to that
spermatogonium. And here, also, in the formation of such a
bivalent chromosome in the synapsis there must be a union of a
paternal with a maternal chromosome. No other explanation
seems possible.

These conclusions, the numerical ratios of certain clearly dis-
tinguishable chromosomes in the spermatogonia, to certain
equally distinguishable ones in the spermatocytes and sperma-
tids, could be established for the heterochromosomes for some
forty species of Hemiptera, and for other chromosomes in the
cases of TricJipcpla scinivittata, Protcnor bclfragci, Pcliopclta ab-
brcviata, Prionidus cristatns, Zaitlia fluminea and Corizus latera-
lis. To make this point clear a few figures of certain of these
cases are given here again. In the spermatogonium of Anasa
(Fig. 16), as well as in the ovogonium (Fig. 15), are recognizable
among the 22 chromosomes, two very much smaller than the
others (A", ;/, heterochromosomes), and two considerably larger
(A, <?); in the first spermatocyte there are eleven bivalent ones, six
of which are shown in Fig. 17, and here we recognize again the
chromosomes N, n and A, a. In Corizns, in the spermatogo-
nium (Fig. 1 8) are two particularly small (A 7 , ;/) and two partic-
ularly large chromosomes ; and these recognizable again in the
first maturation spindle (Fig. 19), Similarly in the case of Tricli-
pcpla (Figs. 20, 21). In the spermatogonium of Protcnor (Fig.
22) are thirteen chromosomes ; a particularly large one (A'), two
next in size (K, k\ and two smallest (N, 11). In the spermatocyte
(Fig. 23) A" is recognizable as being the largest; it is the odd
chromosome and is not paired with any other. A^ and // are
paired and so are K and k.

From these observations I concluded that probably in every
case the chromosomes in the synapsis united to form bivalent
pairs in such a way that the one of each pair was paternal and
the other maternal ; and I was able actually to demonstrate it in
those cases where the differences in volume between the chromo-
somes were sufficient to allow them to be followed from genera-

152 T. H. MONTGOMERY, JR.

tion to generation. And I could also prove that in all cases the
two components of each bivalent chromosome always become
separated from each other in the first maturation mitosis.

Following this came the paper of Sutton (/. c.\ proving con-
clusively that in the spermatogonium of Bracliystola the chromo-
somes occur regularly in pairs of graduated sizes, the two of a
pair being always of the same length ; that in the synapsis stage
bivalent chromosomes are produced by the conjugation of every
two chromosomes of the same length ; and that corresponding
chromosomes became separated from each other in the reduction
division (here the second maturation mitosis). So he concluded
quite rightly that there are two series of chromosomes in the sper-
matogonium, a paternal series, A, B, C . . . n, and a maternal
series, a,b,c . . . n, in which A corresponds to a in size and hered-
itary value, B to b, and so through the series. By A joining with a
in the synapsis, like chromosomes conjugate ; and by these sep-
arating from each other in the reduction divisions, it results that
two chromosomes of like size are not found in the spermatid.
Sutton was the first to demonstrate this for the whole series of
chromosomes, and to argue that such a conjugation, together
with the following reduction division, would operate so that no
spermatid could receive two chromosomes of like hereditary
value, but only one chromosome representing a particular value.

In the present paper I have shown that in Plctliodon and Dcs-
mognathus also one may recognize the two corresponding series
of chromosomes in the spermatogonium. An examination of
Ascaris megalocepliala bivalens shows the same relation. Pole
views of the first cleavage spindle (Figs. 28-30) show each two
larger (A, a) and two smaller chromosomes (B, b}. The differ-
ences in size of the two pairs is not very great, but always recog-
nizable when the chromosomes can be seen in their entirety.
This is then evidently a case parallel to those described above :
that of the larger pair (A, a] one is paternal and the other ma-
ternal, and that of the smaller pair (B, b] the same relation holds.
Now the formation of the tetrads in the ovogenesis of this species
has been described by Boveri as two equation (longitudinal) di-
visions of each bivalent chromosome ; and Braur has reached the
same result for the formation of the tetrads in the spermatogen-

MATURATION PHENOMENA OF (iERM CELLS. 153

esis. But in concluding this these writers do not give a satisfac-
tory explanation either why or how univalent chromosomes unite
to form bivalent ones, and so give no clue to a reason for the
chromosomes being paired in the fertilized egg. This point can
be settled only by a careful reexamination of the changes in the
early growth period, and here I shall simply call attention to cer-
tain appearances that speak for the first maturation mitosis in
Ascans being a reduction division.

In the fertilized egg are two pairs of chromosomes, A, a and
B, b (Figs. 2830), one pair being considerably larger than the
other, and the two composing a pair sometimes differing some-
what in length bat being approximately equal in volume. As
Van Beneden first showed, two of these chromosomes come from
the ovotid, and two from the spermatozoon. Fig. 27 shows a
slightly earlier stage, the chromosomes in two groups, one group
derived from the male pronucleus and the other from the female
pronucleus. In the group to the right is a large and a small
chromosome (A, B} ; in the group to the left also a large and
small one (a, &). But A corresponds approximately to a in vol-
ume, and B to b. Therefore we may say that of the four chro-
mosomes in the fertilized egg (Figs. 27-30) a small paternal one
(from the male pronucleus) corresponds in volume to a small
maternal one (from the female pronucleus), and a large paternal
one to a large maternal one. In other words, of each pair of
chromosomes in the fertilized egg, one chromosome is paternal
and one maternal. Which two come from the male pronucleus,
and which two from the female pronucleus, there is as yet no.
means of deciding, for the two pronuclei appear structurally
alike. But to make my argument clear I will assume that A and
B are paternal, and a and b maternal.

Now in the formation of the first polar body (Figs. 24, 25) we
find the two well-known quadripartite chromosomes. There are
two, not four; hence they are bivalent with regard to the normal
number. In each bivalent chromosome (tetrad) we should ex-
pect then to find two univalent chromosomes. Both these figures
(24, 25) were drawn with great care to get the exact proportions
of the parts of the chromosomes ; both represent the stage
where one plate of chromosomes is passing into the polar body.

154 T. H. MONTGOMERY, JR.

Now in each of these cases we notice in the polar body, as in
the egg, a larger and a smaller bipartite chromosome. Thus
in the polar body the larger dyad A, A, and the smaller B, B ;
in the egg the larger dyad a, a, and the smaller, b, b. As far as
I can determine this relation appears to be constant : one large
and one small dyad in the polar body as well as in the egg ; and
not two smaller (or larger) dyads in the polar body and two
larger (or smaller) dyads in the egg. Now from what we have
found to be the case in other objects, I would judge A, A to be
an entire univalent chromosome that had been paired previously
with the entire univalent chromosome a, a ; and that a similar
relation holds between B, B and b, b. This "would then be a
reduction division, separating entire univalent chromosomes. In
favor of this is the fact that A, A is approximately similar in vol-
ume to a, a, and B, B to b, b ; and we have learned that chro-
mosomes of similar volumes conjugate in synapsis. Two dyads
are left in the egg, a, a and b, b, each of which could be re-
garded as a longitudinally split univalent chromosome. In the
formation of the second pole body (Fig. 26) the two parts of
each dyad separate from each other, and this would be an equa-
tional division. There are then left in the egg the two chromo-
somes a and b, which differ markedly in volume. And this is in
exact accord with the fact shown in Fig. 27, that from each pro-
nucleus comes one large and one small chromosome.

This interpretation would bring Ascaris into close agreement
with the other objects discussed in this paper : it explains why
there are two large and two small chromosomes in the fertilized
egg ; why each pronucleus has one large and one small chromo-
some ; finally, why the two bivalent chromosomes of the first
maturation mitosis differ in volume. The idea that each such
bivalent chromosome has been formed by a double longitudinal
splitting, hence that both divisions are equational, gives no ex-
planation for any of these phenomena, nor yet explains how or
why bivalent chromosomes should be formed. The onus no
longer rests upon us to prove the occurrence of a reduction divi-
sion ; but upon those of the other school to prove that a bivalent
chromosome represents one chromosome that has undergone a
double longitudinal division, and to show that such an interpre-

MATURATION PHENOMENA OF GERM CELLS. 155

tation is explanatory of the kind of phenomena that we have
discussed.

5. CHROMOSOMAL COMBINATIONS AND THE MENDELIAN RATIO.
In his paper on " The Chromosomes in Heredity " ' Sutton

4

argues that the combination of paternal and maternal chromo-
somes in the fertilized egg would result in a Mendelian ratio. It
will be recalled that Mendel 2 in his experiments on crossing
varieties of Pisuin, to determine the law of transmission o
parental characters to the hybrid, found the crosses to result in
the ratio I D:2 Dr : i r, in which D represents the pure char-
acter of one parent, r the pure character of the other parent, and
Dr represents the possession of both characters. In other
words : out of four offspring resulting from such a cross, one
would resemble the father, one the mother, and two combine the
characters of both parents.

Sutton starts with the fact that there are two series of chro-
mosomes in each fertilized egg, A, B, C, ... ;/ and a, b, c, ...
n, the first set of paternal origin (from the spermatozoon) and the
second of maternal (from the ovotid). In these series A is the
homologue of a, B of b, and so on. In the synapsis stage of the
germ cells A would conjugate with a, B with b, and so on, so
there would be formed the bivalent chromosomes Aa, Bb, Cc,
... n. In the reduction division A and a would pass to sep-
arate cells, and such would be the case with B and b and the re-
maining paired chromosomes.

Then Sutton takes the case where there are the two homol-
ogous chromosomes A and a in an ovogonium and A and a in a
spermatogonium ; in the maturation period there would be
formed Aa in the ovocyte, and Aa in the spermatocyte ; the
ovotids would contain then either A or a, and the spermatids
either A or a. There would then result in fertilization these
combinations :

A $ + A 9 = AA

A $ + a 9 = Aa

a $ + A 9 = a A

a $ -\- a 9 = aa.

1 BIOLOG. BULL., 4, 1903.

~ 2 " Versuche iiber Pflanzenhyhriden," Vfrh. nat. \ r er. Briinn, 4, 1865.

156 T. H. MONTGOMERY, JR.

" Since the second and third of these are alike the result
would be expressed be the formula A A : 2Aa : aa which is the
same as that given for any character in a Mendelian case."

But as a matter of fact this can be so only in a case where
there are only two chromosomes in the fertilized egg. For let
us take the case where the normal number of chromosomes is
four ; express by capital letters those chromosomes originally de-
rived from the spermatozoon, and by small letters those derived
from the ovotid ; and assume that A is homologous to a, and B
to b. Then the spermatogonium would have the chromosomes,
A, a, B, b, and the ovogonium have also A, a, B, b. By the
synapsis stage would be formed bivalent chromosomes Aa, Bb
in the spermatocyte, and Aa, Bb in the ovocyte. The reduction
division would separate A from a and B from b in both sperma-
togenesis and ovogenesis. The spermatids would contain then
either A, B, or a, b, or A, b, or a, B ; and the ovotids either A,
B or a, b, or A, b, or. a, B. In the fertilization of one of these
ovotids by one of the spermatozoa, 16 different combinations are
possible : A, B, A, B ; A, B, a, b ; A, B, A, b ; A, B, a, B a,
b, A. B ; a, b, a, b ; a, b, A, b; a, b, a, B ; A, b, A, B ; A, b, a,
b\ A, b, A, b \ A, b, a, B ; a, B, A, B\ a, B, a, b ; a, B, A, b ;
a, B, a, B. But only one of these combinations is of purely
paternal chromosomes, namely A, B, A, B ; and only one of
purely maternal, namely a, b, a, b. The other fourteen combi-
nations show paternal together with maternal chromosomes (six
cases where the paternal and maternal chromosomes are present
in equal number, four cases where there are three paternal chro-
mosomes to one maternal, and four cases where there are three
maternal chromosomes to one paternal).

Hence the ratio is : \P : \j\PM : iM, where P stands for purely
paternal chromosomes, M for purely maternal, and PM for com-
binations of paternal and maternal chromosomes. This is clearly
not a Mendelian ratio of I : 2 : I. And obviously the disparity
would become greater with any increase in the number of chromo-
somes. According to Sutton's own computation, in forms which
have 24 chromosomes, the number of possible combinations of
these in the fertilized egg would be 16,777,216. That would
give the ratio of \P : i6,777,2i4/W : I M.

MATURATION PHENOMENA OF GERM CELLS. 157

But though the combinations of paternal and maternal chro-
mosomes in the fertilized egg do not support the Mendelian ratio
for hybrids, I fully agree with Sutton that "the phenomena of
germ-cell divisions and of heredity are seen to have the essential
features, viz., purity of units (chromosomes, characters) and the
independent transmission of the same."

UNIVERSITY OF TEXAS,
November 29, 1903.

158 T. H. MONTGOMERY, JR.

EXPLANATION OF FIGURES.

FIGS. 1-3. Pole views of spermatogonic monasters of Plethodon ci tier ens.

FIG. 4. Pole view of a spermatogonic monaster of Diemyctilus virescens.

FIGS. 5, 6. Pole and lateral views respectively of spermatogonic monasters of
Desmognathus fusciis.

FIG. 7. Oblique pole view of one plate of daughter chromosomes, early sper-
matogonic anaphase of Desmognathus fuscus.

FIG. 8. Lateral view of a late synapsis (postsynapsis) stage of Desmognathus
fuscus ; four entire bivalent chromosomes shown, and half of another. Nuclear mem-
brane not yet formed.

FIG. 9. Polar view of a spermatocytic nucleus at a slightly later stage in the same
species.

FIGS. 10-12. Successive prophases of the first maturation mitosis in Desmogna-
thus fuscus.

FIG. 13. Lateral view of a spindle of the first maturation mitosis, showing three
bivalent chromosomes in metakinesis ; Plethodon cinereus.

FIG. 14. A heterotypic chromosome of Plethodon cinereus in its definitive form.

FIG. 15. Anasa (undetermined species from California), pole view of ovogonic
monaster.

FIG. 1 6. Anasa sp., pole view of spermatogonic monaster.

FIG. 17. Anasa sp., lateral view of first maturation mitosis, showing six of the
eleven bivalent chromosomes.

FIG. 18. Corpus alternates, pole view of the spermatogonic monaster.

FIG. 19. Corizus alternatus, lateral view of first maturation mitosis, showing five
of the bivalent chromosomes.

FIG. 20. TricJipepla semivitfata, pole view of spermatogonic monaster.

FIG. 21. TricJipepla semivittata, lateral view of first maturation mitosis, showing
all the bivalent chromosomes.

FIG. 22. Protenor belfragei, pole view of spermatogonic monaster.

FIG. 23. Protenor belfragei, late prophase of the first maturation mitosis.

FIGS. 24, 25. Ascaris megalocephala bivalens, formation of the first polar body
(first maturation mitosis), the spindle seen very obliquely in Fig. 25.

FIG. 26. Idem, formation of second polar body.

FIG. 27. Si/,.'Hi, lateral view of the first cleavage spindle showing the two groups
of chromosomes.

FIG. 28-30. Idem, pole views of the chromosomes in the first cleavage spindle.

r /

^ ^ y /

3. T&.Z'i \\

JB-

Vol. 1 1. Mai'di, iqo-f.. No.

BIOLOGICAL BULLETIN

NOTES ON REGENERATION.

T. II. MORGAN.

During the past summer I made at Woods Holl a number of
observations and experiments on the regeneration of several
animal forms. The results are here brought together, although
they have little more in common than that they all deal with
problems of regeneration.

THE LIMITATION OF THE REGENERATIVE POWER OF
DENDROCOZLUM LACTEUM.

The fresh-water planarians show such remarkable powers of
regeneration that it is surprising to find in one of them, Dcndro-
cceluni lacteuui, that this power is much reduced. The question
at once arises whether we can discover anything peculiar in the
relation of this planarian to its surroundings, or in its internal
structure that will give a clue to its exceptional behavior.

There is nothing in its habitat to suggest that it has lost, or
has never acquired to the same degree, the power of regeneration
possessed by other planarians. In the pond at Falmouth where
I collected this species there were also present, sticking to the
under surfaces of the same stones, both Planaria maculata and
Phagocata gracilis. If Dendroccelumis not as subject to injury
as are the other two species, and if, therefore, it does not need
the same .regenerative power, it is remarkable that Dendroccelum
should be so uncommon in comparison with the other two forms.
If it is subject to greater injury, then it has not acquired the
power to meet the situation as have the other species. Consider-
ations of this kind do not have, I believe, any real bearing on the
question of whether an organism has or has not acquired the power
to regenerate, although some biologists lay great stress on this
sort of speculation. The limitations in the power of regenera-
tion of Dendroccelum are peculiar. Lillie found that when only

'59

l6o T. H. MORGAN.

the anterior end of the worm is cut off a new anterior end is
regenerated. This power to produce a new head was found to
extend back to about one-third of the length of the worm, i. e.,
to a region just in front of the pharynx. Behi'nd this level the
posterior piece fails to regenerate a head at its anterior end.

On the other hand, the anterior pieces regenerate a new pos-
terior end from any level, with the possible exception of the
immediate region of the head itself; but the latter point has not
yet been sufficiently examined in this species. It appears a
remarkable fact that this planarian should have such extensive
powers of regenerating posteriorly, and such limited powers of
regenerating anteriorly, especially since, as far as we know, the
same cells produce either a head or a tail according to which end
is exposed ; but this has not been definitely determined, and
would be almost impossible to determine with absolute certainty.
Eugen Schultz has also studied the regeneration of Dendroccelum
lacteum of Europe * and finds that posterior pieces do sometimes
regenerate a head, although the regeneration is very slow, and it
may appear that Lillie did not keep his pieces a sufficiently long
time for the regeneration to take place. He states, in fact, that
most of the posterior pieces died after five or six days. Schultz
believes that these posterior pieces have potentially the power to
regenerate, but that sometimes the piece closes in such a way
that the formation of new tissue is prevented, as I have found to
occur occasionally in Bipalium. Lillie, on the other hand, tries
to account for the lack of power of posterior pieces to form a
head by means of the following hypothesis. He suggests that
the regeneration from the posterior cut surface at all levels is due,
in some unexplained way, to the presence "of the brain and
anterior part of the nervous system in the anterior piece." Con-
versely the absence of these structures in posterior pieces is sup-
posed to account for the lack of regeneration from the anterior
cut surface. A simple experiment would have shown the untena-
bility of this point of view. If the head end is cut off just in
front of the pharynx so that the brain and the anterior part of the
nervous system are removed, and then the tail end of the middle

1 It has been assumed that the European Dendroccelum lacteum and the American
form or forms are identical, but I think this question will bear further examination.

NOTES ON REGENERATION. l6l

piece is also removed, it will be found that the middle piece with-
out regenerating a new head will still regenerate a new tail. This
shows conclusively that Lillie's supposition m regard to posterior
regeneration is erroneous. The remainder of his argument,
which rests on this assumption, also falls, I believe, in the light
of this fact.

The great mortality that Lillie observed in the posterior pieces
is due largely, at least in my experiments in which the same
thing was observed, to the temperature being too high, or possibly
to exposure to light. If the pieces are kept cooler (by sur-
rounding the dishes by the cool, running salt water of the labora-
tory) the mortality is much reduced, and instead of dying after
six days, as in Lillie's experiment, I have kept short posterior
pieces for several weeks. It is only by keeping such pieces for
a long time that one can fairly test their powers of regeneration.

Schultz states that he cut Dendroccslum in two either between
the pharynx and the reproductive region or else in front of the
pharynx. In the former case he found that the posterior pieces
regenerated an anterior end very slowly, and he found it more
profitable in studying the regeneration of the head to use those
posterior pieces that had been cut off in front of the pharynx.
He found that the regeneration of the anterior end often failed to
take place, and he attributes this to fusion of the sides of the
cut surfaces, as I had found to occur not infrequently in Bipaliinn.
Whether this is the whole of the question remains to be seen.
In a marine polyclad, Leptoplana, Schultz found that posterior
pieces, no matter at what level they have been removed, fail to
regenerate an anterior end, even when only a small piece of the
head is cut off. Yet regeneration from a posterior cut surface
takes place at all levels. Schultz attributes the lack of regener-
ation at the anterior end either to the closing over of the " grow-
ing point" by the coming together of the old tissue from the
sides, or to the muscles from the sides uniting and thus prevent-
ing further growth. Both factors he thinks may enter into the
result. This point could be tested, I think, by making the cuts
so that there is left a pointed anterior end, when regeneration
should occur, if Schultz's view is correct. From an experiment
of this sort that I have carried out on Dendroccelum I think it

1 62 T. H. MORGAN.

probable that in Lcptoplana also no better regeneration would
occur, even at a pointed end, 1 and if this proves to be the case
Schultz's explanation is insufficient. 2

In my experiments I first examined whether the form of the
cut surface at the anterior end had anything to do with the lack
of regeneration, for it was possible here, as in the case of Bipa-
lium, that the cross-cut surface closed in such a way that subse-
quent regeneration was prevented. By changing the form of the
cut surface this difficulty should be eliminated. Posterior pieces
were cut off through the region of the pharynx and also behind
the pharynx. The anterior ends of some of these pieces were
very oblique ; others were pointed in the middle, i. e., they were
cut off by two oblique cuts meeting in the middle line. In the
latter case especially it is impossible that the muscles from the
sides could close the anterior cut surface. 3 These pieces were
kept alive for two or three weeks, and although it could be seen
that there was a little new tissue at the anterior cut surface, yet
no further regeneration occurred after the first ten days or there-
abouts, and there is no indication that regeneration w r ould have
gone any further if the pieces had been kept alive for a greater
length of time.

Sections of these pieces were made. The results will be given
below.

In two other series each worm was cut into three pieces. The
head pieces extended to the middle of the region in front of the
pharynx. These pieces should be capable of regenerating at the

1 Loeb says that Thysanozoon regenerates a new head, but he did not determine
whether a new brain is formed. Monti also obtained regeneration in this form and
also in Leptoplana, except when cut far posteriorly. Lang also records regeneration
in marine polyclads.

2 Schultz states in the opening of his paper that I carried out my experiments
without making sections of the planarians, and he intimates that had I done so I
would not have reached certain conclusions in regard to the growth of the new part.
How Schultz obtained this information it would be interesting to know. Probably
he based his generalization on the absence in my earlier papers of reference to histo-
logical details with which I was not then especially concerned. As a matter of fact
I had made and studied many sections. My students also were at work on the minute
anatomy, and one of them published a complete account of the histological changes
taking place during regeneration before Schultz's paper appeared.

3 Whether union of the dorsal and ventral muscles might close these pieces I have
not here considered.

NOTES ON REGENERATION. 163

posterior end. The middle pieces included the next portion of
the worm, and extended to the region of the reproductive pore.
These pieces should be capable of regenerating a head at their
anterior ends and a tail at the posterior ends. The third pieces
were the tail pieces and included the rest of the worm. These
pieces should be incapable of regenerating a head at the anterior
end. The pieces were preserved at intervals of I, 2, 3, 4, 5, 6
days, killed, embedded, stained and examined with immersion
lenses.

A study 9f the sections shows that the changes taking place at
the anterior end of the tail -pieces appear to be similar in all
respects to those that occur at anterior or posterior surfaces at
which regeneration of the missing part takes place. There is
nothing in the sections to show why the regeneration should con-
tinue in the one case and not in the other, and it is difficult to
believe from the evidence of the sections that anterior regenera-
tion from the tail-pieces would not in time be accomplished, yet
after three weeks there was no sign of further regeneration and I
am forced to conclude with Lillie that in the form of Dendro-
ccehtin found at Falmouth regeneration does not, ordinarily at
least, occur behind the level of the pharynx. Sections through
tail-pieces, cut off behind the pharynx and kept for nearly three
weeks, show that the formation of new tissue has not gone much
beyond that of the first six days, and that a new head has not
been produced. Sections of the oblique, and of the pointed tail-
pieces give exactly the same results.

Several writers seem inclined to account for the lack of regen-
eration in certain planarians, and especially from the posterior
region of the body, as due to the absence or small size of the
nerve cords in these regions. With this view I do not agree.
Lillie has used Dendrocoeluni as a case in point. Sections of this
worm show, however, that the cords in the more posterior regions
are as well developed, judging from their size, as they are in
Planaria maculata.

REGENERATION IN PYCNOGONIDS.

In 1895 Loeb published some observations that he had made
on the regeneration of one of the Pycnogonida, PJioxichilidium

164 T. H. MORGAN.

maxillarc. He cut the animal in two between the second and third
pairs of legs, and found in two cases that after a time a new part
suddenly appeared, presumably after a moult. This new part
that regenerated at the posterior end of the anterior piece Loeb
speaks of as a body, and points out that this is the first case
observed in the arthropods in which new body segments have
been seen to regenerate. I have repeated this experiment during
two summers, for it did not appear to me beyond dispute that the
new part that had been observed was necessarily a body, since no
satisfactory evidence that it was such is furnished -by Loeb's
paper. Although sections of the new part were, apparently,
made, no posterior opening of the digestive tract was found, no
ganglia are described as being present in the new body, nor do
new legs appear to have been present at the sides as we should
expect if this new part were really a body.

My first experiments were made in 1901 and, although a
number of pycnogonids were kept for two months or longer,
none of them regenerated at the posterior end. Since I had used
large individuals it seemed not improbable that the lack of regen-
eration might have been connected with the maturity of the in-
dividuals. During the past summer I have repeated the experi-
ment on a large scale, both with large and with small individuals ;
but although many of the pieces were kept for nearly two months
no regeneration took place, with the possible exception of two

instances that will be described.

In a number of cases the individuals were cut in two between

the third and fourth pairs of legs, i. e., nearer the posterior end
than Loeb had cut them, for, from analogy with other cases, it
seemed more probable that if the body could regenerate at all it
would be more likely to do so the nearer the cut was made to
the posterior end. Other individuals were cut in two between
the second and third pairs of legs. In only one case did regen-
eration appear to take place, as shown in Fig. I. Here the
bases of the fourth pairs of legs bulge out as though they had
been formed anew, and it seems possible that the rudimentary
abdomen is also new, although it is also possible that a part at
least of this structure had been left unintentionally when the cut
was made. Sections show that the digestive tract opens at the

NOTES ON REGENERATION.

i6g

end of the abdomen. There is no trace of further regeneration
within the stumps of the legs. At most, the bases of the legs
and the abdomen, or part of the latter, have regenerated.

FIG. i.

The second case is shown in Fig. 2, in which there is only a
bulging out of the end of the body. The cut had been made in
this case between the second and third pairs of legs. Sections
of this individual do not show any indications of the development

FK;. 2.

of legs or of a rudimentary abdomen in the new tissue of the
bulging portion, and there is nothing to indicate that the develop-
ment would ever have gone any further. The digestive tract
ends blindly and is not connected with the ectoderm.

In looking over a large number of individuals I found a few
cases in which a leg on one side was much smaller than its

1 66 T. H. MORGAN.

opposite, and from this it seems probable that the original leg
had been lost at the breaking joint at the base, and a new one
had begun to regenerate. Moreover, I found one case in which
the new leg was clearly a new structure, Fig. 3. The different
segments had not yet been formed in their adult proportions and

FIG. 3.

the leg could not have been functional as yet. There is some
resemblance between this leg and the newly regenerated part
from the posterior end of the body that Loeb saw and figured.
In fact this idea seems to have suggested itself to Loeb for
he writes : " Das Vorhandensein eines ueberzahligen Segmentes
konnte vermuthen lassen, dass das neugebildete Stuck vielleicht
im Laufe der Zeit sich zu einer Extremitat entwickelt haben
wiirde, dass es sich also um die Bildung eines Beines an Stelle
des abgeschnittenen Rumpfstuckes gehandelt habe, ein Fall, den
ichals Heteromorphose bezeichnete. Allein Hoek fiihrt an, dass
bei Ammotheen das Abdomen nicht selten Spuren einer Segmenta-
tion zeigt." Thus in order to explain away the presence of too
many segments in the new part Loeb has recourse to a condition
found in another species a mode of explanation that will
scarcely recommend itself.

A somewhat fuller analysis of these two cases of Loeb's may
not be unprofitable. If the new part is really a body, /. c.,
thorax and abdomen, we should expect to find the digestive tract
opening at the posterior end, but this does not appear to have
been the case, for, Loeb says : " Der Darm setzte sich in clen
vorderen Theil des regenerirten Stiickes fort. Im Uebrigen aber
waren die Gewebe wenig differenzirt." It is to be remembered
that the digestive tract also continues out into the legs in the
pycnogonids as a blind sac. In the second place, while the three
segments of his first example might be interpreted as representing
the two remaining thoracic segments, and the rudimentary ab-
domen, yet in the other case five or six segments appear in the
new part. It is this that led Loeb to suggest that the new part

NOTES ON REGENERATION.

l6/

might represent a leg ; but he withdraws this interpretation at
once as seen above. There is certainly no striking resemblance
between the new part figured by Loeb and the abdomen of Ain-
mothca. Finally, if the new part is a new thorax where are the legs ?
In the light of these considerations we must wait until some
one, favorably situated, has an opportunity to work over the
subject with ample materials. Meanwhile it seems to me that so
far as the evidence goes it is rather in favor of the view that the
regeneration described by Loeb is a new leg and not a part that
replaces the lost segments of the thorax and abdomen.

THE LACK OF REGENERATION OF THE PIGMENT SPOT IN

THE FlN OF FUNDULUS.

If a gold fish having a black band at the end of its tail be
selected, and the end of the tail be cut off proximal to the band,
a new band like the one removed reappears in the regenerated
tail. The presence of black pigment at the cut surface from
which the new part regenerates is clearly not necessary for the
development of pigment in the new part. This result is all the

FIG. 4.

more curious since the occurrence of the pigment band is only an
individual peculiarity. It seemed desirable to try the same ex-
periment in a species in which a characteristic spot or a ring was
present. The dorsal fin of the male of Fundiilus majalis has a
black spot in its posterior part, Fig. 4. The spot is not present
in the female, and it appears, therefore, that this color marking
belongs to the category of secondary sexual characters.

The posterior part of the fin was cut off by an oblique cut ;
the part removed containing all of the black spot. The lost part
was slowly replaced, and in the course of two months the fin was
completed, but the pigment spot did not come back, and there

1 68 T. H. MORGAN.

was no evidence that it would have done so if the fish had been
kept longer. Since the operation had been carried out during the
height of the breeding season, it seemed possible that the spot
might normally fade out later, but other fish, examined in Sep-
tember, showed the spot still present.

The results on Fundnlns appear to be different from those on
the gold fish, and it is not apparent why this difference should
exist. The result does not seem to be connected in any way
with the fact that the spot in Fuudulus is a secondary sexual
organ. The most plausible explanation that suggests itself is
that in the tail of a gold fish that has a black tip there are cells
throughout the tail that can develop pigment should they get
into the terminal portions of the tail, while no such cells are pres-
ent in Fnndnlns, or if present they fail to produce pigment in the
new part. It may be that in Fundnlns all the cells capable of
producing pigment have been already carried into the pigment-
spot itself, and hence when this spot is removed no cells capable
of developing this pigment are present in the remaining part.
Further work will be necessary to determine whether these sug-
gestions have any value.

THE METHOD OF CLOSURE OF THE CUT ENDS OF TUBULARIA.
The peculiar method of closure of the cut ends of Tnbnlaria
has attracted attention since it appears to be different from the
closing observed in other forms. I have already discussed at
some length this process 2 and shall not repeat here what has
been already said, but since I have observed during the past sum-
mer certain processes that seem to throw some light on this ques-
tion I shall briefly refer to them in this connection. Stevens has
figured the closed end of a piece that had been cut through the
hydranth-forming region at the time when the primoidium of the
new hydranth had just been laid down, and when the red pig-
ment lines, that indicate the appearance of the new hydranth,
were present. Over the closed end the red lines radiate to the
center of the bounding membrane. It seemed to me that a
further examination of pieces that closed in this way might throw
some light on the process in general. Pieces were cut off and

1 Morgan, Ronx 1 s Archiv, XIV., 1902.
2 " Regeneration," 1901, p. 69.

NOTES ON REGENERATION. 169

kept about twenty-four hours when the primoidia of the new
tentacles had begun to appear. At this time I cut the ends
squarely off, the plane of section lying across the middle of the
new proximal tentacles. To my surprise the cut ends now
closed in a very different way from that of ordinary cross-cut
pieces. The whole wall contracted from the perisarc and the
cut edges were brought together almost at once, and subse-
quently fused, often showing the radiating lines described by
Stevens over the new end. It was perfectly clear that the result
was due to a contraction of the ccenosarc, and the difference be-
tween this process and that shown by ordinary pieces appears to
be due entirely to the fact that at the time when the tentacle
primoidia are laid down, the ccenosarc has become free from the
outer wall, or perisarc.

From this result it seems to me to follow with great proba-
bility that in ordinary pieces the closure of a cut end is also due
to a process of contraction of the ccenosarc, but ordinarily the
wall of the ccenosarc is so closely stuck to the inner surface of
the perisare that it is not free to pull away as a whole, and
there is a consequent drag that holds back the contracting wall,
and a consequent modification of the method of closure of the
opening. This conclusion also fits in well with some facts ob-
served at the time of closure of the pieces. Certain of the cells
that appear to be more closely stuck to the wall are often left
behind, or are retarded in their progress towards the center of
the newly forming membrane. Thus the peculiar method of
closure of Tnbularia finds its explanation in the unusually close
connection between the perisarc and ccenosarc. I have tried to
show elsewhere * that this same connection may also be respon-
sible for the characteristic " incomplete structures " of Tubidaria,
whose chief peculiarity is that their organs are full sized so far as
they are formed.

TRANSPOSITIONAL OR COMPENSATORY REGENERATION OF THE

LARGE CHEL/E IN SOME CRUSTACEA.

Przibram 2 discovered in 1901 in the decapod Alplieus that it is
possible to cause the small claw (chela) of one side to become

1 " Some Factors in the Regeneration of Tubularia," Roux'' 's Archiv, XIV., 1903.
2 J?oux's Archiv, XL, 1901.

I/O

T. H. MORGAN.

the large claw by the simple operation of removing the large
claw of the other side. At the next moult the small claw be-
comes the big one, and the newly regenerated claw becomes
the small one. Zeleny 1 found in 1902 that a similar throwing
over of the large operculum of the annelid, Hydroidcs, can be
brought about by the same sort of operation. Wilson - in 1903
made some important additions to Przibram's work, using an
American species of Alpheus. He suggested that the small claw
is merely an arrested stage of development of the big claw, and
that when the big claw is removed the check is at the same time
taken away that holds back the development of the small claw.
At the next moult the small claw becomes the large one, and the
new claw the small one.

As yet no one has detected the nature of the correlation that
causes the transposition, and this must obviously be the next step
in advance. Wilson has suggested that the throwing over is con-
nected with the nervous system, but the experiments on which
he bases this suggestion appear to me to be capable also of an-
other interpretation.

During the past summer I undertook some experiments which
I hoped would give results bearing on this question, but the out-
come has been almost entirely negative. Nevertheless, I shall
venture to describe these experiments briefly, because if carried
out on more suitable forms they will very probably throw some
light on this exceedingly important subject.

Several years ago I found that by cutting the nerve of the leg
of the hermit-crab, proximal to the breaking joint, the leg can
then be cut off at any level beyond the breaking joint without the
remaining part being thrown off at the base. By removing por-
tions of the large leg at different levels, after first cutting the
nerve at the base, I hoped to be able to discover whether the
amount removed had any effect on the transposition of the large
claw to the other side. It was also possible that the simple
cutting of the nerve might have some effect, as Wilson's experi-
ment seems to show. The result might also, as Wilson appears
to believe, depend in part upon the degree to which the new nerve

1 Roux's Arc/iiv, XIII., 1902.

2 BIOLOGICAL BULLETIN, IV., 1903.

NOTES ON REGENERATION. I/I

regenerated before the next moult. In practice, however, it is
not possible to cut the nerve without cutting also the blood-ves-
sels, and the injury to the latter may be as important as, or even
more so, than that to the nerve.

The experiments were carried out with the hermit-crab and
with the fiddler-crab, but were unsuccessful in both cases for dif-
ferent reasons. First, the transposition does not occur under any
circumstances in the hermit-crab, as this and other experiments
showed ; and second, in the fiddler-crabs the muscles, etc., be-
yond the breaking joint degenerate after the operation. This
caused the death of most of the crabs, and those that remained
alive had only the outer shell of the leg beyond the breaking
joint, and even this fell off in several cases. Since, however, the
operation can be carried out in the hermit-crab without the outer
part of the leg degenerating, it may be possible, in other forms
that have the power of transpositional regeneration (in Alphcns,
for example), to carry out this experiment successfully.

In both the hermit- and the fiddler-crab I also tried the effect
of removing three of the walking legs on the same side of the
body as the big claw, leaving the big claw uninjured, in order to
see if the absence of the other legs might possibly affect the
transposition. This did not succeed, because in the hermit-
crabs, as I have said, the big claw does not throw over, and in
the fiddlers the experiment had to be brought to an end before
any of the crabs had moulted.

All of the individuals of the hermit-crab that I have examined
were right-handed, and the shells in which they live have also
right-handed spirals. It has been suggested to me that this is
an adaptation, in so far that the right-handed hermit crab is
placed to better advantage in a right-handed shell. Conse-
quently, if this were true (and I am by no means certain that it
is so), it would be disadvantageous for the hermit-crab to have
the power of transposition after the loss of the big claw, and in
consequence this power has not been acquired, or else, if it ex-
isted in the ancestors "of the hermit-crabs, it has been lost. That
there is really no basis for an argument of this kind is shown by
the state of affairs in other decapods ; in the lobster, for example.
In the American lobster I have seen several cases in which the

1/2 T. H. MORGAN.

big claw had been lost and a new one of the same kind was re-
generating on the same side. Przibram has also described cases
of this sort. This result is all the more interesting since in the
lobster the big claw is present in some individuals on one side,
and in other individuals on the other. It cannot be claimed in
the lobster that one kind of claw represents an undeveloped stage
of the other. In the regeneration of the claws, as especially well
seen in the lobster, the particular type of claw is present, although
not always fully developed, at an early stage, as Przibram has de-
scribed, and as I have also found. No doubt the advocates of
the view that all beneficial processes have been acquired because
of the benefit conferred, will find in these cases of transposition of
the big claw from one side to the other evidence of the acquire-
ment of a useful process through natural selection, but I do not
think that there is any connection of this sort in these cases. 1

I have intimated above that the injury to the blood-vessels
that run to the leg may be closely connected with the changes
that take place in the leg, and account for the absence of trans-
position in those experiments of Wilson's in which the nerve of
the small claw was cut (and presumably also the blood-vessel).
My work on the fiddler-crab convinced me that cutting the
blood-vessels, which seems nearly always to take place when the
nerve is cut, brings about important changes in the condition of
the leg. If my suggestion prove correct, namely, that the lack
of transposition in Wilson's experiment is due to injury to the
blood-vessel rather than to cutting the nerve, then it is possible
that the whole phenomenon of transposition may be connected
with the condition of the blood supply to the leg. After re-
moval of the large claw more blood may be thrown into the ves-
sel going to the small claw, and this may be the cause of the
change that takes place.

1 In this respect I am in entire agreement with Wilson.

NOTES ON REGULATION IN STYLARIA LACUSTRIS.

E. H. HARPER.

This paper is not concerned chiefly with regeneration, but
more especially with some processes of regulation accompanying
regeneration which were observed in the living animals. Stylaria
lacnstris L. is a well-known fresh-water oligochaete of the family
Naididae, whose members have the power of reproducing by self-
division. The worm is six to eight mm. in length, and is easily
recognized by its long prostomium or proboscis. The body is
divided into three regions : (i) An anterior specialized region of
five segments, containing the pharynx, which is marked by a yel-
low pigment distinct in appearance from the dark brown chlora-
gogue layer of the rest of the alimentary tract. The first seg-
ment bears the long prostomium and eyes. The mouth is ven-
tral and the pharynx is slightly eversible. The anterior region
is further distinguished by the absence of the capilliform dorsal
setae, ventral setae also being absent from the first segment. (2)
The middle region, with an indefinite number of segments, con-
taining the oesophagus and the crop, a dilatation between the
seventh and eighth segments, and the stomach-intestine. The
middle region ends in a budding zone of incompletely developed
segments and embryonic tissue. (3) The specialized anal seg-
ment, somewhat longer than the rest and tapering in form, devoid
of setae, and with the gut lined with cilia.

Self-division takes place at a region posterior to the middle of
the worm, from the seventeenth to the twenty-fifth segments,
very commonly at the end of the twenty-first. The first indi-
cation of division is the appearance of a band of transparent
tissue divided in the middle by a slight constriction. Em-
bryonic tissue accumulates before and behind the septum between
the segments forming an ectodermal thickening. These regions
lengthen and soon become segmented, that in front of the septum
forming the anal segment of the anterior zooid, and the part
behind developing into a pharyngeal region of five segments for
the posterior zooid. The zooids remain united until completely

173

174 E. H. HARPER.

developed, a continuous band of fecal matter being visible
through the length of the worm. During rapid division another
zone of fission makes its appearance, one segment in front of the
one previously formed, making a chain of three zooids.

This method of self-division is often compared with the more
primitive type called fragmentation in which the parts separate
before regenerating the ends. The physiological regeneration
that takes place in self-division is the same in its results as re-
generation from a cut surface. Asexual multiplication continues
through the warmer months. In October and November the
sexual organs attain maturity. Budding then ceases, and the
po\ver of regeneration is also diminished.

METHODS.

Whole mounts of these worms have been made by the follow-
ing method. As stated above, the results of this paper have
been obtained from the study of the living forms, but when it
has been desired to preserve specimens to show results of regen-
eration, difficulty was encountered in killing the animals in an
extended condition, since they invariably become coiled up on
application of the fixing fluid. A remedy for this was found by
getting the animal extended in the angle between the beveled
edge of a slide and a glass plate. A very little hot sublimate-
acetic applied to the animal in this position will be drawn under
the slide with considerable force and prevent, the animal from
moving. If as little liquid as possible be used, the animals may
be killed in a perfectly extended condition. The animal should
be placed on the glass plate in a small drop of water. If the
slide is then moved up to the drop, the worm will become ex-
tended next to the glass, and held there with sufficient force to
prevent its coiling when the killing fluid is applied.

I. REGENERATION IN THE ASEXUAL FORMS.

Experiments. i. A resume of some general features of re-
generation in Sty/aria.

These forms including 5. laciistris have been studied exten-
sively, both in respect to their method of self-division and their
regeneration. Section within the middle region is followed by

NOTES ON REGULATION IN STYLAR1A LACUSTRIS. 1/5

regeneration of the pharyngeal region of five segments from an
anterior cut surface. Similarly, the anal segment is regenerated
from a posterior cut surface, but within a shorter time. In re-
generation a bud or knob of transparent embryonic tissue is first
proliferated, which increases in length, develops a prostomium,
and becomes segmented within a few days. Section within the
anterior specialized region is followed by the restoration of the
number of segments which were removed, from one to five.

Thus it is seen that the pharyngeal region of five segments is
the unit of regeneration after section within the trunk region,
and the process is identical with the normal or physiological
regeneration that occurs in self-division. On the other hand, the
segments are the units of regeneration when the mutilation is
within the pharyngeal region.

There is a close analogy between these results and the obser-
vations made upon the earthworm, in which four or five seg-
ments are regenerated after more than five are cut off.

The prostomium has great power of regeneration. Frequent
cases are met with of forms with regenerating prostomium. Child
('oo) figured a specimen found in nature with a forked prosto-
mium which was doubtless a product of regeneration. In Fig. 9
is shown a somewhat similar case of regeneration of the pro-
stomium following the severing of the organ at the base ; in this
case the organ is doubled. In later stages the lateral bud was

O ~J

absorbed.

2. Regulation of the intestine behind a cut surface to form the

o

oesophagus and crop.

It was stated at the outset that this paper deals chiefly with
certain processes of regulation accompanying regeneration proper.
As an excellent example of the general regulative changes ac-
companying regeneration in this worm a case of regulation may
be mentioned which occurs normally in the process of self-division
or physiological regeneration as well as in cases of regeneration
after injury. The oesophagus and crop occur in the sixth to
eighth segments, i. c., not in the region which is formed from
new tissue by proliferation. Hence the oesophagus and crop
must be formed by a process of regulative transformation of the
intestine behind the new proliferating tissue. This involves a

176 E. H. HARPER.

narrowing of the intestine to form the oesophagus and a dilata-
tion to form the crop. The histological features are not consid-
ered here. The same process occurs in regeneration from a cut
through the intestine.

3. Inhibition of the zone of fission under the influence of a
regenerating region anterior to the fission plane.

A striking instance of regulation is seen in the effect produced
upon a zone of fission by section anterior to it. Under certain
conditions the formation of a regenerating region will inhibit the
process of self-division and cause the disappearance of the zone
of fission. This involves the rearrangement of tissues and a
process of redifferentiation in a region at a distance from the cut
surface. If the animal is severed in front of and near to a zone
of fission which is in an early stage of development before seg-
mentation has taken place, the constriction and the accumulated
embryonic tissue disappear and the zone is completely rediffer-
entiated (Fig. I, a-c].

The nearer the regenerating region is to the zone of fission the
greater is the tendency for the latter to disappear, but the in-
fluence may be exerted at a considerable distance. The follow-
ing table gives the results in 48 cases :

Number of Segments Between the Cut Number of Experi- Disappearance of Zone of Fission
Surface and the Zone of Fission. ments. in.

1-7 28 60 per cent.

8-15 20 15 per cent.

In all the cases included in the above table the zone of fission
was in an early stage of development, but not all were at exactly
the same stage. If only specimens were taken for experiment in
which the zone was just beginning to form, no doubt a much
larger percentage of cases of disappearance would be obtained
both when the regenerating region was near and when it was far
away.

The writer was informed by Professor Frank Smith of an ob-
servation upon a nearly related form, Pristina sp., to the effect
that the zone of fission may disappear in animals kept in the
laboratory, as a result of insufficient food or other unfavorable
conditions. It is realized that a control experiment would be
desirable, to show what percentage of cases of disappearance of

NOTES ON REGULATION IN STYLARIA LACUSTRIS. 177

the fission zone might be traceable to other causes than the in-
fluence of the regenerating region. The results here given are,
however, relied upon in the belief that the early disappearance of
the zone of fission (in all cases in about two or three days) suffi-
ciently indicated that it was due to the influence of the regenerat-
ing region. There is also the evidence of the cases to be
mentioned later in, which the zone of fission did not disappear
subsequent to a cut behind the zone of fission.

An effect is produced the converse of what has just been de-
scribed when an animal is severed in front of and near to a well-
advanced zone of fission. In such a case its development exerts
a retarding influence on the regenerating region. A small piece
in front of such a zone of fission is soon separated, after under-
going little or no regeneration.

4. Effects of section behind a recently formed zone of fission.

Section behind a recently established zone of fission does not
ordinarily seem to exert an inhibitive influence. In only one
such case observed was the zone redifferentiated. No change is
produced, unless it be a retardation of the development of the
zone of fission. Two considerations seem to have weight in ex-
plaining the difference between this and the case in which the
cut is anterior to the fission plane. First, the regeneration of the
anal segment is a process requiring the withdrawal of less material
from the old parts than the regeneration of an anterior end, and
hence is calculated to interfere less with the development of the
zone of physiological regeneration. Second, the establishment
of a regenerating region behind the zone of fission does not
materially change the conditions already existing in that region,
since in this case a regenerating region posterior to the fission
plane is substituted for the previously existing proliferating re-
gion in front of the anal segment.

Galloway ('99) has noted in respect to Dcro vaga that cutting
behind the fission zone tends to disorganize its growth and the
posterior piece soon drops off.

As a parallel to the results of the preceding experiments upon
Stvlaria it may be mentioned that Dr. Child has informed me
that he has found a similar disappearance of the fission plane in
the Rhabdoccele Stenostoma. under the influence of a regenerating

D O

1^8 E. H. HARPER.

region. There is probably a close similarity in the physiological
processes connected with fission in the two groups.
5. Disorganization in the region behind a cut surface.

() There are a number of phenomena taking place internally
during regeneration which can be plainly seen, owing to the
transparency of these animals. When a portion of the head
segment in front of the eyes is severed, in almost all cases the
pigmented portions of the eyes are found later in a fragmented
condition and the black pigment cells scattered through the
pharyngeal region (Figs. 2, 5, 1 1). This does not seem to be a
direct mechanical effect, since it occurs when the eyes are ap-
parently not directly injured by the mutilation. This observa-
tion, which may seem of slight importance, is mentioned as show-
ing how organs behind the regenerating region may be affected,
apparently disorganized, in this case, and later restored. The
eyes are reformed subsequently and the scattered pigment cells
disappear. In Figs. 5 and 1 1 the eye appears in two parts. It
became normal later. The interpretation, if such it may be called,
which would seem to be indicated by the facts, is that the region
just behind the cut surface tends to return to an embryonic or
disorganized condition as the result of change in physiological
conditions.

() Another observation of a like nature has been made in the
case of the regeneration of the whole pharyngeal region. During
the regeneration numerous brown pigmented cells, similar to the
chloragogue cells of the stomach-intestine, are seen floating in
the ccelomic fluid. These are different from the normal unpig-
mented coelomic corpuscles. As was pointed out above, the
region behind the cut surface is a seat of regulative activity, the
oesophagus and crop being produced in that region by regulation
of the intestine. The free pigment cells may be a product of the
disorganization of the intestine immediately behind the cut sur-
face, as in the case of the pigment cells of the eye.

As stated above, these two observations seem to indicate that
the organs just behind the cut surface tend to return to an em-
bryonic condition similar to that of the new proliferated material.
This breakdown of tissues may also be comparable to the loss of
cell boundaries and return to a syncytial condition in the well-

NOTES ON REGULATION IN STVLAKIA I.ACUSTKIS. 179

known experiments of Loeb with segmenting Fundulus eggs, in
which the cause ascribed was lack of oxygen.

6. Healing of a cut surface without regeneration.

The regenerative power is so strong in the asexual naids that
it would naturally be expected that regeneration would take place
under all circumstances in pieces above a certain limit of size.
Certain instances of failure to regenerate have been met with,
however, which indicate that the power to regenerate is depen-
dent upon certain internal conditions. If an oblique cut be made
in front of the pharynx, removing the prostomium and one side
of the head segment, including perhaps one eye, the cut surface
may frequently heal over and failure to regenerate the prosto-
mium may result (Figs. 4 and 5). A number of such cases have
been obtained. In some instances the animals were kept for
more than three weeks without showing any tendency to regen-
erate the prostomium. Since regeneration ordinarily requires
only a few days, the effects seemed in these cases to be per-
manent.

Internal conditions favorable to proliferation, such as the ex-
posure of cut surfaces of intestine and blood-vessels, are present
in nearly all possible experiments with Stylaria. But after section
of the prostomium and one side of the head without touching the
pharynx, the tendency to proliferate is apparently at a minimum.
The ectoderm may, therefore, close over the wound and cut off
the outlet for proliferating material. Failure to regenerate
seemed to follow only after an oblique cut, but in one instance,
after a transverse cut in front of the eyes, a somewhat similar re-
sult was obtained. In this animal an outgrowth appeared from
the cut surface which was of less diameter than the region be-
hind, appearing like a new head segment superimposed upon the
old. Its appearance suggested that the outlet for the prolifer-
ating material had been narrowed by reduction of the cut sur-
face. It is conceivable that still further reduction of the cut
surface, or even complete closing of the wound, might occur in
case the proliferating tendency was slight (Fig. 3).

The above cases of failure to regenerate were only occasional
instances, showing that the balance between the tendencies to
proliferate and to heal over without regeneration is rather deli-

i8o

E. H. HARPER.

cately adjusted. Cases occurred in which the prostomium was
regenerated, but the side of the head or lobe was not normally
restored, at least within the period of observation (cf. Fig. 13, of
a sexual individual).

The general conclusion is that certain parts which may be
mutilated without exposing cut surfaces of internal organs which
are of importance as internal factors in proliferation are less likely
to be regenerated, since, when the proliferating tendency is slight,
the ectoderm closes over the wound and checks proliferation.

I a.

i i,.

I c.

2 a.

26.

4 a.

5*-

NOTES ON REGULATION IN STYLARIA LACUSTRIS. l8l

DESCRIPTION OF FIGURES.

FIG. I. (a, b, c.) Redifferentiation of a zone of fission. In (a], a zone is
formed and there is a constriction in the body-wall. The intestine in this region is
transparent, devoid of pigment. In (b) and (<:) the zone and constriction have
disappeared and the intestine becomes pigmented. The accumulation of transparent
embryonic tissue before and behind the septum in (), mentioned in the text, is not
indicated in the figure. The ectodermal thickening is indicated at the constriction.
In this and all the figures the dorsal capilliform setae are not represented full length.

FIG. 2. Fragmentation of the eyes after mutilation. The prostomium was sev-
ered and a short longitudinal cut made in the first segment. The direction of the
cut indicated by line. The condition in (/;) was observed three days after the muti-
lation. The slightly curved outgrowth of the bud of the prostomium is an unusual
occurrence.

FIG. 3. Regeneration from first segment, resembling a repetition of the head seg-
ment, of less diameter. The line indicates direction of the cut. The eye is regen-
erated. The condition here represented was fifteen days after the operation.

FIG. 4. Failure of regeneration after an oblique cut through first segment. In ()
the condition twenty-three days after the operation is shown. The eye is not regen-
erated and there is no trace of a prostomium.

FIG. 5. Failure of regeneration in first segment. The eye in (b) is fragmented
into two portions. Later it became normal.

7. Absence of regeneration in short posterior pieces.

With the purpose of finding whether heteromorphosis would
take place, as in the earthworm, in the case of very short pos-
terior pieces a number of experiments were tried with as short
posterior pieces as would survive the operation. No tails appeared
on the anterior ends. A number of pieces which survived the
operation for as long a time as three weeks, did not regenerate
the anterior ends. During the first week these pieces elongated
within the budding region in front of the anal segment. The
immature segments became further differentiated, their setae in-
creasing in size, but no new segments appeared. The pieces then
appeared to be composed of segments nearly equal in size, and
there was no zone of embryonic undifferentiated tissue remaining.
One such piece was obtained by a cut one segment behind the
pharynx of a posterior zooid. It contained at first a region com-
posed chiefly of indistinct segments. At the end of a week these
segments had increased in length and their setae had grown until
twenty segments could be easily counted. Its size was greater
than that of a minimal piece capable of regeneration taken from
the middle of the body. It showed no trace of anterior regen-
eration for three weeks, but was still active in its movements. Un-

1 82 E. H. HARPER.

fortunately, it was not possible to keep it under observation longer.
A number of similar pieces have been kept with the same results
(Figs. 6 and 7). The elongation of such pieces did not seem to be
merely mechanical, since it was accompanied by some differentia-
tion such as the growth of the segments. Possibly the proliferating
tendency at the cut surface was checked by the continued growth
of the budding zone. The latter, containing an accumulation of
embryonic tissue, was perhaps able to check regeneration by
absorbing the available materials for growth. Whether such
pieces \vould in time regenerate needs, of course, to be deter-
mined before the possibility of heteromorphosis would be settled.

8. Regeneration of the posterior end.

It was thought possible that heteromorphosis might occur in
regeneration from short anterior pieces. In no case did the
specialized anterior region of five segments survive long enough
to begin regeneration. An anterior piece of eight segments sur-
vived for six days without showing any posterior regeneration.
This was the shortest piece which was able to survive more than
a short time after the operation. Pieces with only two more of
the trunk segments present on the average regenerated freely,
however, as the following table will show :

Number of Number Abie to Survive Number Able to

Length.

Experiments.

for Several Days.

Regenerate.

8-9 segments.

9

2

O

10-11 "

10

9

7

12-13 "

10

9

9

14-29 "

37

35

33

There is a great average increase in the power to regenerate
correlated with the presence of only two additional segments of
the trunk. The specialized head region, as would be expected,
has the least power of regeneration. Portions of the trunk of
the same length as the first class given in the above table are able
to regenerate freely.

No cases of true heteromorphosis were obtained. The anterior
region of five segments which might perhaps be expected to de-
velop a head posteriorly, contains no nephridia (they are found
back of the sixth segment), and as we have seen, does not sur-
vive the operation long enough to regenerate. Results obtained in

NOTES ON REGULATION IN STYLARIA LACUSTRIS. 183

Planaria, with its diffuse excretory system, could not be expected
to occur in a form like Stylaria, with a head region so specialized
as to be unable to exist independently even for a short time.

A case of duplication of parts was seen in the production of
a double-headed individual, one of whose heads was larger and
in line with the axis of the trunk. The other was lateral, lying
close to it, and smaller. The smaller one was in process of
absorption at the last stage at which it was observed (Fig. 8).

A doubling of the prostomium was noticed in one case as
mentioned above. The two organs lay parallel and were fused
together except at the tip of the shorter (Fig. 9). In later stages
the lateral bud was absorbed.

9. Regulation of growths oblique to the main axis.

It is a common observation that proliferation tends to take
place along an axis normal to the cut surface. After an oblique
cut the new growth continues oblique until it has reached its
full length or nearly so. The straightening of such oblique
growths is evidently not a function of growth alone, but of growth
influenced by certain tensions, which may be exerted by the part
behind the new growth. Rievel ('96) has pointed out the influ-
ence exerted by the peristaltic motions of the intestine upon the
regeneration of the head region. The knob of new tissue is at
first solid and is later penetrated by the slower ingrowth of the
lumen of the intestine into the region. The waves of peristaltic
motion constantly passing along the intestine favor its ingrowth
into the new region. Finally the mouth is formed by the
bulging out of the body-wall, caused by peristalsis. It is broken
through from within, there being no stomodaeal invagination of
the ectoderm in regeneration.

It may be suggested that peristaltic movements of the intestine
have an influence in straightening oblique growths. This is
probable since the straightening occurs not during the prolifer-
ating period, but coincidently with the ingrowth of the pharynx.
Straightening occurs, moreover, while the tissue still appears
embryonic before the complete differentiation of muscular and
nervous systems (Figs. 10 and 13).

184

E. H. HARPER.

8 a.

8 b.

7-

10 a.

10 b.

NOTES ON REGULATION IN STYLARIA LACUSTRIS.

I8 5

10 C.

10 d.

DESCRIPTION OF FIGURES.

FIGS. 6 and 7. Failure of anterior regeneration in case of very short posterior pieces.
Fig. 6 represents such a piece twenty-three days after it was severed, Fig. 7 a piece
eighteen days after it was severed. It measured 1.25 mm. Further observation
was prevented.

FIG. 8. Regeneration of a double-headed individual. The line in (a) indicates
direction of cut. The smaller head was apparently destined to be absorbed at last
observation. The specimen was lost before regeneration was complete. Stages (a)
and (^) were six and ten days after the cut was made.

FIG. 9. Regeneration of abnormal prostomium, a doubling of the organ. Later
the lateral bud was absorbed.

FIG. 10. Regeneration occurring in a direction normal to the cut surface. (&)
represents the condition three days after the operation, (d] the complete regeneration.
In (</) the right dorsal bundle of setae is not restored. In (c) scattered pigment
cells resembling the chloragogue cells are present in the new tissue.

II. REGENERATION OF THE SEXUAL FORMS.
The sexual organs of Stylaria come to maturity during the
months of October and November. In the height of the sexual
stage the animals cease to multiply asexually. In the earlier
stages budding animals are frequently seen. In the fully de-
veloped sexual animal the formation of new segments in the
anal proliferating region also ceases. As a result the animals
attain larger size, since the segments in the budding region
become fully differentiated. The clitellum is conspicuous, cover-
ing the fifth and sixth segments, in which the testes and ovaries
lie. This part becomes more opaque and loses its setae.

1 86 E. H. HARPER.

The results obtained in regard to the regeneration of the sex-
ual forms will be treated under two heads : (i) The regeneration
of the pharyngeal region involving the results following the
severing of 15 anterior segments ; (2) regeneration within the
trunk region.

1. In the regeneration of the pharyngeal region three sorts of
cases are met with :

() Complete regeneration may occur in the less mature sexual
forms.

(&) There may be an incomplete regeneration, resulting in the
production of an outgrowth which is devoid of ventral setae and
seems to be the equivalent of the first segment. The regener-
ated part is distinguishable by its relative lack of pigment and
by the fact that it does not attain the original diameter (Fig. 1 1).
It is comparable to Fig. 3, where a growth took place in an
asexual form from an apparently restricted outlet, and the same
explanation may be offered here, viz., that owing to the slow-
ness of proliferation the partial closure of the cut surface by the
body-wall restricted the outlet. The prostomium does not
reach full length and the lateral lobes of the head are not de-
veloped. Often the eyes are not formed in such cases.

(c) Often complete failure to regenerate occurs. The end
heals over and a mouth is formed (Figs. 12 and 14). In twenty-
one cases of mature sexual animals in which from one to four
segments were removed there were six such cases of failure to
regenerate and the rest regenerated an amount apparently equiv-
alent to one segment.

2. After the removal of five or more segments results followed
which likewise may be divided into three classes.

(a) Normal regeneration in the less mature sexual specimens.

(//) A pharyngeal region of five very short segments with a
diminutive prostomium may be formed. Four pairs of ventral
setae appear showing that the full number of segments is repre-
sented, but the region is remarkable for its dwarfed appearance

(Fig. IS)-

(c] There may also be failure to regenerate. In twenty-nine
cases in which from six to thirty segments were removed there
were six cases of failure to regenerate anteriorly and five cases of
the dwarfed regeneration.

NOTES ON REGULATION IN STYLARIA LACUSTRIS.

187

The regeneration of the anal segment is equally slow in the
advanced sexual forms and the part regenerated is not of full
size (Fig. 16).

3. In a single instance the clitellum was regenerated, but the
specimen was unfortunately not kept under observation long

II a.

ii b.

II c.

II d.

II e.

~)

\

I2,f.

12 b.

12 a.

14 a.

\ /

14 b.

188

E. H. HARPER.

13

I 3 /,.

16.

DESCRIPTION OF FIGURES.

FlG. II. Regeneration of a sexual individual after removal of first three segments.
The clitellum is indicated by the widening of the body. It takes in the fifth seg-
ment, which is a part of the pharyngeal region. The setse are absent from the clitel-
lum. One pair of ventral bundles were left in front of the clitellum. In stage (e)
the head segment is regenerated. As no more pairs of setse bundles were produced,
the regenerated part is regarded as the equivalent of the head segment alone. The
stages (b-e) followed 3, 8, 12 and 19 days after the operation, respectively.

FIG. 12. Failure of regeneration after severing part of first segment in a sexual
individual. In (l>) a side view is given. The condition shown in (l>) and (f)
followed seventeen days after the cut.

NOTES ON REGULATION IN STYLARIA LACUSTRIS. 189

FIG. 13. Oblique growth of bud of prostomium in (/>), and slightly incomplete
regeneration. Stages (b, c, d) were 4, 8 and 18 days after the operation, respec-
tively. The eye was not regenerated. The other eye increased in size.

FIG. 14. Failure of regeneration in a sexual individual after removal of two seg-
ments. This condition existed nineteen days after cut was made.

FIG. 15. Dwarfed anterior regeneration from a sexual individual after removal of
fifteen segments. The prostomium is not regenerated. A pharyngeal region of five
short segments is produced, as shown by the number of setae bundles. In (c) a side
view is given. The level at which the cut was made is shown in (a).

FIG. 1 6. Regeneration from posterior end of a sexual individual. The part pro-
duced is of less diameter.

enough to determine whether the regeneration of the sexual or-
gans was complete. The cut was made just behind the clitellum.

CONCLUSIONS.

1. The formation of a regenerating region will under certain
conditions inhibit the process of asexual multiplication and cause
the disappearance of the zone of fission. This effect may be pro-
duced by a cut anterior to the zone of fission, less often by a cut
posterior to it, and occurs only when the zone is embryonic.
The zone is also more likely to disappear if the cut is near to it.
The band of transparent embryonic tissue redifferentiates and the
energy of growth is transferred to the regenerating region.

2. There is evidence of disorganization and a return to embry-
onic conditions in organs just behind a cut surface. This effect
does not appear to be a direct mechanical result, /. c., due to
crushing. Fragmentation of the pigmented portion of the eye is
one case adduced.

3. Internal conditions favorable to proliferation, such as the
exposure of cut surfaces of intestine and blood-vessels, are pres-
ent in nearly all possible experiments. But if a corner of the
head segment be removed, including the prostomium, without
injuring the pharynx, the ectoderm may close over the surface
and regeneration may fail to take place.

4. Short posterior pieces often fail to regenerate anteriorly,
but no cases of heteromorphosis, such as individuals with tails on
the anterior end, have been obtained. Short posterior pieces
which failed to regenerate within the time of observation may
elongate and show some differentiation in the budding region.

5. The middle portion of the body has the greatest power of
regeneration, the specialized pharyngeal region has the least.

IQO E. H. HARPER.

6. Growth takes place at right angles to a cut surface, and it
the cut is oblique the bud will grow out at an angle to the axis
of the body. Straightening is affected after the penetration of
the lumen of the pharynx into the region, probably under the
influence of the tension produced by the peristaltic motions of
the intestine.

7. Sexual individuals lose the power of regeneration to a large
extent just as they do their power of budding. In an advanced
sexual stage regeneration may fail to occur or be incomplete.

I wish to express my thanks to Dr. C. M. Child for suggesting
to me the subject of this paper and for further suggestions rela-
tive to some of the results described.

HULL ZOOLOGICAL LABORATORY,

UNIVERSITY OF CHICAGO, 1903.

REFERENCES.
Bourne, A. G.

'91. Notes on the Naidiform Oligochseta. Quart. Jour. Micr. Sci., vol. 32, pp.

335-356-
Child, C. M.

'oo. A specimen of Nai's with bifurcated Prostomium. Anat. Anz., Bd. 17, pp.
311-312.

Galloway, T. W.

Observations on Non-Sexual Reproduction in Dero vaga. Bull. Mus. Comp. Zool.,
Vol. XXV, No. 5.

Harper, E. H.

'01. Abstract in "Science," N. S., Vol. XIV, No. 340, pp. 28-29, J u ty 5-

Hepke, P.

'97. Uber histo- und organogenetische Vorgange bei den Regenerationsprozessen
der Naiden. Zeit. \yiss. Zool., LXV.

Mayer, C.

'59. Reproductionsvermogen und Anatomic der Naiden. Ver. Nat. Vereins,
Rheinlande XVI.

Morgan, T. H.

'97. Regeneration in Allolobophora foetida. Arch. f. Entw.-mech. V.

Rievel.

'96. Die Regeneration des Vorderdarmes und Enddarmes bei Einigen Anneliden.

Zeit. f. Wiss. Zool., Bd. 62.
Schultze, Max.

'49. Ueber die Fortpflanzung durch Theilung bei Nais proboscidea. Arch. f.
Naturgesch., Bd. I., Jahrg. 15, pp. 293-304.

VARIABILITY IN THE NUMBER OF TEETH ON THE

CLAWS OF ADULT SPIDERS, SHOWING THEIR

UNRELIABILITY FOR SYSTEMATIC

DESCRIPTION. 1

CARL HARTMANN.

Since the number of teeth on the claws of spiders is often used
as a specific character, the need of testing its constancy sug-
gested itself. It has been pointed out by W. Wagner 2 that the
number of ungual teeth varies with each moult. In the present
study of the variations in the adult this fact was well taken into
consideration, great care being exercised in choosing fully mature
individuals that had undergone the last moult. To my knowl-
edge no one has ever tested the constancy of the number in
fully mature individuals.

The study was made on the claws of the right legs of 70
females and 40 males and comprises, therefore, observations on
nearly 1,320 claws of 440 legs. Representatives of a number
of different families were chosen as follows : Dictynidae {Dictyna
volupis, Keys., West Chester, Pa.), Theridiidae (Theridium tepi-
dariorum, Koch, Philadelphia), Pholcidae (Spermaphora sp .?
Hentz, Austin, Texas), Epeiridae (Epeira marmorea Clerck ;
E. benjaiiiini, Walck.; Acrosoma reduvianum, (Walck), West
Chester, Pa.), Lycosidae (Lycosa nidicola, Emerton, Austin,
Tex.).

The counting of the claws was easy except in the case of
Dictyna volupis and of Spennophora ; but even here, if any mis-
takes in the counting are recorded, they are extremely few, for I
never left a claw until convinced that my count was correct ; or
in a few cases where this seemed impossible the individual was
entirely discarded. To count the teeth the three claws of each
leg, if they were large, were snipped off with a needle (keeping
the foot on a slide in a drop of alcohol) and pressed flat with a
cover-glass. If the claws were too small, the whole tarsus was

1 Contributions from the Zoology Department of the University of Texas, No. 55.

2 W. Wagner, " La Mue des Aragnees," Ann. Sc. Nat., 1888, p. 363.

191

192

C. HARTMANN.

TABLE I. FEMALES.

in

(*-

No. of the Individual.

'o

' V

V

0.

^

i

2

3

4

5

6

7

8

9

10

13,9

13,12

I 3 ,I1

12,11

12,11

12,10

14,12

14,12

12,12

13,"

^

s

5

5

5

5

5

5

5

5

5

1

2

12,10

12,12

12, JO

ii,-

12,12

12,12

12,11

13,13

11,12

1

5

5

5

5

4

5

5

5

5

1

9,10

10,9

-,9

9,9

9,9

8,9

9,10

10,10

10,10

9,9

*

4

4

4

4

3

4

3

4

3

4

c5

9,10

9,10

10,10

9,10

9,10

9,9

11,10

9,10

10,11

10,10

4

4

4

4

3

4

4

4

4

4

i

6,6

6,5

5,8

6,5

6,5

6,6

4,54,5

6,5

6,5

6,6

2

2

2

2

2

2

2 2

-

2

2

ll

2

6,6

6,5

6,5

6,5

6,6

6,5

5,5 -,6

7,5

6,6

6,6

*S-2

2

2

3,2

2

2

2

2 2

2

2

2

V

5,6

5,6

5,5

5,6

5,6

5,6

->5 -.6

5,6

5,6

6,6

s^,

2

2

2

2

2

2

2 2

2

2

i

fc*

4,2

3,2

4,2

4,2

4,2

4,1

3,2 3,2

4,2

3,1

4,2

4

2

I

I

2

2

i

I I

I

2

i

i

11,10

10,10

11,11

10,10

10,10

10,10 11,10 11,10

9,9

11,10

.

I

I

I

I

I

I

I

I

i

I

k

2

10,10

10,10

11,10

12,10

10,10

11,10

II, II

9,9

10,10

,

I

I

I

I

I

I

I

i

I

s

9,10

10,10

II, II

10,10

II, II

9,9

10,9

10,10

9,9

9,12

%

2

I

I

I

I

i

i

I

i

I

A

7,9

7,9

-,9

9,10

7,9

8,10

8,10

8,9

7,8

10,10

T-

o

i

2

i

i

i

i

i

i

I

I

7,8

8,8

7,8

8,9

9,7

7,8

7,8

8,8

8,8

9,9

2

V

2

2

2

2

2

3

3

2

3

3

i

2

7-8

6,7

7,8

9,9

8,8

8,7

7,8

7,8

8,8

8,8

K.

2

2

2

2

2

2

3

2

2

3

i

u

6,6

-,7

6,7

7,6

6,5

6,6

6,7

6,6

8,7

.5

*

2

2

2

2

2

2

i

2

2

A

5,6

6,5

6,8

6,6

5,6

6,5

6,5

6,6

6,7

^

2

i

2

2

2

2

2

2

2

I

4,4

4,4

4,4

4,4

4,4

5,4

5,4

4,5

4,4

4,4

J

o

o

o

o

o

o

o

*N

2

7,6

7,6

5,4

5,4

5,4

6,5

5,4

5,6

5,5

4,5

.12

o

o

o

o

i

o

o

o

o

8

3

7

7,6

6,6

7,6

6,5

7,7

7,6

7,7

7,7

6,6

2

J

o

o

o

o

o

o

o

4

A

"

6,5

9,9

6,7

6,7

5,7

8,8

7,7

8,8

7,7

7,7

o

o

o

o

o

o

o

i

5,8

5,9

8,-

8,8

9,8

8,8

10,8

8,9

7,8

6,8

*
.8

2

2

3

2

3

2

3

2

2

2

i

2

5,9

6,9

5,6

7,9

7,9

8,8

8,9

7,8

6,8

7,8

?

2

2

3

2

3

2

2

2

2

2

"

J

3,6

8,7

7,6

7,7

8,7

7,7

6,11

7,6

7,7

6,6

*1S,

j

2

2

2

2

2

2

2

2

2

2

d.

6,5

7,6

7,6

4,5

6,5

5,5

6,5

6,5

6,5 6,5

T^

2

2

2

2

2

i

2

2

2

2

TEETH ON CLAWS OF ADULT SPIDERS.

193

TABLE I. FEMALES. Continued.

(fl

V
D.
C/3

1*4

No. of Individuals.

'

2

3

4

5

6

7

8

9

10

i

7,6

7,6

7,7

7,6

7,7

7,6

8,7

7,6

7,7

7,7

2

2

2

2

2

2

2

2

2

2

r *

?

7,6

7,6

7,6

7,-

6,6

6,7

7,7

7,7

6,7

5

2

2

2

2

2

2

2

2

2

t i

7

5,6

5,5

6,5

6,5

6,6

5,5

6,6

5,5

6,6

5,5

g < *

2

2

2

2

2

2

2

2

2

2

A

3,2

3;4

3,4

4,4

4,5

5,4

4,2

4,4

4,3

I

2

2

2

2

2

I

2

i

covered, placed under the compound microscope and pressure
applied until all the teeth came into full view.

The results of the counting of the teeth are recorded in the
accompanying tables, Table I. containing the figures for the fe-
males and Table II. for the males. The three claws were always
distinguished from one another and the number of teeth on each

recorded after the formula ----- , where represents the number

of teeth on the anterior, / on the posterior and z on the inferior
claw.

The reduction in the number of teeth seems to take place at
the proximal end of the claw because, firstly, the distal teeth
usually maintain the size and form characteristic of the species,
and secondly because the proximal tooth (or teeth) in some cases
becomes so small as to merit the name tubercle in place of
tooth. This latter fact forced me to establish a criterion to de-
termine what to count as a tooth and I decided to call the
structure a tooth if it had attained a length at least half as great
as its width at the base.. In Dictyna volupis the two distal teeth
are small and are closely approximated to the claw for nearly
their entire length. One of these was counted in some four or
five cases where it was unusually large and stood out from the
claw for at least half its length.

In order to reduce the tables to percentages so as to get at a
simple set of figures for comparison I have adopted what may
be called the " percentage of constancy " method, which may be
illustrated as follows :

In Table I. the anterior claw of the first leg of Lycosa nidicola

194

C. HARTMANN.

TABLE II. MALES.

en

V

*-

No of the Individual

'o

a?

1)

p<

O 1

i

2

3

4

s

6

7

8

9

IO

12,11

12,11

12,10

12,11

12,9

II, II

II, II

10,10

II, II

11,10

.3

I

5

5

5

5

5

5

5

4

7

6

1

12,12

12,11

13,12

n, ii

12,11

12,11

ii,-

II, II 12,11 II, IO

2

6

5

5

5

5

5

5

5

6 6

s

n, ii

11,10

9,9

9,10

9,9

9,9

10, IO

9,9

-, IO IO, IO

>

.Vj

3

4

4

4

4

4

4

5

4

4 5

3

ii, 1 1

10,10

9,9

10,10

10,9

10,10

11,10

9,10

10,11 10,10

4

5

4

4

4

4

4

4

4

5

5

4,4

5,5

5,4

5,5

6,4

-.4

5,-

5,5

5,5

5,5

i

i

2

2

2

2

2

2

2

2

~ s

ft s

V J^

2

4,4

5,4

5,4

5A

5,4

5,4

5,4

5,5

5,5

5,5

It

i

2

2

2

2

2

I

2

2

2

5-2

-A

4,5

5,3

4,5

3,5

4,3

4,5

4,3

4,4

5,5

*-;

i

2

2

2

2

2

2

2

2

2

2,1

3, i

3,1

3,1

4,1

3,-

4,1

4,1

3- 1

4,1

4

I

i

i

i

i

1

i

1

I

I

12,11

12,-

12.11

12,10

11,10

10,9 10,10 ii, 10

11,10

12,11

I

I

1

i

I

i i i i

I

1

2

12,10

11,10

12,11

11,10

12,11

10,9 n, 10 10,10 ii, 10

11,10

^s

1

I

1

i

I

i

i

I

I

I

i

10,11

II, II

II, IO

-,IO

II, II

9,9

10,10

9,9

10,9

10,10

4

3

1

I

I

I

I

I

i

i

I

to

9,10

8,10

8,10

8,10

9,IO

8,9

8,9

8,9

9,10

4

i

i

i

i

I

i

i

I

I

i

7,9

10,11

9,8

8,9

8,9

2

2

2

2

2

2

1

8,8

7,9

8,8

8,8

<s

2

2

2

2

R

6,6

6,6

7,8

6,6

6,6

.*:

2

2

2

2

i

A

6,6

7,-

-,5

6,5

6,6

^

2

2

i

i

i

I

6,6

6,6

6,6

6,7

6,6

3

o

o

o

o

1

2

7,7

8,8

7,7

7,8

7,8

ijj

o

o

o

o

S

i

8,9

9,10

8,8

8,8

9,9

3

J

i

o

o

^

8,9

9,9

9,9

10, 10,

o o

o

o

has 4 teeth 8 times out of 10, the posterior claw has 4 teeth 9
times out of 10. So that the claws have percentages of constancy
of 80 and 90 respectively. On the second leg of the same
species the anterior claw has 5 teeth more often than any other
number of teeth or in other words it has a maximum constancy of

TEETH ON CLAWS OF ADULT SPIDERS. 195

5 in 10 = 50 per cent.; the posterior claw has a maximum con-
stancy of 40 per cent, having 4 teeth 4 times in 10. This method
was pursued throughout. Where only five specimens of a
species were examined the result was calculated to ten. On
the basis of these data the following conclusions were drawn.
Where percentages are cited they are percentages of constancy
and refer only to the superior claws.

1. Claws having larger numbers of teeth show more varia-
tion in numbers than claws having a smaller number and this
holds for both sexes. The percentages of constancy for the
superior claws of male and female are as follows : Spcrmophora
52 per c&nt., Dictyna volupis 54 per cent., Epeira marmorea 59.5
per cent., Lycosa nidicola 62 per cent., Theridiuui tepidarionim
68 per cent. A glance at the tables will convince one that this
order will practically hold for the relative total number of teeth.

The same result is more strikingly shown by the inferior
teeth. In Dictyna volupis, which has normally 4-5 teeth on the
inferior claw, there are 16 variations in 80 cases of both males
and females ; while Spermophora and Lycosa nidicola, which have
normally only I and o tooth respectively have only 3 variations
in 80 cases in the former case and i in 60 in the latter.

Moreover, the superior claws, having many teeth, show many
times as much variation as the few-toothed inferior claw.

2. The teeth on the claws of the first leg show least variation
in number, those on the third most. The percentages of con-
stancy are : First leg, 61.5 per cent.; second leg, 60.0 per cent.;
third leg, 57.0 per cent.; fourth leg, 58 per cent.

3. The number of teeth on the claws of the female varies
slightly more than in the male, the percentages of constancy
being 58.0 and 59.75 respectively. The inferior claw gives the
opposite result, however, for here in the females there are 17, in
the males 19 deviations from the normal in 160 cases. A glance
at the tables will disclose a sexual dimorphism in the total num-
ber of teeth which may be greater in the male or in the female
or the same in both, according to the species. Thus by actual
count 5 females of Epeira marmorea have 318, 5 males 335
ungual teeth on the right side ; but 5 females of Lycosa nidicola
have 318 teeth as against 228 for 5 males; while 10 females and
10 males of Dictyna volupis have each 1,030 teeth exactly.

196

C. HARTMANN.

4. The anterior and the posterior claws vary in nearly equal
measure throughout, the average constancy percentage for the
former being 58.0, for the latter 59.75.

5. In only one case were two individuals found that had the
same number of teeth on the corresponding claws of all the four
legs on the right side (Table II., Theridinni tepidariorum, indi-
viduals 2 and 4).

In view of these facts one would be safe in saying that the
number of teeth on the tarsal claws of spiders is too variable to
be used as a specific character ; it should at least not be used in
a diagnosis until its absolute constancy for a given species has
first been demonstrated.

In addition to this common variation in the number of teeth on
the claws one often meets with additional claws or with double

rows of teeth on the same claw (mutations). Fig. I represents
the first foot of the seventh specimen of Theridium tepidariorum
in Table I. Two complete sets of claws, similar to the ones

TEETH ON CLAWS OF ADULT SPIDERS.

figured for the first leg, were found on each leg of the animal,
both on the right and on the left side. These claws were all of
the normal type but possessed relatively fewer teeth than those
on the claws of other specimens of the same species.

Fig. 2 represents four claws on the second leg of the second
female of Theridimn tepidariomm, the claw bearing three teeth
being an additional inferior one.

Figs. 3 and 4 show on the posterior claws double rows of
teeth, the additional row lying close to the normal and more
regular one. The claws in Fig. 3 are from the third leg of
specimen seven of Epeira benjamini and those in Fig. 4 from the
fourth leg of specimen four of E. mannorea, both in Table I.

In conclusion I wish to thank Dr. T. H. Montgomery for the
many specimens which he placed at my disposal and for his kind
suggestions in my work.

UNIVERSITY OF TEXAS,

AUSTIN, TEXAS, December 24, 1903.

A BIOLOGICAL EXAMINATION OF DISTILLED

WATER.

E. P. LYON.

The accuracy of results obtained in many of the present lines
of physiological research depends so entirely on the purity of the
chemicals used, including water, that I may be pardoned for pub-
lishing an account of work which otherwise would have little sig-
nificance, the toxicity of metal-distilled water having been re-
peatedly shown. The results obtained are, however, applicable
in a practical way at Woods Hole, where it is difficult to get
good distilled water.

Two years ago I arranged an automatic still in which the
water was boiled in a block tin vessel but the dry steam received
and condensed in glass. It was thought that if the water did not
touch metal after condensation, contamination by ions would be
avoided. The distillate proved, however, to be decidedly toxic.
I could not believe that dry steam carried with it an appreciable
quantity of metal and, therefore, decided to find out where the
trouble lay.

In these experiments, sea-water was condensed to a known
fraction of its volume and then measured portions brought back
to the original volume by adding the distilled waters to be tested.
Tap-water was also used for comparison ; sometimes, without
treatment ; sometimes, sterilized without boiling ; in some experi-
ments a portion was boiled away and the remaining portion used.

To the artificial sea-waters prepared as above equal amounts
of fertilized Arbacia eggs were added and the development ob-
served. The percentage of plutei developed in the different so-
lutions as compared with natural sea-water was usually taken as
a measure of purity. In other cases, the percentage of blastula;
was the standard ; or the percentage of segmentation in a given
time ; or the length of time that any larvee remained alive. The
records of some typical experiments follow :

Experiment 6. --250 c.c. sea-water were concentrated to 75
c.c. To each 15 c.c. of this solution 35 c.c. of the waters named

198

BIOLOGICAL EXAMINATION OF DISTILLED WATER.

I 99

in column 2 were added. To the dishes were added equal
amounts of Arbacia eggs in 16 32-cell stage.

No

Description of Water.

20 Hours.

28 Hours.

54 Hours.

I

Double distilled
in glass.

Early gastrulse.
Behind control.

Behind control.

Plutei.

2

Tap.

Blastulae.

Many going to

All dead.

pieces.

3

Tap boiled down
one half.

Slightly behind
No. I. But
much better

Almost as good
as No. I.

Plutei as well de-
veloped as No. I ;
not so numerous.

than No. 2.

4

Control in sea

Advanced plutei.

water.

Experiment n. Two liters of sea-water were evaporated to
250 c.c. To 10 c.c. portions of the resulting solution were
added respectively 70 c.c. of waters described below. Equal
quantities of Arbacia eggs were added. Three days later the per-
centages of plutei were ascertained by first killing the cultures and
then making counts in a watch glass marked off into squares.

Per cent.
Plutei.

I.

Control, 80 c.c. sea-water.

2. Artificial sea-water made from tap-water.

3. " " " " " tap-water heated to 60 in closed flask.
" " " " " distilled water from Metcalf's, Boston.
" " " " " water boiled in copper without H 2 SO 4

but condensed in glass.
6. Artificial sea-water made from water boiled in copper with H 2 SO 4 but

condensed in glass.
Artificial sea-water made from water distilled wholly in glass ; first

part thrown away.
Artificial sea-water made from water distilled in patent automatic

metal still.
Artificial sea-water made from water distilled wholly in glass, with

K 2 Cr 2 O 7 and H 2 SO 4 .

10. Artificial sea-water made from water double distilled in glass, first

distillates rejected.

11. Artificial sea-water made from " Pureoxia " distilled water.

4-
5-

7-
8.

9-

90

o

o
161

32 ,

78*
66

5 3
87

90
3 4

Experiment 8.-- Two liters of sea-water were boiled down to
400 c.c. Ten c.c. of this concentrated sea-water were added to
40 c.c. of each of the waters listed in column 2. To every dish was
added an equal amount of fertilized Arbacia eggs on July 10, 1903-

1 Nearly all small.

2 Some small ; others as good as control.

3 All very small and imperfect.

4 All very small and imperfect. Only experiment with this brand.

200

E. P. LYON.

00

H

Z

w

3

a

W

00

-a -a -o Ti

n

<U D D D

JM

13 "U 13 "O

3

>

3 3 5 3

SP

D
D D

_> .> a

>,

ctf cd c3 o

3

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BIOLOGICAL EXAMINATION OF DISTILLED WATER. 2O I

From the above and other experiments I can make the state-
ments enumerated below, which apply, of course, to Arbacia only
but point to the necessity of caution in using distilled water on
other organisms. It is true also that the results are most appli-
cable to Woods Hole conditions and to the tap-water used as a
basis of the distilled water there. It is probable that the toxicity
is due to ammonia although this was not proven. It is certain
from experiments made that Arbacia larvae are very sensitive to
that substance.

1. Tap-water is decidedly, although variably, toxic. The
toxicity is not lost by sterilization but is greatly reduced by boil-
ing the water for a long time, say until one third has been boiled
away. The residue in such cases is less toxic than some distilled
waters, particularly commercial brands and that from automatic
stills.

2. Water from ordinary automatic stills, whether metal or en-
tirely glass, is toxic.

3. The commercial distilled waters used by me were toxic,
often in high degree.

4. In distilling water in the ordinary way from glass, the first
one tenth distilled over is decidedly toxic, the second tenth less
so, the third tenth still less so. The fourth tenth is of good
quality.

5. The best distilled water used was produced by double dis-
tilling in glass, the first fourth distilled over in each distillation
being rejected.

6. Nearly as good water was produced by single distillation
from tap-water to which H 2 SO 4 and K 2 Cr 2 O 7 had been added.

7. If a little H 2 SO 4 is added to the tap-water to start with, an
excellent quality of distilled water may be produced from an
automatic still consisting of a copper vessel and glass condenser,
the arrangement being such that none of the condensed water
touches the metal. This water is practically free from ions or
toxic volatile substances. It is much better than water double
distilled in glass in the ordinary way, unless in the later case a
large proportion of the product be thrown away. Such an auto-
matic still is recommended for use at Woods Hole.

8. It was noted in a number of cases that Arbacia lived longer

2O2 E. P. LYON.

in an artificial sea-water prepared from a good quality of distilled
water than in natural sea-water. It is probable that the volatile
toxic substance (ammonia ?) exists in sufficient quantity in sea-
water to have an appreciable effect.

HULL PHYSIOLOGICAL LABORATORY,
UNIVERSITY OF CHICAGO.

Vol. VI. April, 1904. No.

BIOLOGICAL BULLETIN

ON THE HABITS AND REACTIONS OF SAGARTIA

DAVISI.

HARRY BEAL TORREY.

Sagartia davisi 1 is the Pacific representative of the 5. luciic of
the Atlantic coast of the United States. The two species appear
to differ only in the absence, in 5. davisi, of the narrow yellow
stripes on the column which are so characteristic of the eastern
form. Both species commonly reproduce by fission, and it is
probable that the following descriptions of some of the other
habits of 5. davisi may be applicable to J>. lucicc also.

5. davisi was first discovered clustered on the valves of a spe-
cies of the bivalve Chione, common in the harbor of San Pedro,
Cal. 2 The clam dwells near or on the surface of the sand. As
it plows its way along, the uppermost regions of the shell are
about the hinge and the siphons. To these regions, invariably, the
polyps cling. The deeper the clam goes the more do their small,
thin-walled bodies lengthen, supported by the surrounding sand,
to a degree I have never seen in an erect and unsupported form.
vS. davisi is not, however, a burrower, nor is the association with
Chione a case of commensalism, as might be concluded at first
sight. It fastens readily to any object which can give it a foot-
hold and keep it out of the sand. On the sand flats at San Pedro

1 Description. Column of largest polyps about I cm. in diameter; spread of ten-
tacles about 2. cm. Foot disk very extensible ; body wall everywhere quite thin and
semitransparent ; a distinct capitulum above a well-defined collar as in Metridium ;
oral disk almost circular, mouth small, oval, lips with about twelve lobes, not prom-
inent ; one or two, occasionally three siphonoglyphs. Tentacles tapering, slender,
pointed, variable in number, most often 45-50 in perfect individuals. Color of column
dark brown, tentacles and disk green. Many individuals with light longitudinal stripe
of variable width on column (zone of regeneration after fission). Mesenteries unusu-
ally variable in number and arrangement. Reproduction sexual and non-sexual ; latter
the cause of irregularities in number and arrangement of tentacles and mesenteries.

2 It has since been found in San Diego Bay, Cal.

203

2O4 HARRY BEAL TORREV.

it was uncovered about half each day by the falling of the tide,
during which periods its tentacles were completely retracted, con-
trary to the custom of such typical sand dwellers as Harenactis
attenuata? and another species of Sagartia from San Pedro as yet
undescribed. It may live permanently submerged, however, and
thrives in aquaria.

Though ordinarily attached to a solid substratum, it is occa-
sionally found free on the sand. Its powers of locomotion are
considerable. By means of multicellular amoeboid processes of
the foot disk, readily seen with a hand lens at the edge of the
disk, it is capable of creeping more than an inch in an hour. The
polyps often leave the clam to which they are attached when
placed in an aquarium, especially when they are on the lower
valve. They may occasionally creep along the surface of the
water, hanging from the surface film.

The inverted position is not long retained, however, for 5.
davisi has a marked tendency to assume as erect a posture as its
situation will permit. An example of this tendency is provided
in the case just mentioned, of the relatively greater haste of the
polyps in leaving the lower than the upper valve of a clam lying
on the aquarium floor. Moreover, the axes of polyps clinging to
the vertical sides of the aquarium are either perpendicular to the
sides or bent upward. They never bend downward if the polyps
are submerged.

The orientation of 5. davisi is, then, partly a result of geotro-
pic stimulation. The same may be said of the locomotion of the
species. There is a definite tendency of the polyps on the walls
of the aquaria to collect near the surface, although the aquaria
may be sealed jars completely filled with water, or furnished with
green water plants evenly distributed (precautions against the
possible influence of oxygen at the surface). The polyps on the
floor of the aquarium, if it be horizontal, move about but little,
and when they do, sporadically and without certainty of direc-
tion. When by chance, however, they reach the angle made by
the floor and a side of the aquarium and begin an ascent, there
is never a retrograde movement, seldom a halt, until they draw
near the surface. This locomotor geotropism is especially inter-

1 Torrey, 1901.

HABITS AND REACTIONS OF SAGARTIA DAVISI. 205

esting from the fact that the major axis of the animal is not par-
allel with the direction of locomotion, a peculiarity which dis-
tinguishes it from the reactions of the majority of animals to
directive stimuli. The major axis is the axis of geotropic orien-
tation, but it can only be the axis of locomotion in swimming
forms ' and those which lack a foot disk and creep on the column
(e. g., Pcachia].

It is possible to reconcile these different cases if we think of
the foot disk merely as a differentiated portion of the body wall.
Edivardsia has no well defined foot, though its aboral end is
rounded and adhesive. The hydroid, Coryuiorpha, again, has no
foot such as is possessed by Hydra, its aboral end coming to a
point ; yet the sides of this tapering extremity are adherent, and
through their amoeboid cells the hydroid, orienting negatively to
gravity, tends also to move vertically upward. It adheres in this
case by a portion of what may truly be called its lateral wall ; in
consequence of which the axes of locomotion and geotropic orien-
tation coincide. 6". davisi is an extreme case in the other direction.
Having a large and well defined foot, it can hardly be said to
cling obviously by a portion of the lateral (i. e., column) wall.
At the same time, when on a vertical surface, its axes of locomo-
tion and orientation are as nearly parallel as the differentiation of
foot and column will allow. This, however, is not equivalent to
saying that the direction of locomotion in response to a directive
stimulus is determined by the orientation of the major axis of the
polyp, for the elements of the foot may be directly affected by
gravity.

Loeb ('91, p. 70) has said that Cerianthus and Actinia equina
went from smooth glass to a mussel shell or piece of ulva more
readily than in the reverse direction in his experiments. This
indicates a certain "contact irritability," which seems to be pos-
sessed also by .$. davisi, as the latter moves about more freely
on smooth glass than on rough surfaces. The reaction to the
contact stimulus, however, is not so strong as the orienting re-

1 Besides the pelagic species which Andres describes among the Minyadidce, all
non-adherent, there is an interesting polyp abundant in the harbor of Honolulu which,
I am told by my friend Mr. Loye H. Miller, leads both a sedentary and a free exis-
tence. It appears to have no pedal float, sustaining itself by means of rhythmic move-
ments of the tentacles which send it along foot foremost at a fair rate of speed.

2C>6 HARRY BEAL TORREY.

action in response to the stimulus of gravity, so that as a result-
ant of the opposing responses, the polyp leaves the shell for the
glass as stated above.

Light does not appear to stimulate 5. davisi in any way. The
polyps neither bend nor move toward the light when it comes
from but one side of the aquarium, in all degrees up to the in-
tensity of bright daylight. Neither do flashes of sunlight
falling upon polyps in a darkened aquarium produce any mus-
cular responses. 5". davisi differs in this respect from Ccrian-
thns mcmbranaceus and Edwardsia lucifuga, according to Nagel
('94, p. 545).

The responses of anemones to mechanical and chemical stim-
uli have been investigated already by Pollock ('82), Loeb ('91
and '95), Nagel ('92, '94^, '94^) and Parker ('96). With most
of the conclusions of these investigators my own observations
accord. I must differ with some, adding also a few facts which
to my knowledge, have not been published heretofore.

Two quite distinct reactions, usually follow the stimulation of
a tentacle of 5. davisi by means of a slight touch with a needle
or glass rod. The first is a bend at and toward the point of
stimulation, whether the latter be near tip or base, on right side
or left, above or below, and appears to be due to the response of
the muscles involved to a direct stimulus. The second is a con-
traction of the whole tentacle, with a simultaneous bending of the
tentacle toward the mouth. Evidently all the longitudinal mus-
cles of the tentacle not previously active are indirectly excited to
produce this reaction, those on the inner (upper) side between
base and point stimulated contracting more strongly than the
outer (lower) muscles. This unequal contraction is probably to
be explained by the greater strength of the inner muscles, which
play the greater part in the chief work of the tentacles carrying
food to the mouth. The hydroid Corymorpha shows this inequal-
ity still more strikingly ; the first reaction of S. davisi is en-
tirely wanting and the outer muscles are in use only when the
tentacles are slowly returning to their expanded condition after
a contraction. But I have been unable to demonstrate histolog-
ically any difference in size between outer and inner muscles,
in either animal.

HABITS AND REACTIONS OF SAGARTIA DAVISI. 2O/

The result of the second reaction is varied. Often the tentacle
merely waves stiffly inward. At other times it may arch so that
its point is directed toward the mouth. On the whole, however,
its movements are less definitely adaptive than those which
Parker describes for Mctridimn.

The second reaction does not always follow the first. The
general contraction does not appear to be induced by contact
alone. If the tentacle be touched lightly and for but an instant,
only the first reaction occurs. If, however, the stimulating ob-
ject rest against the tentacle sufficiently long to allow the latter
to adhere to it, the second reaction immediately follows. Whether
this results from the adhesion itself, or the duration of the stimu-
lus, or a tension in the muscles due to the resistance of the
stimulating object, I am unable fully to decide. Such small ob-
jects are capable of producing this reaction that the third possi-
bility seems to be excluded. Whichever of the other two be
the efficient stimulus, it produces a strong contraction of the
muscles directly affected. This strong contraction probably
serves as a direct stimulus for contiguous muscles, the contrac-
tion of these for others, and so on, until all are involved. In no
case did the evidence enforce the assumption of the presence of
nerves, in the tentacular responses.

So far the movements of but a single tentacle have been con-
sidered, without relation to the others. And it should be said
here that tentacles cut from the polyp behave in all essential
respects as they do under normal circumstances. Often the
stimulus applied to one tentacle is sufficient, unless care be used,
to induce contractions in several. It may be that only a few
tentacles on each side of the one touched will react ; with a
stronger stimulus the entire set of tentacles may contract with
vigor. There is no more evidence, however, that this correla-
tion of parts is attained by the aid of nervous tissue than there
was in the case of the single tentacles. Communication from
one tentacle to the next is largely through the oral disk.
The proper degree of contraction of a tentacle induces a con-
traction in neighboring muscles in the oral disk, and pos-
sibly in contiguous tentacles directly. The vigor of the stim-
ulus, if it be local, appears to determine the extent of the

2O8 HARRY BEAL TORREY.

response, which spreads by the direct effect of the tension of
one muscle on those near it.

The usual response of a tentacle stimulated indirectly in the
manner just described is a waving or arching toward the mouth,
with or without vigorous shortening of the whole tentacle. Occa-
sionally the response is quite opposite to this, the stimulation of
one tentacle producing an outward waving of neighboring tenta-
cles. The anomaly is only apparent, not real, for as a matter of
fact the muscles of the neighboring tentacles are not involved at
all in the latter case. The tentacles neither shorten nor bend.
They move outward stiffly, owing to a local contraction of
muscles in the oral disk or the capitulum.

If the stimulus applied to a tentacle be sufficiently strong, all
of the tentacles may shorten simultaneously, may even be entirely
withdrawn into the body by the contraction of mesenterial muscles
and hidden by the contraction of the sphincter ; the column may
shorten also, and the foot disk may change its shape. All of
these movements seem to be induced by the direct passage of
the stimulus from muscle to muscle without the aid of nerve
tissue.

The oral disk, between tentacles and mouth, is almost insensi-
ble to mechanical stimuli.

Stimuli applied to the column produce the inward movement
of several or all tentacles, the outward movement of a few, or the
contraction of column and foot disk, according to the strength of
the stimulus. Stimulation of the foot disk, either at the edge or on
the lower surface, produces local contraction of the foot and base
of the column, and acontia are usually emitted near the point stimu-
lated. The tentacles may contract also, but always as a whole, the
same general reaction following stimulation at different points of
the disk instead of a local reaction as in the cases of the foot,
column and acontia. This inability of the tentacles to recognize
the direction of the stimulus is also characteristic of the reaction
of the tentacles of Coryuwrpha to stimulation of the column, and
is due, I believe, to the opportunities for diffusion of the stimu-
lation impulse owing to the distance of the reacting structures
from the point at which the stimulus is applied.

The entire surface of 5. davisi, with the possible exception of

HABITS AND REACTIONS OF SAGARTIA DAVISI. 209

a small zone between mouth and tentacles, responds to a mechan-
ical stimulation, the greatest irritability being manifested by the
tentacles, the tactile organs par excellence. The latter exhibit a
very definite adaptive reaction. The preliminary bend of an irri-
tated tentacle at and toward the point stimulated makes it possible
for the polyp in a sense to pursue its prey actively if, indeed, to
but a limited extent. The great advantage of this reaction over
the simple inward movement of the tentacle indirectly stimulated
is obvious. The latter is also adaptive, however, since it is the
most likely movement to clutch food organisms in a polyp whose
tentacles are habitually outstretched. Supporting this idea is the
fact that the tentacles of hydroids react only in this way, whether
stimulated directly or indirectly. It is the simpler, more primi-
tive reaction.

A more efficient adaptive reaction, also indirectly induced, is
the extrusion of acontia at the point stimulated, for purposes of
defense. Though the reaction is always the same-, the acontia
always move in the most desirable direction, which is not always
the case with tentacles.

The passive outward movement of tentacles due to the con-
traction of muscles in the oral disk or capitulum is not directed
by the position of the stimulating object. It may be toward the
latter, but only when the stimulus happens to be applied at a
point external to the tentacles, that is, at some point which is less
likely to be stimulated by a food organism than points on their
inner surface. Wlien a tentacle o'f an outer whorl is moved pas-
sively as a result of the stimulation of a tentacle of an inner
whorl, the movement is away from, not toward the tentacle
stimulated. The reaction in this direction is of no obvious im-
portance to the polyp in- this case, and seems to be of no more
importance, in the sum of all cases, than a movement in any
direction. It appears, therefore, to have no adaptive value
whatever.

By means of its varying sensitiveness to different chemical sub-
stances and its ability to discriminate between mechanical and
chemical stimuli, 5. davisi is enabled to make certain choices in
its quest for food. This capacity, which it possesses in common
with other anemones, has been described as olfactory by Romanes

2IO HARRY BEAL TORREY.

and Nagel, and as gustatory by Jourdain. Such expressions, how-
ever, are essentially psychological, and Loeb ('91) has justly
insisted upon the substitution for them of some physiological
expression, such as chemical irritability. This power of dis-
crimination has been shown by Loeb to reside not only in the
tentacles (Nagel, '92), but also in other regions. Actinia cqnina
discriminated between crab's flesh and small rolls of paper as
definitely after he had removed the tentacles by a transverse
cut as before. Parker ('96) demonstrated later that Mctridiuin
diantlins reacts in different ways to mechanical and chemical
stimuli.

Actinia cqnina, from Loeb's observations, is so definite in
its choices that chemically inert paper pellets were never taken
into the mouth. Parker found that Metridium would swallow
pieces of white india rubber as well as flesh, though the former
were sometimes disgorged before they had passed out of the
oesophagus. Since he has shown that the cilia covering the
lips of Mctndiiun and beating outward in the absence of chem-
ical stimuli, reverse their dominant beat in response to the stim-
ulation of meat juices, their behavior when stimulated by ap-
parently chemically inert india rubber leaves a doubt as to
whether or not they can be reversed by purely mechanical
means. There is, however, no doubt of such a reaction in
S. daiisi, as will be shown in the course of the following ac-
count of my experiments.

It may be well to begin with the effects of various chemicals.
Cane sugar in solutions of various strengths produced no ap-
preciable reactions in any part of the polyps on trial. Strong
picric acid and 4 per cent, formalin caused the retraction of all
the tentacles, indicating stimulation of body muscles. One half
per cent, hydrochloric acid caused a general contraction of
tentacles. From a knowledge of the behavior of Corymorpha,
which, though unable to detect the presence of flesh until
touched by it, yet reacts strongly to strong alcohol and acetic
acid, I am led to suspect that these substances irritate the
polyp in the same way that they irritate one's skin, through the
tactile organs merely.

Crab's muscle, bits of limpet and nnm lid worm were used as

HABITS AND REACTIONS OF SAGARTIA DAVIS1. 2 I I

stimulators with uniform results. Small amphipods were devoured
with avidity. The response differed according as the stimulus
was applied locally or generally. For my first experiment I
placed a small piece of worm on several of the outstretched ten-
tacles of a polyp. The tentacles immediately adhered, bending
at and toward the point stimulated, as though responding to a
purely mechanical stimulus, and then contracted, dragging the
morsel to the mouth. For some seconds the tentacles not in
contact with it remained motionless. Then, one or two at a time,
they waved slowly inward and grasped the flesh, almost every
tentacle finally becoming thus engaged. This experiment was
tried many times with similar results. Apparently the tentacles
not mechanically stimulated were irritated by substances in solu-
tion diffusing out of the flesh, and the reaction was as definite as
it would have been if induced by a mechanical stimulus. The
movement was toward the stimulus.

The possibility of an indirect stimulation of these tentacles
through the oral disk from the tentacles touching the flesh was
eliminated by holding a piece of worm flesh immediately above the
mouth of another polyp. In a few seconds some of the tentacles
began to twitch slightly, and a little later all began to wave slowly
inward, toward the flesh, finally grasping it. A similar result
followed numerous trials.

Next, a bit of flesh was placed on the aquarium floor, near,
but not in contact with the foot disk of another polyp. Would
the tentacles bend in the direction of the flesh now, or toward the
mouth? This experiment, repeated a number of times, did not
give uniform results. In the majority of cases the tentacles
waved toward the mouth, away from the flesh. In the rest they
moved toward the flesh in the most definite and unmistakable
manner. Not only that ; the column, in several cases, bent
toward the morsel which was seized by the tentacles nearest it
and dragged toward the mouth. There is no doubt here that
the movements were in the direction of the stimulating object,
and are thus comparable to the well-known movements of the
manubria of various mendusae toward stimulated points on the
subumbrella. The proboscis of Corymorpha reacts similarly, as
will be shown in a forthcoming paper.

212 HARRY BEAL TORREY.

Pollock ('82) observed this fact but was unable to reconcile his
varying results. The reason for his failure was, I believe, that
he failed to distinguish between general and local stimulation.
When the meat juices of the annelid used previously were dis-
charged gently over a polyp from a pipette, I observed that the
tentacles always waved inward, without regard for the direction
from which the juice was coming. This general chemical stimu-
lation produced the same response from the tentacles that a me-
chanical stimulation of the foot disk provoked. In both cases
then, in which the tentacles waved inward and away from the
flesh, the diffusion of soluble substances from the latter was
probably so rapid that the tentacles were stimulated on all sides
so nearly at the same time that no differential of stimulation
beween opposite sides of the tentacles was established, the neces-
sary condition of a directive reaction. But why the movement
toward the mouth ? Because it is the primitive clutching move-
ment already spoken of as most likely to capture food organisms,
in a polyp whose tentacles are habitually outstretched. It is the
simplest adaptation of the prehensile mechanism, common to
hydroids as well.

The responses of the tentacles to mechanical and chemical
stimuli are essentially the same. The bend is toward the stimu-
lus when the stimulation is local, toward the mouth when it is
general, whether direct or indirect.

If we turn now to the phenomena of swallowing, we shall see
that the cilia of both lips and oesophagus may respond to mechani-
cal as well as chemical stimulation by waving more strongly inward
than outward. I early observed that not only were pieces of
flesh occasionally rejected, but bits of shell and gravel were
sometimes taken in. With the idea in mind that the size and
shape of the object might affect the reaction, several substances,
presumably chemically inert, were given to various polyps, in
pieces varying in these respects. Pieces of very thin paper, from
i mm. to 3 mm. square, when placed npon the tentacles, were
cast off in half an hour. A piece of cork, about one fourth as large
as the polyp, was likewise rejected. A much smaller piece,
capable of being easily ingested, was taken into the gullet and
retained for thirty minutes. A piece of paraffine of similar

HABITS AND REACTIONS OF SAGARTIA DAVISI. 213

size was swallowed in three minutes, and a half-cube of heavy
drawing paper, of about the same size, was also swallowed,
though more slowly. Tiny bits of glass were frequently swal-
lowed.

I can say definitely that these objects were, not carried in by
the beat of the cilia covering the siphonoglyphs and producing
an insetting current, but by cilia covering the lips and oesophagus
between the siphonoglyphs and producing a current which ordi-
narily sets outward. It would seem, then, that chemically inert
substances, if small enough to be taken easily into the mouth
and thus brought into direct contact with the ciliated cells lining
the oesophagus, are ingested under some conditions. Other ex-
periments show that one of these conditions, probably the most
important, is the degree of hunger of the polyp. Starving polyps
were always more ready than well fed individuals to swallow
chemically inert substances. Some explanation of this fact may
be derived from the further fact that hungry polyps are in gen-
eral unusually sensitive to both chemical and mechanical stimuli.
Increased sensitiveness means increased effectiveness of a given
stimulus ; this is equivalent to saying that the stimulus is more
intense. 5". davisi, then, responds only to certain intensities of the
same stimulus, so far as the ciliated cells of the lips and oesoph-
agus are concerned. Under mechanical stimulation of a given
intensity, the cilia do not reverse their beat ; an increase in the
intensity or, if you will, effectiveness of the stimulation may
produce this reversal. To chemical stimuli, or to mechanical
which are above a certain degree of intensity (i. e., when the
stimuli polyp is starving), the response is usually positive ;
to a weakened mechanical stimulus there is less likelihood of
any response.

The positive response to mechanical stimuli is undoubtedly
advantageous to the polyp. It is apparent that substances with
even a very small food value must be of some importance to a
starving polyp although they would not be desirable as food
for a well nourished animal. For the latter they would come
into the category of useless substances, which the ciliary cur-
rents on (Esophagus, lips and tentacles are admirably adapted
to remove.

214 HARRY BEAL TORREY.

The disgorgement of non-nutritious bodies may now be briefly
considered. All harmless non-nutritious bodies, and all food
stuffs from which the nutrient juices have been taken during the
process of digestion, are sooner or later cast out of the mouth.
The cause of the ejection is to be found in the behavior, under
varying stimulation, of the oesophageal cilia. The mesenterial
filaments bordering the mesenteries, and the defensive filamentous
acontia, are ciliated, but probably take no part in the process, for
several reasons. 1

First, the mesenterial filaments pursue excessively meandering
courses along the edges of the mesenteries, and their cilia pro-
duce many currents which are antagonistic instead of proceeding
in one general direction. I have not been able to determine
whether the cilia beat more strongly away from or toward the
mouth. In all parts of each filament, however, they appear to
beat in the same direction ; and this beat is not reversed by con-
tact with meat or meat juices. Second, the cilia on the acontia
beat always more strongly toward their free ends, and they too
do not reverse their beat in the presence of meat juices. Since
the acontia are attached by one end only, have a marked ten-
dency to coil, and occupy without regularity of arrangement any
position in the ccelenteron, they can hardly be concerned with
the phenomena of disgorgement. It may be noted in passing,
however, that when they are thrust through mouth or cinclides,
their cilia, in carrying toward their tips whatever foreign particles
may come in contact with them, are performing what must be in
the long run an advantageous service.

Finally, the oesophagus itself, ordinarily more than half the
length of the column, reaches nearer to the foot disk when the
polyp contracts as it does with food substances within it. The
objects taken into the coelenteron never get far away from the
lower edge of the oesophagus. Under the influence of the mesen-
terial and acont'al cilia, they may, if small enough, rotate aim-
lessly about during the period of digestion and absorption. In
the absence of direct stimulation, the oesophageal cilia resume

1 The following facts concerning the behavior of the cilia on mesenterial filaments
and acontia were obtained from Metri,/im>i, but I feel confident that the same results
would have followed an investigation of S. dbr'/.r/ had the supply of material permitted.

HABITS AND REACTION'S OF SAGARTIA DAVISI. 215

their dominant outward beat, and are able to carry away non-
stimulating objects. At the end of the period of digestion and
absorption, the ingested bodies have reached their minimum of
stimulating power ; and now, no longer able to reverse the
dominant beat of the cesophageal cilia, they are carried out by
the latter just as soon as they come into their sphere of influence.
Why chemically inert bodies, once swallowed, should be dis-
gorged, may be explained, I believe, by assuming inability on
the part of the cesophageal cilia to continue reversing their dom-
inant beat in the presence of a persistent or frequently applied
mechanical stimulus which was originally weakly positive. This
is in entire harmony with Parker's demonstration that after
repeated applications of a weak chemical stimulus to the
lips of MctriJiuin, there comes a time when no positive reaction
results.

Peristaltic movements of the oesophagus may assist the cilia,
but I have no evidence that they take more than a very subordi-
nate part in the phenomena of swallowing or disgorgement.

UNIVERSITY OF CALIFORNIA,
January 1 1, 1904.

2l6 HARRY BEAL TORREV

BIBLIOGRAPHY.

Jourdain, E.

'8g Les Sens chez les Animaux Inferieurs. Paris.

Loeb, J.

'91 Untersuchungen zur physiologischen Morphologic. I, Ueber Heteromor-
phose. Wurzburg.

'95 Zur Physiologic und Psychologic der Actinien. Arch. f. ges. Phys., L1V,
P- 4I5-

'02 Comparative Physiology of the Brain and Comparative Psychology. New
York.

Nagel, W. A.

'92 Der Geschmackssinn der Actinien. Zool. Anz., XV, p. 334.

'94a Experimentelle sinnesphysiologische Untersuchungen an Coelenteraten.
Arch. f. ges. Phys., LVII, p. 495.

'94b Vergleichend Physiologische und anatomische Untersuchungen ueber den
Geruchs- und Geschmackssinn und ihre Organe. Bibl. Zool., Heft 18.

Parker, G. H.

'96 The Reactions of Metridium to Food and other Substances. Bull. Mus.
Comp. Zool., XXIX, No. 2, p. 107.

Pollock, W. H.

'82 On Indications of the Sense of Smell in Actiniae, with an Addendum by
George J. Romanes. Jour. Linn. Soc. Lond., Zool., XVI, p. 474.

Torrey, H. B.

*O2a Papers from the Harriman Alaska Expedition. XXX, Anemones, with
Discussion of Yariation in Metridium. Proc. Wash. Ac. Sc. , IV, p. 373.

'o2b The Hydroida of the Pacific Coast of North America. Un. of Cal.
Publ., Zool., I, p. I.

VARIATION IN BEES.

FRANK E. LUTZ.

In the study of evolution, there is nothing more important
than the investigation of variations, since the whole doctrine rests
upon the premise that organisms do vary. There was a time
when it was sufficient in such an investigation to take a series of
specimens and from the general looks of things postulate theor-
ies. But the world has become more critical now and demands
that when a statement is made concerning some phenomenon,
exact data accompany the statement. Hence, the statistical
study of variation which attempts to exactly measure the varia-
tions and correlations of different organs and to set them down
in figures which "cannot lie." And here we cannot allow the
other proverb which says that figures will prove anything, for
figures truthfully handled can only prove the truth. But there
is, on the other hand, great danger that, having collected a set
of measurements, we make a show of accuracy that will lead us
and others astray by reason of careless or insufficient analysis.
Such work is most troublesome because of its seeming exactness
and the difficulty of detecting errors.

Messrs. Casteel and Phillips, in the December (1903) number
of this BULLETIN, have taken up a very interesting and vitally im-
portant problem. The comparative variability of the drone and
worker bees hits, in a way, at the very root of the variation
question. Accordingly, while we lament with the authors the
smallness of their series, it seems well worth while to consider a
few points about the paper.

In the first place, we have to disagree with the statement that
if the variability is "due to chance," it is "not in accordance
with any law," for it is well known, and has been for years, that
nothing is more bound by law or more expressible in mathe-
matical formulae than "chance." However, we will heartily
agree with them that the "true test of the relative variability"
is the " descent in numbers of individuals " in the different classes
as they are removed from the mean ; but we w r onder greatly

217

218

F. E. LUTZ.

why they did not apply this simple test. It is called the standard
deviation and must be known to everyone who has ever done
any statistical work. The phrases just quoted are taken from
the discussion of the counts of the hooks on the hind wings.
Let us therefore examine them by means of this confessedly bet-
ter measure.

We find that for the drones we have :

Lo>.

No. of Specimens. Standard Deviation.

Probable Error.

I.

50

2.1548

0.1453

II.

IOO

I -5435

0.0736

III. 100

1.7716

0.0845

IV. 100

1.6486

0.0786

V. 50 2.0988

0.1416

VI. 98

1-9377

0.0934

For the workers we have :

Lot.

No. of Specimens.

I.

II.

III.

50

35
100

Standard Deviation.

1.5223
I-55 6 4
I-5523

Probable Error

o. 1027

0.0397
0.0740

This gives an average standard deviation, or variability, for
the drones of 1.8592 0.1028; and for the workers of 1.5437
0.0721. But we see that the difference between the averages
for the two sexes is less than the difference between the two sets
of drones from the same hive (I. and III.) ; and, considering the
probable errors, neither is significant. If we omit the three small
series because of their large probable errors, we see that the dif-
ference between the variabilities of the two sexes is even smaller
and clearly not significant. It is also unfortunate that the work
should have been passed by both the authors and still two of the
nine averages be wrong. The average for lot II. of the workers
is 20.99, n t 21.08 ; and that for lot VI. of the drones is either
22.65 or 22-75, according as we do or do not include the indi-
vidual with 12 hooks, but it is surely not 22.42. This was prob-
ably gotten by including this individual (although he was excluded
by their argument above), and then using 100 as the total num-
ber, but for some strange reason there are only 99 of the 100
said to have been studied which are listed.

VARIATION IN BEES. 2IQ

Passing over the grave error of lumping the different series of
ratios (p. 27) because they seemed to be alike, when really their
only claim to homogeneity is that they are of the same sex and
all bees- - Italians, hybrids, " peculiar strains," ct <?/., from cen-
tral Ohio to eastern Pennsylvania being jumbled together let
us take up the first table (vein R) as we have done the last. We
find that the standard deviation of lot I. of the workers is 1.5637-
0.1055, and for lot V. of the drones--" from the same hive "
is 2.25 17 d= o.i 5 19. There is here a difference of 0.6880. The
probable error of this difference is d= o. 1 849. The standard
deviation of lot I. of drones is 2.4023 d= 0.1620 and that of lot
III., of the same sex, also from the same hive is, 2.9598 =t 0.1412.
The difference here is 0.5575 ^ 0.2149. This is due to the ex-
tremely small size of the series measured. Since the formula
for the probable error of the standard deviation is 0.6745
(stand. dev./i/27z), we see how rapidly an increase of " n '''
the number of individuals measured --decreases the error of the
result. But it is manifest that the differences between the two
sexes, as shown by these data are of no significance ; for, far
within the probable error of our work, we get as great a differ-
ence between two lots of drones (one lot being twice as large as
either of the two considered in the comparison of the different
sexes) as we get between the two sexes.

I have not taken the time to go over the rest of Messrs. Cas-
teel and Phillip's work ; but, having reached the above results
with the first and the last tables will leave them to go over
the intervening ones. It is also probably unnecessary to remark
that, even if it turns out that the greater variability of the drones
can be established, their proofs of their theory to account for
this difference seem rather unsatisfactory.

HULL ZOOLOGICAL LABORATORY,
UNIVERSITY OF CHICAGO.

THE SEXUAL ELEMENTS OF THE GIANT SALA-
MANDER, CRYPTOBRANCHUS ALLEGHENIENSIS.

ALBERT M. REESE.

In the spring and early summer of 1902, the author made
strenuous efforts to obtain embryological material for investigating
the development of the hellbender (CryptobraiiclinsAllcgJicniaisis}.
These unsuccessful efforts have been described in an article en-
titled "The Habits of the Giant Salamander." l

In the fall of 1903, another effort was made to obtain the de-
sired material, and a dozen or more live hellbenders were ob-
tained as the result of a trip to the region of the Allegheny River,
in western Pennsylvania.

These animals were all about the same size, 45 cm. in length,
and were sent by express from their native stream to Syracuse,
N. Y.

Upon opening the box, in which they were shipped, after its
arrival in Syracuse, a number of eggs were found scattered through
the grass that had been placed there to protect the animals dur-
ing their trip. There seemed to be no difference in the colora-
tion of the males and females, and the only way in which they
could be distinguished was by the fact that, in the males, the
lips of the cloaca were considerably swollen by the enlargement
of an elongated mass of glandular tissue on each side.

In handling one of the ripe females of this lot of hellbenders,
the author was bitten on the thumb ; this was the only time in
which any attempt to bite had been noticed, though many dozen
animals had been handled at many different times. The bite
was not at all serious, being merely a painful pinch which
scarcely broke the skin. In removing one of the females from
the box in which they had arrived, an egg, enclosed in its jelly-
like envelope, was seen protruding from the cloaca. By gently
pulling this extruded egg, it was found that a whole string of
eggs could be drawn from the cloaca, without apparently injur-
ing them in the least.

1 I\>f>itlar Scii'iice Monthly, May, 1903.

22O

SEXUAL ELEMENTS OF THE GIANT SALAMANDER.

221

Each egg is a spherical yellow body, about 6 mm. in diameter,
resembling somewhat the yolk of a miniature hen's egg. It is
surrounded by a clear gelatinous envelope, which is arranged in
two distinct layers (Fig. i).

When removed from the gelatinous envelopes, as may easily
be done by cutting through the latter with a pair of fine scissors,
the egg is seen to be enclosed in a very thin and delicate vitel-
line membrane which is easily torn in handling.

The yolk, which is apparently evenly distributed throughout
the egg, is made up of a compact mass of granules of various
sizes (Fig. 2).

O

'f\J

c o o O

o r -^-' / O^o

>a r^oQ

^^ti^s'

FIG. 2.

Fie. i.

The egg is surrounded by a small amount of watery ma-
terial (Fig. /i, 7C') which is, in turn, enclosed in a capsule of
more dense jelly, the inner envelope (Fig. i, /. e.}. The inner
envelope is continued as a solid, more or less tough cord of
jelly (i r . e'.) from egg to egg, and binds them together in the
continuous strings that have already been mentioned. The dis-
tance between two adjacent eggs of the string is usually about
four or five times the diameter of the egg, but the elasticity of
the jelly will, of course, permit the eggs to be drawn much
further apart.

222

A. M. REESE.

The outline of the inner envelope is sharp and even, while
that of the outer envelope (Fig. I, o. e.) is more or less irregular
and uneven. The outer envelope is composed of such trans-
parent jelly that it might easily be overlooked at the first glance.
It forms a continuous layer over the entire mass of eggs.

When the unfertilized eggs are left for some days in water,
they become very much swollen, by the osmosis of water through
the vitelline membrane, and may eventually burst.

There was no apparent swelling of the gelatinous envelopes on

coming in contact with water as is de-
scribed in connection with some other
amphibian eggs.

Several dozen eggs were obtained
from one average-sized female, about
two dozen being^drawn, without appar-
ent injury, from the cloaca, while the
rest were obtained only after killing
the animal and opening the body cavity.
All the eggs obtained in the latter way
were found to be contained in the right
oviduct, the ova of the left ovary being
nearly all in a very immature condition.
Whether or not this was a normal
condition, indicating perhaps, a very
prolonged breeding season, it was not
possible to say.

The spermatozoa were obtained as
a milky fluid from the living males by
the usual process of stripping, though
considerable pressure had, in most cases, to be exerted. They
were immediately examined under the higher powers of the
microscope, but no motion could be detected, though it would
naturally be expected that spermatozoa obtained in this way would
show the usual activity of mature spermatozoa.

An attempt was made to artificially fertilize the eggs by put-
ting them into a dish of water into which a great number of
spermatozoa had been stirred, but the attempt was entirely un-
successful.

FIG. 3.

SEXUAL ELEMENTS OF THE GIANT SALAMANDER. 223

No structures resembling spermatophores were discovered, and
there was nothing that would seem to give any indication of the
method by which the act of fertilization was accomplished.

A single spermatozoon, as seen under a magnification of about
1,300 diameters, is shown in Fig. 3.

Fairly good preparations were easily made by drying them
rapidly on the slide, and staining in haematoxylin and eosin.

The nucleus, //, is very much elongated, so that it makes up
almost one third of the entire length of the spermatozoon. It is
capped, at its anterior end, by a sharp, gradually-tapering apical
body, a, which is plainly differentiated from the nucleus proper
by the fact that it does not take up the stain to any great ex-
tent. No structural details in the nucleus or apical body can be
discerned with the magnification used, nor is any middle-piece
distinguishable. The tail, which is comparatively stout, consists
of a central supporting fiber, s. f., which takes up the stain
slightly, surrounded by a transparent envelope, e, which does
not stain at all. The envelope is usually considerably wrinkled

and twisted, probably by the rough method of fixation.
ZOOLOGICAL LABORATORY,

SYRACUSE UNIVERSITY.

THE RHYTHM OF IMMUNITY AND SUSCEPTIBILITY

OF FERTILIZED SEA-URCHIN EGGS TO ETHER,

TO HC1, AND TO SOME SALTS.

E. G. Sl'AULDING.

INTRODUCTION.

The experiments described in this paper were undertaken
during the summer of 1902 at the Marine Biological Laboratory
upon the suggestion of Dr. A. P. Mathevvs ; their publication has
been delayed because of the pressure of other work and of the
desire to, if possible, get beyond their mere description to their
meaning. This end is believed to have been attained in connec-
tion with the working out of a synthesis of the artificial parthen-
ogenetic methods, the detailed results of which attempt appear in
a preceding paper. 1 The effectiveness of all these methods and so
the special physico-chemical result of normal fertilization and the
nature of cleavage processes can, it is believed, be explained from
a unitary standpoint, viz.: if it is considered that in the process of
cleavage an average decrease in surface tension takes place as a
result of the equilibrating of a potential difference between os-
motic pressure and surface tension, accompanied by such elec-
trolytic changes as cause the constricted form. That this average
decrease in surface tension takes place is a necessary inference
from the change from the approximately spherical to the con-
stricted form of the egg at cleavage, for this means an increase
in surface. It carries with it, therefore, the decrease in that po-
tential, osmotic, which opposes surface tension in direction. The
preceding cause in these events is the creation of a potential dif-
ference by first increasing the osmotic pressure, which is done
artificially by each of the parthenogenetic methods. Accor-
dingly with the equalization of this difference, caused, c. g., by a
splitting up of colloidal particles or molecules, there is in the case
of eggs of marine forms an absorption of water. Both of these

1 Spaulding, E. G. " The special physics of segmentation as shown by the syn-
thesis, from the standpoint of universally valid dynamic principles, of all the artificial
parthenogenetic methods." BIOLOGICAL BULLETIN, February, 1904.

224

IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 22$

last events either alone or together may constitute what is termed
liquefaction.

The results of the experiments given below can, it is believed,
be interpreted in agreement with this view of segmentation.
They were undertaken primarily as an extension of experi-
mental work which had already been done on the action of vari-
ous chemical compounds on protoplasmic bodies, but were limited
to the study of such action on the eggs of Arbacia at successive
periods after fertilization.

The existence of a rhythm of immunity and susceptibility has
been shown already by Lyon in studying the effect of KCN and
of lack of oxygen upon the fertilized eggs and embryos of the
same form, 1 and it has been found also that many eggs do not
segment at all in the absence of oxygen, notably Arbacia 2 and
Ctenolabrus? Lyon also found this summer that Arbacia eggs
required more oxygen during precleavage and gave off more CO,,
during cleavage than at other times. 5

From these results it may be inferred in analogy to a large
number of instances well known in chemistry that at least the
ultimate effect of oxygen on the processes conditioning cleavage
is the causing of analytic chemical changes ; /. c., fermentation,
one might say, occurs and CO 2 is given off, as Lyon found..
Previous to this, however, synthetic processes may take place
which in turn as certain preferments become active 4 give rise to.
molecular splitting. The result of such analytic change is that
increase in osmotic pressure and therefore the creation of that
potential difference between it and the surface tension which we
have found to be necessary and in the equalization of which both
potentials decrease, water is absorbed, and the egg cleaves.

The hypothesis to be deduced from this and which might serve
as a guide in our experimentation is that any method either (i)
of preventing this necessary preliminary increase in osmotic
pressure, or of compensating it after it has been created, or (2)
of increasing it beyond a certain point, will tend to do away with

1 Lyon, E. P., American Journal of Physiology, VII., i.

2 Lyon, loc. cit.

3 Loeb, ]., Archiv fitr die gesammte Physiologie, 1895, LXII.

4 Cf. Hofmeister, " Chemische Organisation der Zelle."

5 Personal communication.

226 E. G. SPAULDING.

the event of cleavage. To the first two possibilities correspond
the effect respectively of lack of oxygen and the use of strongly
hypertonic solutions on the fertilized egg ; to the second method
our own results with ether, HC1, etc. From this hypothesis it
can also be reasonably inferred that the nearer to the point of
termination of the preparatory process that either method is used,
so much the less will its effect in general be, and this supposition
is again confirmed by experimental results.

Lyon l found that the effect of KCN on the fertilized Arbacia
egg, taking, c. g., various strengths of a titrated solution mixed
with sea water, was the indication of " successive stages of
relatively high and low resistance in each cleavage." Putting
the eggs into the solution at successive five-minute periods after
fertilization and allowing them to remain perhaps one hour, then
washing and removing to sea water, he found that "there is a
stage about ten or fifteen minutes after fertilization when the egg
is especially susceptible to KNC." " Again soon offer the first
cleavage comes a second stage of small resistance ; a third fol-
lows the second division." " The resistance of the egg to KNC
increases up to a maximum up to the time of separation into the
two cells." " The effect of KNC is the same as lack of oxygen."

In interpretation of these results Lyon says that the processes
dependent upon oxygen seem to begin about 1015 minutes
after fertilization, for if they are inhibited the egg does not seg-
ment and they recur at each segmentation. To identify them
with the morphological processes of the splitting and separation
of the chromosomes or with the dissolution of the nuclear mem-
brane seems to him to be impossible, for these occur too late to be
directly affected. Wilson and Matthews, 2 he says, mention how-
ever two processes which occur sufficiently near to the suscep-
tible stage to be worthy of consideration in this respect. One is
the growth and division of the sperm aster, the other the growth
of the nucleus. From the part which in order to explain the
constricted form of cleavage 3 must be attributed to, as played by
each of these processes, the supposition that they are affected by
a lack of oxygen, by KCN, etc., receives confirmatory evidence.

1 Lo;. cit.

' 2 Journal of Morphology, 1895, X., p. 319.

;! Lillie, R. S., BIOLOGICAL BULLETIN, IV., March, 1903; and Am. Jour, of
/ v 'r.*/'i'/(;;'T, VIII., 4, Jan. 1903.

IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 22/

But furthermore, whatever the morphological elements may be,
it must also be admitted that in the processes leading up to and
culminating in cleavage, we are dealing with chemical and elec-
trolytic and consequently also with osmotic phenomena, coex-
isting with those of surface tension. That these first two which
condition the other two are, however, not uniform, but, rather,
are varying, /. c., rhythmical, during that period must be ad-
mitted to explain the observed rhythm in morphological changes.
The experiments herein described serve the purpose then of
testing the above-mentioned hypothesis of the existence of a
liquefaction during the event of cleavage, and of a rhythm of in-
creasing immunity up to and of marked susceptibility during that
time. To this end use was made of ethyl ether, HC1, KC1,
NaCl and sodium citrate solutions.

THEORIES OF THE NATURE OF THEIR ACTION.

From the position that has been taken in this and a previous
paper that, inasmuch as in protoplasm we are dealing with col-
loidal (probably also electrolytic) particles in solution, we there-
fore in segmentation necessarily have to do ultimately with the
relations of two kinds of energy, osmotic and surface, and that
the cleavage process itself depends upon the existence of an un-
compensated potential difference between these, from this it fol-
lows that this potential difference might be caused in eitlier of two
ways, viz., at the same time that either one is kept constant, by
changing the other, /. c., either increasing the osmotic or decreas-
ing the tension factor, the former being identical with the energy
of the particles in solution, the latter with that of the solvent.
The theories also which we find advanced in order to explain the
nature of stimulation seems to us to be in complete agreement
with this view. For example, we find the statement that " stimu-
lation consists in the precipitation, /. c., gelation, of colloidal
particles and is due in the case of positively charged particles to
the negative ions, i. c., to the charge; 1 the inhibition of this
stimulation, /. c., what in some cases is termed poisoning, to the
positive ions"; for negative particles the converse would hold

1 Mathews, A. P., "The Nature of Nerve Stimulation, etc.," Science, March 28,
1902. *

228 E. G. SPAULDING.

true. In any case the osmotic pressure would necessarily be af-
fected. Both the stimulating and poisoning effect have further-
more been correlated with the valency. 1 In the case of a com-
pound of anion and kation, both of which are monovalent, like
NaCl which does and KC1 which does not stimulate easily and
when in both therefore the charges might seem to offset each
other, the stimulating effect, c. g., on the nerve has nevertheless
been said to be due to the " overbalancing " of the kation by the
anion, and conversely for the inhibitory effect. This of course
is not real explanation unless the difference in effect can be cor-
related with a difference in some such quality as velocity of dif-
fusion or solution tension, and Mathews has this summer shown
that the poisoning qualities of the metals and non-metals as
well are in fact a function of this latter. Some ground for this
" overbalancing" effect seems to be furnished by the fact that in
the case in which c. g., a divalent anion is combined with a
monovalent kation (2) a greater stimulating effect is observed.
Thus KC1 does not stimulate the nerve at its osmotic pressure,
K.,SO 4 does occasionally, K 3 citrate stimulates in solutions of a
gram molecule to 22,000 c.c. H.,O. But even here the number
of opposing charges is the same. The kations therefore differ in
some way other than in their mere number of charges. This
must also hold true of the anions because of the increasing
stimulating effect on the nerve of NaCl, NaBr, Nal and NaFl.
The suggestion has been made that the difference in effect when
the charges are the same in number is due to a difference in the
translatory path of the charge around the atom ; but as a cause
for this latter difference must in turn be assigned the admission
of an ultimate difference in the atoms themselves would seem to
be necessitated.

While the salts therefore seem to affect the colloidal particles
directly, the known inhibitory action of the anaesthetics would
accordingly have to be identified with a direct effect on the sol-
vent and so only indirectly on the solute. Thus it may be con-
sidered that the anaesthetics as being better solvents in most
cases than water have the same effect on colloidal particles as do
like charges, which repel ; therefore they increase the osmotic

1 Loeb, Afchiv filr die gesch. Physiologic, Bd. 88.

IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 2 29

pressure. It is believed that on this basis the varying effect of
ether on the eggs of Arbacia at successive periods after fertiliza-
tion can be explained.

EXPERIMENTAL.

i. The Effect of Ethyl Ether.

The general methods of experimentation may be outlined as
follows : First, a slightly supersaturated solution of ether in sea
water was prepared, kept tightly corked, and when used a por-
tion was drawn from the bottom, thus ensuring a saturated solu-
tion. The eggs were fertilized in the usual way, good lots from
among a number being selected. At successive periods these
eggs were transferred to staining jars containing 50 c.c. of the
solution used. The strength of the solution actually used was
accurately controlled by starting with twice that strength and
then diluting exactly one half, in part with the sea water neces-
sary for transferring purposes. The eggs were allowed to stand
in these covered jars for the length of time selected ; the solution
was then carefully drawn off; the eggs were thoroughly washed
twice with sea water, which was again added, and given time to
develop. In important experiments the lots were each observed
twice. All the experiments were conducted at the room tem-
perature, about 20 C.

The practical problem presented was to get such a strength of
solution and time of exposure that of the eggs transferred at the
various periods after fertilization some would be stopped in their
development, others not. For each value of the one factor there
would probably be a corresponding value of the other, the prod-
uct being a constant. An abstract record of what were essenti-
ally preliminary experiments is given on the next page.

From these results it seemed that the correct relation between
the two variables, time and solution strength, had been found ;
accordingly in the next experiment a one sixty-fourth saturated
ether solution was used for 25', which gave very satisfactory
results. These are tabulated and plotted on page 008.

In the plotting of the curve of these results the abscissae repre-
sent the times of transferral after fertilization, the ordinates the
per cent, of swimmers found by as accurate observation as possible.

230

E. G. SPAULDING.

Exper. Strength of
Solution.

Period Time of Observation. Control.
After Fertilization. Exposure.

I.

Saturated.

Every 15'. 8 lots.

I hour. All dead in the Well

July 24.

stage of treatment, developed

III. X and /^ sat -

Just before and Various. All dead.

Well

July 26. sol. by dilut-

after each cleavage.

developed

ing with sea

water (same

lot of eggs).

IV., A. Jjc sat. sol.;

At " critical " pe- 30' All dead.

Control

July 29.

one lot of

riods, z. e. , just be-

good,

eggs for A, B,

fore and after each

regular.

and C.

cleavage, as below

in B.

IV., B.

3^2 sat sol.

15 minutes. (Lyon's 45' 50% unsegmen-

Lot (i).

ist critical point. )

ted, but pigmented

and swollen, some

in 4 and S cell

stage ; 2O% swim-

ming.

(2)

50', ist. cleavage 30' So% dead,swol-"

just beginning.

len, pigmented,

20% swimming.

Segmen-

(3)

I hr. 10', toward 30' 87% dead, irreg.

tation go-

end of cleavage. segment. , some

ing on.

in 16 cell stage

13% swimming.

(4)

2 hrs., during 2d. 30' All stopped in 4 Control

cleavage. cell stage. good.

(5)

2 hrs. 40', after 2d. 3C/ All dead.

cleavage.

IV., C.

^L sat. sol.

I5 / , Lyon's crit- 45' 8 hrs. afterward Control

(i)

ical point.

all had segmented, good.

8, 16, 32 cell stage;

25 % swimming.

(2)

50', cleavage just 30' (Like C i.)

beginning.

(3)

70', toward end of 30' Nearly all swim-

cleavage.

ming.

(4)

2 hrs., during 2d. 30' 2-32 cell stages

cleavage. present ; decom-

posed and pig-

mented ; J~ swim-

ming.

(5)

2 hrs. 4O / , after 30' | swimming.

2d. cleavage.

IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 23 I

Exper.
V.
July 30.

Solution
1/64 Sat.

Time After
Fertiliza-
tion.

Lot i

2'

" 2

V

" 3

12'

" 4

17'

" 5

22'

" 6

.

21'

" 7

3 2/

" 8

37 /

" 9

42'

" 10

47'

" ii

5*'

" 12

57'

"13

62'

' H

67'

" 15

72'

" 16

n'

" 17

82'

"18

87'

" 19

92'

" 20

97^

" 21

102'

" 22

IO7'

"23

112'

"2 4

117'

Exposed to
Solution.

Observation.

Control.

25' 20% swimming.

20

1 4

2O

4 4

50

< (

62

^

85

4 i

88

4(

90

4

IO% in 2-cell stage,

44' after fertilization.

95

4 t

4 4

20% segmented.

all

dead, pigmented,

and swollen.

20%

87

5% swimming.

90

i 4

90%

f\ f

4 I

< t I

95

90

ft

90

(C

90

i

95%

90

4 4

95

4 (

95

4 t

33% in 4- cell stage.

55

C 4

7

5

2

( 6

30

+

232 E. G. SPAULDING.

The character of this rather remarkable curve is obvious. Up
to within twelve minutes after fertilization the resistance remains
the same, but from this point on it gradually rises up to either
just before or the beginning of the first cleavage ; during the earl}'
part of cleavage it falls to zero, with a sharp rise afterwards and
a fall at the second segmentation.

The more important question however is to get at its meaning.
To get at this we take, corresponding to the general rise in im-
munity up to the time that cleavage is beginning, the greater
demand for oxygen, established by Lyon in his work this summer.
This might mean in view of the fact that either at least just pre-
ceding or for sonic time before cleavage an increase in osmotic
pressure must take place, as we have shown, either one of two
things to account for this, viz., either that the oxidation process
is at first synthetic and subsequently determines analytic events ;
or that it is analytic from the start. Also to be correlated with
this is the known effect of ether as a better solvent than is water.
This is identical with its causing a greater degree of solution and
consequently an increased osmotic pressure. The inhibiting
effect of the ether on the eggs at the critical period in the above
curve may be ascribed then, we believe, to its augmentation of
the normal predominence in osmotic pressure at that time, i. e.,
to its increase of that difference of potential in the direction of
pressure-tension necessary for cleavage. Accordingly in the
equalization of this augmented potential difference the eggs would
be expected to increase in size more than usual in their attempt
to divide, and this is confirmed by the observed sivollen appear-
ance, even when as in some cases division takes place once and
then stops.

This increasing immunity up to the maximum can be explained
then in two possible ways. If synthetic as simple oxidation proc-
esses precede the analytic then during that period there is some-
thing to oppose the dissolving effect of the ether ; but since this
opposition would seem to exist equally all through the precleavage
period, the rise in immunity would be hard to account for in this
way. On the other hand if analytic processes take place from
the start as a result of the use of oxygen then the longer before
cleavage that the exposure to ether is made the greater should be

IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 233

its effect in augmenting the normally occurring increase in the
pressure, and the point of greatest susceptibility would be at such
points and also at that of the normal maximum pressure, viz., just
before or during cleavage. This explanation therefore accords
best with the sharp rise in the curve, and is supported also by the
evidence from the parthenogenetic methods for Arbada, in which
the pressure is first increased, sometime before the cleavage, which
takes place after the return from the hypertonic solution to the
sea water.

The characteristics of the above curve were confirmed in general
by Experiment IV., B and C, already presented, and more
especially by three subsequent experiments, as can be seen from
the following records :

EXPERIMENT V]I.
August 22, 1/64 sat. sol. Time of exposure, 1/2 hr.

Lot.

Period
After Fertil-
ization.

Observation.

Control.

I

3'

66% unsegmented.

2

19'

90% living.

3

3i'

90% "

4

56'

95% dead, many in 2-cell stage, pigmented.

Middle point of seg-

swollen. mentation.

5

63'

25% swimming.

6 79'

25%

7

96'

All dead in 4-cell stage.

During second cleavage.

8

112'

it t 4

* < < c

Experiments P//7. and IX., August 23 and 29. Solution,
one sixty-fourth saturated ether, one half hour exposure. Lot
i. one half hour after fertilization all living. Lot 2, during- see-

o o &

mentation, all dead in 2-cell stage.

EXPERIMENTS WITH HC1.

Preliminary and theoretical. According to the views that we
have previously discussed the hydrogen ion in the case of the
nerve is held to inhibit the stimulating action of the chlorine ion,
"overbalancing' 1 this more than do either K, Li, NH 4 or Na.
This may be due to the greater velocity of diffusion of H, which
is 325, that of Cl 70.2 at 25 C. 1 On the other hand it has been
supposed that the H ion brings about the parthenogenetic devel-

1 Ostwald.

234 E - G - SPAULDING.

opment in Asterias. These two seemingly contradictory effects
cannot, however, be so in reality and the difficulty may be done
away with if it is borne in mind that the effect depends as much
on the character (electronic) of the colloid as on the agent. For
on positively charged particles the H ion would have a repelling,
/. c., dissolving ; on negatively charged, the opposite effect.

If all the protoplasm of the Arbacia egg was uniformly posi-
tive just prior to or during segmentation it accordingly might be
deduced that H ions would have the same effect in increasing the
osmotic pressure as does ether, and so of inhibiting development.
Lillie, 1 however, has shown that at cleavage the cytoplasm is
markedly electropositive, the nucleus negative. Accordingly at
that time he holds that the periphery repels the free kations
within the egg and the nucleus the anions, so that the kations
then predominate at the center, the anions at the periphery. As
like charges repel each other, this is made to account for the con-
stricted form of the egg at cleavage. In agreement with this
view the effectiveness of the H ions, in parthenogenetic methods,
in the environment might be considered to be due to their induc-
tion of a predominance of negative charges at the surface and
this in turn of positive charges at the astral centers. If this be
so, however, then other kations ought to have the same effect ;
but they do not. This indicates a specific action by the H ion,
which it might have in accordance with its high diffusion velocity.
It alone might therefore be considered to penetrate the egg mem-
brane because of its and the latter's definite chemical make-up ;
yet there remain difficulties even here in explaining why it should
do this, since, if, at least before cleavage, the membrane and cyto-
plasm are themselves positive they would tend to repel the H
ions rather than to attract them. Only provided the surface were
negative from the start could the attraction be explained. If,
however, the egg, when jus f about to divide, were put into such
a medium of H ions, it is reasonable to suppose that since the
surface at least then is negative these might be attracted ; but
again it is difficult to understand how they can go further, since
the cytoplasm is even yet positive. However, if they succeed

1 Lillie, R. S., Am. Jour, of Physiology^ VIII., IV., and BIOI OGICAL BULLETIN,
IV., 4.

IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 235

in acting on the cytoplasm the effect would be a repulsion of its
particles and an increase in pressure, the same as that of ether.
Accordingly it would a priori be probable that the period of least
immunity to HC1 would coincide with that of the greatest nor-
mal pressure, viz., just before cleavage.

EXPERIMENTAL.

Two preliminary experiments with seventeen different strengths
showed that the proper strength of solution, for an exposure of
one half hour, was between a and --i- normal HC1 soluion.

Accordingly a ^ ^ n solution was next tried with the following

results :

EXPERIMENT IV.

August 3, 1/450 n HC1 solution (by diluting with sea water); three series,

2 5 / > 3 O/ an< i 35' exposure.

Lot

Time After
Fertiliza-
tion.

Series A, 25'. Per Cent.

Series B, 30'.
Per Cent.

Series C, 35'.
Per Cent.

Control.
Per Cent.

I

\'

No segmentation at

A few swim-

A few swim-

all. 1

mers.

mers.

2

1 6'

50 swimming.

80 swimming.

50 swimming.

30 blastulre, stopped.

Gastrula;.

3

V

Quite undt-celopt d and

Like AS. Like AS.

disintegrated.

4

41'

80 swimming. 40 swimming. 40 swimming.

Segmentation

began here.

5

46'

All dead in i- and 2- Like A 5 .

Like A 5 .

Segmentation

cell stage.

going on.

6

53'

40 swimming. 20 swimming. 20 swimming. ~l

Toward end

7 59' 5 " So 50 " /

of cleavage.

8 1l' All dead in 2- and 4- Like A s . Like A 8 .

15 were in

cell period.

4-cell stage.

August 5, Experiment V., -^-^ n HC1 solution, used every five
minutes for one half hour. The results are plotted on the next
page, the times after fertilization being the abscissae, the per-
centage of swimmers the ordinates, and are in general confirma-
tory of the previous experiments with HC1.

Two other experiments were made with HC1, which gave a
general confirmation of the two described. These all agree in
giving a rise in immunity soon after fertilization, then a fall at

1 The difference between A 1 , B 1 , and C 1 may be due to the effect of the necessary
manipulation on the eggs transferred so soon after fertilization, viz. A 1 ; B 1 and C 1
were necessarily transferred a little later. Cf. Mathevs, American Jour-nal of Physi-
ology, VIII. , IV., "Impcrterce cf RUcr.rr.ical Shcckon Protoplasmic Activity."

2 3 6

E. G. SPAULDING.

EXPERIMENT V., 1-450 n HC1.

, 6o Q 5 IQ IS 10 If 30 y 40 4ffSOW't>0' 6f 70' 7<f' $0

fcammliCt

30', followed by a second rise and a second fall respectively
before and during segmentation. This fall is repeated at the
beginning of the second cleavage.

Comparison of this with the ' ether curve ' however shows a
difference in respect to this 'thirty-minute fall/ and that this
should be so is to be expected from the possibility of the agent
here used (HC1) directly affecting the colloidal particles as well
chemically as electrically, as the evidence for a specific H ion
action indeed indicates ; while in the case of ether the solvent
alone is first concerned.

Assuming that the H ions in some way penetrate the egg, the
increase in osmotic pressure resulting therefrom might be con-
nected with the dissolution of the nuclear membrane, which
occurs at about the time of the first, the 30' fall, 1 in such a way
that an abnormal increase in pressure results from both, which
inhibits development. This however could not be due alone to
the electronic action of the ions for were this so ether also

i Mathews and Wilson, lac. cit.; cited by Lyon.

IMMUNITY' AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 237

would have the same effect in increasing the pressure abnormally
and causing a fall in immunity at the same time, which it does
not do. Hence from this, together with the evidence, in other
instances, of H acting differently than do other ions, and the
probability that the dissolution of the nuclear membrane is caused
or accompanied by chemical changes, it may be inferred that the
H ions have a specific chemical effect, which, in addition to their
electronic action, accounts for the fall in immunity at about 30'
after fertilization.

Both series of experiments agree, however, in making the
period of greatest susceptibility just at the beginning and during
the earlier part of cleavage, though for the HC1 this drop seems
to come somewhat earlier than for ether. The explanation for
this effect of HC1 we believe to be essentially the same as for that
of ether, viz., that the H ions in some way penetrating the mem-
brane cause a repulsion of at least the cytoplasmic particles,
thereby augmenting the normally increased pressure to such an
extent that further development is inhibited.

EXPERIMENTS WITH KC1, NaCl AND Na CITRATE.
The acceptance of a specific action for the ions is, of course,
by implication not limited to H, but is quite as necessary for
others, like K, Na, Ca, etc. For instance Loeb in one place
holds that the kations (specific) and not the anions are poison-
ous, 1 for the reason that the newly fertilized Fundnlus egg will
develop in KC1 but not in NaCl ~ and because, of fertilized Arbacia
eggs in |-# NaCl only 10, 20, and in one case 50 per cent, be-
gan to segment, the majority stopping in the 2-cell stage, while
in ^u KC1, 7080 per cent, segment to 8 cells. 3 Opposed to
this view of Loeb's is that which ascribes toxic effects also to the
anion, for, e. g., NaCl, NaBr, Nal and NaFl have a different poi-
soning effect. The two views, however, are quite compatible if
the colloids affected are in the two cases of different sign. Loeb
also finds that the toxic effects of Na salts is a function of the
valency, increasing from the acetate to the citrate. 4 Further-

1 Loeb, Am. Jour, of Physiology, III., VII. and VI., VI. and Archh- fiir d.
ges. PA}>siot.,Rd. 88, 1901.

2 Loeb, Am. Jour, of Physiology, III., VIII.

3 Loeb, Am. Jour, of Physiology, III., IX.

4 Am. /'itr. of Physiology, VI., VI.

238 E. G. SPAULDING.

more, as an illustration of the lack of a consistent theory here,
are the views of Loeb, that the antitoxic as well as toxic effect is
a function of specific kations, and of others that where the kations
are toxic the anions are antitoxic. For instance, in 100 c.c.
f NaCl + 8 c.c. -^n CaSO 4 or Ca(NO 3 ) 2 70 per cent, of Fnn-
ditlns eggs develop, while if Na.,SO., be substituted they do not.
Ca is therefore considered to be antitoxic to Na. A1.,C1. { and
Cr (SO 4 ) 3 also have the same inhibitory effect on Na, but in
smaller quantities. Loeb therefore concludes that the toxic and
antitoxic effect of the ions is a function of their valence, but that
only kations are poisonous. The necessity for such " balanced"
solutions also holds good according to Loeb for muscle and for
the contractions of Gonionemus ; " margin and center must con-
tain three ions, Na, K and Ca." 1

The opposite view is that in the instance of, c. g. y NaCl poison-
ing but KC1 not, the difference in effect is due to a greater over-
balancing by the Cl ion in one case than in the other. 2 The effect
is due in any case to both the ions. Both may be either toxic or
antitoxic according as the colloid is like or unlike in charge and
the normal event is one of gelation or of liquefaction.

Accordingly if the normal progress of cleavage in Arbacia de-
mands liquefaction, /. e., increased pressure and absorption of
water, any salt either preventing this by unlike charges or aug-
menting it by like beyond a certain point may be assumed to
interfere with or inhibit division. And from the data at hand we
may expect NaCl to do this to a less degree than KC1.

Experiment IV., 7 pts., |H KC1 sol. to 2 of sea water, the
other conditions as in III., gave exactly tJic same results, and is
tabulated on the next page.

Examination of this record makes manifest a decrease in im-
munity beginning I 5 minutes before cleavage, much as with HC1,
but reaching its maximum during segmentation. The same ex-
planation of this effect that was made for HC1 and ether holds
good, we believe, here ; but the thirty-minute fall obtained with
HC1 is not present in these series, which indicates a different spe-
cific chemical effect of H and of K ions. Two subsequent KC1
experiments confirmed these results.

1 Am. Jour, of Physiology., III., VII.

2 Mathe\vs, Science, March 28, 1902, and May 8, 1903.

IMMUNITY AND SUSCEPTIBILITY OF SEA-URCHIN EGGS. 239
EXPERIMENTS WITH KC1.

Solution.

After Fertilization.

Exposure.

Observation.

Control.

Exper. I.,
Aug. 16.

J;/.

Lots I. -III.
Lot IV.

At critical periods
before cleavage.
During cleavage,
66' after fertili-

I hour.
I "

9-95 % swim-
ming.
75% swimming.

Good.

zation.

Exper. II.,
Aug. 17.
Lots I.-1V.

fan KC1.

Before cleavage.

I hour.

All dead in 2

cells.

Good.

Lot. V.

During cleavage.

Dead.

Exper. III.,
Aug. 19.

8 pts. \n
KC1 -(- 2 of
sea H 2 O in
t r a n s f er-

every 5'

I hour.

ring.

Lot I.

5'

80% swimming.

So % swim-

"II.-VIII.

(ic/-4o')

So

ming.

" IX.
" X.
" XL
" XII.

45'
5'
55'
60'

70
60
5
25
Remainder dead
and disinte-

Segmentation
began here,
60' after.

grated.

" XIII.
and XIV.

6 5 '- 75 '

5-10% swim-
ming, remain-
der pigmented
and disinte-

grated.

Experiment /., August 29, with NaCl, 15 pts. n to i of sea
water, exposure one hour, at chosen critical periods. Observa-
tion showed a slight rise in immunity from 70 to 95 per cent, up
to the beginning of segmentation, then a fall to 70 per cent, during
that process, but as opportunity for further experimentation was
lacking this result is hardly conclusive, though by itself it is con-
firmatory of the supposed greater immunity to NaCl than to KC1.

Experiment /., with the Na citrate, August 13 ; 3 series, viz.,
2 c.c. of seven-fourths ;/ Na citrate to 300, 400, 500 c.c. respec-
tively of sea water, diluted one half at transferral. 15' periods,
I hour exposure. Parallel observations showed that the eggs
so treated were ahead of control both in beginning to segment

240 E. G. SPAULDING.

and in the number segmented at all cleavages. No period of
susceptibility was indicated. This acceleration effect might
according to the views above discussed be ascribed to the so-
called "overbalancing effect" of the trivalent radical, but the
experiment was not repeated because of lack of time.

SUMMARY AND CONCLUSIONS.

1. There is a pronounced rise in iunn unity of fertilized sea-
urchin eggs to ether up to eithery/^Y before or the beginning of
segmentation ; the exact point is impossible to determine owing
to the unevenness of the cleavage. A sharp decrease then occurs,
followed by a sharp rise toward the end of the cleavage. A repe-
tition of this occurs at the second segmentation.

2. Similar changes are found resulting from the use of HC1,
KC1, and NaCl with the difference that the fall in immunity
comes somewhat earlier with KC1 than with HC1 and with this
than with ether. All of these differ therefore from Lyon's inter-
pretation of the susceptible points with KNC as occurring "after
division," nor is a 15' period found, although with HC1 there is
a fall in immunity at 30' after fertilization.

3. The marked decrease in immunity " at cleavage" caused
by the four agents employed seems to be explainable on the
basis that all, in one way or another, augment beyond a certain
point the increase in osmotic pressure normally necessary for
cleavage. The results obtained seem therefore to be confirma-
tory of the position presented by the author elsewhere * that
cleavage is due to the equalization (Ausgleichung und Umfor-
mung) of an uncompensated potential difference between osmotic
pressure and surface tension, accompanied by electronic phe-
nomena which cause constriction.

The author wishes to acknowledge his appreciation of the
many opportunities offered him by the Laboratory and of the

kind advice and suggestions of Dr. Mathews.

COLLEGE OF THE CITY OF NEW YORK,

DEPARTMENT OF PHILOSOPHY, January, 1904.

1 UIOLOCICAL IJri.i.ETiN, February, 1904.

BUDDING TENTACLES OF GONIONEMUS.

GEORGE T. HARGITT.

While looking over specimens of Gonioncnnts for class work,
and being somewhat on the lookout for any cases of variation,
etc., my attention was arrested by an unusual appearance of a
tentacle. Near the distal end was present a small knob which
at first glance appeared simply as a protuberance, apparently
without any very definite form or structure.

On further and more careful examination it seemed to me to
warrant a careful study. It was somewhat similar to some of
the conditions found by Hargitt l in his work on the variation of

FIG. i.

FIG. 2.

this form. The conditions referred to are the presence of bifur-
cated tentacles. He found a number of tentacles which had
double, or in one case triple tips. In all these cases the extra
tip or bud seemed to arise either from the suctorial pad or else
immediately proximal to it. Fig. I represents a rather typical
example of bifid tentacle. The specimen is one I found in C.

111 Variation among Hydromedusce," BIOL. BULL., Vol. II., No. 5, 1901.

241

242 GEORGE T. HARGITT.

W. Hargitt's collection, but which he did not use in his paper on
variation. The extra tip is seen to be much shorter than the
main tip. Each tip is supplied with one of the suctorial pads,
but the shorter one arises from a point considerably proximal to
the pad on the main tentacle and directly from the tissue of the
tentacle. No sign of injury is present. The appearance of this
tentacle suggests the probable result of further growth of the
bud shown in Fig. 3, except of course the lack of the pad at the
base of the bud. Fig. 2, showing a trifid tentacle, is taken from
Hargitt's paper on variation (pp. cit.}. It shows two branches
arising from near the end of the main tentacle which seems to be
degenerate as mentioned later. The buds here do not seem to
arise from the suctorial pad of the main tentacle, which is not
shown, but each bud is supplied with a pad near its tip. The
knob on the tentacle under consideration, however, had more
the appearance of a bud than a bifurcation. This was due
chiefly to its small size which rather suggested that it was a very
early stage in the formation of an extra tip to the tentacle.

The bud arose from a definite base which presented almost
exactly the same external appearance as the normal suctorial pad.
This similarity consisted not only in the smooth appearance, due
to the absence of the ectodermal ridges found on the other parts
of the tentacle, but also in its concave form, and the further
presence of a bend or " knee " in the tentacle at this point ; all
of which are characteristic of the normal suctorial pad (Figs.
3-5). The bud arose from a depression in the base (Fig. 4) due
to the cup-like shape of the pad already mentioned.

No external sign of injury was found either in this pad or in
the surrounding tissue. The pad was of course not functional
as an adhesive organ, another functional one being present
nearer the distal end of the tentacle (Figs. 3 and 5). Whether
this new pad formed after the beginning of the development of
the bud from the old pad, whose functional activity would thus
be destroyed ; or whether a second pad formed first, and a bud
began to develop from the old one (which would not then be
necessary) simply as a result of the capacity for regeneration, or
rather duplication of parts, inherent in the tentacles, is an ex-
tremely interesting question. Of course no direct answer can be

BUDDING TENTACLES OF GONIONEMUS.

243

made without a considerable body of facts before us, coming
from actual observations on this particular point ; facts which it
would be very difficult if not impossible to obtain. It seems to
me, however, that we can suggest a probable answer from con-
ditions observed in other cases, which bear more or less directly
on this point. A comparison of -Figs, i and 2 with some of
those in Hargitt's paper show that buds do not always arise
from pads. Indeed of the six figures of bifid and trifid tentacles
shown in that paper and in this, only two show the bud as
arising from the pad. This alone would show quite conclusively
that there is no necessary or regular connection between the bucl

FIG. 3.

Fn:. 4.

and the suctorial pad. This is further emphasized by the fact
that F found nine tentacles having each three pads, apparently
well developed and functional, and yet there was not the least
sign of buds arising from any of the pads. These facts all point
directly toward an unusual predisposition to a duplication of
organs, and this perhaps offers the most satisfactory explanation
of the budding and bifurcation of tentacles.

C5

The bud showed a constriction near its middle region, but did
not present externally any signs of annulation, or rather of the
ectodermal ridges present on the old tentacle. However, nemato-
cysts were present in abundance. The general appearance of the

244 GEORGE T. HARGITT.

bud is similar to that of the old tentacle, with the exception noted
above, and is undoubtedly of the same structure.

Hargitt (op. cit., p. 244) in referring to bifid and trifid tenta-
cles suggested that they might have orginataed is the result of
some injury to the distal end of the tentacle. This seemed to
be especially indicated by the one trifid tentacle found. In this
case there seemed to be a degeneration or atrophy of the median
branch, which was probably the end of the original tentacle.
From the sides of this tentacle two branches arose opposite each
other which were considerably longer than the median tip (cf.
Fig. 2). He says concerning the cause of this : " The degenerating
middle tip would very naturally suggest the probability that an
injury might have been the predisposing cause of the secondary
tips ; on the other hand, it must not be overlooked that in each
of the other specimens with double tips no such cause seems at
all evident."

It was with the thought of trying to determine whether there
was any sign of injury which might have influenced the forma-
tion of a bud in that region, as well as to determine the histo-
genic changes involved in its formation, that I was led to under-
take a careful study of this budding tentacle.

The tentacle was stained in toto with borax carmine. Sections
were cut transversely across the tentacle, thus making the sec-
tions of the bud longitudinal.

Fig. 6 represents a section of the entire tentacle showing the
bud in its general relations. The entoderm of the bud is seen to
be directly continuous with the entoderm of the tentacle. The
bud is solid with the exception of a cavity at the distal end and
there is no connection between this cavity and the cavity of the
tentacle. It will be noticed, however, that the cells are arranged
more or less definitely in two rows with the dividing line quite
distinctly marked in the proximal region, as though in further
growth these would pull apart and thus connect the distal cavity
of the bud with the cavity of the tentacle. On either side of the
bud are masses of the rather dense tissue which makes up the suc-
torial or adhesive pad already mentioned. This tissue resembles
very much muscular tissue rather than glandular tissue, suggest-
ing that the pad acts by virtue of its muscularity, rather than by

BUDDING TENTACLES OF GONIONEMUS.

245

means of a secreted adhesive substance as suggested by Perkins. 1
At the edges of this pad is present the collar-like expansion of
the ectoderm which is characteristic of this structure normally.
Indeed, the shape, size and contents of these cells, as well as their
method of staining and general appearance, is almost exactly the
same as in the normal pad. Thus there seems little doubt that
there was originally present here a functional suctorial pad. So

FIG. 6.

far as we can tell from the sections the pad might have formed after
the bud began its development, though this is very unlikely, since
in such case the function would be quite limited. Indeed the pad
would lose its power of functioning normally if the bud increased
in size to any extent. The ectoderm of the bud is thrown into
the folds or ridges, which are characteristic of the normal tenta-
cles, and nematocysts are limited to these ridges (cf. figures).

1 " The Development of Gonionema Murbachii,' 1 '' Proc. Acad. Nat. 6V/., Phila-
delphia, p. 764, 1902.

246

GEORGE T. HARGITT.

Figs. 7, 8, 9 show only the bud and the tissues immediately
adjoining. The same general features already noted are also seen
here. In Fig. 7 the muscular pad on each side of the base of
the bud is nearer the same size than in Fig. 6. The relation of
the^entoderm of the bud and tentacle is shown in about the same

j

way, but the arrangement of the bud entoderm into two rows is
not so distinctly marked. In Fig. 8 this arrangement of ento-
dermfis scarcely indicated, the cells being more or less massed
together and not showing any apparent regularity. The charac-
ter of the tissue of the muscular pad is shown better as are also
the ectodermal ridges. The cavity at the distal end of the bud

FIG. 7.

is larger than shown in the other figures. In Figs. 8 and 9 the
muscular pad is shown on only one side of the base of the bud,
showing that the bud is not completely surrounded by it, a feature
also indicated in Figs. 3 and 5, where the pad is seen to have a
notch or sinus on one side. Fig. 9 represents a section cut one
side of the long axis of the bud, so that the entoderm of the
bud and tentacle is not continuous.

It will have been noticed that in all the figures the bud is solid
with the exception of a cavity at the distal end. Perkins (of. cit.,
p. 785) referring to the development of the normal tentacle states

BUDDING TENTACLES OF GONIONEMUS.

247

that it is at first solid, but that later " the cavity of the circular
canal is drawn into it." "The entodermal cells, arranged radi-
ally about the central axis, thicken until they are forced away
from the center and a tubular cavity is left." This process, he
states, begins at the proximal end and the cavity is gradually
" carried out along the axis of the tentacle toward the tip." In
this bud there seems to be present the cavity at the distal end so

FIG. 8.

that the method just mentioned does not apply, at least not
wholly. In Fig. 6 the cells are evidently arranging themselves
in rows next the supporting layer, with their edges meeting near
the center, suggesting a drawing away from the axis and a forma-
tion of a cavity (as Perkins suggests) connecting the cavity of
the tentacle with the cavity already formed in the bud. Fig. 9,
however, would seem to indicate a somewhat modified process.
The cell outlines are not distinct, so that their arrangement cannot
be definitely determined, but in the central part of the core are a

248

GEORGE T. HARGITT.

number of irregular cavities. This suggests the possibility of
these cavities enlarging and running together, the cells at the
same time taking up a regular position next the supporting layer,
and thus the cavity of the bud being formed. In neither case,
however, would the process necessarily begin at the proximal
end. Furthermore, it is not quite certain just how the cavity at
the distal end of the bud forms, or why it should form so early
and not involve the proximal portion of the bud.

In regard to the early method of formation of the bud little
can be suggested since it has developed beyond the initial stage.
It can be said, however, that not the slightest trace of injury was
found, which might be a predisposing cause. Alb. Lang l from

Fir,. 9.

his work on budding in Hydra, Endcndnmn and Plumularia tried
to show that the bud originated from the ectoderm entirely, that
the previous view that both layers were active could not be main-
tained ; ectoderm cells migrated through the supporting layer and
formed the bud entoderm, the old entoderm being absorbed.
Seeliger 2 and Braem, 3 however, working on Hydra, Eudendrium,
Plumularia, Obelia and Sertularclla claimed that Lang's results
were entirely incorrect and the conclusions drawn from them

1 Zeitschr. f. wiss. ZooL, Bd. 54, 1892, pp. 365-385.
* Zeilsckr. f. wiss. ZooL, Bd. 58, 1894, pp. 152-188.
3 Biol. Centralbl., Bd. 14, 1894, pp. 140-161.

BUDDING TENTACLES OF GONIONEMUS. 249

not warranted. They found dividing cells in the entoderm as
well as in the ectoderm, a condition which Lang did not find.
They found no trace of ectoderm cells migrating into the ento-
derm even in the earliest stages, of the two layers running into
each other