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JOURNAL

OF

MORPHOLOGY.

EDITED BY

C.°O: WHITMAN,

PROFESSOR OF ANIMAL MORPHOLOGY, CLARK UNIVERSITY,
WORCESTER, MaASss.
With the Co-operation of

EDWARD PHELPS ALES; Junr:,

MILWAUKEE.

Vow. inn.

BOSTON, U.S.A. :
GINN & COMPANY.
18QI.

p46

1

at.

IV.

VY.

if.

CONTERIS OF VOL IV.

No. 1.— July, 1890.

PAGES

Isaac NAKAGAWA, B.Sc.
The Origin of the Cerebral Cortex and the
Homologies of the Optic Lobe Layers in the

Lower Vertebrates: 0) 3 2) i ee I-10
Ox Pi EEAye
The Skeletal Anatomy of Amphiuma during tts
Earlier S1ageS NO) Re et ey RIB

Cyarctes F. W. McCuure, B.A., E.M.
The Segmentation of the Primitive Vertebrate
Beate EL EE UR = 50

W. H. Howe tt, Pu.D.
The Life History of the Formed Elements of the
Blood, especially the Red Blood Corpuscles . 57-116

W. H. Howe tt, Pu.D.
Observations upon the Occurrence, Structure,
and Function of the Giant Cells of the
NIAPHOD SD Wis) ld cul ea) aay SM De eam oe

No. 2.— October, 1890.

J. PrayrarrR McMourricu.
Contributions on the Morphology of the Actinozoa, 131-150

FREDERICK TUCKERMAN.
On the Gustatory Organs of Some of the Mam-
PEG OE OV ON ake e erat yy Ces ED

III.

II.

III.

VI.

Vil.

CONTENTS.

T. H. Morcan, Pu.D.

The Origin of the Test-Cells of Asctdians
Epmunp B. WILSON.
The Origin of the Mesoblast-Bands in Annelids,
Howarp AYERS.
Concerning Vertebrate Cephalogenests
No. 3.— January, 1891.
S. WATASE.

Studies on Cephalopods. 1. Cleavage of the

Ovum
J. Prayrair McMorricu.

Contributions on the Morphology of the Actin-
ozoa. 2. On the Development of the Hex-
actimi@ .

G. Baur.
On Intercalation of Vertebre
WILLiAM M. WHEELER.
Neuroblasts in the Arthropod Embryo
G. Baur.
The Pelvis of the Testudinata; with Notes on
the Evolution of the Pelvis in General
C. O. WHITMAN.

Spermatophores as a Means of Hypodermic

Impregnation .
C. O. WHITMAN.
Description of Clepsine Plana .

Typocrapny By J. S. Cusuinc & Co., Boston, U.S.A.

Presswork By Ginn & Co., Boston, U.S.A.

PAGES

. 195-204

205-219

. 221-245

. 247-302

>» Ose ie

331-336

- 337-344

- 345-360

. 361-406

. 407-418

Volume IV. Fuly, 1890. Number z.

JOURNAL

OF

MORPHOLOGY.

THE ORIGIN OF THE CEREBRAL CORTEX AND
THE HOMOLOGIES “OF SiH, OPTIC / LOBE
LAYERS IN THE LOWER VERTEBRATES.

By ISAAC NAKAGAWA, B.Sc.,

MORPHOLOGICAL LABORATORY, PRINCETON COLLEGE.

' EpINGER’S discovery of the cortical gray as distinct from the
ventricular gray in the Reptilia (1, p. 111) leads naturally to the
supposition that the rudimentary cortex, if nothing more, must
be present in the amphibian cerebrum, in which the mantel is,
in its relative size at least, not so very different from that of the
reptiles.’ And if we are able to find the homologous structure
in Amphibia, what is the extent of that homology when we take
into comparison the air-breathing vertebrates as a whole? The
first part of this paper contains the result of my observations
along that line. The second part consists of investigations
directed to determining the homology, and in part the functions,
of the several layers described by Osborn and others in the
tectum opticum of Rana (2, p. 82) in comparison with those of
the reptiles and birds.

The researches have been made under the supervision of
Professor Osborn in the Class of ’77, Biological Laboratory
of Princeton College.

‘This is not admitted by Edinger (1, p. 108). “Das erste was an diesen
Schnitten auffalt, ist, dass keine Spur von einer Hirnrinde zu sehen ist.”

I

2 NAKAGAWA. Vor WIV.

PART 1 “THE CEREBRAL) CORTEX:

1. AmpHipiA.—In Amphibia the gray matter is mainly con-
fined to the ventricular area, extending not more than one-third
of the way out toward the surface (Fig. 1, vg). But at the inner
corner of the cortex we find a number of cells which must be
considered as constituting the rudimentary cortical layer. My
first reason for taking this to be the rudiment, is the fact that
the portion of the hemisphere where these cells are found coin-
cides with the portion in the reptilian brain, where the differen-
tiation of the layers is most complete ; and the inference is, that
the scattered cortical cells must be developed first in this portion
before they make their appearance continuously in the mantel,
in the form of a cortical layer. It is highly probable, therefore,
that we shall find in the reptilian embryo a stage corresponding
to the amphibian structure as just described. This statement is
further justified by my observations in mammalian embryos,
which, at a certain stage, present the reptilian structures as de-
scribed subsequently ; in short, by Von Baer’s law, by which the
higher forms repeat the structure of the lower in their ancestral
history. The second and conclusive argument is, that these cells
are situated superficially to the fibres of the corpus callosum, and
consequently cannot be regarded as ventricular gray substance
(Fig. 1, cg). In Rana catesbiana and Menobranchus these cells
are irregularly arranged; but in Spelerpes ruber, from which the
figure has been taken, a decided tendency to arrangement into a
layer can be seen. Rows of from four to six cells are joined in
lines parallel to the surface, making a sort of disconnected layer.

2. ReptiLia. — In Tvopidonotus (Fig. 2) the cortical layer is.
distinctly developed, and in close examination it will be seen
that there are two cell-layers in the cortex, specially marked at
the inner corner. Thus making, in all, four layers for the cortex
of Tropidonotus, viz. : first, the superficial white layer (Fig. 2, 7’) ;
second, the first gray layer (cg") [Edinger’s]; third, the second gray
layer, or the layer of cells found among the fibres of the corpus
callosum (cg"'); and lastly, the ependyma (ef). This disposition
of the two gray layers is especially interesting, because it corre-
sponds to the typical structure of the cortical gray substance
in Aves as well as in Reptilia, and thus appears to be the charac-
teristic feature of the cerebrum of Sauropsida (Fig. 2, cg’, cg").

No. 1.] OPTIC LOBE LAYERS IN LOWER VERTEBRATES. 3

There is in the Zvopzdonotus cortex a striking change in the
appearance of the first gray layer, at the point midway between
the inner and outer edges of the cortex. Outward from this
point, the cells are considerably larger than those inside; they
take a deeper carmine stain, and their cell-processes are very
much more distinct. The change is rather abrupt. These cells
continue downwards to the ventral outer corner of the hemi-
sphere, and at this point terminate close to the surface.

Much the same structure obtains in Emys (Fig 3), excepting
the last-mentioned characteristic, viz., the change in the size
and appearance of the cells of the first cortical layer. There
is also an indefinite fibre-layer between the first and the second
cortical layers (f").

3. Aves.—In Columba livia (Fig. 4) there are four layers
corresponding approximately to those of the reptiles, but the
cells composing the gray layers are very much more numerous
than in reptiles; and, in fact, than in any other forms I have
studied, not excepting Didelphys. First, there is a thin super-
ficial fibre-layer ; then comes the first gray layer, occupying not
less than three-fifths of the entire thickness of the mantel.
Some of the cells in this layer present a triangular appearance,
but as far as I could ascertain, they are not of the pyramidal
variety found in the mammalian cortex. The second gray
layer is composed of cells running in the same direction as the
fibres of the corpus callosum. Most of them are lenticular in
shape, but there are a number that are less elongated.

4. Mammatrs. —A lower type of the class—the opossum —
was selected in order the more easily to find the homology,
should any exist, to the preceding forms. In the opossum there
are seven layers altogether: I, superficial fibre-layer; II to V,
four gray layers; VI, fibre-layer of the corona radiata; VII, the
ependyma (Fig. V).

The first gray layer (or the layer II of the figure) consists of
numerous pyramidal cells among which are also seen some of the
ordinary spherical cells. The pyramidal cells have each a nucle-
olus, and their apices are turned toward the surface. The layer
III is composed exclusively of ordinary nerve-cells, and their
processes present no definite trend. In this layer vertical fibres
are visible, as is also the case with the layer II. The layer IV
resembles the II in its composition; only the pyramidal cells

4 NAKAGAWA. [VoL. IV.

are slightly larger than those of the II, and they are very much
less in number. Next is the layer of ordinary cells, through
which pass the fibres of the corpus callosum.

It seems very likely that the first three (II-IV) of the four
gray layers have been developed from the first cortical layer of
the Sauropsida (cg‘); in other words, I am led to homologize
these with each other, and also the layer V with the second cor-
tical layer (cg"). I find in the young squirrel embryo (Sczzrus)
a stage corresponding to the sauropsidan structure; the first
gray layer is very thick, and the second is not so rich in cells as
the first. I was not fortunate enough to obtain a stage in which
the first gray would be in the process of differentiation into
three, but the general disposition was such as to justify, in
my opinion at least, the conjecture stated above.

To summarize: we find in the cells of the gray matter sur-
rounding the ventricle a tendency to migrate toward the super-
ficies. This process, as we have seen, is in progress in the
Amphibia, and initiates the reduction of the ventricular gray,
from its condition as the main cellular element of the hemi-
spheres to its entire absence in the adult condition of the

higher forms.
PART lL. OPTIC. VOBES:

1. AMPHIBIA.— The mesencephalon in the Urodela is a tub-
ular structure, — gray substance lining the central cavity, the
mesoccele, and showing, in most cases, no differentiation. In
some salamanders (Spelerpes), however, the cells are arranged
in concentric rows, and at places these rows, leaving little spaces
between them, anticipate the more complex structure of the
Anura. Inthe latter the mesencephalon assumes a bilobular
structure and forms a prominent portion of the brain. Professor
Osborn (2, p. 82), in his ‘Internal Structure of the Amphibian
Brain,” has described eight distinct layers in the tectum opticum
of Rana, —four white layers alternating with the four gray
layers, and also traced the optic nerve-tract into the first and
second white layers.

These layers in Rana are so beautifully differentiated that
they may be taken as a standard of comparison for those of
the Sauropsida. Taking them in order from without inwards, the
characters of these layers are as follows: The layer [A] (Fig. 6)

No. 1.] OPTIC LOBE LAVERS IN LOWER VERTEBRATES. 5

consists chiefly of fibres which are traceable to the optic nerve.
Some of these fibres sweep over the tectum opticum and pass
into the Thalamencephalon (and ultimately into the cerebrum).
[B.] This is the first gray layer, consisting of loosely arranged
cells. These cells are of the spherical description, and the
majority of the polar processes point toward the mesoccele.
[C.] The second white layer differs from the first in two respects :
it contains vertical fibres descending from the layer B, and
secondly, none of its fibres seem to pass toward the hemispheres.
The layer [D] is a compact cell-layer. These cells are somewhat
spindle-shaped, the processes running radially. They are ar-
ranged in from seven to nine concentric rows, and at places one
or more of these rows are seen separated from the rest. Ante-
riorly, in front of the mesoccele, this layer forms a wide band owing
to the interspaces of fibres formed between each cell-row, and
along the proximal side of the lobe it is pushed apart to admit
the exit of the Trigeminal nerve-tract, which proceeds from the
layer #, as will be described further on (Fig. 10), and also for
the fibres of the commussura tecti optict. [E.] The fibres descend-
ing from the layer D to F the commissural fibres apparently
connecting the layers D and Fof the opposite sides, and prob-
ably fibres coming from the cerebrum constitute the afferent por-
tion of the third white layer (Fig. 6, Z) (Fig. 10). The efferent
portion is composed of fibre-tracts which, according to my obser-
vation, partly contribute to the III, IV, and V (probably the
VI also) nerves, starting from the layer F The tract con-
tributing to the third nerve arises from the ventral portion
(below the mesoccele), that of the fifth nerve from the proximal
side, and those of the fourth and sixth from the dorso-posterior
portion. The tract of the VI probably passes through the
“nucleus magnus”; but my observation as to the course of
this tract has been very incomplete. [/-] The layer F is marked
by the presence of large multipolar cells, constituting the well-
known mesencephalic nucleus of the Trigeminus. They each
have a nucleolus, and are situated mainly in the anterior and
proximal portions, where the fibres of the III and V nerve-
tracts, respectively, are very distinct (Figs. 10 and 11).2 The
greater part of the cells composing this layer are rather spindle-

? The multipolar cells are drawn rather too large in these figures. The real pro-
portion is represented in Fig. 6.

6 NAKAGAWA. [Vou. IV.

shaped and proximal to the mesoccele ; are in single row pressed
side by side. Posteriorly the cells are more numerous, and in
the anterior and ventral portions they expand to form a wide
band, as has been mentioned in reference to the layer D.

[G.] In this layer fibres or striations are seen connecting the
ependyma below with the layer / above. There are also some
fibres running around the ependyma layer (7, Fig. 6).

2, HoMOLOGIES IN THE CORPORA BIGEMINA OF THE SAUROP-
sipa. — Although in reptiles and birds the structure of the optic
lobes is not so clearly marked as it is in the frog, yet the homology
of the layers in these forms is not difficult to trace. Beginning
with the layer A, we find it constant in 7ropidonotus, Emys, and
Columba (Figs. 7, 8, 9, 10, A). These fibre-layers are trace-
able to the optic nerve, as is the case in Rana. They are, how-
ever, much reduced in thickness, especially in Columba. In
Tropidonotus (and in most places in Columba also), this layer
is not well differentiated from the layer 4, which, in turn, is not
marked off from the layers below (Fig. 7). In the turtle it is
slightly better marked; but, owing to the presence of cells in
the next layer, it is not so clearly brought out as in Rava. In
Columba, layer PB is distinct at the inner corner of the tectum
opticum from which Fig. 9 has been taken, Layers 2; C,and
D are not differentiated in ZTropidonotus, while the latter two
(C and D) are not distinguishable in the turtle and pigeon.
The layer CD consists of fibres and cells, representing the two
distinct layers in the frog. The cells are less spindle-shaped
than in the frog, although the fact that their processes run
mainly upwards and downwards (or strictly speaking, radially)
makes them appear rather elongated in the same direction. In
Tropidonotus cells are irregularly scattered about, but in Emys
they are more numerous toward the mesoccele; the rows of
cells bordering the layer E being somewhat larger than the
rest (Fig. 8, CD; Fig. 7, BCD). Columéa exhibits a high degree
of differentiation toward the inner edge of the tectum opticum.
This layer is divided up into three sublayers of cells, with fibre-
laminze intervening between them (Fig. 9, CD). The character
of the cells is similar to what is found in preceding forms.

E, or the layer of the commissura tecti optici, is conspicuous
in all the specimens. It is thicker in Z7opzdonotus than in
either Emys or Rana. Tropidonotus also presents a singular

No. 1.] OP7IC LOBE LAYERS [IN LOWER VERTEBRATES. yi

arrangement of £. This fibre-layer in its lower half is crowded
by cells which apparently have extended outward from the layer
below. These intruding cells are lenticular in shape, with their
longest diameter arranged radially (or vertically in Fig. 7). In
Columba it is subdivided into two strata, —an outer stratum of
horizontal fibres (of the commissure), and an inner stratum of
vertical fibres. These strata are further distinguished by the
presence of cells different in each. But as I am inclined to con-
sider these cells as properly belonging to the layer F, they will
be described later on.

In Zropidonotus the layer F is well provided with large multi-
polar cells, with nucleoli (mesencephalic nucleus of the Trigeminus),
and the ordinary cells are much more abundant than in Rana,
but are not packed side by side, as we have seen them in the
latter. Besides, there are, as has been mentioned, in connection
with £,a number of lenticular cells proceeding upward from
F (Fig. 7, #). Although in Emys the large cells are not nearly
so numerous as in 7vopidonotus (being mainly confined along
the junction of the lobes), their scarcity is compensated, as it
were, by the elaborate stratification observed in this layer. In
most places there are five strata divided by fibre-lamina. These
strata are composed of ordinary cells which are somewhat spindle-
shaped. Returning now to those cells found in the layer Z of
Columba, which form the first two sublayers of 7, we observe,
first, in the horizontal stratum, or among the fibres of the Com-
missura tecti opticis, that the large multipolar ganglion cells are
elongated in the direction of the fibre-tract ; but the smaller cells
do not present any definite direction. Secondly, in the vertical
fibre-stratum, ganglion cells do not present any definite direction,
while on the other hand the lenticular cells run vertically ; that
is, in the trend of fibres. The third stratum, or the layer F
proper, consists of ganglion and small spherical cells with nucleoli
and with processes extending in all directions.

The layer G has no marked feature, except in Columba, where
it presents more of the appearance of molecular structure than
distinctly fibrillar. The ependyma is thickest in Raza, consist-
ing of from five to six rows, while in 7vopidonotus and Columba
it is very thin (Figs. 6, 7, 8, 9, #).

8 NAKAGAWA. [Vou. IV.

3. FUNCTIONAL RELATION OF THE OPTIC Lose LAYERs.

AFF EFF
ws
A _ It
Cc
8 Tt
= Ivt
ve
“
G
It It’

Jit

eS —

Diagram showing the supposed relations of the layers of the optic lobes. Each lobe is
composed of afferent and efferent portions; but the figure represents (for the
sake of clearness) the afferent portion of the one and the efferent portion of the
other side.

The above diagram is intended to represent the conclusions
I have reached by the preceding investigations.

The optic nerves decussating at the chiasma sweep around and
enter the lobes in the opposite sides, at the distal and posterior
portions (//¢"). These tracts can be traced to layers A and C;
as has been described above. Many of the fibres (//#'") of the
layer A pass over the tectum opticum and enter the thalami,
and thence probably into the cerebrum.? Ass there are descend-
ing fibres (4) from the layer JZ, it is natural to infer that they
are those of the optic tract of the layer A turned out of their
course by the agency of cells constituting the layer 4. Most,
if not all, of the fibres of C are turned from their course by the

8] have observed in 7vofidonotus the tract going direct (without passing through
the tectum opticum) to the thalami (Fig. 12, Z/¢’). “In other forms my observation
has not been complete.

No. 1.] OPZ/C LOBE LAYERS IN LOWER VERTEBRATES. 9

layer D, and a great portion is reflected so as to enter the cell-
layer F of the opposite lobe. The reflected fibres from the
opposite sides together constitute the commussura tecti optica.
So far, the nerve-impulse through the fibres has been sensory.
In layer # the conversion into, or relation with, motor impulses
must take place, because we find fibres and cells from this layer
supplying directly the oculo-motor and trigeminal nerves, and
indirectly, probably, through the xzcleus magnus, the trochlear
and abducens. The presence, constant in all the forms, of the
large ganglion cells of the mesencephalic trigeminal nucleus
confirms this hypothesis. In the layer /, therefore, we are to
look for reflexes of the III, IV, V, and VI nerves (Fig. 12,
diagram). And consequently the eye-muscles and other parts
supplied by those nerves may be excited into activity in a reflex
manner. Furthermore through the relations these tracts have
with the centres in the Medulla, this may serve to explain the
co-ordinated reflex movements exhibited by animals in which
the cerebrum has been removed. I have also observed fibres
which appear to show that the tracts coming from the cerebrum
enter into the layer / In this case the cerebral voluntary
impulses would also run through this tract. This point, how-
ever, must be confirmed by further observation.

This is of course a tentative hypothesis of the complex re-
lations of the optic lobe layers to each other, to the cerebrum
and to the origin of the cranial nerves ; it is, nevertheless, along
a line of observation which has not to my knowledge been
attempted before, and which it is evident will lead to definite
results.

PRINCETON, August, 1889.

BIBLIOGRAPHY.

1. EDINGER. Untersuchungen iiber die Vergleichende Anatomie des Ge-
hirns. I. Das Vorderhirn. Frankfort, 1888.

2. OspornN. A Contribution to the Internal Structure of the Amphibian
Brain. Journal of Morphology. Vol. II, No. 1, July, 1888.

IO NAKAGAWA.

EXPLANATION OF PLATE.

Fic. 1. Cerebrum of Sjelerpes ruber. Transverse section. x40:
Fic. 2. The same of 77ofidonotus. Xe lst
Fic. 3. The same of EZmys. SnD:
Fic. 4. Of Columba livia. xX 80.
Fic. 5. Of Didelphys. X 120.
Fic. 6. Part of the transverse section of the tectum opticum of Rana. X 110.
Fic. 7. The same of 77opidonotus. X 180.
Fic. 8. Of Emys. XiL5O:
Fic. 9. Of Columba livia. X 90.
Fic. 10. Transverse section of the optic lobes of Rana. < 18.
Fic. 11. Vertical section of the same. SS A:
Fic. 12. Vertical section of the optic lobe of 7ropidonotus. x22.

A, Ist white layer of the tectum opticum. J, Ist gray layer. C, 2d white layer.
D, 2d gray layer. £, 3d white layer. , 3d gray layer. G, 4th white layer. , 4th
gray layer. BCD, layer corresponding to the layers 2, C, and D,in Rana. CD,
layer corresponding to the layers Cand D in Rana.

Itt, Iit', Tit'', [it!'', tracts of the optic nerve. J///¢, tract of the oculo-motor
nerve. JV¢, Vt, Vit, tracts of IV, V, and VI nerves respectively. Vx, nucleus of
the Trigeminus. 4, fibres descending from B to D. cal, corpus callosum. cg, cortical
gray. cg’, Ist cortical gray layer. cg’’, 2d cortical gray layer. c. ¢ 0. commissura
tecti optici. Dz, diencephalon, or the optic thalamus. £/, ependyma. // super-
ficial white layer. /'', 2d white layer. “& J. 4. basal prosencephalic tract. For,
fornix. msc, mesoccele. J/¢, metencephalon. frc, proceele. pres, anterior com-
missure. vg, ventricular gray.

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THE SKELETAL ANATOMY OF AMPHIUMA DUR-
ING [Ts "BARLIER STAGES:

By O. P. HAY.

In the American Naturalist, Vol. XXII (1888), p. 315, the
writer has published an account of the finding of the eggs of
Amphiuma, and accompanied it with a short description of the
anatomy of the embryos contained in those eggs. In the
present paper it is proposed to enter somewhat more into details
in describing the structure of the skeleton of the young Amphz-
wma and to illustrate the descriptions by drawings.

As stated in the communication referred to, these eggs were
found in a cypress swamp at Little Rock, Ark., on Sept. 1,
1887. They had been deposited in a small excavation under
an old log, which was lying at a distance of some rods from the
nearest water; and were being cared for by the mother, who
was lying coiled up around them. The mass of eggs was about
as large as one’s fist, and, being connected in strings, they
greatly resembled a mass of large beads. When the eggs were
put into alcohol, the young were seen to move about within the
eggs.

The egg-strings were so entangled that it was found to be im-
possible to separate them, for the purpose of determining the
number of strings and of eggs. However, since there were four
ends visible, it is supposed that there were two strings, one for
each oviduct. The number of eggs is estimated to be at least
one hundred and fifty. They are globular in form, and have an
average diameter of 9g mm. (See Fig. 1.) They are separated by
from 5 to 12 mm. of string. Fourteen eggs were counted ona
piece of string 225 mm. in length. Each egg, in the condition
in which they were discovered, consists of the contained larva
and an external capsule. This latter is of a condensed gelati-
nous material, thin as paper, becoming brittle in strong alcohol,
but swelling somewhat in weaker alcohol and in water. The
connecting cords are of the same materials, and have a diameter

12 HAY: [ VoL. IV.

of from 1.5 mm. to one-half that size. The capsules are almost en-
tirely filled up by the young amphiumes ; but in their fresh state
they probably contained also some water. The young are coiled
within the eggs in various positions, so as to form about three
turns of a spiral. On being taken from the eggs and extended,
they are about 45 mm. in length (Fig. 2). So far as I have
been able to discover, they are all at the same stage of develop-
ment. The color above is dusky, with a slightly darker dorsal
streak and a similar lateral band. Below, the color is pale. In
the alcoholic specimens the belly appears yellow on account of
the great amount of yolk that is contained within it.

In form and proportions the young amphiume is stouter than
the adult, and the head is broader and more depressed, and the
snout more rounded. The head, therefore, resembles more
nearly that of the typical Urodeles than does that of the adult.
The eyes also are more conspicuous than are those of the more
mature animals, and would doubtless, for some time after hatch-
ing, be of more service. The fore and the hinder limbs are
present, but are diminutive in size. On the anterior limb,
three toes are indicated; the hinder limbs give little evidence
of separation into digits. The tail differs from that of the
adult, inasmuch as it has on both its upper and lower edges a
distinct membraneous fin.

The larvze possess conspicuous gills; and since they are
evidently near the period of hatching, it becomes quite probable
that these gills will be retained for some time after the young
have betaken themselves to the water, their native element.
The gills consist of three pairs, and are of the simply pinnate
form. The second gill is the longest, measuring about 9 mm.
in length, and gives off from the main stem ten delicate twigs.
Only once have I observed any of these lateral filaments to
divide. The first and third gills are somewhat shorter, and
have about eight lateral branches each. In all the main stems
and the lateral twigs may be seen arteries and veins filled with
the coagulated blood. Three gill-slits are still open, the first
and second of which become closed in the adult.

We cannot but be struck by the close resemblance that exists
between the breeding habits of the Amphzuma and those of
Epicrium glutinosum of Ceylon, as these are presented to us
by Messrs. P. B. and C. F. Sarasin in the “ Avbeiten aus dem

No. I.] SKELETAL ANATOMY OF AMPHIUMA. 13

Zoologisch-zootomischen Institut in Wiirsburg,’ Bd. VII, 1885.
According to these observers, the female of Epicrium excavates
a cavity in the earth a little below the surface, and there de-
posits a mass of eggs, which are connected by means of an
albuminous cord, and thus resemble a string of pearls. These
eges, when found in the oviduct, are of an oval form, about
9 mm. through the longer axis and 6 mm. through the shorter ;
but, when found in the earth, they were about twice as large,
having probably during their development absorbed considerable
water. Unlike Amphiuma, the eggs seem to have something
of a regular arrangement in the mass, the connecting strings
being bent in toward the centre of the mass and cohering there
into a viscous knot. Around this mass of eggs the female lies
coiled, and gives them protection. The embryos at the stage
described were 4 cm. long, and moved actively about. On
each side of the neck were three plume-like gills, the longest of
which was about 2cm. The eyes were relatively large and
distinct ; while the tail was surrounded by a strongly developed
fn-membrane. These eel-like young must greatly resemble
those of Amphiuma. Other larvee of Epicrium were found
by the Messrs. Sarasin swimming in the neighboring brooks.
These are without gills, possessed yet gill-clefts and a caudal
fin, and are said to attain a length of perhaps 16cm. At
length their transformation is completed, and they leave the
water. These facts bearing on the mode of embryonic develop-
ment and others derived from anatomical structure have made
it evident to the writers named above, as well as to others, that
the Ccecilians must be arranged very closely to the Urodela, if
not consigned to the same order. The habits of oviposition
and incubation and the course of development which have
now come to light as characterizing Amphiwma, must tend to
strengthen this opinion.*

It is not yet known how the young amphiumes get to the
water after they have been excluded from the eggs. Consider-
ing the nature of the ground where the eggs were found, it
would appear impossible for them to travel any considerable
distance. It might well be, however, that, like many other

* Dr. C. O. Whitman kindly sends me the interesting information that the Giant
Salamander of Japan (Megalobatrachus maximus) lays its eggs in a string exactly
like that of Amphiwma, and the eggs are of about the same size.

14 HAY. [VOL. IV.

Amphibia, their development would be retarded until some fortu-
nate day when a heavy rain would make it possible for them to
reach permanent water.

I now proceed to describe the structure of the larval Amphiuma
as disclosed by the contents of the eggs under consideration.
In my attempts to unravel this structure, I have depended
partly on dissections made by means of lens and needle, but
mostly on stained sections cut and mounted serially.

I. “THe SKULL.

In Fig. 3 we have a view of the cartilaginous cranium seen
from above, and in Fig. 4 a view of the same from the side.
What will probably first strike the attention of the observer
is the existence, in the basilar region, of two fontanelles in the
cartilage, one on each side of the middle line. They are of an
elongated oval form, and are of such size that they leave only
a narrow strip of cartilage between them, and a similar ledge
along the inner and lower border of each otic capsule. Both
before and behind these fontanelles, the cartilage passes from
one side of the skull to the other. This portion of the primitive
skull is worthy of comparison with the adult skulls of Mecturus
and Szvex. In WNecturus the trabeculze cranii are not connected
by cartilage behind the pituitary region, and have but a narrow
band connecting them in the exoccipital region; so that there
is no cartilage in the floor of the brain-case behind the ethmoidal
region until immediately in front of the foramen magnum. In
Siren there exists, opposite the middle of the otic region, a
band of cartilage that passes from side to side. Behind this,
there is in the middle line a single fontanelle ; and this is limited
behind by the cartilage of the basioccipital region. The anterior
ends of the two fontanelles of Asphiuma come much further
forward than does the single one of Szren.

In the narrow strip of cartilage between the two basal fonta-
nelles is seen the anterior end of the notochord extending well
forward toward the pituitary space.

In the exoccipital region the condyles project prominently
backward, after the manner of those of the adult. The deep
notch between them is occupied by the tooth-like process of the

No..1.] SKELETAL ANATOMY OF AMPHIUMA. 15

atlas. On each side, at the base of the eondyles, are the fo-
ramina for the vagus and the glossopharyngeal nerves. The
occipital condyles are invested with a thin ectostosis, which
continues as far forward as the foramina mentioned. This is
the only cartilage-bone that is found in the skull, except that in
the hyobranchial apparatus, soon to be described.

In the supraoccipital region, there is a narrow strip of carti-
lage which arises from the posterior end of the otic capsule, and
extends inward toward the middle line, but it lacks much of
reaching the corresponding cartilage of the other side. Along
its upper surface, therefore, the brain, from one end to the
other, has no other protection than that of the integument.

The otic capsules are large, well-developed, and of a long-
ovoidal form. They occupy about one-third of the total length
of the cartilaginous skull. The upper surface is somewhat flat-
tened, and slopes outward and downward. Anteriorly they pass
by a narrow band of cartilage into the upper edge of the tra-
beculz in front of the foramina for the fifth pair of nerves.
Behind, each capsule is rounded, and in the angle between it
and the projecting condyle, is found the vagus ganglion. The
membraneous canals are well-developed, and may be seen through
the walls of the capsule. They are enclosed within correspond-
ing cartilaginous canals. On the outer wall of the capsule is
found the large fenestra ovalis. It is partially occupied by the
cartilaginous stapes, as shown in Fig. 4. All round this stapes
is a tract of membrane, except anteriorly, where it is articulated
to the otic wall. This stapedial cartilage is confluent with the
hinder end of the columella, which will come up for considera-
tion in its place. With the exception of the fenestra ovalis,
there is no interruption in the cartilage of the outer wall of the
otic capsule. The facial foramen lies immediately in front of
the fenestra ovalis. At the anterior end of the capsule, and on
the lower floor, is found the entrance of one portion of the audi-
tory nerve into the labyrinth. Farther back, about opposite
the fenestra ovalis, there are three openings in the cartilage of
the mesial wall of the otic capsule. The smaller one, high up,
is for the passage of the ductus endolymphaticus into the brain-
cavity. A second larger foramen in the cartilage, immediately
below the last mentioned, admits into the labyrinth the branch
of the auditory nerve going to the sacculus and lagena. This

16 HAY. [VoL. IV.

nerve will receive further attention. Immediately behind the
foramen just considered is a third break in the cartilage, the
purpose of which I have not been able to determine. It pos-
sibly gives passage to some of the lymph sinuses.

In sections made previously to decalcification, there are seen
abundant otolithic deposits.

In front of the otic capsules, the trabecular walls are low and
slope gently downward and inward. The foramen for the
branches of the trigeminal nerve is large, and is traversed by
the long, slender, ascending process of the suspensorium (Figs.
3 and 4, As.f.), which process passes between the orbito-nasal
and the other branches of the fifth nerve. Anteriorly to this
foramen the cranial walls, becoming lower, approach each other
gradually until they finally meet and coalesce, and thus enclose
the ovate-acuminate pituitary space. A little further forward,
the trabeculz again separate into the cornua.

In the low trabecular wall, just behind the eye, are found two
foramina. Through the most anterior passes the optic nerve.
The posterior possibly admits the passage of the oculo-motor ;
but this I have not been able to demonstrate.

In front of the optic foramen, there is given off from the
upper border of the trabecular wall a rod of cartilage which
extends outward and forward to a point just in front of the eye,
and above the hinder end of the nasal-sac. Here it expands
into a rudimentary capsule for this organ (Figs. 3 and 4, WVa.C.’).

From the point where the above-mentioned rod leaves the
cranial wall, the trabeculz are slender and rod-like, but increase
somewhat in size to their coalescence in the ethmoidal region.
The lateral halves of the ethmoidal cartilage slope downward
and outward. There is no trace of a naso-septal lamina. The
trabecular cornua are bilobate, one portion of each (Figs. 3 and
4, C.t.'), forming a plate that curves outward under the nasal-
sac; the other running forward, at first a little outward, then
downward and inward, until it terminates in a point close to
the base of the ascending process of the premaxilla (Figs. 3
and 4, C.t.).

Just below the eye there is a short piece of cartilage that
stands outward and forward from the trabecule, to which it is
joined by means of connective tissue. This is the antorbital
(Fig. 4, Azz¢.). Running parallel with the trabecula along its

No. 1.] SKELETAL ANATOMY OF AMPHIUMA. 7.

outer border, and between the antorbital cartilage and the tri-
geminal foramen, is a slender club-shaped tract of cartilage, whose
position is shown in Fig. 4, Pt. It appears to represent the
pterygoid cartilage of other Urodeles; but it has not yet formed
a connection with the suspensorium. It lies about its own
diameter outside of the trabecula.

The eye is wholly devoid of any cartilaginous capsule.

Another cartilage that I have sought for with great interest is
that which appears in the roof of the mouth of the adult, in
front of and between the anterior ends of the vomers and below
the palatine process of the premaxilla. Nota trace of this car-
tilage is seen in any sections that I have examined.

The floor of the brain-case just described is concave from side
to side. Proceeding forward from the foramen magnum, the
floor, as shown in a longitudinal section along the middle line,
slopes rapidly down beneath the hind-brain, then horizontally
forward to the middle of the cerebrum, where the slope is again
upward.

Coming to the post-oral structures, we observe first that
Meckel’s cartilage comes forward and meets its platetrope,
while posteriorly it projects behind the articulation with the
suspensorium, and gives insertion to the digastric muscle. This
cartilage is ensheathed by membrane-bones, which will be con-
sidered further along; but it shows no signs of a deposit of
calcific matter to form the articular.

The suspensorium (Fig. 4) is of a quadrate form, is directed
slightly forward, and in transverse sections is broader below
than above. It articulates with the auditory mass by means of
the otic process and the pedicle. Starting from the lower end
and inner border of the suspensorium is the long and slender
ascending process, which runs upward and forward, and co-
alesces with the cranial wall at the anterior side of the trigemi-
nal foramen. I find no trace of a pterygoid process. From the
hinder border of the suspensorium (Fig. 4) starts out a short
process which articulates with the columella auris. No ossifica-
tion has as yet appeared in the suspensorium.

The columella is a short rod of cartilage, which, articulating
with the suspensorium anteriorly, runs backward and coalesces
with the outer surface of the stapes. Its relation to the facial
nerve will be discussed later.

18 HAY. [VoL. IV.

The hyobranchial apparatus (Figs. 4 and 5) consists of the
hyoid arch and four branchial arches. The hyoid arch presents,
on each side, a hypohyal and a ceratohyal. The basihyal has
not as yet become chondrified. There is a single basi-branchial
as in the adult. The four branchial arches are much as in the
mature animal. There is no second ceratobranchial present.
Huxley states that there is one present in the adult, but it
is not represented in Wiedersheim’s figure. The ossification
connected with the hyobranchial arches will be referred to
immediately.

Ossifications. — It has been already stated that the exoccipi-
tals are undergoing ossification. These ectostoses do not meet in
the lower middle line in front of the foramen magnum.

The only other ossification of this kind occurs in the first
branchial arch. A delicate but easily distinguishable layer of
bone invests the slender portion of the lower end of the carti-
laginous bar, as shown in Fig. 5.

The following parostoses occur in this skull: premaxillary,
vomers, parasphenoid, frontals, parietals, squamosals, dentaries,
angulars, and a hyoidean splint. In my determination of the
presence and the relations of these bones, as well as of the two
cartilage bones, I have carefully compared them with the ossifi-
cations found in the skulls of larval Amblystomas. I have also,
in the case of nearly all of them, been able to dissect them out,
clean them, and apply chemical tests.

The premaxillary of the adult is a very remarkable bone ; it
is no less so in the case of the embryo. In the adult the lat-
eral halves are so completely consolidated that no evidence is
afforded by them that they ever have been distinct. It is com-
posed of two alveolar processes: an ascending process, which
runs backward between the nasals and the frontals to a point
a little behind the line joining the anterior borders of the
orbits ; and a palatine process, which appears in the roof of the
mouth between the vomers nearly as far back as their hinder
ends and underlying the parasphenoid. It appears to be this
process which has been described by several authors as a sphe-
noidal ossification. For nearly half their length anteriorly these
two processes are connected by a thin plate of bone which
functions as a nasal septum. Nearly the whole remaining
space between them is occupied by cartilage. Dr. Wiedersheim

Noi 1.] SKELETAL ANATOMY OF AMPHIUMA. 19

(Kopfskelet der Urodelen) seems to have been the first to de-
scribe correctly this structure, especially the relation of the
palatine process to the premaxilla. He endeavors to explain
this remarkable bone by suggesting that we have in it a com-
posite of morphologically different elements. In the ascending
and the alveolar portions of the bone there is supposed to be
the proper premaxillary. In the osseous nasal septum and pal-
atine process we have an ectosteal (ferichondrostotisch) bone
formed from the originally hyaline nasal septum, which bone
has become confluent with the proper premaxillary. The fact
that the parasphenoid pushes itself between the ethmoidal car-
tilage and the posterior end of the palatine process causes Dr.
Wiedersheim to suggest a doubt as to the correctness of his
own theory ; and he says that amid these doubts nothing will
clear up the difficulty except a knowledge of the embryology
of this Urodele.

In my specimens the premaxillary is already well ossified ;
and there is, even in this early stage, no trace of any original
separation into two centres. The alveolar processes are long
and comparatively strong. Situated on their border is a number
of teeth, eleven, as I count them, one being accurately in the
middle line. An examination of the adult in my possession
shows that it has the same number and arrangement of the
teeth. In the young this median tooth, and one on each side
of it, are especially large, sharp-pointed, and directed nearly
backwards. There are long ascending and palatine processes,
although, as might be expected, they do not extend so far back-
ward as in the adult. The palatine process reaches back nearly
to the point where the trabecular cornua diverge from each
other. It has no connection with any cartilage, and there is at
this stage no cartilaginous nasal septum. It seems quite evi-
dent, therefore, that the premaxillary is not a composite struc-
ture, but that the palatine process continues to grow backward
as a membrane-bone until it attains the dimensions that it has
in the adult.

As has already been stated, the anterior lobes of the trabec-
ular cornua, like a divided prenasal process, end in the angle
between the alveolar and the palatine processes quite close to
the lower border of the latter, and close to one another. To
me it now appears quite probable that these cartilages grow

20 HAY. [VoL. IV.

downward until they meet below the palatine process and then
coalesce. Afterward, by the expansion medially of the maxil-
laries and the vomers, the portion of the cartilage below the
plane of the vomers becomes cut off from the cornua, and forms
the unpaired piece that appears so anomalous. But it will
require older specimens than those in my possession to settle
this matter fully.

The frontals are long, slender splints which first appear, pos-
teriorly, in those cross-sections which pass through the hinder
border of the eye. At their hinder ends they are comparatively
thick, and overlap the parietals. They lie entirely above the
level of the eyes. At the anterior border of the eyes, the
frontals descend to near the level of the cartilage overlying
the nasal-sac, the lower edge lying a little nearer the middle
line than the upper border of the nasal-sac. They may be
traced forward as very thin films as far as the perpendicular
section just in front of the divergence of the trabecular cornua.
Such sections show the anterior end of the cerebrum, and the
hinder ends of the two median processes of the premaxilla. (See
Fig. 6.) This anterior end of the frontal passes forward over
the olfactory nerve, which is directed laterally into the nasal-
sac, and the bone may be followed to the anterior border of the
nerve. Wiedersheim (of. cz¢., pp. 52-53) has shown that the
anterior end of each frontal forms a sort of ring or ferrule
(Knochenzwinge) around the olfactory nerve, and through which
this nerve makes its exit from the brain-cavity. According to
his descriptions, there is a flat process of bone sent down from
the frontal on the outside of the nerve; then in front of this
outer process a similar one descends on the inner side of the
nerve; then the two are united under the nerve. Already in
my specimens the frontal has come into close relations with
the nerve, but has not yet enclosed it by means of its processes.

The parietal extends from the perpendicular sections through
the hinder border of the suspensorium forward to that passing
through the anterior border of the lens. It is better developed
than the frontals. Its lower border lies upon the upper border
of the auditory capsule and trabecula to the eye, where it rises
somewhat above the cartilage. In the latter region also it lies
somewhat below the hinder end of the frontals.

Neither the frontals nor the parietals, along their inner or
upper borders, approach at all near the middle line.

No. 1.] SKELETAL ANATOMY OF AMPHIUMA. OI

The vomers, or vomero-palatines, are present as a pair of
thin, narrow splints, which extend from the middle of the sub-
nasal bands of cartilage backward almost to the antorbital carti-
lages. They lie parallel with the trabecule and a little outside
of them. Each is accompanied by a row of dental papilla, five
or sixin number. These lie a little to the mesial side of the
bone. Some of the papilla are already undergoing calcification.

There are no deposits of bone to represent the maxilla; but
two rows of tooth-papillz, five or six in each, which extend
backward from the hinder ends of the alveolar processes of the
premaxillz, show where these maxillze will soon appear.

No bony pterygoids, prefrontals, or nasals are yet to be seen.

The parasphenoid is a broad but very thin and delicate film
of bone underlying the brain from just in front of the foramen
magnum forward nearly to the coalescence of the trabecule,
and passing laterally from one trabecula to the other.

The squamosal is a curved bone that overlies the suspenso-
rium and runs upward and backward upon the otic capsule.
Its lower border is applied closely to the columella along the
anterior half of the latter.

The lower jaw is furnished with two strongly developed bones.
One of these is the dentary. It meets its fellow in front to
form a symphysis, and extends backward on the outer side of
Meckel’s cartilage nearly to the articulation with the suspenso-
rium. Arranged along each dentary are about fifteen teeth,
only the anterior one of which is anchylosed to the bone. This
tooth and the one immediately following it are large and fang-
like, and correspond in that respect to the large teeth of the
premaxillary.

The second bone of the mandible, the angular, lying along
the inside of the cartilage, extends from the angle of the man-
dible half way to the symphysis.

There is no trace of a splenial bone, and none of an articular.

As before stated, there is a parostosis connected with the
ceratohyal. It lies along the inner and lower side of the carti-
lage, running nearly the whole length of the latter. This slender
splinter of bone I have repeatedly been able to dissect off ; and
having under a cover-glass treated it with hydrochloric acid,
have obtained satisfactory effervescence. Wiedersheim (of. c7z.,
Tafel I., Fig. 8) represents the ceratohyal as having a strip of

22 HAY. [ VoL. IV.

bone running its length; and an examination of the adult at
hand shows that the cartilage is only partly ensheathed by bone.

The lower end of the first branchial arch is meanwhile under-
going ossification of a different kind, being overlaid, as before
mentioned, with bone deposited ectosteally.

To the foregoing on the cartilaginous and bony skull, I make
the following notes on other structures belonging to the head : —

The common ganglion of the facial and auditory nerves lies
wedged in between the otic capsule and the outer bar of the
basicranial cartilage. It gives origin, as usual, to the facial
nerve, which runs outward to escape by the facial foramen, and
to the auditory, which enters and supplies the anterior portions
of the labyrinth. Further back the ganglion, or what appears
to be a portion of it, seems to be crowded through the mesial
wall of the capsule so as to appear to lie partly within the cap-
sule. Here it lies in close relation with the mesial wall of the
sacculus, to which it distributes nerve-fibres, as it does also
probably to the rudimentary cochlea. This branching of the
auditory nerve before it enters the capsule I have observed also
in Amblystoma and Spelerpes. The acoustic nerve in the frog
enters the labyrinth by two or more foramina (Owen, Azazt.
Vert., Vol. I, 312).

The facial nerve, after emerging from the cranial cavity,
courses outward and passes below the columella. My sections
show this plainly. Dr. Wiedersheim undoubtedly errs when he
announces (0/. cz¢., p. 137) the rule that the facial nerve in all
Urodeles, without exception, makes its way out over the suspen-
sorio-stapedial ligament, whether this consists of fibrous tissue
or cartilage. And unless the relation of the facial nerve to the
columella in JZenopoma is variable, it, too, offers an exception
to his rule, despite the figures which he gives to illustrate these
parts (Kopfskelet, etc., Fig. 24). Messrs. Parker and Bettany
(Morphology of the Skull, p. 132) state that the cartilage pass-
ing between the stapes and the suspensorium lies over the facial
nerve, and a dissection made by myself is confirmatory of this
statement.

Mention has already been made of the foramen of the ductus
endolymphaticus. This latter is a narrow tube which enters the
brain-cavity, having taken its origin in the sacculus. On the
upper and outer surface of the brain it expands into a saccus

No. 1.] SKELETAL ANATOMY OF AMPHIUMA. 23
endolymphaticus of considerable size ; but those of the opposite
sides do not come into contact.

There are at this stage rudiments of two nasal glands. Each
consists of a single duct, which opens into the floor of the cor-
responding nasal-sac and passes directly inward so as to lie
finally upon the outer edge of the anterior end of the ethmoidal
plate. Here it divides into two tubes, which may be traced for
a short distance backward along the inner side of the nasal-sac.

its. Top AxiaAL SKELETON.

The vertebre are undergoing ossification, and this is more
advanced in the anterior, than in those of the pelvic, region.
The bodies of the vertebrz are invested with a layer of bone
which closely surrounds the notochord. Toward the ends of
the vertebra the bony sheath expands a little, so that the verte-
bral body is somewhat hour-glass shaped. There is in the
centre of each vertebral body a portion of vertebral cartilage, as
represented by Dr. Wiedersheim in his Comparative Anatomy as
belonging to the vertebra of Gyrinophilus porphyriticus. Out-
side the notochordal sheath, at the ends of each vertebra, is a
ring of much-modified intervertebral cartilage. The cartilagi-
nous arches of the vertebrae come down upon the bony sheath
of the centra, and the bone rises up from the centra upon these
arches two-thirds the distance to their upper ends.

There are no traces of ribs. Above the base of each lateral
half of some of the anterior vertebral arches there stands out a
process to which the future rib will possibly be attached.

III. THe APPENDAGES.

The shoulder girdle consists of two lateral masses of carti-
lage, in each of which may be distinguished a scapula, a cora-
coid, and a precoracoid. The scapula is slender, and is directed
downward and forward. The precoracoid is somewhat longer
than the coracoid. Both are considerably broader than the
scapula. They are widely removed from each other in the
middle line below. There is no suggestion of a sternum.

The humerus has its shaft ensheathed in a thin layer of bone.

24 LTA, [VoL. IV.

The ulna and radius, carpal, metacarpal, and phalangeal ele-
ments are present in cartilage.

The pelvic girdle consists of a plate of imperfectly differen-
tiated cartilage on each side, which has no connection with the
vertebral column above nor with its fellow below. Femur,
tibia, and fibula are present in cartilage, as well as some portions
of the foot.

IV. OBSERVATIONS ON A LARGER SPECIMEN.

Since the above was written, I have received from the collec-
tions of the United States National Museum a specimen of
Amphiuma six inches long, one of the smallest in the collection.
This has been secured in the hope that it might throw some
light on the origin of certain structures which had not yet made
their appearance in the very young, and might furnish, in the
case of other structures, stages intermediate in development
between those of the already described larva and those of the
adult. Of such structures the most interesting, perhaps, are
the unpaired piece of cartilage which is found in the roof of the
mouth, and the various portions of the premaxillary bone. The
specimen has been decalcified, stained, cut, and mounted seri-
ally; and such results as I have been able to obtain are now
presented.

As might have been anticipated, this specimen is already too
far advanced in development to be of the highest value for the
solution of the problems before us. The skull is nearly as
thoroughly ossified as it is inthe adult. Nevertheless, the prep-
aration is, I think, a very instructive one.

An examination shows that the cartilage which was found in
the hinder part of the floor of the brain-case of the unhatched
larva has been extensively removed, so that there is now none
of this tissue in the middle line between the ethmoidal plate
and the narrow basioccipital cartilage. The base of the skull,
therefore, as regards the primordial elements, is much like that
of NMecturus, except that such cartilage as remains along the
borders of the otic capsules is more extensively ossified in the
Amphiuma. Inthe region about the anterior end of the pro-
Otic, where in the larva a band of cartilage is sent from side to
side, a shelf of bone, now a process of the prootic, extends in-

Non SKELETAL ANATOMY OF AMPHIUMA. 25

=<

ward from one-third to one-half the distance to the middle line.
On this shelf is supported the trigeminal and facial nerve
ganglia. With the central band of cartilage have disappeared
also all traces of the notochord from the base of the skull.

The prootics are quite thoroughly ossified. Two points, how-
ever, as Wiedersheim has observed, even in the adult, remain in
a cartilaginous state, viz. : those to which are articulated the ped-
icle and the otic process of the suspensorium. A broad band of
cartilage, running transversely through the otic capsule in the
region of the fenestra ovalis, separates the prodtic from the
opisthotic. The latter send inward toward the middle line each
a process of bone which grows wider as we proceed backward.
These, however, nowhere come into contact, but are connected
by a considerable basioccipital cartilage.

The foramen magnum is bounded above by the opisthotics,
which for a short space come into contact in the middle line.
More anteriorly, beneath the hinder ends of the parietals, the
opisthotics are separated by a mass of cartilage which may be
regarded as the supraoccipital.

The inner wall of the auditory capsule is well ossified. In
this inner wall I find anteriorly a foramen for the branch of the
auditory nerve, which is distributed to the upper portions of the
labyrinth. Further back, a much larger branch of the auditory
nerve enters the labyrinth through about three closely placed
foramina, and is distributed to the sacculus and probably the
lagena. On the inner wall of the sacculus, we find a large
macula, and immediately outside of this a very large otolith.
(See Fig. 10, O¢.) This otolith reminds us of that of some of
the fishes. The opening for the escape of the ductus endolym-
phaticus is situated at the upper border of the wall immediately
above the foramina for the saccular branch of the auditory
nerve. Just behind the last-mentioned foramina is an opening
in the cartilage, as in the larva, through which I have supposed
a lymph sinus to pass. This foramen lies just mesiad of the
lagena.

The ossification of the trabecula lying mesiad of the prodtics
is carried forwards, anterior of the foramina for the escape of
the fifth nerve. It soon, however, becomes reduced to a mere
shell of bone surrounding the cartilage. Then begins the orbito-
sphenoidal bone. This is more or less completely ossified as

26 HAY. [VoL. IV.

far forward as the section passing through the lens, at which
point the frontals and the vomero-palatines begin to enter into
the side walls of the brain-cavity. Contrary to Dr. Wiedersheim’s
statement concerning the orbitosphenoidal bone in the adult, it
is in my specimen higher in front than behind.

Following the trabecular walls forward, we find that before
the ossifications have disappeared the cartilage has divided
itself into two bars, an upper and a lower, corresponding to those
of the larva, which are designated by /Va.C’ and 7». in Figs.
3 and 4. The lower bar is the continuation of the trabecula.
Opposite the eye, the upper bar is a very slender rod, which
does not lie nearly so far to the outside of the middle line as it
does in the embryo, a circumstance due probably to the narrow-
ing of the snout. As soon as the nasal-sac is reached, this rod
expands outward, while its inner edge lies against the descend-
ing process of the frontal bone. Just where the olfactory nerve
pierces the frontal, the cartilage again divides into an inner and
an outer portion. The inner division runs forward in the angle
between the facial and the descending processes of the frontal
and coalesces with the X of the cartilaginous nasal septum.
The outer division extends forward over the upper outer side
of the nasal-sac until opposite the bony internasal septum, where
its outer edge unites with the cartilage that underlies the nasal-
sac; its inner edge meanwhile extending inward meets and
unites with the advancing border of the cartilage that covered
the inner and upper wall of the nasal-sac. In other words, we
may say that the nasal-sac is roofed over with a cartilage that
has in it a large fontanelle, and that this roof mesially coalesces
with the cartilaginous nasal septum, while externally it coalesces
with the band of cartilage which expands beneath the nasal-sac.

Where the internasal septum is formed by the premaxilla,
the cartilage is missing on the inner side of the nasal-sac, but
above, below, and on the outer side, the cartilage is unbroken.
When the alveolar process of the premaxilla is reached, no
cartilage is found immediately over it; but on the outside of
the sac and above it, the cartilage continues to the borders of
the external nostrils, while just before the nostril is reached, the
cartilage is expanded so as almost to surround the passage.

On its lower inner side the nasal cartilage sends a prong into
the angle between the body and the alveolar process of the

No. 1.] SKELETAL ANATOMY OF AMPHIUMA. 27

premaxillary, as has been shown in the case of the larva, Below
the premaxillary is found the unpaired piece of cartilage which
has already been referred to. There is no cartilaginous con-
nection between it and the processes from the nasal cartilages
ending in the angle between the body and the alveolar processes
of the premaxillary. Hence, my theory of the origin of the
unpaired cartilage is not demonstrated by the specimen in hand.
However, it is not disproved; while the apparently transitional
character of the intervening tissues is favorable to the opinion
that the cartilage has but recently undergone conversion into
connective tissue. It is greatly to be desired that a specimen
of this species may soon be obtained of intermediate age, so
that the origin of this structure may be definitely determined.

The premaxillary has the same features as that of the adult.
I can, however, see no grounds for accepting Dr. Wiedersheim’s
view as to the origin of the median descending plate and the
palatine process from the interseptal cartilage. There is no-
where to be seen such a transition from bone to unossified car-
tilage as might be expected, were the bone derived from the
cartilage. The relations between these portions of the premax-
illary are no more intimate than is that between these cartilages
and the descending processes of the frontals. There is thus no
sphenoidal ossification in this animal.

It appears to me that many of the peculiar structures of the
Amphiuma may be explained by considering its habits. It is
eminently a burrowing animal, as has been shown by many
observers. Such a mode of life would require and, in time, lead
to the production, probably, of a narrow and pointed snout,
instead of the rounded snout, so common among the Urodeles.
The ability to thrust the body rapidly into the earth at the
bottoms of rivers and swamps would also call for a solidly con-
structed cranium; and accordingly, we find the skull of the
Amphiuma as thoroughly ossified as in the higher members of
its order. In the act of burrowing the premaxillary would be
especially exposed to pressure, and it would be essential that
this pressure should be transmitted to and sustained by the
other bones of the skull. This result is secured in a beautiful
and effective manner through the structure and connections of
the premaxillary. Its solidity is, first of all, secured by its
being composed of but a single piece. At the sides its alveolar

28 HAY. [VoL. IV.

processes are joined to the strong maxillaries, which, instead of
being directed widely outward, as in C7xyptobranchus, are turned
backward with their palatine processes lying close to the long
and strong vomers. The latter, in their turn, run far back
beneath the parasphenoid, and all these bones are firmly bound
together by connective tissue. On the upper surface of the
skull, too, the maxillary is closely joined to the nasal, and
through the prefrontal with the strongly developed frontal.
This, however, seems not to be enough. The premaxillary has,
above, a process that extends back between the nasals and ends
by being wedged in between the frontals for half their length.
Below, the premaxillary sends backward a similar spine, which
is firmly bound to the vomers and the parasphenoid. The pre-
maxillary is further strengthened by having the two backwardly
directed spines connected at their bases by the plate of bone
which functions as a partial internasal septum. The whole
structure of the skull is in strong contrast to that of the skulls
of NMecturus and Stren, both exclusively swimming animals.
Reference has already been made to the peculiar structure of
the anterior ends of the frontals, as these have been described
by Dr. Wiedersheim. My observations on my largest specimen
do not wholly confirm his descriptions. I do not find that the
frontal forms anything that can properly be called a ring or
ferrule around the escaping olfactory nerve. That the olfactory
nerve does leave the brain-case through the frontal is very true.
What I do find is this: the anterior ends of the frontals send
down each a descending process, which at length touches the
ethmoidal cartilage. For a space the processes form the side-
walls of the brain-case, and when they have come into contact,
they function as a portion of the internasal septum. Where
they form the walls of the cranium, the olfactory nerves of
course lie mesiad of them. As the processes approach each
other like the sides of a wedge, the nerves at length pierce them
and enter their respective nasal-sacs. Fig. 7 represents a sec-
tion made across the head at the point where the nerves are
either passing or are about to pass through the frontals. On
the right the olfactory foramen has not yet been reached, though
the bone is thinning. On the other side the nerve is in the act
of escaping through the process. Fig. 8 shows the condition of
things only two-thousandths of an inch further forward. Here

No. I.] SKELETAL ANATOMY OF AMPHIUMA. 29

=<

we find both nerves on the outside of the descending processes,
and yet these processes have undergone no change, except that
they have approached each other more closely. These parts
may undergo some modifications by the time the animal has
reached adult size, so as to justify the distinguished author’s
description, and his Fig. 20, Tafel II; but it seems more
probable that he has been misled by not having closely consec-
utive sections. With my sections the thousandth of an inch in
thickness I have no difficulty in making out the changes in
position of the processes. It would almost appear that in the
process of lengthening which the snout has undergone the
orbitosphenoid and the cartilaginous internasal septum have not
been able to keep pace with the other structures, and that their
deficiencies have had to be made good in the one region by an
extraordinary development of the frontals and in the other by
the production of the perpendicular plate of the premaxillary.
Dr. Wiedersheim (of. czz., p. 136) states that “das cartilagi-
ndse Operculum zu einem kurzen ebenfalls knorpeligen Stiel
auswachst,” etc. My specimen, young as it is, tells a different
story. The rim of the operculum is wholly cartilaginous; but
both the inner and the outer surfaces of the central portion of it
are converted into bone. The head of the columella is codssi-
fied to the centre of the operculum; but almost immediately
after it has freed itself, the columellar rod becomes cartilagi-
nous. The thickened lower border of the squamosal then de-
scends upon the columella, and is continued upon it to a point
somewhat in front of the fenestra ovalis. Here this rod once
more becomes surrounded by bone, which passes forward into
that of the quadrate. Sva. in Fig. 10 points to the bone-
incrusted operculum. From the anterior bony portion of the
columella a broad process of bone rises up between the squa-
mosal and the otic capsule ; and this may be traced backward and
upward for some distance, until at length it ends in a point.
The termination of this point may be seen in Fig. 10, Cap.
For a part of the way anteriorly this process rises nearly to the
upper border of the squamosal. Such is seen to be the case in
Fig. 9, which passes through the foramen for the facial nerve.
Here the axis of the columella is seen to be cartilaginous, but
surrounded by bone which passes into a plate lying inside the
squamosal. We might say, in other words, that the quadrate

30 HAY. [VoL. IV.

sends upward and backward a strong process between the
squamosal and the otic capsule, and that the lower border of
this involves the greater portion of the columella. As far for-
ward as the quadrate the squamosal rests on the columella, the
process just mentioned springing from the inner border of the
columella. This relation is shown in Fig. 9.

If we consider how firmly the quadrate is clamped to the
skull by means of the squamosal, how greatly movements of the
columella must be restricted by its close connection with the
squamosal, and how the backwardly directed process of the
quadrate adds to the stability of the parts, we can easily believe
that the delicate structures of the labyrinth will be but little
disturbed by movements, even the most violent, of the jaws.
Since the Amphiuma, as has been shown both by the observa-
tions of Dr. Shufeldt (Sczence, Vol. TI, 163) and myself, attacks
its enemies with great vigor, seizing them between its jaws and
turning about its long axis like a drill or whirling around ina
spiral, it would appear necessary to protect the delicate organ
of the ear from such agitation as might during such conflicts be
imparted to it through the columella.

The facial nerve is plainly seen to escape beneath the col-
umella, This is shown in Fig. 10, V/Z.

The principal part of the ossification of the quadrate is found
on the outer surface of the cartilage. It is overlapped by the
squamosal, and along its outer border sends out a Jedge which
supports this bone. The remarkable process sent backward by
the quadrate has already been mentioned. It may be called the
columellar process of the quadrate.

The pterygoid cartilage, which in the larva consisted of a
slender rod unconnected with the suspensorium, has now joined
the lower border of the ascending process about one-third of the
distance back of where the latter unites with the trabecular car-
tilage. The pterygoid bone is present as a very thin and
slender splint, which posteriorly forms a suture with the inner
side of the quadrate, and runs forward beneath first the ascend-
ing process of the suspensorium and then the proper pterygoid.

Posteriorly the groove for the temporal muscle and tendon is
along the lower border of the parietal bone. When the prootic
bone is reached, the parietal overlaps it. Farther forward the
two bones form a harmonia suture. Near the anterior end of

No. 1.] SKELETAL ANATOMY OF AMPHIUMA. St

the labyrinth the prodtic rises so as to overlap the parietal, and
at length it alone forms the outer wall of the groove. This pro-
cess of the prootic continues thus to its termination at the very
tip of the beak-like process of the parietal, which Wiedersheim
has figured on Tafel II, Fig. 17.

The antorbital is almost entirely cartilaginous, but posteriorly
coalesces with the ossification of the orbitosphenoid.

I find no cartilage strengthening the capsule of the eye.
While such a support seems usually to be present in the eye of
the Urodela, I find none in that of Spelerpes longicaudus. The
integument passes over the eye of Amphzuma, and the connec-
tive layer is very dense and thick. The animal probably enjoys
very limited powers of vision.

In the lower jaw we find the articular undergoing ossifica-
tion; but this seems to be due rather to an extension of the
bone of the angular first around, and then into, the territory of
the articular, than to an independent centre.

As already observed in the case of the larva, the ossification
of the hyoid seems to be rather a parostosis than a cartilage bone.
The bone lies on the mesial side of the cartilage. At the ante-
rior end of the hyoid, the bone seems almost immediately to press.
itself through the cartilage, so that there is cartilage on both
sides of the bone. Further back, the bone thickens, becomes
crescentic in section, and partially encloses the outer and main
portion of the cartilage. In Fig. 9 at Ce.h.0. Ihave represented
the section and position of this bony portion of the hyoid. The
letters Ce.4.c. point to the portions of the cartilage. These
relations continue nearly to the upper end of the hyoid. For
the greater distance, the cartilage on the mesial side is a very
slender rod, and at one point it disappears entirely, but almost
immediately it comes into view again. As there is no trace of
this inner rod of cartilage in the larva, it must grow from the
extremities of the outer cartilaginous rod. Near the posterior
end of the hyoid, the two portions of the cartilage reunite into
one mass.

About the ends of the hypohyal are located several nodules
of cartilage, which probably represent the basihyal. Dr. Wieder-
sheim has figured these as he has observed them in the adult.

A large portion of the basibranchial is ossified. The first
branchial arch is ossified from end to end. Here, as in the case

32 HAY. [VoL. IV.

of the basibranchial, the calcific deposit forms a shell surround-
ing the rod of cartilage, but the deposit is also invading the
cartilage extensively.

For the opportunity of making my investigations on this
extremely interesting animal, I am indebted to Dr. John C.
Branner, Director of the Arkansas Geological Survey, and to
the liberality of the management of the National Museum.

No. 1.]

Ant.
Asp.

Bor.
Bue.

SKELETAL ANATOMY OF AMPHIUMA.

EXPLANATION OF LETTERS USED IN THE FIGURES.

Antorbital.

Ascending process of suspenso-
rium.

Basibranchial.

Middle strip of basicranial carti-
lage.

Br. I, 11, 111, 7V. Branchial arches.

by.
Ce.br.
Ce.h.

Ce.h.c.
Ce.h.o.

Co.
Cond.
Co.p.
(Cue
Gee

Dp.
Eth.

Brain. [Rhinencephalon. ]

Ceratobranchial.

Ceratohyal.

Ceratohyal cartilage.

Ceratohyal ossification.

Columella.

Condyle.

Columellar process of quadrate.

Cornu trabeculz, anterior lobe.

Cornu trabecule, lobe beneath
nasal-sac.

Dental papillee.

Ethmoidal cartilage.

Frontal bone.

Olfactory nerve.

Optic foramen.

Pe
Ma.
Nac.
Na.s.
Not.
Ot.
Pa.

PMx.a.
PMx.n.

Pix. p.

IER
Pro.
Ps,
NG.
iS.G:
Sq.

Oculomotor (?) foramen.
Maxillary bone.

Cartilage roofing nasal-sac.
Nasal-sac.

Notochord.

Otolith.

Parietal.

Alveolar processof premaxillary.
Ascending process of premaxil-

lary.

Palatine process of premaxillary.
Prefrontal.

Prodtic.

Parasphenoid.

Pterygoid.

Semicircular canal.

Squamosal.

Fifth nerve and foramen.

Facial nerve and foramen.
Auditory nerve.

Saccular branch of VIII nerve.
Vomers.

Foramen for vagus nerve.

34 HAY.

EXPLANATION OF PLATE II.

Fic. 1. View of two eggs, containing young and showing the connecting cords.

Fic. 2. View of the young taken from the egg, and enlarged to twice the natural
size.

Fic. 3. View of the cartilaginous skull of the larva, as seen from above. Enlarged
Io diameters.

Fic. 4. Same skull seen from side. Enlarged as Fig. 3.

Fic. 5. Hyobranchial apparatus. Enlarged 10 diameters.

Fic, 6. Transverse section across the snout of the larva. Enlarged 56 diameters.

Fic. 7. Section across the snout of the larger specimen, six inches long. This is.
designed to show the relations of the descending processes of the frontal bone to the
olfactory nerves. The nerve is seen, on the left side, passing through the descending
process. One branch is passing outward to the walls of the olfactory organ. The
section is cut somewhat obliquely, so that on the right side the olfactory nerve yet
lies mesiad of the descending process. Enlarged 32 diameters.

Fic. 8. Section taken 7,45 of an inch anterior to the preceding. Both nerves.
have passed through the frontals, but the processes continue on with little change.
Enlarged 32 diameters.

Fic. 9. Section across head of same individual as the last. Passes through the
foramen for the facial nerve. This figure illustrates the ossification of the columella,
and the broad process of bone that arises from it and passes up between the squa-
mosal and the prodtic. The ceratohyal ossification lying between the two portions of
cartilage is seen, as well as the ossification of the first ceratobranchial. Increased 32
diameters.

Fic. 10. Section through same skull and with the same enlargement. It passes
through the anterior edge of the stapes. Shows more especially the columella, car-
tilaginous at this point, the facial nerve passing below it, the posterior extremity of
the columellar process of the quadrate, and the passage of a portion of the saccular
branch of the eighth nerve into the labyrinth, and the large otolith.

Figs. 5, 6, 7, 8, 9, and 10 were outlined under the camera and the details filled
in from the slide under higher power. Figs. 3 and 4 were .partly drawn under the
camera and partly reconstructed from the sections.

PLIL.

Jowmn. Morph. VoLIV.

a. a. Asp. Vi. Col.

i 4 red.

BMeisel ith Boston

Hay, del ad nat

THE SEGMENTATION OF THE PRIMITIVE VER-
TEBRATE BRAIN.

By CHARLES F. W. McCLURE, B.A.,

E. M. FE.LiLow 1n Brotocy aT PRINCETON.

PART ot:

THE primitive segmentation of the vertebrate brain is a
problem which has probably attracted as much of the attention
of morphologists as any one of the great, unsettled questions of
the day, and many views have been advanced which have, it is
true, reached one important point of agreement; namely, that
the primitive brain was undoubtedly a segmented structure.
But beyond this, in regard to the character of these segments
and the number of segments of which the brain originally con-
sisted, I think it can be said with perfect freedom that nothing
whatever has been definitely proved. It is the purpose of this
paper to add a few more links to the chain of evidence neces-
sary for the elucidation of this important question.

The majority of investigators on this subject have made use
of the cranial nerves as a means of determining the number of
segments of the primitive brain. Investigations in this line are
good as far as they go, but as far as the determination of the
original number of segments and the character of these seg-
ments by this method is concerned, it is largely conjectural for
the following reasons :

1. We have positive proof that the degeneration of certain
branches has taken place.1. This being the case, we have every
reason to assume that whole segmental nerves may have once
existed, which have completely degenerated, leaving no trace
whatever of their previous existence. If such be the case, the
segments originally connected with these degenerated nerves
must necessarily be overlooked, if the existing nerves are made

1 Marshall states that the IV nerve possesses a sensory branch in Selachians and
Amphibians. Gegenbaur notes the same for Selachians.

36 MCCLURE. [Vou. IV.

use of as a means of determining the original number of
segments.

2. Furthermore, the vagrant changes in the position of some
of the cranial nerves must necessarily cause confusion. For
example, take the VI nerve which in the frog and tadpole stages
is situated between the first and second roots of the IX. nerve,ta
position somewhat posterior ‘to its place of origin. This remark-
able shifting clearly shows not only what great changes in position
the cranial nerves are capable of undergoing, but it also goes
to prove that we can find no reliable means of determining the
primitive segments by means of their connection with the exit
of the existing cranial nerves. Beard in taking up this problem
made use of an important series of sense organs for which he
- has proposed the name of “ Branchial Sense Organs,” from their
development from thickenings of the epiblast over each bran-
chial cleft. The dorsal branches of certain cranial nerves fuse
with these epiblastic thickenings; the superficial part of the
thickening giving rise to a branchial sense organ, while the
deeper portion becomes the ganglion of the dorsal root of
the cranial nerve. This close relation which exists between the
dorsal branches of the cranial nerves and their corresponding
sense organs is undoubtedly of segmental character. But this
line of research is beset by a great difficulty, namely, that the
degeneration of certain branchial sense organs would, in time,
involve the degeneration of their corresponding cranial nerves,
and such degeneration has certainly taken place, in part or in
whole, leaving in doubt the primitive segments with which they
were connected. As far as I have been able to compare Beard’s
investigations with my own, I think they are correct when he
considers the I, III; V., VIL; Vill; [X., and 56 nenvesein
connection with their corresponding branchial sense organs, as
representing respectively the remains of primitive segments.
In fact, my own observations lead me to the same conclusions,
but in addition to these I find intermediate encephalic segments
between I. and VIII. nerves which Beard’s method has led him
to pass over entirely.

These investigations were carried on in the Morphological
Laboratory of Princeton, under the direction of Dr. Henry F.

1] am indebted to Mr. Strong of Princeton for this point.

No. 1.] THE PRIMITIVE VERTEBRATE BRAIN. 37

Osborn, to whom I feel greatly indebted for his kindness in
furnishing me with everything necessary for the accomplish-
ment of this work, as well as for valuable advice in connection
with it. I also wish to express my thanks to Dr. Henry Orr
for the use of his sections of the Lizard. Also to Professor
Ryder for some fish embryos which he kindly sent me.

The following types were studied in connection with this
subject, which, though not the most desirable, were the only
ones obtainable at the season : —

Amphibia, Amdlystoma punctatum.
Reptilia, Axolts sagroet.
Aves, chick embryos.

The general object of this paper is to show that the symmet-
rical constrictions or folds found in the lateral walls of the
embryonic brain are remains of the primitive segmen-
tation of the neural tube, in part atavistic, extend-
ing into the primary fore-brain.

Literature. — The folds in the side walls of the medulla or
hind-brain have been frequently noticed and commented upon,
but only recently has their importance as segmental structures
been recognized. Remak in 1850 observed these folds in the
medulla, and rightly considered them as structures formed in
connection with the “Anlagen” of the cranial nerves. They
were observed by Von Baer in 1828 and Dursy in 1869: the
latter counted six folds in the hind-brain. In 1875, Dohrn
pointed out the segmental significance of these folds with rela-
tion to the mesoblastic somites, and in the joint resemblance to
the segmentation of an insect embryo. In 1876, Foster and
Balfour, and in 1877, Mihalkovics, inclined to give a
mechanical explanation to these medullary folds. Béraneck
quite recently observed five folds in the medulla of the lizard,
and described and figured their connection with the origin of
some of the cranial nerves. Kupffer finds in the mid and
hind-brains of the trout and salamander at least eight segments,
and, if I understand him correctly, says these segments not
only correspond to the lateral somites (p. 476), but that there
is something similar to these brain segments to be observed in
the spinal cord. He concludes, however, by expressing the
opinion (p. 477) that the fore-brain is not to be reckoned in the

38 MCCLURE. [Von. IV.

segmented region. He does not, in his brief paper, give any
of the histological characteristics of the segments. I am also
indebted to this paper for many bibliographical references.

Gegenbaur has recently expressed the following opinion:
«So interessant und so vielversprechend diese Thatsachen sind,
so wenig scheinen sie mir gegenwartig geeignet, zur Beurthei-
lung der Metamerie des Kopfes selbst als Faktoren in Geltung
gebracht zu werden. Das wird erst eintreten konnen, wenn
ihre Beziehung zu anderen, den Kopf aufbauenden Organen
erkannt ist.” ‘

In 1887, Orr described six folds in the hind-brain of the lizard,
five of which are of equal size, and the 6th, from which the
1oth nerve originates, somewhat longer than the others. He
described the mid-brain as consisting of one fold, and in addi-
tion to this described two folds in the primitive fore-
brain. He gave the name “neuromeres”’ to these folds, —a
name previously used by Ahlborn with a somewhat different
significance. Orr found that the V., VII, VIII, IX., and X.
nerves each originated in connection with a neuromere which
degenerated after the nerve was formed. He fully described
the typical structure of a “neuromere,” which I quote, as it
bears directly on my own work:

1. “Each neuromere is separated from its neighbors by an
external dorso-ventral constriction, and opposite this an internal
sharp dorso-ventral ridge, —so that each neuromere (7.e. one
lateral half of each) appears as a small arc of a circle.”

“The constrictions are exactly alike on each side of the
brain.”’

2. “The elongated cells are placed radially to the inner
curved surface of the neuromere.”’

3. “The nuclei are generally nearer the outer surface, and
approach the inner surface only towards the apex of the ridge.”

4. “On the line between the apex of the internal ridge and
the pit of the external depression, the cells of adjoining neuro-
meres are crowded together, though the cells of one neuromere
do not extend into another neuromere.”’

“This definition of adjacent neuromeres presents, in some
sections, the appearance of a septum extending from the pit of
the external depression to the summit of the internal ridge

(Sp2).”’

No. I.] THE PRIMITIVE VERTEBRATE BRAIN. 39

Dr. Hoffmann, of Leiden, published in the Zoologischer An-
seiger, June 24, 1889, an article on the segmentation of the hind-
brain in the reptiles, which appeared after my abstract of June
14th had been sent to the same journal. (See Bibliography.)
He refers to his previous article in Bronn’s “ Reptilien” (pub-
lished in 1888), p. 1967, where he considered the hind-brain as
consisting of seven metameres or segments, each of which is
connected with a nerve in substantially the same manner as
described by Orr in his ‘Embryology of the Lizard,” 1887.
In his more recent article, as I understand him, he considered
the IV. nerve to originate from the first segment of the hind-
brain, and to gradually shift its position forward into the mid-
brain. I will show that Dr. Hoffmann is probably wrong in
considering the hind-brain as consisting of seven segments, and
that the segment considered by him as the first segment of the
hind-brain is rather the posterior segment of the mid-brain; in
other words, it is the second neuromere of the mid-brain (my
neuromere Trochlear, Nm. IV.).

In addition to the above statement, Dr. Hoffmann gives the
following important evidence in connection with the Trochlear
nerve, which I quote in full. ‘Aus alledem scheint also mit
Bestimmtheit hervorzugehen, dass der N. trochlearis einen
dorsalen Kopfnerven bildet, denn er besitzt bei Embryonen
von Lacerta in jungen Entwicklungsstadien ein ziemlich mach-
tiges Ganglion, welches einen bis unmittelbar an die Epidermis
tretenden Fortsatz abgiebt, der aber, wie das Ganglion, bald
wieder vollstandig abortirt, ja es fragt sich selbst. Ob der Ner-
vus trochlearis vielleicht nicht als der vorderste, segmentale
Kopfnerv zu betrachten ist, der dem 1, vordersten Segment
zuge hort: fiir diese Meinung spricht auch die Thatsache, dass
Ganglion, sobald es sichtbar zu werden anfangt, fast vollstan-
dig allein dem 1. Segment aufsitzt, und spater auch auf
das Mittelhirn ibergreift.”’

From an examination of longitudinal horizontal sections of
Amblystoma, Anolis, and chick embryos, the latter ranging from
30 hours to five days old, I find that the lateral walls of the Mye-
lon and Encephalon (hind, mid, and primitive fore-brain) consist
of a series of constrictions which are exactly alike on each side of

40 McCLURE. [Vor avs

the brain; and that the constrictions of the Myelon
gradually pass or merge into those of the Enceph-
alon, thereby forming a continuous series of constrictions
throughout the entire length of the neuron, which increase in
size anteriorly.

For sake of clearness I have classified the constrictions of the
neuron as follows:

Constrictions of the Myelon = Myelomeres

fie Neuromeres.
Constrictions of the Encephalon = Encephalomeres

The number of encephalomeres! actually observed in the
types examined is as follows:

HB MB FB
AaB iystOma se) i Bey a ne 5 2
Anolis and’ Chicky. = xu. eles 6 2

I do not, with Orr, consider the mid-brain as equivalent toa
single encephalomere,” but rather relying upon the observations
of Kupffer, as equivalent to two (or even three) which have de-
generated in the above-mentioned forms, but persist in the
Teleosts, and probably in other fishes. The total number of
encephalomeres was thus probably ten, divided as follows :

Fore-brain, 2 and possibly a portion of a third.
Mid-brain, 2 or 3.
Hind-brain, 6 or 5.

In order to avoid confusion when speaking of the encepha-

lomeres individually, I have given them names which I think
for the present will answer the purpose.

I. Olfactory Neuromere. III. Oculomotor Neuromere.
The most anterior neuromere of Mid-brain neuromere.
the primitive fore-brain. IV. TZrochlear Neuromere.

Il. Optic Neuromere. Second neuromere of the mid-
The second neuromere of the brain (demonstrated in Petro-
primitive fore-brain. myZon).

1 Term proposed by Wilder for the large encephalic vesicles which we cannot
now consider in any proper sense segmental. See article “ Brain” by Wilder in
Reference Handbook of the Medical Sciences, Vol. VIII 8., § 23, prop. X., p. 113.

* For mid-brain neuromeres, see Appendix.

No. 1.] THE PRIMITIVE VERTEBRATE BRAIN. 41

V. Trigeminal Neuromere. VIII. Auditory Neuromere.

The first and most anterior neu- The fourth neuromere of the hind-
romere of the hind-brain. brain.

VI. Abducens Neuromere. IX. Glossopharyngeal Neuromere.
The second neuromere of the The fifth neuromere of the hind-
hind-brain, absent in the Newt. brain.

VII. Facial Neuromere. X. Vagus Neuromere.

The third neuromere of the hind- The sixth neuromere of the hind-
brain. brain,

Comparative Structure of the Myelomeres.

The spinal cord is of clearly segmental character, and at a
certain period of its embryonic development, at the time of for-
mation of the mesoblastic somites, we see that its lateral walls
are constricted in a manner similar to those of the encephalon,
and that the transition from the former to the latter is a gradual
one.

Gross mounts of chick embryos ranging between 35 and 46
hours old clearly show this structure; also Figs. 4, 4a, which
are longitudinal horizontal sections of Amdblystoma.

Pind thatthe structure of the Myclomeres in the
Newt, Lizarp and Cuick, conforms in every respect
Lothe, foun characteristics which) Orr sives tas
found by him in the’ neuromeéeres: of the hind-bram
of the Lizard. These four characteristics are quoted in full
on a previous page.

An examination of Figs. 1, 2, 3, all of which are camera draw-
ings of neuromeres of the spinal cord, shows —

1. ‘That the neuromeres have the appearance of small arcs
of circles, z.e. one lateral half of each (Vm). And that the con-
strictions are exactly alike on each side of the brain.”

2. “That the cells are elongated and are placed radially to
the inner curved surface of the neuromere”’ (in).

3. “The nuclei are generally nearer the outer surface (oz/),
and approach the inner surface (zz) only towards the apex of the
ridge’”’ (a).

4. “On the line between the apex of the internal ridge (ap)
and the pit of the external depression (ex) the cells of adjoining
neuromeres are crowded together, though the cells of one neu-
romere do not extend into another neuromere.”’

42 MCCLURE. [VoL. IV.

The Relation of the Myelomeres to the Mesoblastic Somites.

The Myelomeres are intersomitic; that is, the centre of each
Myelomere is opposite the space between two somites (Figs. 1,
2 and 3). The dorsal branches of the spinal nerves pass from
the external surface of the Myelomeres to the space between two
somites, which is opposite their point of origin, and fuse with
the épiblastic thickenings to form the spinal Ganglia.

Comparative Structure of the Neuromeres.

In the hind-brain of the lizard and chick six neuromeres
are distinctly seen. Figs. 5, 52, 6, and 6a, which in each case are
of exactly the same size with the exception of the Vagus Neu-
romere (Vm X.) which is slightly longer than the others. In
Amblystoma, Figs, 4, 4a, only five neuromeres are found in the
hind-brain. The Abducens Neuromere (Vm V7.) is not present.
The remaining neuromeres are of equal length except the
Vagus Neuromere (Vm XX.) and the Trigeminal Neuromere
(Nm V.), which are somewhat longer than the others. We have
already seen that the Vagus Neuromere in the lizard and chick
is somewhat longer than the others, but that the Trigeminal
Neuromere does not vary. In Amblystoma, the Trigeminal
Neuromere is equal in length to about two of the three remain-
ing neuromeres of the average dimensions. (Figs. 4, 4a.)

This variation in size of the Trigeminal Neuromere is due in
all probability to the coalescing of the Abducens Neuromere
with the Trigeminal Neuromere to form one neuromere. The
fact that the recent Amphibia are somewhat removed from the
main vertebrate line, and that their development has been
influenced by the great quantities of food yolk present, may
account in some degree for the varying structure of the Tri-
geminal Neuromere in Amdlystoma.

So much for the similarity and points of difference which
exist between the neuromeres of the medulla of Azolzs, Am-
blystoma and the chick, so far as their relations of size are con-
cerned. Now in regard to their histological structure, I find
that the four characteristics given by Orr for the neuromeres
of the medulla of Azolis are represented in every respect
in the structure’of the neuromeres of the meauia
of Amblystoma and the chick; that is, the cell arrangement of

NO. I.) THE PRIMITIVE VERTEBRATE BRAIN. 43

the neuromeres in the medulla of all three classes is the same.
It has already been shown that the structure of the Myelomeres,
in all three of the types studied, conforms in every respect to
the typical neuromere of the hind-brain. Thus we see that a
conformity of structure exists between the neuromeres of the
spinal cord and those of the hind-brain in the three forms studied.

By applying the description given for the structure of the
neuromeres in the spinal cord to Figs. 40, 4c, 5, and 64, which
are camera drawings of the neuromeres in the medulla of
Amblystoma, Anolis, and the chick, it will be seen that the
SEEuctuUre (Or the newromeres! of -the medulla and
spinal cord in the Amphibia, Reptilia and Aves is
identically the same.

Comparative Structure of the Neuromeres of the Primitive
Fore-brain.

So far as known to myself, Orr was the first to notice the
presence of two neuromeres in the primitive fore-brain of the
Lizard, but he did not compare their cell-structure with that of
the neuromeres of the hind-brain. In addition to confirming the
presence of two neuromeres in the primitive fore-brain of the
Lizard, I have also found that the primitive fore-brain of the
Newt and Chick consists of two neuromeres. Also between
the mid-brain and optic neuromere (Vm //.) of the Lizard,
Fig. 8a, there is a structure (Vm J//.') which resembles a
portion of a neuromere. Its form is that of an arc of a circle,
but the radius of its arc is less than that of either of the two
remaining neuromeres of the primitive fore-brain, which I have
already said resemble arcs of circles. I make merely a passing
mention of this, for the reason that from the existing data noth-
ing but conjecture can result as to its neuromeric value; while
on the other hand if it is a neuromere, it ought to be present in
toto in some of the lower vertebrates. (See Appendix.)

The fore-brain neuromeres of the Lizard and Chick, so far
as their external character and histology is concerned, are true
neuromeres. By external character I mean their form and
position with respect to each other. Figs. 8a, g, illustrate the
following description of the neuromeres in the primitive fore-
brain of the Lizard and Chick.

44 MCCLURE. (VoL. IV.

1. One lateral half of each neuromere is an arc of a circle.

2. The elongated cells are placed radially to the inner curved
surface of the neuromeres (77).

3. The nuclei are generally nearer the outer surface (ow/), and
approach the inner surface (zz) only towards the apex of the
ridge (a).

The arrangement of nuclei in the neuromeres of the primary
fore-brain does not always conform to the typical structure.

4. On the line between the apex of the internal ridge (7)
and the pit of the external depression (ex) the cells of the
adjoining neuromeres are crowded together, though the cells of
one neuromere do not extend into another.

The fore-brain neuromeres of the Lizard and Chick persist
up to a certain stage in the embryo and finally disappear.

The stage in which the fore-brain neuromeres of the Lizard
are fully developed is represented by Fig. 8a. In the Chick
these neuromeres are prominent in embryos from 36 to 96
hours old. The external character of the neuromeres in the
primitive fore-brain of the Newt is not found to be as perfectly
developed as those in the Lizard and Chick ; that is, each lateral
half of a neuromere does not form as perfect an arc of a circle
as in the latter, (Fig. 7). I am, however, in doubt whether this
variation from the general form is due to the fact that I did
not study the stages in which the neuromeres were most fully
developed, but rather those in which degeneration had already
begun but not been completed. In any case this was unavoid-
able, as the stages of this species which I possessed were limited
to a few. Possibly their development may have been arrested by
external means, due to the presence of yolk spherules, which
were found present in such great quantities, mixed in among
the cells, that it was a difficult task to make out the structure
of the neuromeres. It seems probable that one of the above-
mentioned reasons may explain this variation of form in the
fore-brain neuromeres of the Newt. But that these structures
are neuromeres Or remains of neuromeres I think there can be
no doubt whatever, since their structure in most respects con-
forms to the typical structure. The cells have a radial arrange-
ment (Fig. 7), and between the neuromeres they are crowded
together, but the cells of one neuromere do not enter into
another neuromere. The arrangement of the nuclei is vari-
able and does not always conform to the typical one.

No. I.] THE PRIMITIVE VERTEBRATE BRAIN. 45

The fore-brain neuromeres of the Newt persist up to a
certain period and finally disappear, leaving no trace whatever.
This we have already found to be the case in the Lizard and
Chick.

Up to this point we have seen that the structure of the
folds in the lateral walls of the myelon (myelomeres)
conforms in every respect to the four characteris-
tics which are found in the hind-brain and primi-
tive fore-brain folds of all three forms studied (with
one exception in the Newt), which goes to prove that the
encephalomeres are not only remnants of neural
segments similar to the myelomeres, but that they
were originally continuous.

The mid-brain has been purposely omitted up to this point,
but will be considered further on.

Relation of the Auditory Vesicles to the Neuromeres of the
Hina-brain.

The importance of this relationship will be seen further on
in connection with the nerves of the hind-brain. The auditory
vesicle (awd ves) in the Newt and Lizard is opposite, in a
transverse line, to the auditory neuromere (Vm V///.,; Figs. 4,
4a, 5, and 5a). In the embryo Chick it holds the same position
as in the Newt and Lizard up to the 96th hour, or slightly later;
after this its position is shifted backwards to a point between
the auditory and glossopharyngeal neuromeres (Figs. 6, 62,
Nm VIIT. and Nu IX).

Relation of the Myelomeres and Encephalomeres to their
Respective Nerves.

All the neuromeres of the spinal cord give off (on each side)
from their dorsal half a mass of ganglion cells, which constitute
the dorsal or sensory roots of the spinal nerve (SfV), Figs. 1, 2,
and 3. In a like manner I find that four neuromeres in the
hind-brain and one in the primary fore-brain give rise to dorsal
or sensory roots of cranial nerves.

The myelomeres on giving rise to the spinal nerves, in the
manner stated above, degenerate soon after the nerves are

46 McCLURE. [VoL. IV.

formed. The encephalomeres, after giving rise to their respec-
tive nerves, likewise degenerate.!

All nerves mentioned as originating from the centre of a
neuromere have reference to the dorsal or sensory root, unless
otherwise specified. Orr states that the Ist, 3d, 5th and 6th
neuromeres in the hind-brain of the Lizard (neuromeres, 7¢7-
geminal, facial, glossopharyngeal and vagus) give off (on each
side) from their dorsal half a mass of ganglion cells which con-
stitute the roots of the V. (VII., VIII.), 1X. and X. nerves respec-
tively, and that the 4th neuromere (auditory neuromere) gives
off no nerve, but the space opposite to it is occupied by the
auditory vesicle. He also states that the VI. nerve arises,
though at a much later period than the others, from the ventral
portion of the 2nd neuromere (abducens neuromere). Béraneck
previously to Orr mentioned the fact that certain of the hind-
brain neuromeres of the Lizard held a definite relation to the
origin of the V., VII., VIII., and [Xth nerves.

My own observations upon the Lizard confirm the above
statements of Béraneck and those of Orr in every instance but
one; that is, in regard to the origin of the VI. nerve? from the
ventral portion of the 2nd neuromere of the hind-brain (abdu-
cens neuromere). That it originates ventral to the origin of
the other nerves, somewhere defween the origin of the V. and
VII. and VIII. nerves, there is no doubt, but I cannot defi-
nitely confirm its point of origin as stated by Orr from the
ventral portion of the 2nd neuromere. (See Fig. 5a, which is
a camera drawing of the hind-brain neuromeres of the Lizard.)
From an examination of this figure it will be seen that the V.
nerve arises from the trigeminal neuromere (Vm V.); that the
abducens neuromere (Vm V7.) gives rise to no dorsal root ; that
the facial neuromere (Vm V//.) is connected with the origin
of the VII. and VIII. nerves; that the auditory neuromere
(Nm VIII.) gives off no nerve, but the space lateral to it is

1 Orr states that the degeneration of the encephalomeres takes place in the hind-
brain of the Lizard as soon as the nerve fibres begin to develop. This point I did
not satisfactorily make out, either for the neuromeres of the spinal cord or those of
the medulla, but I am inclined to think that Orr’s statement is correct.

2 The preliminary announcement of this paper, which appeared in the Zot/og-
ischer Anzeiger, No. 314, 1889, states incorrectly on Orr’s authority, that the VI.
nerve originates in connection with the most anterior neuromere of the hind-brain.
It should read that it arises from the ventral portion of the 2nd neuromere.

No. 1.] THE PRIMITIVE VERTEBRATE BRAIN. 47

occupied by the auditory vesicle; that the glossopharyngeal
neuromere (Viz LX.) gives rise to the IX. nerve; and that the
vagus neuromere (Vm .X.) gives rise to the X. nerve. I find
in the hind-brain of the Chick an exact correspondence in struc-
ture to that of the Lizard; that is, in the hind-brain of the
Chick the auditory vesicles and nerves hold exactly the same
relation to their respective neuromeres as the corresponding
auditory vesicles and nerves in the hind-brain of the Lizard do
to their respective neuromeres, I think this relationship of
neuromeres to nerves has been fully described for the Lizard,
and I refer the reader to Fig. 6a, which is a camera drawing of
the hind-brain of the Chick, that comparisons may be made.
We have already seen that the abducens neuromere (Mm VI.)
is absent in the Newt. I find also, with this one exception,
that the remaining neuromeres in the hind-brain of the Newt
hold exactly the same relation to the auditory vesicles and
nerves as do their corresponding neuromeres in the hind-brains
of the Lizard and Chick.

(See Fig. 4, which is a camera drawing of the hind-brain of
the Newt, for comparison with the above-mentioned figures of
the Lizard and Chick.)

It has been shown in the preceding pages that there are two
neuromeres in the hind-brain of the Lizard and Chick, which do
not give rise to dorsal or sensory roots (abducens neuromere)
(Vm VJ.) and auditory neuromere (Vm V//I.; Figs. 5a, 6a).
It seems probable that these two neuromeres must have once
been connected with sensory roots when we consider similar
structures in the spinal cord and hind-brain and their systematic
connection with dorsal roots. The fact that the abducens neu-
romere is absent in the Newt may be accounted for in the
following manner: namely, that the degeneration of the sensory
nerve of this neuromere has resulted in the consequent degener-
ation of the neuromere itself. But this is pure conjecture,
and then the fact still remains, that these two neuromeres have
not degenerated in the Lizard and Chick, both of which are
representatives of much higher forms than the Newt. Again,
the VI. nerve may be the motor element of the primitive seg-
mental nerve of this neuromere (abducens neuromere), its
sensory branch having become degenerate. The position of its
origin, somewhere between the neuromeres, trigeminal and

48 MCCLURE. [VoL. IV.

facial, may give credence to this view. It is also possible that
the VI. nerve is a motor branch of the V. or VII. nerves: the
persistence of the VI. nerve and the absence of the abducens
neuromere in the Newt certainly imply as much.

Most of the early investigators are agreed concerning the
origin of the VII. and VIII. nerves from a primitively single
trunk, based on the relations of the VII. and VIII. in Mammals.
The opposed view of their separate nature has been steadily
gaining ground, and IJ think at present the latter theory has the
greater number of supporters. The double nature of these
nerves certainly suggests the probability that they were prim-
itively of separate origin, and the following theoretical evidence
may throw some light on this theory. The auditory neuromere
(Nm VIII.) has no nerve connected with it, and it is situated
posterior and adjacent to the facial neuromere (Vm VI7.), which
gives rise to the VII. and VIII. nerves. (Vm VIL, Nm VIIL;
Figs. 4, 5a, 62a.)

The auditory vesicle (on each side of the brain) is situated in
the space lateral to the auditory neuromere, (awd; Figs. 4, 5a,
6a), but the dimensions of the vesicle occupy so much of this
lateral space, that the space left between the neuromere and
the vesicle is very narrow; so narrow, in fact, that a nerve aris-
ing from the neuromere could not possibly obtain a growth in it
sufficient to perform the functions required of the auditory
nerve. Thus it is possible that the VIII. nerve may have been
primitively connected with the auditory neuromere before the
auditory vesicle became so prominent, and that the gradual
growth of the vesicle has pushed it from its original position
anteriorly into the facial neuromere, where the fusion of its
root with that of the VII. nerve has taken place.

Mid-brain Neuromeres (Nm III. and Nm IV.).

In the WVew?, Lizard, and Chick the mid-brain has the appear-
ance of being an enlarged neuromere, larger than any one of the
remaining neuromeres of the brain, but equal in size to about
three or four of the first five neuromeres in the hind-brain, and
not quite as large as the three neuromeres in the primitive fore-
brain. Its cell structure is radial, but its nuclear arrangement
does not conform to that of a typical neuromere, except that at

NOS I.) THE PRIMITIVE VERTEBRATE BRAIN. 49

its anterior and posterior limits the cells are crowded together
and do not enter the adjoining structures. Two nerves are
connected with this neuromere of the mid-brain, —the Ocz/o-
motor, and Trochlear,—each of which, according to the recent
investigations of Gaskell, conforms to the type of a complete
segmental nerve, in that each contains remnants of the primitive
sensory elements ; that is, they possess “nerve fibres and groups
of ganglion cells corresponding in position, and doubtless also
in function, with the nerve fibres and nerve cells of the station-
ary ganglia on the afferent root of a spinal nerve.” Gaskell
suggests that both of these nerves (III. and IV.) are probably
complete segmental nerves of the type which Balfour supposes
to have been the original type, when mixed motor and sensory
roots were the only roots present. I do not consider Gaskell’s
investigations with respect to the III. and IV. nerves as conclu-
sive without further evidence on the subject, but I agree with
him, and on entirely different grounds, that the III. and IV.
nerves represent two separate segmental nerves. Taking into
consideration the size of the mid-brain neuromere in compari-
son with the remaining neuromeres of the brain as well as its
“neuromeric”’ characteristics, also the fact that two nerves arise
from it, which are probably either two segmental nerves or parts
of the same, also the investigations of Kupffer, previously men-
tioned, in which he states that he found at least eight segments
in the hind and mid-brains of the Trout and Salamander, there
can be little doubt left but that the mid-brain originally consisted
of at least two neuromeres, and that in all probability the III.
and IV. nerves were the segmental nerves of these neuromeres
respectively. (See Appendix.)

The Primitive Fore-brain Neuromeres and thetr Nerves.

The optic neuromere (Figs. 7, 8a, 9; Vm JI.) has no connec-
tion whatever with any segmental nerve. The optic nerve is
undoubtedly secondary in its nature, and is, I believe, consid-
ered by all as outside the series of segmental nerves. It seems
probable that the primitive segmental nerve of this neuromere
degenerated as soon as the vertebrate eye came into existence,
the latter requiring a nerve better suited to perform its functions
than the nerve which primitively belonged to the neuromere.

50 MCCLURE. [Von. IV.

The olfactory neuromere (Figs. 7, 8a, 9; Vw J.) is connected
with the olfactory nerves, which arise from the neural crest,
according to Marshall, in exactly the same manner as the sen-
sory roots of segmental nerves. He also states that the olfac-
tory nerves arise before the cerebral hemispheres, and in the
Dog-fish, Trout, Salmon, Axolotl, Frog, Lizard, Turtle, and
Chick their development is fundamentally the same.

Orr states that in the Lizard the olfactory nerves spring
laterally from the anterior dorsal (nasal) tip of the primary fore-
brain, and run a very short distance direct to the nasal thicken-
ings of the epiblast, in which they end. In the Chick it is
fundamentally the same. In addition to confirming Orr’s state-
ment in regard to the origin and course of the olfactory nerve
in the Lizard, I find an exact correspondence in the Newt
(Figs. 7a, 76, 7c). Thus it is seen that in the Lizard, Newt, and
Chick the olfactory neuromere (anterior dorsal tip of primary
fore-brain) gives off (on each side) a mass of ganglion cells
which constitute the roots of the olfactory nerves. This mode
of origin, as we have already seen, is exactly the same as that
described for the sensory roots in the segmental nerves of the
spinal cord and hind-brain. Therefore I think it is safe to say
that the olfactory nerve is the sensory division of the segmental
nerve which belonged to the olfactory neuromere, which accords
with Marshall and Beard, who upon entirely different grounds
consider this a true segmental nerve.

General Summary.

It has been my endeavor in the preceding pages to show that
a continuous and symmetrical series of folds (neuromeres), in-
creasing in size anteriorly, extend from the lateral walls of the
embryonic brain, throughout the entire length of the neuron,
and that these neuromeres are the remains of the primitive
segmentation of the neural tube.

1. By proving that a conformity exists in the structure of
these neuromeres throughout the entire length of the neuron.
(See typical structure of neuromeres.)

2. That all of the neuromeres in the spinal cord, four in the
hind-brain, and one in the primitive fore-brain, give rise to
dorsal or sensory roots.

No. 1.] THE PRIMITIVE VERTEBRATE BRAIN. 51

(a) That the relation of the neuromeres to the origin of their
respective dorsal or sensory roots is fundamentally the same in
all three regions of the brain in which neuromeres give rise to
sensory roots.

(0) That all the neuromeres of the brain, whether giving rise
to sensory roots or not, degenerate before the adult stage of the
animal is reached.

It has also been shown (see Appendix).

3. That in all probability the mid-brain originally consisted
of two neuromeres, and that the III. and IV. nerves were the
segmental nerves of these segments.

4. That the number of primitive Encephalic segments was
probably ten (six in the hind-brain, two in the mid-brain, and
two in the primary fore-brain).

5. That the neuromeres of the spinal cord, opposite the meso-
blastic somites, are “‘intersomitic”’; that is, the centre of each
neuromere is opposite the space between two somites, or vice
versa; hence it is seen that nine mesoblastic somites exactly
correspond to the nine spaces between ten neuromeres.

It is now a well-known fact that the segmented mesoblast of
the trunk extends into the head region, and according to the
investigations of Van Wijhe it is there divided into nine meso-
blastic head segments, or ‘‘ Myotomes,” as he calls them, which
theoretically correspond to the nine spaces between the ten
Encephalomeres.

Conclusions.

I consider that the primitive vertebrate brain consisted of a
series of segments similiar to those found in the embryonic
spinal cord, and that the encephalomeres probably held the
same relation to the mesoblastic head segments as the myelo-
meres do to their respective mesomeres; that is, they were inter-
somitic, the centre of each neuromere being opposite the space
between two somites and giving off a mixed nerve from the
apex.

The region known as the Encephalon is the result of a great
differentiation and specialization of the anterior segments of this
primitive structure. That differentiation first began and has
been the greatest in the most anterior segments, which may
account for the greater size of the folds in this region than in

52 MCCLURE. [VoL. IV.

the hind-brain, which, less differentiation and specialization
having taken place, naturally conforms more to the primitive
vertebrate type. I am aware that the forms examined are in-
sufficient to enable us to reach any positive conclusion in regard
to the exact number of segments, but I feel confident that the
method which I have adopted is the one by which this vexed
question of the primitive segmentation of the head region, both
of the neural tube and indirectly of the surrounding mesoblast,
will eventually be decided.

In conclusion, I may say that I feel confident that the full
number of primitive encephalomeres will be found in Elasmo-
branch, Ganoid, or Teleost embryos, the investigation of which
will form the second part of this paper.

APPENDIX.
The Mid-brain Neuromeres.

Just before sending this paper to the press my attention was
called by Dr. Osborn to an article published in the Journal
of Morphology by Dr. W. B. Scott, on the Embryology of
Petromyzon, in which two distinct neuromeres are figured in
the mid-brain (Figs. 10, 11). Dr. Scott makes no mention of
these structures as having any segmental value, and in Fig. 11
the cell structure shown is evidently purely schematic. My
Figs. 10 and 11 are taken from Dr. Scott’s plates. The gradual
transition of the hind-brain neuromeres into those of the mid-
brain is clearly shown in Fig. 10. The gradual transition of
the myelomere into the neuromere of the hind-brain in the
Newt, Lizard, and Chick has already been mentioned. Thus I
think that an examination of various stages of Petromyzon
embryos will show a continuous series of neuromeres through-
out the entire length of the neuron.

BIBLIOGRAPHY.

1. Ahlborn. Ueber die Segmentation des Wirbelthier-Korpers. Gottingen,
4 Januar, 1884.

2. Beard. The System of Branchial Sense-Organs and their Associated
Ganglia in Ichthyopsida. Studies from the Biological Laboratories of
the Owens College. Vol. 1. 1886.

No. 1.] THE PRIMITIVE VERTEBRATE BRAIN. 53.

3. Béraneck. Recherches sur le developpement des nerfs craniaux chez les
Lézards. Recueil Zodloique suisse. T.1. p. 557.

4. Dursy. Entwicklungsgeschichte des Kopfes. Tiibingen, 1869. Atlas.
Taf. IIl., Fig. 15.

5. Dohrn. Der Ursprung der Wirbelthiere und das Princip des Functions-
wechsels. Leipzig, 1875. p. I.

6. Foster and Balfour. Grundziige der Entwicklungsgeschichte der Thiere.
Aus dem Englischen iibersetzt von N. Kleinenberg. Leipzig, 1876.

7. Gaskell. Relation between the Structure, Function, Distribution, and
Origin of the Cranial Nerves. our. of Phys. Vol. X. No. 3.

8. Gegenbaur. Die Metamerie des Kopfes und die Wirbeltheorie des Kopfs-
kelettes. Morph. Fahrb. 13 Bd. p. 37.

g- Hill. The Grouping of the Cranial Nerves. Reprinted from “The
Brain.” XXXIX. and XL.

Io. Hoffmann. Ueber die Metamerie des Nachirns and Hinterhirns, und
ihre Beziehung zu den Segmentalen Kopfnerven bei Reptilien Embry-
onen. Zodlogischer Anzeiger. No. 310. 1880.

11. Kupffer. Primare Metamerie des Neuralrohrs der Vertebraten. Sitzung
der Mathphys. Classe (Akad. Miinchen) vom 5 December, 1885.

12. Marshall. Cranial Nerves of Birds. Your. of Phys. and Anat. Vol. XI.

13. Idem. The Segmental Value of the Cranial Nerves. Your. of Anat.
and Phys. Vol. XVI.

14. Idem. The Morphology of the Vertebrate Olfactory Organ. Quart.
Four. Micros. Sct. Vol. XIX.

15. Marshall and Spenser. Cranial Nerves of Scyllium. Studies Jrom the
Biological Laboratories of the Owens College. Vol.1. 1886.

16. McClure. The Primitive Segmentation of the Vertebrate Brain. Zod/-
ogiscther Anzeiger. No. 314. 18809.

17. Mihalkovics. Entwicklungsgeschichte des Gehirns. Nach Untersuchun-
gen an hoheren Wirbelthieren und dem Menschen. Leipzig, 1877.

18. Orr. A Contribution to the Embryology of the Lizard. our. of
Morphol. Vol. 1. No. 2. 1887.

Ig. Remak. Untersuchungen ueber die Entwicklung der Wirbelthiere.
Berlin, 1850-1855. § 28.

20. Scott. The Embryology of Petromyzon. Your. of Morphol. Vol. 1.
Noe 2h) )1S07-

21. Van Wijhe. Ueber die Mesodermsegmente und die Entwicklung der
Nerven des Selachier Kopfes. Matuurk Verhandelingen Koninkl.
Akademie. Amsterdam. Deel XXII. 1882 (Sept.).

22. Von Baer. Entwicklungsgeschichte der Thiere. I Th. p. 64.

23. Wilder. ‘‘ Brain.” Reference Handbook of the Medical Sciences. Vol.
VIII. 8.

34

McCLURE. [VoL. IV.

EXPLANATION OF PLATES.

INDEX LETTERS.

in, IIn, IlIn, IVn. First, second, third, fourth, etc., cranial nerves.

Ant.

Ap.
Aud. Ves.

MB.
Mes. Som.
Myl.

Na.

Nm I.
Nm LT.
Nm IT!
Nm V.

Nm VI.
Nm VII,
Am VIII.
Nm IX.
Nm X.
Nu.

Out.

Op. Ves.
Spn.

Spt.
Thal.

Anterior.
Apex of internal ridge of neuromere.
Auditory vesicle.

. Epiblast.

. Pit of external depression of neuromere.

. Rudiment of secondary fore-brain.

. Hind-brain.

. Inner surface of neuromere, or that surface of the neuromere which

lines the neural canal.
Mid-brain.
Mesoblastic somite.
Myelomere.
Nasal thickening of epiblast.
Olfactory neuromere. First neuromere of the primitive fore-brain.
Optic neuromere. Second neuromere of the primitive fore-brain.
Possibly a portion of a third neuromere of the primitive fore-brain.
Trigeminal neuromere. First and most anterior neuromere of the hind-
brain.
Abducens neuromere. Second neuromere of the hind-brain.
Facial neuromere. The third neuromere of the hind-brain.
Auditory neuromere. Fourth neuromere of the hind-brain.
Glossopharyngeal neuromere. Fifth neuromere of the hind-brain.
Vagus neuromere. Sixth neuromere of the hind-brain,
Nuclei.
Outer surface of neuromere.
Optic vesicles.
Spinal nerve.
Neural septa.
Thalamencephalon.

All figures of sections have been drawn with the Abbey camera lucida and a Zeiss
microscope. Z. 2 A means Zeiss ocular 2, and objective A, etc.

No. 1.] THE PRIMITIVE VERTEBRATE BRAIN. 55

Fic. 1. Longitudinal horizontal section of spinal cord of Amélystoma, showing
the neuromeres of the spinal cord and their relation to the mesoblastic somites. Also
the typical nuclear and cell arrangement. Z. 4 D.

Fic. 1a. Longitudinal horizontal section of spinal cord of 77zton, showing the
same features as in Fig. 1. Z. 4 D.

Fic. 2, Longitudinal horizontal section of spinal cord of Axolis sagrai, showing
same features as in Fig. 1. Z. 4 D.

Fic. 3. Spinal cord of Chick. Longitudinal horizontal section, showing same
features asin Fig. 1. Z.2D.

Fic. 4. Longitudinal horizontal section of hind-brain of Amdélystoma puncta-
tum, showing the five neuromeres in the hind-brain. Also the gradual transition
of the neuromeres of the spinal cord into those of the hind-brain; the relation
of the auditory neuromere (Vm V///.) to the auditory vesicle (aud ves), and the
intersomitic relation of the myelomeres to the mesoblastic somites (#es som). Also
the relation of the neuromeres to the origin of the V., VII., VIII., IX., and Xth
nerves. Z. 2 A.

Fic. 4a. Longitudinal horizontal section of a slightly later stage than Fig. 4, in
which the neuromeres have begun to degenerate. Z. 2 A.

Fic. 44, Longitudinal horizontal section of the hind-brain of Amélystoma puncta-
tum, in the region of the glossopharyngeal neuromere (Vm /X.), showing the typical
cell and nuclear arrangement. Z. 2 E.

Fic. 4c. Longitudinal horizontal section of the hind-brain of Amélystoma puncta-
tum, between the glossopharyngeal (Vm /X.) and vagus (Vm X.) neuromeres,
showing the cell and nuclear arrangement between two neuromeres (.Sf¢). Z. 2 E.

Fic. 5. Horizontal longitudinal section of the hind- and mid-brain of Axolis
sagrat, showing the six neuromeres in the hind-brain and the relation of the audi-
tory neuromere (Vm V///.) to the auditory vesicle (aud ves). Z.2 A.

Fic. 52. Longitudinal horizontal section of Azolis sagrei, showing the six neuro-
meres in the hind-brain and their relation to the origin of the V., VII., VIII., IX.,
and Xth nerves.

Fic. 54. Longitudinal horizontal section of the hind-brain of Anolis sagrei, in
the region of trigeminus (Vm V.), abducens (Vm VZ.), and facial (Vm VJ.) neuro-
meres, showing the typical nuclear and cell arrangement. Z. 2 D.

Fic. 6. Longitudinal horizontal section of the hind-brain of a five-day Chick
embryo, showing the six neuromeres in the hind-brain and the position of the audi-
tory neuromere with respect to the auditory vesicle (aud ves). Z.2 A.

Fic. 6a. Longitudinal horizontal section of a four-day Chick embryo, showing
the relation of the six neuromeres in the hind-brain to the V., VII., VIII., IX., and
Xth nerves. This section is not cut exactly in a longitudinal horizontal plane.
Ji MIN

Fic. 64. Longitudinal horizontal section of the hind-brain of a four-day Chick
embryo in the region of the neuromeres, abducens (Vm V/.), facial (Vm V/Z.),
and auditory (Vm V///.), showing the typical cell and nuclear arrangement of the
neuromere facial and the septum (.Sf7) between the adjoining neuromeres. Z.2 D.

Fic. 7. Longitudinal horizontal section of the primitive fore-brain of Amdlys-
toma punctatum, showing the two neuromeres, — olfactory (Vw J.) and optic
(Nm /f.). Also the typical cell and nuclear arrangement of the neuromeres.
Z.4 A.

56 MCCLURE.

Fics. 7a, 72, 7¢. Longitudinal horizontal sections through the primitive fore-brain
of Amblystoma punctatum, showing the origin of the olfactory nerve from the olfac-
tory neuromere (Vm /.) and its final fusion with the nasal thickening of the epiblast.
These sections are two or three apart. Z. 4 A.

Fic. 8a. Horizontal section of Anolis sagrei, dorsal, and parallel to the axis of
the primary fore-brain, showing the neuromeres of the thalamencephalon (Vm J.
and Wm J/.). Also Vm I/.’, which has the appearance of a portion of a neuromere.

Fic. 9. Horizontal section of a four-day Chick, dorsal, and parallel to the axis of
the primary fore-brain, showing the neuromeres of the thalamencephalon. Z. 2 A.

Fic. 10. Horizontal section through the superior portion of the brain of Petro-
myzon. 22 mm. larva. Taken from Dr. Scott’s plates on “The Embryology of
Petromyzon.”

Fic. 11. Horizontal section through the anterior portion of the head of a
Petromyzon embryo just before hatching. After Scott. This section does not show
the primitive fore-brain, as it lies at an angle to the mid-brain, due to cranial flexure.
The oculomotor and trochlear neuromeres (Vm ///. and Nm JV.) lie anterior to
the trigeminal neuromere (Vm V.), between it and the primitive fore-brain.

is Mes Som...

MesSom.|}.-

psi

+ Nill

rah

y

yi
Bay
(ey

ke vie
aN

Tih Anstv Werner & Winter, Fraxkfart.

Me. Clare delin. ad nat.

THE LIFE-HISTORY OF THE FORMED’ ELE-
MENTS OR THE, BEOOD,+ ESPECIALLY
THEOREDY BLOOD: CORPUSCEES.

W. He HOWELL, PHD MD;

PROFESSOR OF PHysIoOLoGY AND HistoLoGy, UNIVERSITY OF MICHIGAN.

I. Tue Rep BLoop CorRPUSCLES.

THE origin and the fate of the mammalian red corpuscles
have been the subjects of an extraordinary number of scientific
papers from workers in various fields of biological research.
Contributions have been made from the side of pathology, of
normal histology, and of embryology, so that to discuss the
subject in all its aspects becomes a difficult undertaking. The
results of investigation along these different lines are not at all
in agreement, so that many theories radically different from one
another have been proposed. Indeed, the embryologist, the
pathologist, or the histologist often works at the subject without
any reference to the results made known by the investigations
of the other, inasmuch as the journals in which these results
appear are likely to be read only by the specialists in whose
interest they are published. To one who reads over the liter-
ature even incompletely, the conviction comes, I think, with a
good deal of force, that the various phenomena which have
been observed and described, and which have served as a basis
for the divergent theories, might all find a simpler and better
explanation under some one theory. One cannot help believ-
ing, in other words, that in the mammalia the method of pro-
duction of the red corpuscles is essentially the same in disease
as in health and in the adult as in the foetus; and furthermore
that the formation of these elements takes place not in a num-
ber of different ways, but according to some one scheme of
reproduction, as in the development of tissue elements in gen-
eral. Many authors, on the contrary, have advanced one theory
of the formation of the red corpuscles as the result of their
own work, but have admitted at the same time that the differ-
ent veiws advocated by others might hold good under certain

58 HOWELL. [Vou. IV.

conditions of health or age. Onda fpriori grounds it seems to
me that most persons will incline rather to the other view, that
the production of the red corpuscles takes place in one way
under all conditions of life, though in some cases the process
may be accelerated or abbreviated, and in others retarded. In-
asmuch, then, as decisive proof that ‘the red corpuscles are
formed by two or more different methods is wanting, it is
allowable to examine critically the different theories which
have been proposed, and to endeavor to discover whether the
phenomena they are intended to explain cannot be grouped
under a common theory. My own investigations have ex-
tended over a period of two years; and though they are in
many respects incomplete, yet in a number of points satisfac-
tory conclusions have been reached, and it seems to me, as I
shall endeavor to show, that the phenomena which I have ob-
served help to some degree at least in reconciling different
theories, and indicate that one general plan of formation holds
good in all cases.

Since 1838 it has been known that the red corpuscles of the
foetal animal are at one period nucleated cells. This was first
shown by Rudolf Wagner (1) for the embryo bat and by E. H.
Weber (2) in a human foetus of twelve weeks. Most of the
observations made upon these nucleated red corpuscles of foetal
blood, their occurrence and their relative numbers at different
periods of foetal life, we owe to Kolliker (3) and to Paget.
Kolliker found that in a sheep’s embryo of three and one-half
lines the red corpuscles are all nucleated, and Paget makes the
same statement from the human embryo of four lines (fourth
week). In a human embryo of three months the nucleated
corpuscles in the circulating blood make up from one-sixth
to one-eighth of the total number of corpuscles, while at five
months they are still quite numerous. In a human foetus of
this age (five months) which came under my own observation,
and which had been born about five or six hours, and was
brought to me still enclosed in the amniotic sac, I found that
the majority of the red corpuscles were not nucleated, though
nucleated forms were still very abundant. Among the nucle-
ated corpuscles some were found with the nucleus fragmented
incompletely into a number of pieces, forming what has been

INOW Ic] BLOOD CORPUSCLES. 59

described by Kélliker and by Neumann as the first stage in
the disappearance of the nucleus. In foetal cats I have found
nucleated red corpuscles in the blood even at birth, though they
were few in number. In cat foetuses of an earlier age they
are proportionally more numerous. The youngest cat embryo
which I examined was one measuring 2.5 cms. from the crown
of the head to the root of the tail. A drop of its blood taken
from the heart and stained in methyl green (a r per cent solu-
tion in 0.6 per cent NaCl) showed a number of interesting
things which, so far as I know, have not been noticed, or at
least not dwelt upon before. In the first place, there were two
distinct forms of red corpuscles present in the blood: one,
large, oval, and nucleated, resembling somewhat the corpuscles
of the reptiles or amphibia. In shape, they were biconcave,
irregular, or apparently, in some cases, biconvex, and were so
extremely plastic as to appear semi-liquid. When treated with
staining reagents, they took on an oval biconvex form. These
corpuscles were distinguished, moreover, by the deeper tint
of the hemoglobin which they contained. Their nuclei were
variable in size, were usually placed eccentrically, and were
characterized by the fact that without exception in the em-
bryo of this age they stained a homogeneous bluish color
with the methyl green, showing no trace of nucleoli or intra-
nuclear network, The size of these corpuscles in their long
diameter varied from about two to four times the diameter
of the red corpuscle of the adult mammal. The second form
was circular in outline and of the usual size of the cat corpus-
cles —some of them were nucleated, and some had lost their
nuclei (see Fig. 1). The nuclei of the nucleated forms were
in some cases stained a uniform green blue color from the
methyl green, like the oval corpuscles just described; but in
other cases the nuclei showed an intra-nuclear network or
granulation. It is worthy of special emphasis that all the red
corpuscles which were non-nucleated belonged to this class.
Diligent search through a number of preparations failed to
reveal a single large oval red corpuscle which did not have a
nucleus,!

1 After this article was in the hands of the printer, Hayem’s extensive work on
the blood, “Du Sang et de ses Altérations Anatomiques, 1889,” came into the
author’s possession. In it is found a reference to these large corpuscles. He
speaks of them as giant nucleated corpuscles, discoid and concave in shape, though
occasionally irregular and flat or subglobular.

60 HOWELL. [Vou. IV.

It is permissible, perhaps, to suppose that the large oval
corpuscles represent the form of the red corpuscle character-
istic of the ancestors of the mammalia, and to speak of them
therefore as ancestral corpuscles, while the smaller circular
corpuscles of the usual size of the nucleated red corpuscles of
the mammalia exhibit a modification of this ancestral form
which has become characteristic of the blood of most of the
mammalia. These latter corpuscles under normal conditions
lose their nuclei and become changed to the biconcave red
corpuscles of the circulating blood, the transition, in the young
embryo, taking place in the blood itself. It is not probable
that there is any essential difference in the way in which the
two forms of red corpuscles are produced; but it is possible
that the large (ancestral) form represents an entire embryonic
cell, in which hemoglobin has become developed, while the
true mammalian form arises from similar cells after they have
broken up by karyokinesis into smaller daughter-cells, in each
of which the nucleus is larger relatively to the size of the whole
cell. A similar difference in the red corpuscles of the very
young embryo has been recorded by Erb (4). He states that,
in the blood of two young pig embryos (1 in. long) he found
some nucleated red corpuscles of great size and elliptical form,
having a superficial resemblance to the red corpuscles of the
frog. Most of the red corpuscles had nuclei, and those that
had not were always the smaller variety, similar in outline to
the corpuscles of the adult animal.

The nuclei of the nucleated red corpuscles of the young
embryo (except the larger variety) are lost while in the circula-
tion, and the presumption is that this fate is met by all the
truly mammalian red corpuscles. As the embryo grows older
and the production of new corpuscles becomes localized in
different organs, — liver, spleen, and marrow, — more and more
of the early history of the corpuscle is passed over while still
in the blood-forming organ, and more and more of the red
corpuscles are sent into the blood stream in the non-nucleated
stage. After birth, and throughout adult life under normal
conditions, we find only non-nucleated red corpuscles in the
circulating blood. The nucleated period in their life-history
has passed while they were in the blood-forming organ (the
red marrow).

INO} 2.) ILOOD! CORPOSCLES. 61

Under certain conditions, however, as I shall endeavor to
show later, such as severe hemorrhage or anemia from patho-
logical causes, some of the nucleated red corpuscles escape
from the blood-forming organ before the loss of the nucleus
has taken place. Neumann (5%) states that, in the pig, —un-
like other mammals, —one can always find in the normal adult
animal some few nucleated red corpuscles in the blood. I
have met with a similar exception in the opossum. In several
animals which I have examined I was able in each case to find
a few nucleated red corpuscles in the blood.

The loss of the nucleus is one of the most important events
in the life-history of the red corpuscle, and has naturally been
the subject of much discussion. It is usually believed, as
taught by Kolliker (3), for the embryo, and by Neumann (sf),
for extra-uterine life, that the nucleus disappears within the
corpuscle by absorption, which may be preceded by fragmenta-
tion. I desire to come back to this subject under another
heading, and will not discuss it fully now. It is my belief that
the nucleus is lost not by absorption within the corpuscle, but
by migration or extrusion from the corpuscle, as shown in
Fig. 2. I mention this view at this time to call attention to
some evidence in favor of it found in the examination of the
blood of the young cat embryos of 2.5 cms. In the blood of
this embryo I obtained a number of specimens, such as are
shown in Fig. 2, in which the nucleus was seen in the act of
passing out of the corpuscle. This appearance has been seen
under different conditions by a number of observers, but has
usually been explained as a post-mortem change or as the effect
of mechanical pressure, action of reagents, or some similar
cause. Now, it seems to me that these explanations will not
hold in this case, because none of the nuclei of the large oval
corpuscles were found extruding, though they were submitted
to the same treatment exactly; and, furthermore, amongst the
smaller true mammalian corpuscles, only those were found with
the nucleus extruding in which the nucleus stained a homo-
geneous tint with the methyl green. No nucleus showing an
intra-nuclear network was ever found escaping from the cell,
though there were many such cells in the preparation, and they
had passed through the same treatment as the corpuscles with
the other kind of nucleus. This last fact, as I shall show later,

62 HOWELL. [Vou. IV.

holds good for the nucleated red blood corpuscles in the blood-
forming organs of cats of all ages, and presumably for other
mammals also. In face of these two facts it is hard to believe
that the extrusion of the nucleus is in any sense an accidental
occurrence. On the contrary, it is the normal means by which
the nucleated corpuscle passes into its non-nucleated form.

The place and manner of origin of the first red corpuscles
of the embryo have given rise to a number of different
theories. Reichert (6) taught that the first .corpuscles are
formed from the mass of cells from which the heart is devel-
oped, the central cells of the mass becoming the blood cor-
puscles, while an intra-cellular liquid which forms represents
the blood plasma. Kolliker (3) afterwards extended this theory
so as to take in the great blood-vessels as well as the heart.
He believed that the first blood corpuscles of the embryo are
colorless nucleated cells like the other embryonic cells, and
at first are found in the solid cords or masses from which the
heart and first blood-vessels are developed. The peripheral
cells form the walls of the blood-vascular organs, while the
central cells are floated away in the plasma which forms
between the cells. The red corpuscles are spherical nucleated
cells which multiply by division in the blood stream. Within
recent years a somewhat similar view has been proposed by
Ziegler and by Wenckebach. Wenckebach’s (7) first observa-
tions were made upon embryos of Perca fluviatilis. These
embryos are so transparent that they can be examined entire
under the microscope. Ziegler (8) worked chiefly with the
salmon. Both state that the heart begins to beat and forces
into circulation a colorless plasma before the red corpuscles
appear. These are seen later, and are formed first, according
to Wenckebach, from a mass of cells lying in cross-section
between the aorta and intestine, and outlining the position of
the future posterior vertebral or cardinal vein. The blood
plasma, percolating through this mass of cells, washes off the
central ones, to form the first red corpuscles, while the periph-
eral cells form the walls of the future vein.

Ziegler also dscribes a mass of cells found in cross-section
between the notochord and intestine, which he calls the inter-
mediate cell mass. The central cells of this mass become the
first blood corpuscles in the way described, while the peripheral

No. 1.] BLOOD CORPUSCLES. 63
cells form the walls of the single median cardinal vein, which
anteriorly splits into the two posterior cardinal veins. More-
over, from this central mass of embryonic corpuscles cords of
similar cells branch off toward the yolk on either side, forming
the outlines of new blood-vessels (veins), and serving as centres
of origin for new red corpuscles. Both authors believe that
these embryonic blood corpuscles are capable of multiplication,
since karyokinetic figures are not unfrequently seen. The
first red corpuscles, according to this view, are true mesoblastic
cells, set apart for the formation of certain veins as well as
red corpuscles. Most English embryologists, on the contrary,
adhere to the view proposed by Klein (9), Balfour (10), etc.,
a general account of which is given in Foster and Balfour’s
Embryology of the Chick. According to this theory, the blood-
corpuscles are formed endogenously within large mesoblastic
cells found chiefly in the area vasculosa and area pellucida.
These cells send out processes which unite, forming thus a
protoplasmic network. At the nodal points of this network
the nuclei of the original cells multiply, to form groups. The
protoplasm around each of these assumes a red color from the
development of hemoglobin, and the groups eventually break
up to form nucleated red corpuscles. By a similar method,
blood corpuscles are formed in the connecting processes, while
some of the protoplasm remains granular and uncolored, form-
ing the walls of the future vessels. The nuclei scattered along
the walls are also derived, like the corpuscles, from the nuclei
of the original mesoblastic cells. The plasma in which the
corpuscles float is a secretion from the protoplasmic walls of
the newly formed vessels.

Gensch (11) in a communication giving the results of some
work done under Kupffer upon the teleosts (Esox luctus and
Zoarces viviparous) states that the first blood corpuscles de-
velop out upon the yolk beyond the boundary of the mesoblast.
In this region there is a layer of large plasmodium like cells
lying beneath the epiblast, but not forming a continuous epithe-
lium. The cells become united to one another by processes,
and the blood corpuscles are constricted off from them, forming
the blood islands seen in the blastoderm. This layer of forma-
tive cells is called by Kupffer the “secondary endoderm.” The
theory differs from those previously mentioned not only in the

64 HOWELL. [Vo.t. IV.

way in which the first corpuscles are formed, but also in the
fact that the original cells are not derived from the mesoblast.
In this last respect Kupffer’s theory bears some resemblance to
the well-known parablastic theory of His (12). His believes
that the mesoderm in the sense of the term used by Remak can
be separated into an axial and a peripheral portion. The axial
portion is formed in the higher animals in the neighborhood of
the primitive streak ; in the lower animals, in the groove of the
blastopore. From it are derived the muscles, the chorda, the
generative epithelium, the duct of the pronephros, etc. It falls
into two layers, which taper off laterally, but do not extend
beyond the body proper of the embryo. The peripheral por-
tion of the mesoderm forms what His calls the parablast ; and,
when first formed, it lies outside of the body of the embryo,
arising in fact from the white yolk. The parablast gives rise
to the blood corpuscles and blood-vessels as well as the general
connective tissues. Though arising outside of the body, it
afterwards grows in from the periphery, penetrating the germ
layers, so that the parablastic cells become inextricably mixed
with the cells of the germ layers.

To which of these various theories the balance of evidence
tends it is impossible to say. In my own work I have not
attempted at all to follow the development of the first cor-
puscles in the germ layers of the embryo. If we suppose that
the method of formation of the first red corpuscles in the germ
layers is similar to that which prevails in later embryonic life
and in extra-uterine life, then it seems to me highly proba-
ble, for reasons which I will give presently, that the general
method described by Reichert and Kélliker, and afterwards
extended and modified by Wenckebach and Ziegler, is most
worthy of belief. According to this view, the primitive blood
corpuscles form one variety of mesoblastic cells, which become
arranged in masses or strings that mark the position of future
blood-vessels (veins). The central cells become red blood
corpuscles; the peripheral cells form the walls of the vessels.

Development of the Red Corpuscles in the Later Stages of
Embryonic Life.

Kolliker (3) first proved that the liver, as soon as it is formed,
becomes the seat of production of new red corpuscles. This

No. 1.] BLOOD CORPUSCLES. 65

fact has been abundantly confirmed by all observers since Kol-
liker’s time, and is capable of easy demonstration. The way in
which the red corpuscles develop in the liver has not, as far as
I know, been described in any detail. Kolliker held simply
that the liver contained certain nucleated white corpuscles
which become transformed to red corpuscles by the develop-
ment of hamoglobin, and which subsequently lose their nuclei.
Neumann (5@) states that he finds in the liver nucleated red
corpuscles in greater numbers than can be accounted for by
supposing that they are carried there by the splenic veins and
other vessels opening into the liver. They must be formed in
the liver then de zovo, and he suggests at least two methods by
which they are produced. First by endogenous formation in
certain large cells. A number of nuclei arise in these cells by
a process of endogenous division, and a homogeneous yellow
substance collects round each nucleus. Each nucleus with its
surrounding colored protoplasm constitutes a red corpuscle, and
this is afterwards liberated and undergoes its further develop-
ment. In addition he finds in the embryonic liver a number
of free nuclei which are undoubtedly the same in structure as
the nuclei of the nucleated red corpuscles. How these free
nuclei arise, and what becomes of them, he leaves undescribed,
but supposes that they represent one step in a second method
of production of nucleated red corpuscles. He seems to sug-
gest, indeed, that the free nuclei form round themselves a pro-
toplasmic envelope in which hzmoglobin is afterwards devel-
oped, and in this way they are converted to nucleated red
corpuscles, — a view which, as we will see, has been proposed
by others to account for the formation of new corpuscles in the
marrow in post-natal life. Neumann says, moreover, that a
new development of capillary blood-vessels is taking place in
the liver throughout almost the whole of embryonic life, and
in some way the formation of the new red corpuscles is con-
nected with the existence of these newly forming blood-vessels.
Foa and Salvioli (13) believe that the nucleated red corpuscles
found in the liver are derived from colorless corpuscles, — “ hya-
line cells,” — which in turn arise by constriction or segmentation
from the large giant cells found in the liver. It is undoubtedly
true that in the embryonic liver the nucleated red corpus-
cles are formed from colorless cells. Whether one studies

66 HOWELL. [Vou. IV.

the liver from sections or from teased specimens, he finds
abundant proof for this in the transitional forms, which occur in
considerable numbers. With reference, however, to the origin
of these colorless cells, I cannot agree either with Neumann or
with Foa and Salvioli. In sections of liver in the later periods
of embryonic life one finds the nucleated red corpuscles and
their colorless predecessors lying between the rows of liver
cells, scattered irregularly, and without any very apparent
relationship to the other elements of the liver. But in the
earlier periods of embryonic life, —in the embryo, for instance,
2.5 cms. long, —sections of the liver show the origin of the
blood corpuscles quite distinctly. One sees in such sections
that the blood-forming cells are not scattered without order,
but are grouped into cords or strings lying between the col-
umns of true liver cells which are just beginning to show a
typical structure and arrangement. The cords of blood-forming
cells resemble those described by Wenckebach, Ziegler, etc.,
in the germ layers of the young fish embryo, and here also
undoubtedly mark out future blood-vessels. One often sees the
solid mass of cells stop more or less suddenly while the channel
becomes filled with coagulated plasma containing here and
there fully developed red corpuscles with or without nuclei.
A drawing showing the appearance described is given in Figs.
13 and 14. I have obtained similar cords of blood-forming
cells, evidently developing blood-vessels, in longitudinal sections
through the posterior limb of the same embryo, as shown in
Fig. 15, which indicates that though the production of red
corpuscles at this time is most active in the liver, it is also
going on in other parts of the body, probably wherever new
blood-vessels (veins) are forming. If we accept the theory
proposed by Kolliker, Wenckebach, and others, as to the
method of formation of the first blood corpuscles in the embryo,
then their production in later embryonic life is seen to follow
essentially the same plan. One might suppose, indeed, that
the cords of blood-forming cells in the young liver are directly
or indirectly derived from the original median mass of blood-
forming cells which first appears in the embryo, though I have
no observations at all which can be taken as evidence for such
an hypothesis. A similar method of origin of the red cor-
puscles in the marrow of birds during extra-uterine life has

INGOs a. BLOODY CORPUSCLES. 67

recently been described by Denys (14), as I shall have occasion
to mention later on.

During the second half of intra-uterine life the spleen also
takes part in the formation of red corpuscles. This was first
made known by Kolliker, and has been confirmed by a number
of observers since his time. In the foetal cat the sequence in
which the blood-forming organs enter upon their function is as
follows. First, the liver; then, as the production of new red
corpuscles in the liver becomes diminished, the function is
taken up by the spleen. So that at the time of the maximum
activity of the spleen the liver takes but little part in the proc-
ess. Still later in embryonic life, after the long bones of the
limbs have been formed, one finds that the young marrow has
begun to produce new red corpuscles, while the activity of
the spleen in this respect has suffered a decided diminution.
Shortly before birth it is easy to prove that at least three
organs are taking part in the formation of red corpuscles, —
namely, the liver, the spleen, and the bone marrow, —and even
after birth for a certain short time the same is true; in the cat,
for as long as three or four weeks. Later than this, however,
nucleated red corpuscles showing signs of active multiplication
are found only in the red marrow of the bone. The liver cer-
tainly takes no part at all after this time in the formation of
red corpuscles; and in the spleen one finds under normal condi-
tions no indication of the presence of nucleated red corpuscles.
Whether or not the spleen plays any part in the formation
of the colorless cells from which the nucleated red corpuscles
are afterwards produced will be discussed later.

While it must be accepted that during embryonic life red
corpuscles are made in the three organs mentioned, it seems
to me quite certain, also, that they are formed during this
period in other parts or organs of the body, — wherever, in fact,
developing blood-vessels (veins) are found, —though I have no
evidence for this other than the section already described,
which passed through the long axis of the posterior limb and
showed a developing blood-vessel with its young corpuscles
lying in the muscular tissue. In describing the development
of the red corpuscles at this period in the life of the animal, —
that is, just before and shortly after birth,— mention should be
made of the discovery of the vaso-formative cells by Ranvier

68 HOWELL. [Vor. IV.

(15) and by Schafer (16). Both of these observers have found
in the foetus, or in the new-born mammal (rat), large connective
tissue cells, within which red corpuscles are produced endoge-
nously. The cells become elongated, and connect with one
another to form capillary blood-vessels. The newly formed
corpuscles are never nucleated, and in this respect differ from
the corpuscles produced endogenously in the germ layers of the
embryo according to Balfour and others. In his recent book,
Hayem confirms the work of Ranvier, and states in addition
that blood plates as well as red corpuscles can be seen in the
vaso-formative cells. The hamatopoietic value of these cells
cannot be very great, as they have not been found at any other
period in the animal’s life, except at birth or shortly afterward.
It seems to me that more extended observations are needed
before we can accept such a peculiar method of production as
one of the normal means by which new red corpuscles are
formed. Most of the recent work has shown that the red
corpuscles pass through a nucleated stage, and are not formed
endogenously within larger cells, so that the isolated observa-
tions even of such distinguished histologists cannot be weighed
against the combined work of so many other investigators.
Possibly the appearances upon which the theory is based may
be capable of another explanation.

The White Corpuscles and Blood Plates during Embryonic Life.

I have little that is new to add to our knowledge of these
two elements of the blood during embryonic life; but the little
I have is worthy, perhaps, of being placed upon record, espe-
cially as it is a subject which seems to have attracted very
little attention and about which our information is deficient.
In the youngest embryo which I examined (cat, 2.5 cms. long),
no ordinary white corpuscles could be found, though the blood
was thickly crowded, of course, with nucleated and non-nucle-
ated red corpuscles. Occasionally a colorless corpuscle was
found; but these differed so much from the usual white cor-
puscle of the circulating blood, from both the uninucleated
and multinucleated form, that it seemed probable that they did
not belong to the class of leucocytes, but were embryonic cells
which had got into the blood accidentally, either in opening the

INO: Tell BLOOD CORPUSCLES. 69

heart or in some other way. In any case, they were extremely
few in number, and did not resemble the white corpuscles of the
grown animal. The blood plates also were entirely absent from
the blood of this embryo. Nota single specimen could be found,
though a number of preparations were examined. It seems to
me that this fact has a bearing upon the theories of the origin
of this element, and I shall refer to it again when discussing
that subject. In a human embryo of five months, both white
corpuscles and blood plates were found, though both were pres-
ent in small numbers. The white corpuscles were of two
kinds, as in the adult, — one variety of small size, with a single
vesicular nucleus resembling the lymphocytes; and the other
of larger size, faintly granular, with several nuclei, or, more
correctly, with a fragmented nucleus. At this age in the
human embryo, the great majority of the red corpuscles have
lost their nuclei. In a cat embryo of 9 cm. length the leuco-
cytes and blood plates were both found, though the former
were present in small numbers. I have not been able to find
any special reference to the occurrence of these elements in
the foetal blood, except in a paper by Neumann (57). In the
examination of human fcetuses made by Neumann, he states
that generally the white corpuscles were very few in number,
but makes no reference to the variations with the age of the
foetus. The fact that the white corpuscles are so late in
appearing is important, not only in its bearing upon the old
theory that they become changed into red corpuscles, but also
in the fact that it furnishes a means of determining their influ-
ence upon the chemical composition of the plasma.

II. ForRMATION OF RED CORPUSCLES DURING EXTRA-
UTERINE LIFE.

Historical Review.

The greater portion of the literature of the red blood cor-
puscles bears upon this side of the subject. Very many dif-
ferent views have been proposed; and a brief presentation of
the most important of them may be of value, both as an indica-
tion of the drift of opinion upon the subject, and also to enable
me to present my own work afterwards in the briefest possible
way, without detailed reference to or comparison with other

70 HOWELL. [Viou.4 vi.

views. I shall not attempt to make the review as complete as
the material which I have accumulated might enable me to do,
since some of the work published does not need special men-
tion. I have added, however, an appendix, giving the complete
list of articles which I have been able to consult.

Before 1869 it was quite generally believed that the red
corpuscles are formed from the white corpuscles, most probably
while in the circulation. This theory found its way into the
text-books, and, to a certain extent is still advocated by some
histologists. In fact, some of the most recent investigations
favor this view, although the evidence is so overwhelmingly
against it.

Feuerstack (17) in a recent series of observations, made
upon animals with nucleated red corpuscles, describes in the
circulation transitional forms between the white and the red
corpuscles. The colorless cell from which the other forms are
derived has a relatively large nucleus and small cell body.
The cell substance increases while the nucleus becomes smaller,
and not unfrequently takes a peripheral position. Hzemoglobin
develops in the cell, which gradually changes in shape from a
spherical to an oval form. Most of the transitional stages are
found in the bone marrow and spleen, though in these organs
they occur not in the parenchyma, but within the blood-vessels.
Feuerstack gives no sections to show how these developing
corpuscles are placed within the blood-vessels of the marrow
and spleen. When he says that the red corpuscles are derived
from white corpuscles, he differs somewhat from the older
observers, who thought that the white corpuscles might change
to red anywhere in the circulation, —— were, indeed, continually
undergoing such a change, — while Feuerstack limits it to the
blood-vessels of the marrow and spleen. On the other hand,
his conclusions, if not taken too literally, agree very well with
some new views of Denys upon the formation of red corpuscles
in the marrow of birds. While the older observers accepted
this view of the origin of the red corpuscles without much
question, no one was able to obtain satisfactorily the transi-
tional forms, so that Kolliker (3) was forced to say that the
question was still undecided. Erb (4) asserted, however, that |
he was able to get transitional forms in the circulation by
means of a certain method of treatment. Blood after treat-

No. 1.] BLOOD CORPUSCLES. 71

ment with acetic or picric acid gave him peculiar red cor-
puscles which contained fragments or granules of what ap-
peared to be nuclear matter. These granules might be many
or few in number and varied greatly in size. He believed that
the cells represent the transitional forms between the white
and red corpuscles, and thought that they were more numerous
after a severe hemorrhage, during the period of regeneration
of the blood. In rabbits, moreover, after a starvation of seven
to nine days, the transitional forms could no longer be found.
His complete theory of the origin of the red corpuscles is as
follows. White corpuscles arise in the spleen and lymph
stream and get into the blood first as small uninucleated cells,
with a scanty cell substance. While circulating in the blood,
these leucocytes increase in size, the increase affecting both
the nucleus and the cytoplasm. The nucleus then begins to
fragment, and finally breaks up into granules, while haemoglobin
develops in the cell, making thus one of his transitional forms.
The fragments of nuclear matter gradually disappear; the cell
becomes smaller and takes the shape of a normal red corpuscle.
The transitional forms of Erb can undoubtedly be found in
the circulation under certain conditions, but Erb was in error
in believing them to form an intermediate stage between the
ordinary white corpuscles of the blood and the red corpuscles.
Their real significance I shall describe later. A number of
other theories proposed during this period found but little sup-
port. For instance, Wharton Jones (18) thought that the red
corpuscles are the liberated nuclei of the white corpuscles, but
seems to have had no stronger reason for this belief than an
alleged agreement in size. Gerlach, Funke, Schaffner, ef a/,
taught that the red corpuscles are formed endogenously within
certain large colorless cells found in the spleen. But the cells
containing red corpuscles, which they found, and upon which
the theory was based, were afterwards shown by Kolliker to
be not the mother-cells of the red corpuscles, but, on the
contrary, their destroyers. At present, there can be no doubt
that the white corpuscles of the blood are never transformed
into red corpuscles, though it must be borne in mind that
this does not mean that the red corpuscles of the blood are
not derived from white or colorless cells. On the contrary,
as we shall see, it is now the general belief that the red cor-

72 HOWELL. [Vier ARV:

puscles spring from colorless cells found in the blood-forming
organs; but these colorless cells are not the white corpuscles
of the blood, indeed never under normal conditions get into the
circulation.

The most important discovery of the century with reference
to the development of the red corpuscles was made simultane-
ously and apparently independently by Neumann (5a) and by
Bizzozero (192). In 1868 these observers found that in the
red marrow of the bone nucleated red corpuscles occur, which
are similar to those found in the embryo, and that they are pres-
ent throughout the life of the animal. A nucleated red corpus-
cle can only be interpreted as the predecessor of a non-nucleated
red corpuscle, and the discovery therefore meant that the red
marrow of the bones is an organ for the production of new red
corpuscles throughout extra-uterine life. In his first papers
Neumann spoke of the nucleated red corpuscle as being de-
rived from colorless lymphoid cells, and described transitional
forms; but in his later papers he does not lay so much stress
upon the transitional forms, while still believing without
doubt that the red cells are derived from colorless ones. With
reference to the change from the nucleated red corpuscle to
the ordinary form, he agrees practically with Kolliker’s view
of the nature of this change in the embryo. The loss of the
nucleus takes place by a process of absorption within the cell,
and may be preceded by fragmentation. Neumann (5%) be-
lieves that in the adult the bone marrow is the sole organ for
the production of new red corpuscles, and gives a number of
experiments to prove that the spleen takes no part in their
formation, either under normal conditions or after severe hem-
orrhage. Bizzozero also found the nucleated red corpuscles in
the marrow, and afterwards showed that these cells are capa-
ble of multiplication by indirect division. This latter obser-
vation has been confirmed abundantly by later investigations,
and makes a second important step in our knowledge of the
origin of the red corpuscles. Bizzozero placed too much weight
apparently upon this growth of the nucleated red corpuscles,
and overlooked the importance of the colorless cells from which
the red corpuscles arise. The nucleated red corpuscles of the
marrow are derived, he thinks, from the similar embryonic
cells occurring in the liver and spleen during foetal life. When

No. 1.] BLOOD CORPUSCLES. 73

these organs begin to lose their hamatopoietic function, some
of the nucleated red corpuscles found in them are carried in
some way to the marrow, where they form centres of growth
for similar cells throughout life. Bizzozero, like Neumann,
thinks that the red marrow alone possesses this function dur-
ing extra-uterine life, but, unlike Neumann, he believes that
the spleen may temporarily resume its blood-forming functions
after severe hemorrhage when the marrow alone is unable to
regenerate new corpuscles with sufficient rapidity. As evi-
dence for this statement he publishes experiments made by
himself (19¢) and also in connection with Salvioli, in which it
was shown that, with dogs and guinea-pigs, nucleated red cor-
puscles can be found in the spleen of the adult if the animal
has been subjected previously to a severe bleeding, or, better,
to a number of successive bleedings: under such conditions,
not only were the simple nucleated forms found, but nucleated
cells in process of division by karyokinesis. In man, also,
after death from anzmia there are several cases recorded in
which nucleated red corpuscles have been found in the spleen
(Foa[22], Pellacani). On the other hand, Neumann (5%) contends
that even after severe hemorrhage nucleated red corpuscles are
not found in the spleen, or are found in such small numbers
that their presence may be accounted for by the fact that they
occur also. in the circulating blood, especially in the vena
azygos, which brings back blood from the red marrow of
the ribs. With reference to this last point, it is undoubt-
edly true that in the blood of animals after severe and re-
peated hemorrhages, nucleated red corpuscles may be found,
and similarly in the human subject it is known that in per-
nicious anzemia, leukaemia, etc. (Osler and Gardner [23],
Laache [25]), nucleated red corpuscles may be found in the
circulation. But it must be borne in mind that in the spleen
of animals after strong hemorrhage one may find nucleated
red corpuscles in cases when they are absent from the general
circulation ; and furthermore they may occur in the spleen in
large numbers, and showing every sign of an active multiplica-
tion. Neumann (5/) himself admits that in one casein which the
animal (dog) had been bled a number of times, and in which
septic infection had developed, he could find nucleated red
corpuscles in the spleen, but not in the circulating blood. He

74 HOWELL. [Vo IV.

accounts for this exception by attributing it to the septiczemia ;
but this does not seem to be a satisfactory explanation. Sev-
eral experiments of my own on this point I will describe in
the proper place: it is sufficient to say here that they confirm
the view of Bizzozero and others that the spleen may be made
to resume its hematopoietic activity..

While the fundamental discovery of Neumann and Bizzozero
has been generally accepted so far as it fixes the function of
producing red corpuscles in the marrow, a number of observers
have differed from them and from one another as to the
method by which the red corpuscles are formed in that organ.
Rindfleisch (26) describes the nucleated red corpuscles, but
differs from all others in believing that these cells lose their
nuclei not by a gradual absorption, but by an extrusion.
Rindfleisch is generally quoted as saying that the nucleus is
extended naked from the corpuscle; but this is an error. He
says that “the nucleus, surrounded by some colorless proto-
plasm, leaves the cell, which remains as a bell-shaped body of
a reddish yellow color.” The further fate of the extruded nu-
cleus, with its envelope of protoplasm, he leaves undiscussed.
He describes and figures the nucleus in the act of escaping,
but was not able to watch the process in a living cell, though
he used all sorts of means — heat, electricity, reagents of dif-
ferent kinds —to act upon the corpuscles. After the extru-
sion of the nucleus, the red corpuscle has first a bell shape,
and is afterward moulded into a biconcave disc by the move-
ment of the circulating blood. Malassez (27) believes that the
nucleated red corpuscle is derived from an undifferentiated
marrow cell, which contains little or no hemoglobin, and in
which the nucleus is diffuse. He describes three interme-
diate stages in the transformation which he is able to recog-
nize constantly in the marrow: 1. Spherical cells of large
size, which stain very feebly with eosin or haematoxylin, and
contain no hemoglobin, or only a trace. The nucleus in these
is not a distinct morphological structure, the nuclear matter
being diffused throughout the cell. He designates these cells
as protohzmatoblasts.

2. Cells of the same size, with a granular protoplasm, and
still containing little or no protoplasm. A nucleus is now
present, and is spherical, large, and uniformly granular.

No. 1.] BLOOD CORPUSCLES. 7s

3. Cells of smaller size, containing haemoglobin. The nu-
cleus, also, is smaller, and shows a reticular structure. The
next stage is the nucleated red corpuscle proper, which differs
from (3) in the deeper tint of the haemoglobin and the smaller
size of the nucleus. The nucleus is distinguished further
by the fact that it stains more deeply with haematoxylin.
Malassez differs from other histologists in his explanation of
the way in which the ordinary non-nucleated red corpuscle
is derived from the nucleated form. According to him, the
latter do not lose their nuclei at all, but give rise to the ordi-
nary red corpuscles by a process of budding. The buds are
constricted off, and are first spherical, but afterward become
biconcave, partly from the mechanical action of the circulating
blood, partly because of an unequal diminution in bulk. Foa
and Salvioli (13) also derive the nucleated red corpuscle from
a colorless or “hyaline cell.” This latter cell originates both
in the embryo and the adult from the large giant cells found
in the marrow during extra-uterine life, and in the liver and
the spleen of the foetus during the period when these organs
are producing red corpuscles. The giant cells, myeloplaques,
of Robin are of two kinds, at least, in the red marrow. One
variety is large, finely granular, and contains a number of oval
separate nuclei, which correspond to the myeloplaques as
usually described. The second variety is not multinucleated,
but has a very large coiled, or twisted, nucleus, made up,
apparently, of a number of smaller nuclei, which are, how-
ever, still in connection with one another. This variety
Bizzozero described as the “giant cell with budding nuclei” ;
and Foa and Salvioli believe that they give rise to the hya-
line cells, from which the nucleated red corpuscles are after-
wards formed. They give to this kind of giant cell, therefore,
the name of hzmatoblast. The hamatoblasts separate into
a number of smaller hyaline cells, the large nucleus breaking
up into separate ‘‘ buds,” each of which becomes the nucleus of
a hyaline cell. The hyaline cells change to nucleated red cor-
puscles by the development of hemoglobin within the cell sub-
stance; and these latter pass to the non-nucleated form in
consequence either of an absorption or an extrusion of the
nucleus. In a later paper, Foa (22) expresses his belief that
the nucleus disappears within the cell by absorption. Osler

76 HOWELL. [Vou. IV.

(28), in his Cartwright lectures, describes in the adult marrow
seven different kinds of cells. He derives the nucleated red
corpuscles, in the first place, from a colorless cell 9 to 12 p.
in diameter, with a smooth, homogeneous protoplasm and a
finely granular nucleus. This cell shows, moreover, a pecu-
liar flexibility. These cells, in turn, are derived from what he
calls “ protoleucocytes,” which are solid-looking lymphoid ele-
ments, 2.5 to 5 uw. in diameter, resembling free nuclei, though
some of them may have a narrow rim of protoplasm. The
nucleated red corpuscle is transformed to the non-nucleated
corpuscle by the gradual disappearance (absorption) of the
nucleus, after which the corpuscle becomes condensed to the
flattened disc shape.

The most elaborate, and probably the most important, con-
tribution to our knowledge of the development of the red and
the white corpuscles which has been made recently is found
in a series of papers by Lowit (29). The most important con-
clusions at which he arrives are as follows: the blood-forming
organs of the adult, among the cold-blooded as well as the
warm-blooded animals, are the bone marrow, the spleen, and
the lymph glands. In all of these organs we meet with two
kinds of colorless cells. One of these he calls “leucoblasts ” ;
and they are destined to form the leucocytes of the blood and
lymph. To the second he gives the name of “erythroblasts”:
from these the red corpuscles are developed. These two sorts
of cells are distinguished from each other by differences in the
structure of the nucleus, in the method of multiplication, and
in the properties of the cell protoplasm. The leucoblasts have
a nucleus which is relatively quite large. It contains one or
more small heaps of chromatin, sometimes irregular in shape,
from which a system of delicate lines and bands radiates toward
the nuclear membrane. This latter consists of a distinct, often
doubly contoured, band of chromatin substance, on the inner
side of which one frequently finds irregular projections con-
nected with the intra-nuclear network. The leucoblasts mul-
tiply by a process less complicated than ordinary karyokinesis
and more complicated than simple direct division, The chro-
matin granules during division show some movement, though
of an irregular character, from the equator toward the poles.
He proposes to call this divisto per granula. The erythro-

No. 1.] BLOOD CORPUSCLES. Vi);

blasts have a nucleus which shows always a chromatin reticulum,
but no true nucleolus. They never make amoeboid movements
nor ingest foreign particles, and finally they develop hamo-
globin in the cell substance, passing thus into nucleated red
corpuscles. Cell division takes place with the formation of the
usual karyokinetic figures. L6wit designates this method of
multiplication as dtvisto per fila, in contradistinction to the
method found in the leucoblasts. He states that he has never
been able to find transitional forms between the two kinds of
cells, though in the organs in which they occur they are found
freely intermingled with each other. The leucoblasts enter
the lymph stream, and eventually reach the blood as uninucle-
ated leucocytes. These are rather small, and are devoid of the
power of making amceboid movements,—a fact which was
pointed out long ago by Schultze. In the blood stream, they
increase in size, the nuclei become elongated and constricted,
and finally fragment to form the so-called multinuclear leuco-
cytes. He believes, then, with many others that the multinu-
clear leucocytes are not cells in the act of multiplication, but,
on the contrary, are disintegrating ; and the multinuclear stage
so-called is probably followed by a complete dissolution of the
cell.

In the veins coming from the blood-forming organs the uni-
nucleated leucocytes predominate greatly in number. In the
right heart the number of uninucleated forms is still relatively
large, while in the left heart they become less numerous, and
in the peripheral arteries they show a striking diminution. In
other words, the transition from the uninucleated to the mul-
tinucleated forms takes place chiefly in the venous system
during the brief interval of time required for the blood in the
veins to pass from the lymphoid organs to the left heart. The
erythroblasts, after the development of hzmoglobin, become
nucleated red corpuscles. In the marrow of the bone all the
intermediate stages may be obtained without difficulty. So in
the liver and spleen of the foetus and in the spleen of the adult
in some cases after severe hemorrhage similar transitional
forms are found. But in the lymph glands transitional forms
between erythroblasts and nucleated red corpuscles cannot
be obtained. Hence he concludes that the transition in this
case takes place in the lymph stream or the blood or both,

78 HOWELL. [Vot. IV.

or, as a third possibility, the erythroblasts are carried to the
marrow and there undergo the final changes. The nucleated
red corpuscles pass into the usual red corpuscles by a loss of
the nucleus. This, he thinks, occurs in the way described by
Kolliker and by Neumann; that is, by disintegration and
absorption within the cell. In his latest paper Lowit describes
some new and rather remarkable observations upon the erythro-
blasts. He finds that he can obtain erythroblasts easily from
the veins which bring back blood from the blood-forming
organs; while in the superior vena cava they occur rarely,
and in the left heart and arterial system they are entirely
wanting. Though few erythroblasts are found in the superior
cava and right heart, nevertheless blood from these portions of
the vascular system, when treated for a number of hours with
a modified Pacini’s liquid, shows a considerable number of red
corpuscles which contain a granular body of the shape and
general appearance of a nucleus. The granules may be few
or many, and in some cases they are connected by a sort of
nuclear network. Léwit’s description corresponds very well
to the “transitional forms” of Erb which have already been
mentioned. He interprets these structures as erythroblasts in
which the nucleus is disappearing. Apparently, then, he be-
lieves that an erythroblast may develop its hamoglobin and
lose its nucleus by absorption while in the venous blood and
during the time required for that blood to flow from the blood-
forming organ to the left heart. The blood that flows from
the lungs to the left heart must contain, therefore, a number of
newly formed corpuscles. Nevertheless, comparisons made be-
tween the blood of the left and the right heart showed that the
former contained fewer corpuscles and less haemoglobin than
the latter. Hence, during the passage of the blood through
the lungs there must occur also a more or less important de-
struction of red corpuscles.

It has been generally believed that in the marrow the
nucleated red corpuscles are not arranged in any definite way,
but are mingled indiscriminately with the other elements of the
marrow. Denys (14) in a recent very interesting paper states
that this is not the case, at least not in the marrow of the
bird. He accepts the terms erythroblast and leucoblast pro-
posed by Lowit, and states that in sections of the marrow

No. 1.] BLOOD CORPUSCLES. 79

which have been treated with a double stain of fuchsin and
methyl green the nuclei of the erythroblasts stain green, while
those of the leucoblasts stain red. Moreover, this method of
staining shows that the two kinds of cells are not intermixed
without order; but on the contrary they are sharply separated,
the erythroblasts lying in cords or strings which are clearly
marked off from the masses of leucoblasts. These cords of ery-
throblasts form in reality a part of the vascular system of the
marrow in the following way. Between the well-defined arteries
and veins of the marrow there are two capillary plexuses. One,
a system of arterial capillaries comparatively few in number, is
connected with the larger arteries, and is composed of long,
narrow vessels with distinct, doubly-contoured walls. These
open suddenly into large venous capillaries which are nearly
filled with erythroblasts, and form, in fact, the cords of ery-
throblasts found in the marrow. The blood stream flows
through these imperfectly formed vessels in a central channel
which is more or less open, while the plasma probably percolates
through the whole mass of erythroblastic cells. These capil-
laries have a very delicate endothelial wall which marks them
off from the leucoblasts, and the erythroblasts filling them
are so arranged that the youngest lie next to the wall and
the most matured next to the central channel, where they can
be floated off by the blood current. The similarity of these
cords of erythroblasts or developing veins to the developing
veins found in the germ layers of the embryo by Wenckebach
(7) and Ziegler (8), and described and figured in the liver and
posterior limb of the embryo by me, will be apparent at once.
It would seem that the manner of development of the red
corpuscles is the same in the adult as in the foetus. Unfortu-
nately, Denys has not as yet shown that the same arrange-
ment is found in the marrow of the mammal, while others
positively state that no regular grouping of the blood-forming
cells occurs: so that this point remains to be investigated.
I shall have occasion to refer to it again. Feuerstack (17), it
will be remembered, held that in the birds and other animals
with nucleated red corpuscles the development of the corpus-
cles takes place in the blood-vessels of the marrow; but he
gives no definite description of how this occurs. So, more
recently, Geelmuyden describes for the marrow of the frog

80 HOWELL. [Vot. IV.

what seems to be an arrangement similar to that given by
Denys for the pigeon. He says that in the injected marrow
of frogs the blood corpuscles do not lie free in the marrow, but
are contained in definite vessels. Within the lumen of these
vessels there are a great number of narrow cells which lie
along the walls of the vessels, while the blood corpuscles of
the circulating blood pass through the middle. Hayem (31)
holds an entirely different view of the origin of the red
corpuscles. Hayem, as is well known, deserves the credit of
giving the first elaborate description of the blood plates. Al-
though these elements had been mentioned, and to a certain
extent studied, before his time, Hayem’s investigations into
their structure and meaning seem to have given the impulse
to the great amount of work which has been directed to them
within recent years. He attributed to the blood plates the very
important function of forming the new red corpuscles. The
blood plates, in fact, are in his opinion only young red corpus-
cles possessing the shape of the red corpuscles, — biconcave
discs, —and in many cases having a greenish tint from the
hemoglobin which has begun to form in them. He speaks of
the blood plates, therefore, as “ hamatoblasts.” As proof for
this view, he states that intermediate forms can be found be-
tween the typical blood plate and the ordinary red corpuscles,
and these intermediate forms are especially numerous after
severe hemorrhages when we should expect a rapid regeneration
of new corpuscles. These statements, however, have not met
with confirmation from the work of others. Most of those who
have studied the blood plates agree in the conclusion that they
do not develop into red corpuscles, however much they differ
on other points. It is rather interesting that Zimmermann
(32), who was one of the first to notice the blood plates, to
which he gave the name of “ elementary particles,” also thought
that they develop into red corpuscles.

Gibson (33) believes with Lowit that the spleen and the
lymph glands as well as the marrow take part in the production
of red corpuscles throughout extra-uterine life. To establish
the fact that the spleen makes red corpuscles he removed that
organ from three dogs. In two of them he was able to demon-
strate a slight diminution in the number of red corpuscles,
while the effect upon the number of white corpuscles was not

No. 1.] BLOOD CORPUSCLES. Sr

constant. His results were not striking, but were sufficient to
convince him that the spleen has a distinct though subordinate
part to play in the production of red corpuscles. As proof that
the lymph glands also produce red corpuscles, he cites an ex-
periment in which the thoracic duct was ligated for thirty-seven
days before the animal was killed. Post-mortem examination
showed that some of the lymph glands, especially those of the
mesentery, had a reddish appearance, and contained a number
of nucleated red corpuscles. Moreover, enumeration of the red
corpuscles of the blood of this animal proved that a diminu-
tion of about 13 per cent. had taken place. Gibson’s theoretical
views of the way in which the red corpuscles are formed are as
follows: In some of the colorless marrow cells the nucleus
begins to increase in size, while haemoglobin develops in the
body of the cell. Later, as the haemoglobin becomes fully
formed, the cell shows a diminution in size which affects the
nucleus also, so that finally one of the small typical nucleated
red corpuscles is produced. Just how this becomes changed to
the non-nucleated corpuscle is not stated very clearly. In one
place he seems to agree with the view of Kolliker and Neu-
mann that the nucleus fragments and is absorbed, while in
other places he speaks of the nucleus becoming a blood plate.
He describes the blood plates under the name of “colorless
microcytes,” and thinks that they are formed in part from the
fragmented nuclei of the white corpuscles and in part from the
fragmented nuclei of the nucleated red corpuscles. In addition
to the “colorless microcytes,” he describes in the blood what
he calls “colored microcytes,” which he believes to be the same
as the “haematoblasts” of Hayem. These he considers to be
simply fragments of red corpuscles formed in some way or
other in the circulating blood. Gibson seems to be describing
here the microcyte of pathological literature, small, spherical,
deeply colored corpuscles very common in the blood in progres-
sive pernicious anzemia, leukaemia, chlorosis, etc. [See Osler
(24), Laache (25), Eichorst (34), e¢ a/.]

Obrastzow’s (35) theory bears some resemblance to that of
Olser already described. The nucleated red corpuscles are
derived from colorless cells, which in turn are formed from free
nuclei, or little spheres of nuclear matter (protoleucocytes), each
of which develops round itself a layer of protoplasm. The

8 HOWELL. [Viox. Juv:

colorless cell thus produced may change either into a nucleated
red corpuscle or into an ordinary marrow cell. According to
Obrastzow, the nucleated red corpuscles of most authors —
hamatoblasts, according to his nomenclature — possess in the
living state no nucleus, the nuclear matter being diffused
throughout the cell. After the death of the cell, the nuclear
material becomes condensed to form a typical nucleus such as
is always described for the cell. The process of condensation
or separation of the nuclear matter resembles very much the
coagulation of blood, nuclear substance having properties
similar to though not identical with those of fibrin. The
transformation of the hematoblasts to red corpuscles consists
chiefly in the disappearance and absorption of the nuclear
matter. Obrastzow has seen in his preparations nucleated red
corpuscles, or hzmatoblasts, with the nuclei partially or com-
pletely extruded from the cell in the way described by Rind-
fleisch. He explains this, in accordance with his theory, as the
result of post-mortem changes brought about by the condensa-
tion of the protoplasm after death. Arndt also believes that
the nucleus of the nucleated red corpuscle does not exist in
the living cell, but is formed in consequence of post-mortem
changes. Indeed, he goes further than this and denies that
any nucleus is present in the living red corpuscles of the lower
vertebrates, — birds, reptiles, amphibia, etc. The apparent
nucleus so easily seen in these cells is caused by the action
of reagents or by post-mortem changes. The nucleus seen
in the nucleated red corpuscles after the death of the cell
consists histologically of a gelatinous ground substance con-
taining a number of granules. He speaks of these granules as
‘‘elementary corpuscles,” and thinks that they are of the same
nature as the granules found in protoplasm generally.
Afonassiew (36) concludes that red corpuscles may be regen-
erated in three different ways: 1. Nucleated red corpuscles
multiply by division and are finally changed to non-nucleated
red corpuscles. 2. The blood plates increase in size; each
forms round itself an envelope of protoplasm in which hzmo-
globin becomes developed, making a nucleated red corpuscle.
This loses its nucleus by extrusion and becomes an ordinary
red corpuscle. Under normal conditions this series of changes
takes place only in the marrow. He seems to think that the

No. 1.] BLOOD CORPUSCLES. 83

extruded nucleus in this case again becomes a blood plate and
may enter upon a similar course of development. 3. In cases
of strong anemia one finds occasionally that certain of the red
corpuscles (the poikilocytes, apparently, of the pathologist) con-
strict off small bits of their substance to form small red cor-
puscles (microcytes ?) somewhat larger than the blood plates
which afterwards develop into normal red corpuscles while in
the circulation. Boettcher (37) contends that the red corpuscle
of the blood in man and the mammalia generally is nucleated,
though the nucleus under ordinary conditions is not visible.
His evidence for this belief is not at all conclusive: it seems
to rest chiefly upon the fact that reagents which dissolve the
hemoglobin out of the corpuscles, especially chloroform, leave
behind a colorless sphere, considerably smaller than the orig-
inal corpuscle, which he takes to be the nucleus. When the
action of chloroform upon a red corpuscle is watched, it can
be seen, he says, that the reagent dissolves off the peripheral
colored portion of the corpuscle, leaving behind the colorless
nucleus. Efforts to bring out this nucleus by the action of
ordinary staining reagents failed except in two cases, once from
the blood of a person who had died from leukzmia, and once
from the blood of a tuberculous woman. It is fair to suppose
that in both of these cases he was dealing with nucleated red
corpuscles which had passed into the circulation.

Sappey (38) also asserts that the mammalian red corpuscle is
nucleated, and that to bring out the nucleus one must treat
the blood with some reagent which will make the corpuscles
spherical. He recommends the following liquid: water, 500
grms.; sodium sulphate, 40 grms. Add to this solution acetic
acid in the proportion of 1 to 49. Quite recently, Cuenot (39)
has advanced a theory of the development of the red corpus-
cles which in some respects is more fanciful than any yet de-
scribed. He believes that the red corpuscles are formed in
the spleen, and in mammals that the whole development is car-
ried on in this organ, while in the lower vertebrates a certain
portion of the development takes place in the circulation. The
spleen, according to Cuenot, contains two kinds of colorless
corpuscles,— some of large size and but little refractive, which
are destined to form the white corpuscles; and some of smaller
size, which are very refractive, and become the nuclei of

84 HOWELL. [Vou. IV.

future nucleated red corpuscles. These are not naked nuclei,
but are surrounded by avery thin envelope of colorless proto-
plasm. The protoplasmic layer becomes enlarged, and small
granules are constricted off from the nucleus, and set free in
the cell. In some way these nuclear granules start the forma-
tion of hemoglobin, either because they contain the necessary
iron or because they act as a sort of hemoglobin ferment. As
the hemoglobin develops, the granules disappear, and the nu-
cleus becomes smaller. In the mammals the nucleus becomes
entirely absorbed in the process, so that the fully formed mam-
malian corpuscle is non-nucleated.

If we attempt to sum up the facts with reference to the de-
velopment of the red corpuscles which seem to be fairly well
established, we will be obliged, as one can readily see from the
foregoing review, to confine ourselves to a few fundamental
points. In the first place, it is perfectly well proved that
during extra-uterine life the red corpuscles are developed in
the red marrow of the bones. Whether or not the spleen and
the lymph glands participate in this function is not definitely
determined. In the second place, it is generally admitted that
the red corpuscle is first a nucleated cell, and that it loses its
nucleus in the marrow or other blood-forming organ. Whether
the nucleus is lost by extrusion or disappears within the cell
by absorption is not settled; but the majority of writers cer-
tainly favor the latter view. In the third place, it is pretty
conclusively shown that the nucleated red corpuscle is derived
from a colorless cell —erythroblast, to use Lowit’s term —
which is formed in the marrow. The origin of this cell is the
point about which, perhaps, there is least agreement. Finally,
none of the recent work supports the theory that the red cor-
puscles are derived from the white corpuscles (leucocytes) of
the circulating blood, so that this time-honored theory must be
definitely abandoned.

Experimental Work.

My own work has been confined almost entirely to one
mammal, the cat, partly because there was not sufficient time
to make a complete series of parallel experiments and observa-
tions upon other animals, and partly because, by confining
the work to a single mammal, a thorough familiarity with the

No. 1.] BLOOD CORPUSCLES. 85

different kinds of cells was obtained, and observations made
upon different individuals were capable of a closer comparison.
It cannot be doubted that in its essential features, certainly,
and in all probability in most of the minor details, the genesis
of the blood corpuscles in the cat is the same as in man or in
any of the higher mammalia.

In the course of the work I have made use of many different
methods of treatment; but the methods which I have used
most, and which have given me the best results, are these.
When studying fresh specimens of liver blood, marrow, etc.,
the reagent invariably used was a I per cent solution of
methyl green made up with 0.6 per cent solution of sodium
chloride. The tissue was teased either in normal salt solution
or in its own plasma, and then further teased in a drop of
the methyl green. I did not use acetic acid in combination
with the methyl green, as this reagent quickly dissolves out
the hemoglobin from the nucleated red corpuscles, while
with the methyl green alone this does not happen unless the
quanity used is too great relatively to the amount of tissue
teased. The blue-green color given by the methyl green to
the nucleus of the nucleated red corpuscles served to make
the hamoglobin in the cell protoplasm more distinct by con-
trast. The fresh tissue was examined also without the addition
of any staining reagent after teasing in its own liquid, in
normal salt solution, or, best of all, in blood serum which had
been previously prepared from the same animal.

The marrow, spleen, and liver of the foetus as well as the
adult were studied in section, and specimens were taken from
normal animals, from animals which had been bled, starved,
injected, etc.

The tissue was usually hardened in a cold saturated solution
of mercuric chloride according to the directions given by Gaule.
Sections were cut in paraffin, and were stuck to the cover slip
by the alcohol method, using 70 per cent alcohol. The sec-
tions were then stained by two or more different methods.
The stains usually employed were: first, a triple stain of
heematoxylin, eosin, and saffranin, used successively according
to Gaule’s method; ‘second, alum carmine; third, Biondi’s
triple stain of acid fuchsin, methyl green, and orange used in
mixture; fourth, the Shakespeare-Norris stain for hemoglobin,

86 HOWELL. [Vou. IV.

consisting of a mixture of borax carmine and indigo carmine.
This stain was subsequently abandoned, as it was found not
to work as a differential stain for hemoglobin after mercuric
chloride hardening. In several cases where sections were
made of a foetal femur, with its contained marrow, the tissue
was fixed in Flemming’s solution, and afterwards decalcified
in saturated picric acid solution. These sections treated with
the indigo-carmine solution gave very beautifully the apple
green stain to the hemoglobin in the red corpuscles. Another
method which I used frequently, both for the blood itself and
the blood-forming tissues, is one recommended by Flemming, as
follows: the fresh tissue is quickly teased upon a slide in its
own liquid, and a large drop of diluted Flemming solution is
dropped upon it, and the specimen then kept for twenty-four
hours in the moist chamber. By that time a number of the
cells have become firmly adherent to the slide, so that it can
be washed in water. It is then covered with saffranin for
twenty-four hours, being kept in the moist chamber. The
saffranin is washed off with absolute alcohol, with or without
acid, according to the depth of the stain, and the specimen
treated successively with oil of cloves, xylol, and balsam.
This method gave excellent results.

Development of the Red Corpuscles during Extra-uterine Life.

The importance and even the existence of the nucleated red
corpuscles has been denied by some authors, as I have at-
tempted to show in the historical review of the subject. But
that these cells are found in the red marrow of the bones
throughout healthy life, and that they give rise to the red cor-
puscles of the circulating blood, has been proved beyond any
reasonable doubt, and upon the whole is as well accepted as
most of the facts of physiology. What we desire, then, is a
complete knowledge of the life-history of the nucleated red
corpuscle, its origin, its method of growth or reproduction, and
the way in which it is changed to the non-nucleated corpuscle.
These corpuscles are found chiefly, if not exclusively, in the
adult in the red marrow. Hence most of the work has been
done upon that tissue.

No. 1.] BLOOD CORPUSCLES. 87

Origin of the Nucleated Red Corpuscle.

Most authors agree that the nucleated red corpuscle is de-
rived from a colorless cell existing in the marrow, but there is
considerable difference of opinion as to the characteristics and
origin of this cell. Léwit (29), it will be remembered, gives
to it the name of erythroblast, and describes certain histologi-
cal characteristics which enable him to recognize the cell
wherever seen. Others derive the nucleated red corpuscles
from what are known as the ordinary marrow cells, and others
still, as Osler (28), describe a peculiar kind of cell in the mar-
row from which the nucleated red corpuscles are derived, and
which correspond more or less closely to the erythroblasts of
Loéwit. Before speaking of my own view, it will be necessary
to describe briefly the different sorts of cells found in the red
marrow of the cat. In teased specimens of the marrow we
meet, in the first place, with the morphological elements of the
blood, the red corpuscles, white corpuscles, both uninucleated
and multinucleated, and the blood plates. Of the marrow ele-
ments proper, we have, first, the nucleated red corpuscle. By
this term is meant a nucleated cell colored with haemoglobin.
The size of these cells is quite variable, and they are fre-
quently found in different stages of cell division, as described
by Bizzozero (192), the most common figure being the diaster.
But the most marked peculiarity in the structure of the nucle-
ated red corpuscles is found in the nucleus. In some of these
cells, which for the sake of clearness I will speak of as the
immature nucleated red corpuscles, the nucleus is characterized
by an intra-nuclear network of chromatin, at the nodal points
of which are found conspicuous granules of a similar material,
which stain, however, more deeply than the reticulum. In
badly preserved specimens, therefore, the nucleus seems to be
composed of a number of fine or coarse granules imbedded in
a clear or slightly colored matrix. The cell protoplasm of
these immature forms is, as a rule, only slightly tinged with
hemoglobin, and makes a relatively thin envelope round the
nucleus (see Fig. 8). Others of the nucleated red corpuscles,
which may be distinguished as the mature forms, have a nucleus
which shows no sign of a reticulum when stained with methyl
green, hematoxylin, saffranin, etc. The nucleus, when stained,

88 HOWELL. [Vox. IV

shows usually, indeed, no structure whatever, but takes a deep
uniform tint, as though the chromatin material were evenly
diffused throughout (see Fig. 8). The nucleus of this form is
generally smaller, both relatively and absolutely, than that of
the immature cells; and the cell protoplasm is more deeply
tinged with hemoglobin. It is very common to find these
cells with the nucleus either placed eccentrically or partially
extruded, while in the immature cells no such appearance is
ever seen. As the names I have chosen indicate, I consider
these two forms the two extremes in the life of the nucleated
red corpuscle. Intermediate stages between the extremes are,
of course, of frequent occurrence; for instance, corpuscles with
a nucleus which stains deeply and nearly uniformly, but shows
large or small irregular clumps of a deeper staining material,
like the granules of the nucleus in the younger forms, or others
in which the nucleus contains smaller granules staining deeply
and some indication of a reticulum between the granules; while
the material between the granules and reticulum, the nuclear
liquid, also takes the stain to a certain extent. The morpho-
logical difference between the two extreme types of nucleus is
associated with a difference in chemical structure, as far as this
can be determined by staining reagents. When sections of the
marrow are treated with the triple stain, — hzematoxylin, eosin,
saffranin, —the nucleus of the immature forms takes the h@ma-
toxylin, while that of the mature forms stains a brilliant red
with the saffranin ; and the nucleus of the intermediate stages
shows a combination tint of some shade of purple (see Fig. 9).
The distinctness with which this difference in staining comes
out depends, of course, upon the time of exposure to the differ-
ent dyes. If the section has lain too long in the hematoxylin,
all the nuclei of the preparation may be stained a dark blue or
purple; while, if the exposure to the haematoxylin has been too
short, the saffranin color predominates to the exclusion of the
others. In some degree, however, the difference between the
nuclei may be discovered in all cases; and when the staining
has been properly regulated, it comes out with great distinct-
ness. The time for the action of each dye varies naturally
with the thickness or character of the sections; but usually a
minute to a minute and a half was found to be the proper time
of immersion in each of the staining reagents. It is worthy of

No. 1.] BLOOD CORPUSCLES. 89

mention that the nucleoli of the marrow cells and giant cells,
as well as the nuclei of cells during karyokinesis, when treated
with the triple stain, take the saffranin in preference to the
hzematoxylin, like the nuclei of the mature nucleated red corpus-
cles; whereas the reticulum of the resting nucleus of most cells,
unlike the nucleolus, stains most easily with the hematoxylin.
A similar difference in the behavior of the nucleolus and the
dividing nucleus has been noticed before by Steinhaus (48) for
epithelial cells, and by Hodge (40) for nerve ganglion cells.
With the triple stain of Biondi, the nucleus of the mature
nucleated red corpuscles stains an even solid green, and in the
nucleus of the immature forms the reticulum and granules at
the nodal points stain a light green, while the nuclear material
between the meshes of the reticulum remains unstained.

2. The next most important element of the marrow from
our standpoint is a colorless cell, similar in structure to the
immature form of nucleated red corpuscle, from which it dif-
fers in fact only in the absence of hzmoglobin from the cell
protoplasm. The nucleus is granular without anything like a
definite nucleolus. In well-preserved specimens the granules
are connected by an intra-nuclear reticulum, which stains less
deeply than the granules. This form of cell has been de-
scribed by Osler (28), and also by Lowit (29) and others, as the
progenitor of the nucleated red corpuscle. Lowit has given to
the cell the name of erythroblast. It seems to me that the
name is a convenient one, and I shall make use of it hereafter.
At the same time, I wish to say that I do not accept Lowit’s
theory of the origin and permanent histological characters of
these cells, which has been described in the historical review.
On the contrary, my investigations have brought me to quite
different conclusions, as I shall show in the proper place.
Drawings of this form of cell are shown in Fig. 8.

3. The ordinary marrow cell is a large, colorless cell, with a
characteristic nucleus and a faintly granular protoplasm. The
nucleus is of a vesicular character, having an oval shape, a
doubly contoured nuclear membrane, and one or more conspic-
uous nucleoli. From the nucleolus a scanty reticulum stretches
out toward the peripheral membrane (see Fig 12, a and 8).

4. Wandering cells. These are like 3 in structure, except
that the nucleus, instead of being oval, is pulled out to an

go HOWELL. [Vou. IV.

elongated strap shape, and may be bent into a horseshoe, or
may be coiled upon itself one or more times, like the leucocytes
found so abundantly in the cat’s blood. These cells, are, how-
ever, larger than leucocytes; and it is probable that they
belong to the same class as the ordinary marrow cells (Fig.
12, c and @).

4. Some of the ordinary marrow cells have their protoplasm
loaded down with coarse granules which stain readily with
eosin, methyl green, etc. (Fig. 12, ¢ and z). Sometimes these
cells are very numerous: they evidently play some important
part in the metabolic changes going on in the marrow. They
do not appear to be confined to the marrow, since Heidentain
has described what seems to be the same cell in the lymphoid
tissue of the intestine, though he was unable to arrive at any
satisfactory conclusions as to its function.

6. The so-called giant cells. In the red marrow of grown
animals these are always of the kind described by Bizzozero
as giant cells with budding nuclei to distinguish them from the
multinucleated giant cell, or myeloplaque, found in develop-
ing bone, in pathological formations, etc. A more detailed
description of these cells with a discussion of their functions
is given in an accompanying paper.

7. Free nuclei are found sometimes in considerable numbers.
In size and in the way in which they stain, they resemble
exactly the nuclei of the matured nucleated red corpuscles,
and there can be but little doubt that they arise from these
cells.

With reference now to the origin of the nucleated red cor-
puscles, there seems to be little doubt that they are derived in
the first place from the colorless cells (No. 2) known as erythro-
blasts. There has been some difference in the description of
these cells as given by various observers ; but there is enough
agreement to justify one in believing that the same cell is
meant by all, and that the erythroblast is converted to the
nucleated red corpuscle by the development of haemoglobin
in the cell protoplasm. This point might be regarded as gen-
erally accepted. The real difference of opinion lies in the
theories as to the derivation of the erythroblast. While Lowit
(29), Denys (14), and others believe that it constitutes a dis-
tinct variety of cell found in the marrow and other blood-

No. 1.] BLOOD CORPUSCLES. gI

forming organs, that it multiplies by indirect division, — dviszo
per fila,—and is not derived from any other element of the
marrow, Osler (28) and Osbratzow (35) think that it develops
from naked nuclei found in the marrow, and Foa and Salvioli
believe that it is constricted off from the giant cells. The
theory of Lowit is the best supported by observations and ex-
periments, and has met with most corroboration. While I
with others before and after Lowit have satisfied myself of
the existence of the erythroblasts, I cannot agree with him
that they are not derived from other simpler cells found in
the marrow.

In sections and teased specimens of the liver of the embryo
and of the marrow of the embryo and adult, I have obtained
evidence to show that the erythroblasts are derived from cells
of the marrow similar in structure to the ordinary marrow
cells; that is, large cells with oval vesicular nucleus and a
faintly granular protoplasm. Drawings intended to illustrate
the way in which these cells give rise to the erythroblasts are
given in Fig. 11. The marrow cells themselves have the char-
acteristics of embryonic cells; and those from which the ery-
throblasts are derived are undoubtedly descendants, but little
if any changed, of the original mesoblastic cells from which
the marrow is formed. In the embryonic liver, as well as
in the embryonic marrow, these cells are found, together
with the transitional stages to the typical erythroblast. This
derivation is particularly well marked in the developing blood-
vessels of the liver of the young embryo. As I have already
said, these vessels consist of a mass of cells destined to become
red corpuscles ; and some of them are typical erythroblast,
while others are of the character of the marrow cells or corre-
spond to what Lowit calls leucoblasts, and others still repre-
sent intermediate stages. None of these cells can be regarded
as leucoblasts, according to the definition of Lowit, since at
this time no typical leucocytes are found in the circulating
blood. The embryonic cell from which the erythroblast is
derived is found in the marrow of the adult as an ordinary
marrow cell. In fact, the marrow cells seem to be undiffer-
entiated cells, like the cells of the original mesoblast; and,
while some may change to erythroblasts, others become loaded
with coarse granules or develop into the fat cells of the yellow

92 » WHOWELL: [VOES LV:

marrow. Of course, there may be a difference in structure in
these apparently similar cells, according to the fate which
befalls them; but, if so, it is not apparent as a morphological
characteristic. The ordinary marrow cell, as has been de-
scribed, is characterized histologically by its vesicular nucleus,
which has one or more prominent nucleoli and a scanty reticu-
lum. In the forms intermediate between this and the erythro-
blast we find that the nucleoli, or nucleolar matter, becomes
scattered throughout the nucleus in the form of smaller gran-
ules ; while the reticulum becomes more pronounced, and unites
with the granules to give the characteristic nucleus of the
erythroblast. While in the latter cell, therefore, we have no
distinct nucleoli, we do have a number of small granules of
nucleolar material situated at the nodal points of the reticulum.
This transformation from a marrow cell to an erythroblast does
not take place by gradual changes going on in one cell, but
makes its appearance more or less gradually in successive gen-
erations. The original marrow or embryonic cell multiplies
by indirect division; and the daughter-cells, instead of having
a single large nucleolus, have several smaller ones scattered
throughout the nucleus and connected with its reticulum,
showing thus an approximation to the structure of the erythro-
blast, the cells also being of a smaller size. These cells in
turn multiply ; and their offspring either become erythroblasts
or at least resemble them more closely. One cannot say how
many generations—one or more—are necessary for the
change. All that can be observed is that between the large
embryonic cells and the smaller erythroblasts there are found
cells intermediate in size and in the structure of the nucleus;
and it seems more reasonable to suppose that thesé changes
take place after successive divisions during the re-formation of
the nucleus from the chromatin filaments rather than from a
process of condensation and alteration going on in each cell.
Denys (14) has found in the marrow of birds that the erythro-
blasts are separated from the other elements of the marrow,
and lie in cords, which are in reality a part of the vascular
system of the marrow. I have described a similar arrange-
ment in the liver of the young embryo cat. But if such an
arrangement of the erythroblasts exists in the marrow of the
cat, it is certainly very much obscured, as repeated examina-

No. 1.] BLOOD CORPUSCLES. 93
tions of sections of the marrows of cats of all ages has not
revealed a separation of this character. On the contrary, the
erythroblasts seem to be scattered among the other elements
of the marrow without any apparent regularity. It is possible
that careful injection of the marrow will throw more light upon
the subject. On @ priori ground, I should think that in the
mammalian marrow there must be some such arrangement
as that described for the bird and the embryo, as it would
furnish the simplest explanation of the way in which the
newly formed red corpuscles develop and gain entrance into
the circulation, and would prove that the process of formation
in the adult and foetus and among the chief classes of verte-
brates is essentially the same. The embryonic cells from
which the erythroblasts are formed must also, of course, lie
in the unformed vessels with the erythroblasts, as is the case
in the embryo.

Growth and Reproduction of the Nucleated Red Corpuscles.

Since the observations of Bizzozero (19) it has been known
that the nucleated red corpuscles multiply by indirect division
(karyokinesis) like most of the other cells of the body. Though
his observations have not been disputed, other writers have
described different methods of growth, some of which have
been mentioned already. Foa and Salvioli (13) believe that
the nucleated red corpuscles are recruited continually from
the giant celis, Lowit (29) that they are developed from the
erythroblasts, and Malassez (27), Osler (28), and others take
a similar view. None of them, except Bizzozero, seem to lay
much stress upon the independent reproduction of the nucleated
red corpuscles themselves. It is quite easy to show, never-
theless, that Bizzozero’s observations are perfectly correct, and
that not only the erythroblasts, but the nucleated red corpus-
cles also, multiply by indirect division. Simple examination of
teased specimens of the marrow, especially of kittens which
have been bled severely, gives usually a number of corpuscles
undergoing division, such as are shown in Figs. 5 and Io.
Specimens teased in methyl green solution show sometimes a
portion of the spindle, as indicated in the figure; but the chro-
matin filaments are not well preserved. The reagent seems
to swell the filaments into a mass, but, in spite of this, it is

94 HOWELL. [Vou. IV.

not difficult to recognize the chief stages of karyokinetic divis-
ion. When the marrow is preserved in Flemming’s solution,
and the sections are stained in saffranin, the nuclear figures are
very well preserved, and undoubted nucleated red corpuscles,
showing the skein, monaster and diaster, can be obtained with-
out trouble, as shown in Fig. 10. Nucleated red corpuscles
with two nuclei and the cell partially constricted between, —
that is, the last step in the process of division, —are especially
common. We must admit, then, that the nucleated red corpus-
cles have the power of independent multiplication. But this
power of reproduction is not unlimited; and this, it seems to
me, is an important fact which has hitherto been overlooked.
It is not difficult to determine when the cell has lost its power
of reproduction: it is indicated plainly by the appearance of
the nucleus. The changes in the structure of the nucleus of
the nucleated red corpuscle have been described already in
detail, especially the two extremes designated as the mature
and immature form of the nucleus. The immature nucleated
red corpuscles have a nucleus like that of the erythroblast,
preserving a definite reticulum, and, like the erythroblast, it is
capable of karyokinetic division. But the offspring or daughter-
cells of this form have nuclei belonging to the intermediate class,
in which the reticulum is less marked, and the whole nucleus
shows a tendency to diffuse staining. These cells are very
common in the marrow, and it is probable that they also are
capable of multiplication. But sooner or later the offspring
of these cells show nuclei with no reticulum at all, and stain-
ing diffusely and deeply with the different dyes. This is the
mature form, and is further characterized by the deeper color
of the hemoglobin in the cell substance. This cell is now
ready to lose its nucleus, and become an ordinary red cor-
puscle; and, as far as I can determine, nucleated red ‘cor
puscles which have reached this stage are incapable of any
further multiplication. The mature corpuscles are usually
smaller than the immature forms, as the successive offspring
show a gradual diminution in size both of the nucleus and the
cell substance. It is impossible to say how many generations
intervene between the youngest nucleated red corpuscle, in
which hzmoglobin has just appeared, and the mature form,
with its peculiar nucleus and greater haemoglobin contents.

No. 1.] BLOOD CORPUSCLES. 95

The number, of course, may not be constant, at least not for
different conditions of life. All that one can actually observe,
and this point I wish to emphasize, is that the cells which
I have described as the mature and immature forms of the
nucleated red corpuscle really exist in the marrow at all times,
that the latter undoubtedly multiply by karyokinesis, and that
the former bear every indication of being nearer the condition
of the non-nucleated red corpuscle, both in size and depth of
color, and in the fact that they are no longer capable of repro-
duction. The theory which I have suggested offers a simple
explanation of these phenomena. One other hypothesis which
might be suggested, and which has in fact been proposed, is
that the nucleated red corpuscle, after it has been formed from
the erythroblast by the development of haemoglobin, begins to
undergo a process of condensation which results in making
both the cell and the nucleus smaller. But this theory does
not take into consideration the fact that what I have called
the younger forms of the nucleated red corpuscle are without
doubt capable of active multiplication, and that the offspring
seem to show in general a diminution in size and a definite
change in the character of the nucleus.

The Transformation of the Nucleated Red Corpuscle to the Red
Corpuscle of the Blood.

The essential factor in the transformation is the loss of the
nucleus. After it was known that in the foetus the nucleated
red corpuscle loses its nucleus and changes to the non-nucleated
form, Kolliker (3) proposed the theory that the nucleus is de-
stroyed by absorption within the cell. The absorption may be
preceded by a fragmentation of the nucleus more or less com-
plete, such as one often sees in examining the blood of a young
embryo. Kdlliker does not seem to have given any microscopic
proof for his view other than the partial disintegration of the
nucleus. Neumann (50), after he had clearly shown that the
nucleated red corpuscle exists also in post-natal life as the pre-
cursor of the non-nucleated form, adopted the theory of Kolliker
to explain the disappearance of the nucleus. He was able to
follow the process best in the human foetus (five months), and
describes the nucleus as becoming smaller, more homogeneous,
and finally notched or indented. In addition, he describes red

96 HOWELL, [Vot. IV.

corpuscles with only one or two small granules of nuclear
matter, which he takes to represent the last step in the disap-
pearance. There is very little satisfactory proof, then, for the
theory, since no one, of course, has been able to follow the
process through all its changes, and the appearances described
above might easily be explained in other ways. Nevertheless,
the theory has been generally adopted by those who believe in
the nucleated red corpuscle and its functions. Malassez, of
course, upon his theory of budding, is not obliged to explain
the loss of the nucleus, nor are those who believe in an endog-
enous formation of the red corpuscles; but, outside of these
theories, which cannot be said to have a strong support at
present, the general belief among histologists is that the nucle-
ated red corpuscle loses its nucleus by absorption in the way
described by Kolliker and Neumann. There seems to be, in-
deed, only one other alternative: if the nucleated red corpuscle
changes to the non-nucleated form, the nucleus either disap-
pears by absorption within the cell or by extrusion from the
cell. This latter view has been seriously supported only by
Rindfleisch (26). As I have stated in the historical review,
Rindfleisch believes that the nucleus escapes from the nu-
cleated red corpuscle surrounded by a small layer of colorless
protoplasm, and leaves behind a bell-shaped corpuscle which
eventually becomes a biconcave disc. He figures corpuscles
in which the nucleus was seen in the act of escaping from the
cell. Others have seen similar examples of extruding nuclei,
but have concluded that it was an accidental and not a, normal
phenomenon. The chief result of my own work has been to
obtain what seems to me indisputable evidence that the extru-
sion of the nucleus is the normal method by which the nucle-
ated red corpuscle loses its nucleus and passes into the non-
nucleated form. Unlike Rindfleisch, I have never been able
to discover with the highest objectives (Zeiss Hom. im. 4 and
apochromatic im.) that the escaping nucleus has an envelope
of protoplasm round it. On the contrary, it goes out of the
corpuscle entirely naked, and can be found as a free nucleus
in sections and teased specimens of the marrow, and also in the
embryonic liver, as has been previously described by Neumann
(see Fig. 2). In many cases in the marrow, and especially in
the foetal liver, I have seen the homogeneous nucleus partially

No. 1.] BLOOD CORPUSCLES. 97

segmented or notched in the way described by Kolliker (?) and
Neumann, and interpreted by them as an indication that the
process of absorption had begun. Nevertheless, I have seen
nuclei of this character already partially extruded from the
cell, showing that the partial fragmentation of the nucleus is
not conclusive proof that it is in process of absorption. To
show that the escape of the nucleus is a normal and constant
phenomenon we have the following facts :

In specimens of the marrow of kittens and adult cats, espe-
cially after repeated bleedings, and also in the blood-forming
organs of the embryo when teased out in their own serum and
stained with methyl green, one can easily find very many
examples of nucleated red corpuscles in the act of losing their
nuclei. In some animals the number of examples is striking
—a dozen or more may be seen in a single specimen; while at
other times, especially in unbled animals, it may be difficult to
find a single example. But in bled animals, especially bled
kittens, in which it is fair to suppose that the process of blood
formation is greatly accelerated, no difficulty will be found in
obtaining a number of examples showing all the steps in the
act of extrusion, from the time when the nucleus has only an
eccentric position up to the period when it lies completely
outside the cell, as shown in Fig. 2. The frequency with
which this phenomenon occurs, especially when the production
of red corpuscles is increased, requires that it should be ex-
plained. Now it must be a normal occurrence, or else it comes
from the action of the reagents, or possibly it is the result of
post-mortem changes taking place in the cell after removal
from its normal environments.

There are a number of facts which may be adduced to show
that the phenomenon is not an accidental or post-mortem
change, but a normal occurrence. In the first place, most of
the specimens were obtained from pieces of the marrow (or liver
in the embryo) which were taken as quickly as possible from
the animal after killing, and treated with methyl green, so that
only a few minutes intervened between the death of the animal
and the action of the methyl green. This reagent, as is well
known, is an excellent fixative. It preserves fairly well the
nuclear figures of karyokinesis, and fixes the blood plates quite
as well as osmic acid. It is not likely, then, that such a re-

98 HOWELL. [Vou. IV.

agent would cause in one of the cells of the marrow an expul-
sion of the entire nucleus, and in others preserve the delicate
karyokinetic figures; and, on the other hand, the fact that
the marrow was submitted to the action of the reagent so
quickly after the death of the animal, probably before the
death of the marrow cells, precludes the possibility of post-
mortem changes of the nature required to expel the nucleus
from a cell. So in several cases, both in the adult and the
kitten, after severe bleeding, and also in the foetus, I have
found examples of extruding nuclei in the circulating blood. In
these cases, the drop of blood was taken from the living animal
and mixed at once with the methyl green, so that there was
no opportunity for post-mortem changes (see Fig. 2). More-
over, I have obtained cases of extrusion frequently in sections
of marrow which had been taken from the animal as quickly
as possible after bleeding, and hardened in mercuric chloride.
Here, again, we have an excellent fixative quickly applied,
which ought to have prevented post-mortem changes on the
one hand, and on the other should not have acted with such
violence upon one of the kinds of cells found in the marrow
as to force out the nucleus. To adopt either one of these hy-
potheses to explain the extrusion is not permissible in the light
of our knowledge of the action of this reagent on cells in
general.

In the second place, all the red corpuscles which I have seen
with the nuclei extruding belong to the class of mature nucle-
ated red corpuscles. Never have I seen a nucleus extruding
from a nucleated red corpuscle which showed a nuclear net-
work. This indicates that the escape of the nucleus is not
owing to any accidental or post-mortem changes, since there is.
no reason under such conditions why all kinds of nucleated red
corpuscles should not have been affected in the same way. It
shows, also, that the extrusion of the nucleus is the normal
end to the life history of the nucleated red corpuscle, since it
is found only among those which seemed to have reached full
maturity and are prepared, as far as size, color, etc., are con-
cerned, to become ordinary red corpuscles. It seems to me
that this fact is a very important one in its bearing upon the
question under discussion, and, so far as I know, it has not
been noticed before. I have been impressed with this pecu-

Nos I:] BLOOD CORPUSCLES. 99

liarity of the extruding nucleus, not only from the study of
teased specimens stained in methyl green, but also from an ex-
amination of sections of marrow stained with hzmatoxylin,
eosin, and saffranin. It is not difficult to find in these sections
a nucleus in the act of extruding, and in all cases such nuclei
belonged to the mature nucleated red corpuscles as shown by
the fact that they stain with saffranin in preference to the
hematoxylin in the way that I have described. Osler (28),
who has figured and described the extruding nuclei, but does
not think they occur normally in the living tissue, states that
they are more abundant in the marrow twenty-four hours after
death than in the fresh cadaver. This may well be, even if
the phenomenon is a normal occurrence, since the marrow
cells probably survive some hours after somatic death, and the
mature nucleated corpuscles may lose their nuclei partially or
completely as in life, and the stoppage of the circulation would
lead to an accumulation of such examples in the marrow.
However, in the cat, at least, under the conditions mentioned,
they can be found in abundance immediately after death.
Whether or not with this animal the number is increased
twenty-four hours after death I have never determined. The
presence of granules within a newly formed red corpuscle has
been taken as a proof that the nucleus is absorbed within the
cell, the granules being looked upon as remnants of a former
nucleus. The existence of such cells cannot be questioned ;
but, taken alone, they cannot be considered as strong proof for
the theory of absorption nor as any objection to the theory of
extrusion; for I have in a number of cases found red corpus-
cles containing these granules in which, nevertheless, the
nucleus was in the act of extruding, as shown in Fig, 2.
The granules in such cases evidently did not mean that the
nucleus had been absorbed. Erb (4), it will be remembered,
described such corpuscles in the circulating blood; they form
his transitional stage between the white and red corpuscle.
Lowit (29@) has newly discovered them, especially in the blood
of certain veins after treatment with a modified Pacini’s liquid,
and has laid great stress upon them as transitional forms be-
tween the erythroblasts and red corpuscles. Foa (41) also has
recently described granulations of this character as part of the
normal structure of every red corpuscle and easily brought out

100 HOWELL. [Vor. IV.

by appropriate treatment with methyl blue and chromic acid.
I have met with corpuscles containing granulations very fre-
quently, particularly in the blood-forming organs. In sections
or teased specimens of the blood-forming organs, the newly
formed red corpuscles are often characterized by the ease with
which they lose their haemoglobin. Under such conditions
the granulations come out very distinctly. Sometimes the
granules — which stain, by the way, like nuclear chromatin —
are so arranged as to represent the outline of the nucleus, and
I have obtained such cells in which the nucleus at the same
time was fixed in the act of extrusion (see Fig. 7). It is an
interesting fact with reference to the corpuscles containing
granules that they are usually newly formed corpuscles, and on
that account occur most abundantly in the foetal blood or in
the blood-forming organ (marrow) of the adult. There is no
evidence to show that the granules are the last remaining frag-
ments of an absorbed nucleus. On the contrary, all that we
know about them is opposed to suchaview. They must be
looked upon, it seems to me, as bits of the nuclear chromatin
(membrane) left behind when the nucleus leaves the cell.
What their fate is, whether finally absorbed or whether they
last throughout the life of the corpuscle, is not known.

In this connection I may refer to a curious phenomenon
which has come under my notice and upon which I am now
working. On one occasion, after bleeding a medium-sized cat
very severely (a loss of 90 cc. of blood), it was found upon
examining the blood twenty-four hours afterward that the ma-
jority of the corpuscles in the animal contained a single good-
sized piece of nuclear matter, too large to be called a granule,
but having the shape and appearance of a large nucleolus.
This fragment stained readily with methyl green just like the
nucleus: it could be seen also in the unstained corpuscles as
a refractive particle (see Fig. 4). I cannot recall ever having
seen anything corresponding to this described, except, perhaps,
the first stage of the malarial germ as pictured by Marchiafava,
with which, indeed, the appearance seen by me seemed to be
identical. Closer examination of the corpuscles showed that
the fragment of nuclear matter, as I shall call it, always lay
imbedded in the periphery of the spherical corpuscle after
treatment with the methyl green. When care was taken to

No. 1.] BLOOD CORPUSCLES. IOI

make the corpuscle rotate in the liquid, I found no exceptions
to this position of the fragment. A remarkable thing about
the phenomenon was its persistence. Even two weeks after
bleeding, a drop of the blood taken from the ear showed a
number of these corpuscles. I was successful afterwards in
getting the same result from other cats, though I had many
failures. The necessary condition seems to be that the ani-
mal should be bled quickly and severely. At first, I supposed
that the objects in question were simply large granules floating
in the blood which had adhered to the corpuscles; but I was
soon convinced that this was not the case. The fragments
could not be detached from the corpuscles either by shaking
or by the addition of water, acetic acid, and other reagents,
which dissolve out the haemoglobin from the corpuscles. More-
over, a number of corpuscles were without the fragments, and
in normal cats no such appearance could be obtained. The
only satisfactory explanation of the phenomenon which has
occurred to me is that the fragment is a bit of the nucleus
left adhering to the corpuscle at the time that the nucleus
escaped. Under the conditions necessary for the appearance
of the phenomenon, we may suppose that the process of pro-
duction of new red corpuscles was vastly accelerated, and that
therefore the extrusion of the nucleus was not as perfect as
under normal conditions. The portion remaining in the cor-
puscle is not absorbed at all, but probably remains with the
corpuscle up to the time of its dissolution. Whether or not
my view as to the origin of the fragment is correct, there
can be no doubt that it is not absorbed in the corpuscle
while in the blood, but remains with it up to the time of its
destruction. At the suggestion of Dr. Bowditch, I had hoped
to use the phenomenon to measure the average length of life
of the red corpuscle of circulating blood, but have hitherto met
with certain difficulties which I hope soon to overcome.

After I was convinced from a study of teased specimens and
sections that the nucleated red corpuscle loses its nucleus by
extrusion, it seemed to me that it might be possible to watch
the process taking place in the living cell. The experiments
that I made for this purpose were not very numerous, for
reasons that will be given below; but they were successful in
a measure, at least. The method employed was to use the

TO2 HOWELL. [VoL. IV;

marrow of very young kittens, about a week old, which had
been bled rather severely from the jugular vein some twenty-
four hours previously so as to increase the processes of blood
formation. The marrow was teased out quickly in an indifferent
solution of some kind upon a slide, the edges of the cover slip
were sealed with paraffin, and the slide was kept at a tempera-
ture of 37-38° C., by means of a warm stage. Various indif-
ferent solutions were tried, such as normal salt solution, am-
niotic liquid, aqueous humor, and blood serum; but successful
experiments were obtained only when the serum of the same
animal was used as the teasing liquid. The other liquids were
given only one or two trials; but as far as the experiments
went, they indicated that even such liquids as normal salt solu-
tion and amniotic liquid are sufficiently abnormal to cause a
suspension of the living activities of the nucleated red cor-
puscles. Two experiments were made with the animal's own
serum as the teasing liquid. In the first I saw two cases of
extrusion, in the second only one, in which I was able to
follow the process in part at least. In picking out the cor-
puscle to be observed I found it was necessary to choose one
in which the nucleus already showed signs of extrusion, for
otherwise it would be impossible except by accident to select a
cell which had reached the proper stage. It was not difficult
to find a number of corpuscles with the nucleus beginning to
extrude. Many of them showed no further change, though
watched for some time; but in three cases I was able to follow
the last stages of extrusion until the nucleus lay completely
outside of the cell. Sketches were made of one of these suc-
cessful cases, though unfortunately it was the most incomplete
of the three. The drawings are given in Fig. 2. The experi-
ments were discontinued because of the improbability of obtain-
ing a cell in which the process could be watched from the
beginning to the end. The results, as far as they went, were
still further proof to me that the extrusion of the nucleus is a
normal phenomenon, since it was obtained only when the con-
ditions were most favorable for preserving the life of the cell.
I have spoken of the escape of the nucleus as an extrusion, but
it is quite possible that migration would be a more accurate
term. I was not able to convince myself that the escaping
nucleus in the living cell showed definite amoeboid movements,

No. 1.] BLOOD CORPUSCLES. 103

though the sketches made (see Fig.) seem to indicate that such
movements occur. The figure shows, indeed, that the cor-
puscle as wellas the nucleus undergoes changes in shape; but
this was caused in part at least by the rolling of the cell so as
to present different surfaces in successive drawings. A priori,
it seems much more likely that the extrusion should result
from some active movement on the part of the nucleus rather
than from contractile changes in the cell substance. For it
seems to be generally admitted now that in certain cells —
lymph cells especially (Arnold) — not only movements of the
nucleus may take place, but movements of the granules and
filaments in the nucleus. After the escape of the nucleus, the
spherical red corpuscle eventually becomes a biconcave disc,
I have not attempted to follow this change, though I feel con-
vinced that the bell shape which Rindfleisch ascribes to the
corpuscles which have just lost their nuclei is a mistake. The
red corpuscles even of the circulation, as is well known, fre-
quently take this shape when treated with reagents of any kind,
or even when examined without the addition of any liquid. It
seems to me very natural to suppose that the biconcavity of
the mammalian corpuscle is directly caused by the loss of the
nucleus from its interior. Certainly as long as the corpuscles
in the foetus and the adult retain their nuclei, they remain
more or less spherical, and after they lose their nuclei they
become biconcave. The mechanical conditions of the circu-
lation undoubtedly have some influence upon this change, but
the initial cause lies apparently in the migration of the nuclear
mass from the middle of the cell, so that the viscous material
of the corpuscle is permitted to sink in. The biconcavity is of
course a decided physiological advantage, as the absorptive
surface is thereby considerably increased, so that upon the
doctrine of natural selection, one can readily understand why
such a variation should have become permanently established.
Among the Camellide, it is true, we have biconvex non-nu-
cleated corpuscles. So far as I know, no one has investigated
the haematopoietic function in these animals, but it is possible
that small spherical erythroblasts are not formed in them as in
the other mammalia.

If we grant that the nucleated red corpuscle loses its nucleus
by extrusion when it passes to the non-nucleated form, then

104 HOWELL. [Vox. IV.

we are in a position to explain the budding corpuscles of
Malassez. In several instances, when examining the marrow,
I have met with appearances which seemed to justify Malas-
sez's theory. Nucleated red corpuscles were seen with one or
more non-nucleated corpuscles apparently budding out from
them. Sketches of such cells are given in Fig. 3. They seem
to me, indeed, to be better examples, as far as the drawings
go, of the process of budding than those figured in Malassez’s
(27) own paper. I cannot say that these examples of budding
are common; on the contrary, I.obtained them clearly only
in two cases, in both of which the notes of the experiments
record that the animal had been bled so severely that it did
not make a good recovery, but remained weak and anemic;
and it is possible that this is sufficient to explain their occur-
rence. I was at first inclined to believe that we must admit
that, under certain conditions at least, new red corpuscles may
be produced by budding in the way described by Malassez.
But a simpler explanation of these forms suggested itself.
What seem to be examples of budding are most probably cases
of multiplication of nucleated red corpuscles by division, in
which the process was not carried out to the complete sepa-
ration of the newly formed corpuscles, though from one or
more of the new cells formed the nucleus has escaped, leaving
the non-nucleated corpuscle as an apparent bud on its sister-
cell. As evidence for this explanation, one may find in the
apparent buds granules of nuclear matter staining blue with
the methyl green, such as I have described as occurring some-
times in the newly formed red corpuscle after the extrusion of
its nucleus. Moreover, one frequently meets with two, three,
or more mature nucleated red corpuscles joined in a cluster
or chain as the result of recent division, and such as would
produce apparent examples of budding if one or more of the
cells lost their nuclei. This would be more likely to happen,
of course, in animals in which too severe a bleeding had im-
paired the processes of cell development in the marrow as in
the other tissues of the body. The explanation that I have
adopted seems to me to be preferable to supposing that in the
marrow new blood corpuscles are formed from the same cells
by two entirely different methods of reproduction.

No. 1] BLOOD CORPUSCLES. 105

Fate of the Extruded Nucleus.

If the nucleus of the nucleated red corpuscle is extruded,
the next point to be determined is what becomes of it. Naked
nuclei, similar in all respects to the nuclei of the mature nu-
cleated red corpuscles, can be found easily in the marrow, where,
indeed, several observers have called attention to them, and
also in the foetal liver at the time of its haematopoietic activity,
where they have been noticed before by one writer, at least, —
Neuman (5@),—who has described them very carefully and
attributed to them some function in connection with the
production of new corpuscles. It is fair to assume that the
free nuclei are turned out into the blood stream along with
the new red corpuscles. In that case, one of two fates awaits
them. Either they persist as a morphological element of the
blood, or they are dissolved in the blood plasma. Upon the first
hypothesis, we can only suppose that the free nuclei become
the blood plates, as no other element of the blood resembles
them in size or structure. This theory has, in fact, been pro-
posed by Gibson, though as far as I can see, he gives no proofs
in its favor. I was also at first impressed with this idea; but
the only experiment which suggested itself to me to test the
hypothesis gave me unfavorable results. The nuclei of ma-
ture nucleated red corpuscles, when treated with the triple
stain of haematoxylin, eosin, and saffranin, show a preference
for the saffranin, while other nuclei take the haematoxylin.
If the blood plates are derived from these nuclei, they ought
to show something of the same behavior toward the triple
stain. On the contrary, specimens of blood plates treated
with the triple stain always take the hematoxylin, though they
do not stain deeply. The method of preparing and staining
the blood plates was as follows. A drop of blood was placed
upon a slide, a cover slip was dropped upon it, and moved
round once or twice. The slip was then taken off, and by this
time a number of blood plates had adhered to its under side.
It was next immersed in Hayem’s liquid, to fix the blood plates
and wash off the excess of blood plasma, and was then hard-
ened like a piece of marrow in mercuric chloride, followed by
alcohol, and afterwards stained. I obtained in this way good
specimens of blood plates, somewhat deformed, of course, in

106 HOWELL. [Vou. IV.

consequence of the time which elapsed before getting the slip
into Hayem’s liquid. The method also gave beautiful per-
manent specimens of fibrin reticulum and of red corpuscles,
which retained their normal shape and stained deeply with
eosin. The negative result of this experiment, together with
certain other facts which will be given later in speaking of the
blood plates, convinced me that there is no connection between
the blood plates and the nuclei of the mature nucleated red
corpuscles. There remains, then, only the theory that the
liberated nuclei are dissolved in the blood plasma, and go to
form in all probability one of the proteids of the blood.

It is, perhaps, unwise to speculate further upon the fate of
the dissolved nucleus without some experimental basis to reason
upon. However, my idea is that the free nuclei are dissolved
in the blood plasma while still in the blood-forming organ. I
have seen appearances in the marrow in sections which may
represent this process of dissolution; that is, one meets occa-
sionally with what seem to be globules of varying size from
tiny drops to spheres larger than a white corpuscle which, like
the free nuclei, stain deeply with saffranin, though of a different
tint. Usually these are found in clusters of different sizes,
and possibly they represent the free nuclei, undergoing
changes preparatory to solution, though I have not found in-
termediate stages. These globules are evidently not a fat of
any sort, as one might suppose from their general appearance,
since otherwise they would have been dissolved during the
process of imbedding. With reference to the material produced
by the nuclei after solution, there seemed to me certain reasons
for believing that the fibrinogen of the plasma is the product
formed. Influenced chiefly by this idea, I asked Mr. Dreyer
of the Johns Hopkins University, and formerly assistant in
physiology, to investigate the changes in the blood plasma
caused by severe bleeding. His results, which are very inter-
esting in a number of ways, have not yet been published. It
may be said, however, that with reference to the fibrinogen,
he found that its percentage in the plasma was always increased,
sometimes nearly as much as 100 per cent., over what it had been
in the same animal before bleeding, the analysis in all cases
having been made twenty-four hours after the bleeding. This
striking increase in the fibrinogen is more remarkable because

No. 1] BLOOD CORPUSCLES. 107

at the same time there was usually a diminution in the total
proteids of the blood. As far as it goes, this result is in accord
with the hypothesis that the fibrinogen is formed from the
liberated nuclei of the nucleated red corpuscles. I have in
progress other experiments for the purpose of further testing
the hypothesis.

The Hematopoietic Function of the Spleen.

All the facts bearing upon this question have already been
stated in various parts of this paper. It may be convenient,
however, to bring them together in the form of a brief statement
of the different views held. It is well known and universally
admitted that for a certain period during embryonic life, the
spleen takes part in the formation of red corpuscles, as is shown
by the fact that numerous nucleated red corpuscles, some of
them in the act of multiplication, may be found in it. Shortly
after birth, the spleen no longer contains nucleated red cor-
puscles and for this reason the majority of writers who believe
that these cells are the predecessors of the ordinary red cor-
puscles, have concluded that under normal conditions the spleen
during extra-uterine life takes no further part in the production
of new red corpuscles. This function is relegated entirely to
the red marrow. On the other side, a number of investigators,
while admitting the absence of nucleated red corpuscles from
the spleen under ordinary conditions, have nevertheless classed
it with the lymph glands under the head of the hamatopoietic
organs, because they hold that the colorless corpuscles from
which the nucleated red-corpuscles are formed are produced in
this organ. The most elaborate form of this theory is found
in the works of Lowit (29) upon the origin of the erythroblasts,
an account of which is given in the historical review. For my
own part, I have not been able to convince myself that erythro-
blasts are continually forming in the spleen or lymph glands,
as I have not been able to get any intermediate stages between
them and the nucleated red corpuscles, and therefore take sides
with those who think that the red marrow is the only organ as
yet discovered, in which new red corpuscles are produced dur-
ing post-natal life. This statement applies, however, only to
the spleen under ordinary conditions of life. Bizzozero (19)

108 HOWELL. [Vou. IV.

was the first to discover that in a number of animals, after
severe and repeated bleedings, the spleen again might contain
nucleated red corpuscles showing signs of active multiplica-
tion. This was denied by Neumann, who held that after such
an operation, the nucleated red corpuscles found in the spleen
were not more numerous than those present in the circulating
blood. But Bizzozero’s observations have met with confirmation
at the hand of others, — Gibson (33), Foa (22), e¢ a/.; and I also
in several cases have been able to show without any difficulty
that in the cat, after severe and repeated bleedings, and in some
cases after a single strong hemorrhage, nucleated red corpuscles
can be found in the spleen with every indication that they are
multiplying there. The balance of evidence is strongly in favor
of this power of the spleen to resume its embryonic function
when the demand for new red corpuscles is very urgent. In
what way severe anemia stimulates the spleen to a renewal of
its haematopoietic actively is not known. It is very interesting
in this connection to find that, when the spleen of the adult
is partially excised, it is regenerated, and during the regenera-
tion not only nucleated red corpuscles, but giant cells are found
just as in the developing spleen of the embryo (Foa [42],
Tizzoni [43], Griffini [44]). It may be that in the adult spleen
a number of undifferentiated or erythroblastic cells are contained
which become aroused to activity in consequence of severe
anzemia, for the same reason, whatever it may be, that the cells
of the marrow are stimulated to increased growth and multipli-
cation by the same conditions.

Life-History of the White Corpuscles and Blood Plates.

It is quite generally agreed that the origin of the white cor-
puscles of the blood is to be found in the lymph leucocytes, or
lymphocytes, to borrow a convenient term, which in turn are
formed in the lymphoid tissues of the body, and especially in
the so-called compound lymphatic glands. The lymphocytes
are characterized by a vesicular nucleus, usually with a nucle-
olus and a scanty reticulum, and by a very small protoplasmic
envelope. In the blood we meet with two chief varieties of
leucocytes, — uninucleated and multinucleated. The uninucle-
ated forms do not all have the same structure: some of them

No. 1] BLOOD CORPUSCLES. 109

resemble exactly the lymphocytes, and may be regarded as
lymphocytes newly arrived in the circulation and as yet un-
changed in structure (Erb, Lowit). These are characterized
physiologically, as was pointed out some years ago by Schultze
(45), by not possessing the power of making amoeboid move-
ments. A second form of uninucleated leucocyte is character-
ized by its large, finely granular, protoplasmic envelope. This
form is amoeboid, and it seems most reasonable to suppose that
it is derived from the first form, or lymphocyte, since this
latter cell is the only or chief form in which the leucocytes of
the lymph enter the blood. The first variety of uninucleated
leucocyte passes into the second in consequence of a growth
in the cell protoplasm while in the blood current, the proto-
plasm meanwhile acquiring the power of contractility. A third
variety of uninucleated leucocyte, and what seems to repre-
sent a third stage of development, is like the last, except that
the nucleus is no longer oval or spherical, but is drawn out to
an elongated strap shape, and may take either a horse-shoe
form or may be more or less coiled into a spiral. This form
of cell is especially abundant in the cat’s blood, and possesses
the most active amoeboid properties. The origin and meaning
of the multinucleated forms has been for some time a subject
of dispute among histologists. Formerly it was generally
thought that they represented cells in process of multiplication
by direct division; and this view is still warmly supported by
Arnold and others. The normal fate of the multinucleated
cell, according to this view, is to divide into a number of cells
corresponding to the number of nuclei. Others, and especially
Lowit (29), have urged that the multinucleated forms are cells
on the way to disintegration, and the so-called nuclei are made
simply by the fragmentation of the nucleus of a uninucleated
leucocyte, and represent the first step in the process of de-
struction. As far as my observations go, they support Loéwit’s
view. I have never seen any indication of the multinucleated
cells segmenting to form new cells. On the contrary, there is
every reason to believe that they are undergoing a course of
retrograde changes, the normal termination of which will be
the disintegration and dissolution of the cell. With reference
to the derivation of the multinucleated forms from the uni-
nucleated by fragmentation of the nucleus, I have been able

110 HOWELL. [Vot. IV.

to find all intermediate stages in the process as shown in
Fig. 16. They are derived always from the third variety or
third stage in the life of the uninucleated leucocyte, the
elongated nucleus breaking up into the smaller fragments ;
and it is not difficult to find cells such as are shown in the
ficure in which the fragmentation is going on. According to
this view, the different varieties of leucocytes found in the
blood are in reality different stages in the life-history of the
white corpuscle, and pass one into the other. To complete
the life-history, one other stage must be described, —that of
the disintegration of the multinucleated form. A close exami-
nation of the multinucleated cells, especially when in the act
of disintegrating, has impressed me with the belief that the
fragmented nuclei persist for a certain time in the circulation
as the blood plates, though doubtless the blood plates also,
sooner or later, go into solution.

This view of the origin of the blood plates is not new.
Gibson (33) supports it, and gives some evidence in its favor;
and Hlava (47) especially has given a number of arguments —
none of which, however, are very conclusive —to prove this
derivation.. One is led, at first, to such a theory by noticing
the very striking resemblance between well-preserved blood-
plates and the fragmented nuclei as far as size, shape, and
general appearance are concerned. This resemblance is still
further increased when the blood plates are examined in the
blood of an animal which has been repeatedly bled. Under
such conditions, one gets, or may get, blood plates which have
one or more granules within them staining more deeply than
the rest of the plate, and resembling very closely the chromatin
granules found in the fragmented nuclei of the leucocytes, as
shown in Fig. 6. Something similar to this seems to have
been obtained by Afonassiew. We may suppose in this case
that the increased activity in the processes going on in the blood
in connection with the regeneration, not only of its formed
elements, but of its characteristic proteids, have led to a more
rapid breaking down of the leucocytes, and that some of the
fragmented nuclei are liberated as blood plates before reaching
the usual degree of maturity. There is, moreover, a very close
similarity in the way in which the fragmented nuclei and the
blood plates stain. As far as I have been able to test them,

No. 1] BLOOD CORPUSCLES. Il

they stain alike, except that the blood plates take the stain
more feebly. In the case already mentioned, in which the
preserved blood was treated with a differential stain, successive
staining in hematoxylin, eosin, and saffranin, the blood plates,
like the nuclei of the leucocytes, took the hematoxylin. The
same is true of methyl violet (Gibson) and methyl green. If
this view of the life-history of the leucocytes of the blood is
correct, it seems probable that they play an important part
in the formation of the blood proteids. The young lympho-
cytes increase in size by the formation of new protoplasm ;
and in the end this again passes into solution in the plasma.
Schmidt long ago stated that the paraglobulin of the blood is
derived from disintegrated leucocytes. In fact, if I under-
stand him correctly, he believes that the paraglobulin is all
formed in this way after the blood is shed. Later investiga-
tions of the serum and plasma have shown that this latter
statement is not correct, though there is apparently an increase
in the amount of paraglobulin in the serum over that in the
plasma. Still, it may be considered probable that the para-
globulin of the blood is derived wholly from the breaking down
of the leucocytes, and that the constant supply of paraglobulin
in the blood is derived from the continual disintegration of the
multinucleated leucocytes. The fibrinogen, on the other hand,
is possibly derived from the liberated and dissolved nuclei of
the mature nucleated red corpuscles, and perhaps of the blood
plates also, if they, too, represent nuclear material. We
know little or nothing at present of the genesis and relation-
ship of the blood proteids or of the nutritive value and signifi-
cance of each. The fact that their percentage amounts in the
plasma remain practically constant under many different con-
ditions of nutrition indicates that they are regenerated contin-
ually in proportion as they are used up; but how this happens
is one of the darkest as well as one of the most interesting
points in the physiology of the blood. It seems to me that
the question must be studied, in part at least, upon the hy-
pothesis of their derivation from the formed elements of the
blood in the manner here suggested, somewhat as we look
upon the ground substance, or matrix, of the connective tissues
as having its origin from the cellular elements.

112 HOWELL. [Vot. IV.

SUMMARY.

The chief conclusions to which the investigation has led
may be briefly summarized in the order in which they are pre-
sented in the paper as follows : —

1. In the very young embryo two forms of red corpus-
cles are found,—one large, oval, and always nucleated, re-
sembling the corpuscles of the lower vertebrates, and one
small, biconcave, circular in outline, and found both nucleated
and non-nucleated. The latter are the true mammalian cor-
puscles; the former represent possibly ancestral corpuscles.
The true mammalian corpuscles lose their nuclei by extru-
sion.

2. In the first part of embryonic life new red corpuscles are
produced in the liver from groups of mesoblastic cells outlining
the position of future blood-vessels (veins). The central cells
of these cords become red corpuscles, while the peripheral
ones form the walls of the veins. Similar developing blood-
vessels are found in the embryonic muscular tissue of the
posterior limb. It is probable that new red corpuscles are
formed in all parts of the body where blood-vessels are being
developed.

3. In the second half of the embryonic life red corpuscles
are formed in the liver, the spleen, and the marrow of the
bones, the function being most active first in the liver, then in
the spleen, and finally in the red marrow. In the cat the liver
and spleen lose this function three or four weeks after birth,
and henceforward the red marrow alone produces new red cor-
puscles.

4. The white corpuscles (leucocytes) and blood plates do not
occur in the circulating blood of young embryos, but make
their appearance in later embryonic life. In the human fcetus
of five months both are present.

5. In the healthy animal during extra-uterine life the red
corpuscles are produced only in the red marrow. They occur
first as nucleated cells, the nucleated red corpuscles, found
only in the red marrow of the bones. These cells differ in
structure with their age, and two extreme types may be distin-
guished,—one mature and ready to be converted to a non-
nucleated corpuscle, and one immature, as shown by the char-

No. 1.] BLOOD CORPUSCLES. 113

acter of the nucleus and the amount of hamoglobin. This
latter form multiplies by karyokinesis, and the daughter-cells
sooner or later appear as mature nucleated red corpuscles,
which then lose their nuclei by extrusion, and become non-
nucleated red corpuscles. The biconcavity of the red corpus-
cles is probably caused in the first place by the removal of the
nucleus from the middle of the spherical cell. The liberated
nuclei go into solution in the blood plasma, and probably form
or help to form the fibrinogen of the plasma. The immature
or young nucleated red corpuscles are derived from spherical
colorless cells, erythroblasts, having a definite histological struc-
ture and found in the marrow. These cells multiply actively
by karyokinesis. The erythroblasts in turn are derived from
larger embryonic cells, usually described in the adult as ordi-
nary marrow cells. The structure of the nucleus differs from
that of the erythroblast. The erythroblasts are not derived
each from one of these larger cells by a process of condensa-
tion, but the embryonic cells multiply by karyokinesis, and the
daughter-cells of the first or following generations acquire the
structure of erythroblasts. The chief point in the paper is
the proof that the mature nucleated red corpuscles lose their
nuclei by extrusion, and not by absorption, in changing to the
ordinary red corpuscle of the circulation. The act of extrusion
can be observed in part in the living cells.

6. Very severe and sudden bleeding (in cats) is followed by
the appearance in the circulation of red corpuscles containing
a large fragment of nuclear material. This fragment persists
until the corpuscle disappears. Apparently the greatly accel-
erated production of new corpuscles causes a too rapid extru-
sion of the nuclei, so that a portion remains entrapped in the
corpuscle.

7. The apparent gemmation of non-nucleated red corpuscles
from the nucleated forms, as observed by Malassez, is probably
owing to the multiplication of the nucleated cell and the sub-
sequent loss of a nucleus from one or more of the daughter-
cells before the complete separation of the cells has been
effected.

8. While the spleen of the adult mammal does not take part
in the production of new red corpuscles under normal condi-
tions, it may be made to resume this function in consequence

114 HOWELL. [Vou. IV.

of prolonged or extreme anemia produced by repeated bleed-
ings,

9. The leucocytes of the blood are derived from the lymph
leucocytes (lymphocytes). The latter enter the circulation
as small corpuscles with vesicular nuclei and scanty proto-
plasm, and are not ameeboid. They develop into larger cells,
with finely granular protoplasm which possess amoeboid move-
ments. These have at first an oval vesicular nucleus, which
afterwards becomes elongated and assumes a horseshoe or
spiral shape. From this last form the multinucleated cells are
derived by fragmentation of the nucleus. The fragmentation
of the nucleus is probably followed by the disintegration of the

whole cell.
10. The fragmented nuclei after the disintegration of the cell
persist for a time in the circulation as the blood plates.

LITERATURE:

Wagner. Quoted from Neumann (Zeit. f. Klin. Med., 1881, Vol. III., 411).
Weber. Quoted from Neumann (Zeit. f. Klin. Med., 1881, Vol. III., 411).
Kdlliker. Mik. Anatomie. II., 2, S. 589, 1854.

Erb. Arch. f. Path. Anat. u. Physiol. u. f. Klin. Med. (Virchow). Bd.

34, S. 138, 1865.
5. Neumann. a. Berlin Klin. Wochenschrift. No. 4o, 1868.
. Arch. der Heilkunde. Bd. X., S. 68, 1869.
. Arch. der Heilkunde. Bd. XI., S. 11. ’
Arch. der Heilkunde. Bd. XV., S. 441, 1874.
. Arch. f. d. ges. Physiol. (Pfliiger). Bd. IX., S. 110, 1874.
. Archiv f. Mik. Anatomie. Bd. XI., S. 169, 1875.
. Archiv f. Mik. Anatomie. Bd. XII., S. 793, 1876.

hs Zeit. f. Klin. Med. “Bd: 3,5. AT; 188r.

6. Reichert. Beobachtungen ueber die ersten Blutgefasse u. deren Bildung
so wie ueber die Bewegung des Blutes bei Fischembryonen. 1857.
(Quoted from Feuerstack.)

7. Wenckebach. a. Journal of Anatomy and Physiology. Vol. 19, 1885.

&. Archiv f. Mik. Anat. Bd. 28, 1886.

8. Ziegler. Archiv f. Mik. Anat. Bd. 30, S. 643, 1888.

9. Klein. Sitzungsberichte d. K. Akad. d. Wiss. Bd. 63, 1871.

1o. Balfour. Foster and Balfour. Elements of Embryology. London, 1883,
p02:

11. Gensch. Archiv f. Mik. Anat. Bd. 19, S. 144.

12. His. Archiv f. Anat. u. Physiol. 1882, S. 62.

13. Foa and Salvioli. Archivio delle Scienze Mediche. Vol. IV., p. I.

14. Denys. La Cellule. IV., 203.

15. Ranvier. Arch. de physiol. path. et norm. 1874, 429; 1875, I.

BW N A

FR’ ar Qa &

S

& WN

NN NNN N

NN WN
cM

iS)
\O

30.

eT BLOOD CORPUSCLES. 115

. Schafer. Proceedings of the Royal Society. 1874, 243.

. Feuerstack. Zeitsch. f. Wiss. Zoologie, 38.

. Wharton Jones. Phil. Trans.

. Bizzozero. a. Gazz. Med. Ital. Lombard. 1868, 46; 1869, 24. (Quoted.)

6. Centralbl. f. d. Med. Wiss. 1881.

c. Archives Ital. de Biol. 1, 5, 1882.

d. Archives Ital. de Biol. 4, 329.
Bizzozero and Salvioli. Centralbl. f. d. Med. Wiss. 1879, 273.
Bizzozero and Torre. Centralbl. f. d. Med. Wiss. 1882, 577.
Foa. Arch. Ital. de Biologie. 1, 463.
Osler and Gardner. Centralbl. f. d. Med. Wiss. 1877, 258.
Osler. Centralbl. f. d. Med. Wiss. 1877, 498.
Laache. Deutsche Med. Wochenschrift. 10, 695, 1884.
Rindfleisch. Archiv f. Mik. Anat. 17, 1880.
Malassez. Archives de Physiol. norm. et path. 9, 1882, 1.

. Osler. Cartwright Lectures (Medical News). 1886.
. Lowit. @. Sitzungsberichte d. K. Akad. d. Wiss. 88, 1883.

6. Sitzungsberichte d. K. Akad. d. Wiss. 92, 1885. __
¢. Sitzungsberichte d. K. Akad. d. Wiss. 95, 129, 1887.
d. Sitzungsberichte d. K. Akad. d. Wiss. 95, 227, 1887.
Geelmuyden. Arch. f. path. Anat. u. Physiol. u. f. Klin. Med. (Virchow).
105, 136.

. Hayem. a. Archives de Physiol. norm. et path. 1878, 692.

b.

. Zimmermann. Archiv f. path. Anat. u. Physiol. u. f. Klin. Med. (Vir-

chow). 18, 221.

. Gibson. Journal of Anatomy and Physiology. 20, 100, 1885-86.
. Ejichorst. Centralbl. f. d. Med. Wiss. 1876, 465.
. Obrastzow. Arch. f. path. Anat. u. Physiol. u. f. Clin. Med. (Virchow).

84, 359, 1881.

. Afonassiew. Deutsches Archiv f. Klin. Med. 35.
. Boettcher. Arch. f. path. Anat. u. Physiol. u. f. Klin. Med. (Virchow).

XXIV., 606, 1862, and XXXVI., 342, 1886.

. Sappey. Quoted from ‘‘Eléments figurés du Sang,” Variot. Paris,

1886.

. Cuenot. La Sem. Med. 1888, March 7.

. Hodge. American Journal of Psychology. I., p. I.

. Foa. Abstract in Archives Ital. de Biol. IX., 28, 1888.

=) Hoa.) Arch» Italde Biol)- IV. 299:

. Tizzoni. Arch. Ital. de Biol. IV., 306.

. Tizzoni and Griffini. Arch. Ital. de Biol. IV., 303.

. Schultze. Archiv f. Mik. Anat. I., 1, 1865.

. Arnold. Archiv f. Mik. Anat. XXX., 205.

. Hlava. Archiv f. Experimentelle Pathologie u. Pharmakologie. XVII.,

392.

. Steinhaus. Archiv f. Anat. u. Physiologie (Physiologische Abtheil.).

1888, 311.

116 HOWELL.

EXPLANATION OF PLATE.

Fic. 1. Blood from the heart of a fcetal cat, 2.7 cms. long, stained with methyl
green, shows the large nucleated corpuscles (ancestral form) and the ordinary
circular biconcave mammalian corpuscles. One of the latter is shown with its
nucleus escaping.

Fic. 2. Shows the way in which the nucleus escapes from the nucleated red
corpuscle. I, 2, 3, 4, represent different stages of the extrusion noticed upon the
living corpuscles; the drawings are colored to correspond with the rest of the figure.
a. Specimen from the circulating blood of an adult cat bled four times. 6. Speci-
mens from the circulating blood of a kitten forty days old, bled twice. c. Specimens
from the blood of a fcetal cat g cms. long. Others from the marrow of adult cat,
two of the figures showing the granules present in the corpuscle which have been
interpreted erroneously as a sign of the disintegration of the nucleus. All the speci-
mens stained with methyl green.

Fic. 3. Examples of apparent budding of the nucleated corpuscles, resulting from
the extrusion of a nucleus from one of the cells after division. From the marrow of a
cat. Stained with methyl] green.

Fic. 4. Examples of the large nuclear granules found in the newly formed red
blood corpuscles (cat) after severe and sudden bleeding.

Fic. 5. Multiplication of the nucleated red corpuscles. Methyl green. Marrow
of young kitten after bleeding.

Fic. 6. White corpuscles and blood plates, stained with methyl green, from the
blood of an adult cat, bled once to 90 cc., and treated with methyl green and
acetic acid. To show the origin of the blood plates from the nuclei of the multi-
nucleated leucocytes.

Fic. 7. Newly formed red corpuscles from section of marrow of femur in a foetal
cat 9 cms. Shakespeare-Norris stain of indigo carmine. To show the granules
with outline of nucleus seen in the newly formed corpuscles after extrusion of the
nucleus and the dissolution of the hemoglobin.

Fic, 8. Nucleated red corpuscles stained with methyl green, to show the mature
and immature forms and the intermediate stages and the colorless erythroblasts.

Fic. 9. Nucleated red corpuscles from sections of the marrow, stained in hema-
toxylin, eosin, and saffronin, to show the preference of the nucleus of the mature
form for saffranin, and of the immature form for hematoxylin.

Fic. 10, Karyokinetic figures of the nucleated red corpuscle, from a specimen of
young marrow teased in Flemming’s solution, and afterwards stained in saffranin.

Fic, 11. To show the origin of the erythroblasts and nucleated red corpuscles
from the embryonic cells (marrow corpuscles). From the liver of a foetal cat 2.7
cms., teased in Flemming and stained in saffranin.

Fic. 12. To show the marrow corpuscles. a@ and 6 with oval nuclei, ¢ and d with
coiled nuclei, and e, 7 with the protoplasm loaded with coarse granules. Specimens
teased in Flemming and stained with saffranin.

Fic, 13. From a section of the liver of a foetal cat 2.7 cms., showing the develop-
ment of the liver vessels and the nucleated red corpuscles. To the right of the figure
the newly formed vessel contains a number of non-nucleated red corpuscles, sur-
rounded in the section by the coagulated plasma.

Fic. 14. A second section from the same liver.

Fic. 15. White corpuscles from the blood of a young kitten bled once. Treated
with methyl green and acetic acid, to show the origin of the multinucleated from the
uninucleated forms.

of Morphology Vol We

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Seas SD

Kage

y Wexner # Winter, Pra

OBSERVATIONS UPON THE OCCURRENCE,
STRUCTURE, AND FUNCTION OF: THE
GIANT CELLS OF THE MARROW.

W. H. HOWELL, Pu.D., M.D.,

PROFESSOR OF PHYSIOLOGY AND HistoLocy, UNIvERSITY oF MICHIGAN.

THE observations which this communication is intended to
record were made in the course of a study of the hematopoi-
etic function of the marrow, the results of which have been
given in the previous paper. The giant cells have been sup-
posed by some, notably by Foa and Salvioli (1), to have a
direct connection with the production of new red corpuscles ;
so that an investigation of this last subject necessitated a more
or less thorough consideration of the giant cells. My observa-
tion convinced me that no direct connection exists between the
giant cells and the newly formed red corpuscles. The reasons
upon which this conclusion is founded, together with certain
interesting facts not heretofore observed relating to these pe-
culiar cells, forms the excuse for the present brief paper, which
for the rest does not attempt to give any final solution to the
problem of their function in the marrow. My observations
have been confined to the cat, as this was the animal which
was found to be most convenient for the study of the blood
corpuscles. The giant cells, moreover, seem to show certain
differences in structure in different mammals (Werner), so that
by restricting the work to one animal the different preparations
could be more easily compared amongst themselves.

Before attempting to give a description of these cells as
found in the cat’s marrow, it is necessary to insist upon a
division of them into two classes, —a distinction which has
been emphasized before by certain writers, but which is for-
gotten or denied by others. We have, in the first place, giant
cells containing a variable number of separate nuclei. These
form what are generally described as giant cells or myelo-
plaques, and have been found not only in the marrow, but also

118 “HOWELL. [Vou. IV.

in a number of pathological formations, tubercle, syphilis, etc.
In the second class, the cell contains not many, but one huge
nucleus, often bent or coiled upon itself, or imperfectly seg-
mented or notched so as to form a complicated structure.

These have been described as giant cells with budding
nuclei (Bizzozero [2]); but it seems to me that they are worthy
of a more distinctive name. I shall speak of them here-
after as megakaryocytes, or large nucleated giant cells, while
the first class might be named polykaryocytes, or multi-
nucleated giant cells. Some of the German histologists, espe-
cially Arnold (3), have held that transitional forms can be
found between the two classes, and look upon them therefore
as two stages in the life-history of a single cell. The first stage
is supposed to be the large nucleated form, and this by frag-
mentation passes into the multinucleated form. My observa-
tions upon the cat have led me to believe that we have here
two entirely different cells, probably with different functions,
and that what are described as transitional forms, which I have
also seen, though very rarely, are such only in appearance, and
can be accounted for more easily upon other grounds, possibly
as cells undergoing degenerative changes.

In the cat the polykaryocytes are found in great numbers in
the developing bone of the foetus, and are usually seen in sec-
tions lying upon the spicules of bone while in process of forma-
tion. In extra-uterine life they can be found also, but only in
the marrow of the spongy bone, in the neighborhood of the
bony dissepiments. I have never met with them, in the cat,
lying in the mass of red marrow which fills up the interstices
of the spongy bone and forms solid plugs at the ends of the
medullary cavity of the long bones. It is well known, also (4),
that when pieces of sponge or other porous substances are
introduced into serous cavities, the leucocytes swarm into the
interstices of the foreign body, and in a short while multi-
nucleated giant cells are found lying upon the partitions, just
as in the bone they lie upon the bony spicules.

Very many hypotheses have been made as to the origin of
these cells, the chief question being whether they are derived.
from the growth of a single small cell or from the fusion of a
number of separate cells. It is not necessary for me to give
here any detailed account of the views that are held upon this

No. 1.] GIANT CELLS OF THE MARROW. 11g

question, as I have no special observations of my own to offer
which might help to solve the problem. My own opinion has
been that this form of giant cell is produced by the fusion or
amalgamation of a number of smaller cells. I have been led
to this view chiefly from the fact, already mentioned, that they
seem to be found only when lying upon some solid substratum,
such as the septa of sponge or the spicules of spongy bone.
In the latter locality, when the spicules of forming bone are
covered with an epithelium of osteoblastic cells, it seems plaus-
ible to think that these closely packed cells might become
forced to form a polykaryocyte, and a number of apparently
transitional steps in this process can be seen in sections of the
femur in foetal cats about 9 cms. long. The rows of osteoblastic
cells are found in the same localities with the polykaryocytes,
and the nuclei of the cells have the same vesicular character in
both kinds of cells. As far, then, as my observations upon
this cell have gone, they have induced me to side with those
who believe they are derived from the fusion of small cells.
The function of these cells is unknown. The common view
that they are concerned in the absorption of bone (osteoclasts)
seems to me to rest upon very slight evidence. If we find
them in developing bone lying upon the cartilage trabecule
which are being absorbed, we find them also on the partitions
of sponge or pith, introduced into serous cavities where no
absorption is taking place; and the conclusion in the first case
that the absorption which is going on is due to the giant cells
(osteoclasts) is illogical. Absorption of tissues is an occurrence
common enough in the body, and it is difficult to understand
why the absorption of bone or cartilage should require the
activity of a special cell, when the absorption of other tissues
does not. It would seem more probable that this form of cell
has no specific function, and that its formation is, in fact, acci-
dental or, in a certain sense, pathological: that the presence
of a solid substratum leads to an abnormally rapid growth of
lymphoid cells, leucocytes, osteoblasts, as the case may be,
and the fusion of some of these to produce multinucleated
giant cells. The same explanation might hold, as far as I can
see, to the occurrence of this form of cell in pathological for-
mations, except that in these cases the too rapid growth is
brought about by other conditions.

120 HOWELL. [VoL. IV.

The large nucleated giant cell, or megakaryocyte, unlike the
multinucleated form, is found, and found abundantly, in the
midst of the red marrow filling up the ends of the long bones
and the spaces between the trabeculz of the spongy bone. It
does not lie upon the spicules of bone or cartilage, but away
from them, surrounded by marrow cells, nucleated red corpus-
cles, blood corpuscles, and the other cells characteristic of
marrow. It appears in the marrow with its first formation,
as shown by cross and longitudinal sections of the femur in an
embryo cat 9 cms. long, and is then surrounded by the ele-
ments of the marrow. Throughout the rest of the animal’s
life it can be discovered in sections or teased specimens of the
red marrow. It is evidently a peculiar kind of cell, which has
some definite function to perform. Not only is it found in the
marrow, but throughout embryonic life it is met with in abund-
ance in the liver and the spleen as long as these organs have
any distinct connection with the production of red blood cor-
puscles; and it occurs more abundantly, the more active the
blood-forming function of the organ. In histological structure
it is the same in the embryonic liver and spleen as in the
adult marrow. This fact has been stated by others (Foa and
Salvioli [1], etc.), and is perfectly evident to any one who will .
take the trouble to look. It is certain, therefore, that the
function of this cell is the same in the embryo spleen or liver
as in the adult marrow. That it is not simply one stage in
the life of the multinucleated giant cell seems to be demon-
strated by the fact that typical multinucleated cells are never
found in the mass of red marrow, in the cat at least, lying in
the cavity of the long bones, nor in the embryonic liver or
spleen, though the megakaryocytes are so numerous.

The structure of the megakaryocytes has been clearly de-
scribed by a number of observers, especially by Arnold (3).
They are giant cells, each with a huge nucleus. The body of
the cell is finely granular, and shows no special peculiarities in
structure. It is interesting to note that it does not possess the
power of amoeboid contraction which is so marked in many of
the marrow cells, especially those with elongated nuclei. The
nucleus is the characteristic part of the cell, but varies consid-
erably in size, complexity, and minute structure (Fig. 11, a, 4,
c,d@). Frequently it is crescent-shaped, or even makes a ring;

No. 1.] GIANT CELLS OF THE MARROW. I2I

at other times it is coiled upon itself, or appears simply as a
large, central mass, with projections from its surface; but in
most cases it shows incomplete constrictions of or partitions
from the peripheral membrane or layer of chromatin, which
tend to separate it into small nuclei comparable to those of the
typical marrow cells. The nucleus is granular, and in well-pre-
served specimens shows a distinct chromatin reticulum, with
conspicuous nodal points. In addition, one or many nucleolar
masses may be present, sometimes one apparently for each
incomplete small nucleus into which the large mass is divided,
sometimes only one for the entire nucleus, or in some cases
none at all. What I have called nucleolar masses or nucleoli
are distinguished from the large granules or nodal points of the
chromatin reticulum by their staining. In the triple stain of
haematoxylin, eosin, and saffranin, the chromatin reticulum
takes the hematoxylin stain as in nuclei generally, while the
nucleoli, like the nucleoli of the marrow cells, show a prefer-
ence for the saffranin, staining bright red when the time of
exposure to the different stains is properly adjusted. In a
number of instances, in the sections of the normal marrow of
adult cats, megakaryocytes were met with in which the nuclei
showed no chromatin reticulum at all, but stained diffusely
or almost so with the different dyes employed, taking the
stain like the nuclei of the matured form of nucleated red
corpuscle described in my paper upon the development of the
red corpuscles. In such cells where the chromatin was dif-
fusely scattered throughout the nucleus it frequently happened
that the nucleus was fragmented (Fig. 1,2, f). It seems to me
that this appearance of the nucleus here as in the nucleated
red corpuscles is a sign of old age and death, and that these
cells are in process of dissolution, hence the fragmentation of
the nucleus. Arnold takes the directly opposite view, and con-
siders this appearance as one of the initial changes leading to a
fragmentation of the nucleus and division of the cell into
smaller marrow cells.

According to Arnold, the following successive changes in
the structure of the giant cell occur, leading up to its fragmen-
tation. 1. The first stage is characterized by an increase in
the chromatin substance, the chromatin filaments become more
numerous, form networks, etc., and toward the end of the stage

122 LfO ae [Vou. IV.

a division of the chromatin occurs, leading to a more or less
diffused coloration of the nucleus. 2. The periphery of the
nucleus becomes indented, and in many cases there is such an
important increase in the diffused chromatin substance that the
filaments can no longer be distinguished. The indentations of
the periphery of the nucleus occur at many points and advance
toward the middle, forming the complicated nuclear figures
so characteristic of these cells. 3. The chromatin substance
becomes concentrated at different points, forming small, dark-
colored nuclear bodies which are united by colorless bands.
By a continuation of these changes a number of entirely in-
dependent nuclei are formed. 4. The protoplasm segments
round the newly formed nuclei, either endogenously or by
constriction from the periphery. This makes up the process
designated by Arnold as “indirect fragmentation,” and it is,
according to him, the normal method of development or multi-
plication of the giant cells. He admits in addition, and quotes
from Martin and Waldstein to support the statement, that the
multinuclear giant cells may reproduce also by true mitotic
division of the nucleus, but thinks that this method of division
is very rare. I have never myself seen any indication what-
ever that the nucleus of the giant cells divides by karyokinesis,
though I have examined many sections of marrow from cats of
all ages, normal, bled, and starved, so that with this animal at
least it must be an exceedingly rare occurrence. Moreover,
my observations upon the giant cells have never given me any
evidence of the correctness of Arnold’s view that these cells
normally undergo indirect fragmentation. Foa and Salvioli
also thought that the megakaryocytes break up by segmenta-
tion to form a number of colorless or hyaline cells, which in
turn develop into nucleated red corpuscles, and I shall speak
further of their theory in discussing the function of this form
of giant cell.

On the other hand, that the megakaryocytes multiply by
division, like other cells, giving rise to two daughter giant cells,
has been clearly proved by my sections. In quite a number of
cases the sections have shown me megakaryocytes with two
large nuclei at the ends of the cells, and a constriction begin-
ning between them, or, more frequently, two megakaryocytes
lying side by side with the line of demarcation between them

No, 1.] GIANT CELLS OF THE MARROW. 123

complete, but the cells still adherent to each other (Figs. 2
and 3). Similar appearances have been seen and figured by
Werner (5). In none of the cases of division observed by me
was there any indication of karyokinetic figures; hence it is fair
to conclude that this form of cell multiplies by direct division.
The clear proof furnished by these observations that it does
increase by division is also another indication that the mega-
karyocyte is a definite cell form, and not one stage in the
formation of the multinucleated giant cell.

What now is the origin and function of this cell? Believing
that the megakaryocyte has no genetic connection with the
polykaryocyte, the theories of the origin of the latter have no
bearing upon the former. My sections, especially those made
through the femur of a foetal cat, 9 cms., at a time when the
marrow was just beginning to form, gave me a number of
apparently transitional forms between typical megakaryocytes
and the small marrow or embryonic cells. A series of draw-
ings, showing apparently the gradual development of the small
cell into the giant cell, is given in the Fig. 4. The drawings
were made from different portions of the section, and the
theory they suggest is that the small cell enlarges, the increase
affecting both the nucleus and the cell substance, and, after
reaching a certain size, indentations of the periphery of the
nucleus appear, or in many cases ingrowths of the peripheral
chromatin, which give the incompletely segmented appearance
to the nucleus that is so characteristic, and which has given
to them the name of giant cells with budding nuclei. The
megakaryocyte is not formed, then, by the fusion of a number
of smaller lymphoid cells, nor from a single cell which increases
in size by engulfing other similar cells. Both these theories
might apply to the polykaryocytes, but certainly not to the
megakaryocytes. These latter are developed by the steady
growth in size of a smaller lymphoid cell, and the curious
structure of the nucleus follows after it has reached a certain
size in consequence of partial constrictions or divisions, which
are never carried so far, however, as to lead to a complete
separation.

With reference to the function of these cells, several differ-
ent views have been proposed. Arnold and others, who believe
that the megakaryocyte becomes ultimately a multinucleated

\

124 HOWELL. [Vou. IV.

giant cell, believe that the latter constricts off or separates into
smaller marrow cells. From this standpoint, the function of
the large nucleated giant cell is simply that it forms one stage
in a peculiar method of development of the lymphoid cells of
the marrow. Léowit (6) speaks of the giant cell— including
both varieties —as having some connection with the degenera-
tive changes of the leucocytes, though as far as I know the
exact nature of the relationship is not described. He gives
three reasons for this view. 1. They are less frequent in the
embryo than in the marrow of the adult, and the younger the
embryo, the fewer the number found in the liver and spleen.
The first part of the statement I cannot corroborate, as in the
embryo liver, especially when at the maximum of its hzemato-
poietic activity, the giant cells (megakaryocytes) are quite as
numerous as in the adult marrow. The second portion of the
statement is true to a certain extent. The number of giant
cells (megakaryocytes) varies directly with the blood-forming
activity of the organ, so that they are not numerous in the
liver at its first formation nor toward the end of fcetal life.
2. He has never been able to find them in the lymph glands,
even after severe bleeding. In this, Lowit is confirmed by a
number of other observers who have stated their inability to
find giant cells in the lymph glands. Indeed, this assertion
may be accepted as satisfactorily demonstrated, but its bearing
upon Lowit’s theory of a connection of the giant cells with
the degeneration of the leucocytes seems to be very remote.
While it is true that we do not find in the lymph glands either
giant cells or degenerating leucocytes, that does not in any
way prove that the giant cells have any relation to the degen-
erative changes of the leucocytes. 3. In sections of the mar-
row, the giant cells are not found where erythroblasts and
leucoblasts are in active formation, but rather where the latter
show signs of degenerative changes. This statement I cannot
confirm; indeed, it seems to me that the reverse is true, as
far, at least, as the erythroblasts are concerned, while with the
leucoblasts I have not been able to notice that in the neighbor-
hood of the giant cells there is any increase in the number of
them undergoing degeneration. Foa and Salvioli have thought
that they were able to demonstrate a connection between the
giant cells and the nucleated red corpuscles. Their view, as

No. 1.] GIANT CELLS OF THE MARROW. 125

=

has been stated, is that the giant cell breaks up into a number
of hyaline cells, — erythroblasts, to use Lowit’s terminology, —
which in turn develop into nucleated red corpuscles. Their
strongest evidence for this view is the fact that whenever, in
the foetus or in the animal after birth, there is an undoubted
formation of nucleated red corpuscles, there the giant cells —
megakaryocytes —are also found. This connection has been
noticed by a number of observers, and is certainly very con-
stant and striking. In the embryo liver, the embryo spleen,
the adult marrow, and in the spleen of the adult during regen-
eration after partial excision (7) we find megakaryocytes and
nucleated red corpuscles side by side. Nevertheless, I have
never been able by the most careful and thorough observation
to find any actual connection between these two histological
elements, or between the giant cell and the erythroblast. Foa
and Salvioli picture a group of nucleated red corpuscles sup-
posed to be derived from the breaking up of a megakaryocyte,
but groups of the kind figured are in reality derived from the
multiplication of nucleated red corpuscles by division. Arnold
has stated that white corpuscles are constricted off from the
giant cells, but supposes that these corpuscles are not progeni-
tors of the nucleated red corpuscles. In my own sections and
teased preparations I have never been able to find any indica-
tion that the nucleated red corpuscles are budded off from the
megakaryocytes, and no satisfactory example of the derivation
of a lymphoid corpuscle of any description from them. In the
marrow of the adult, after repeated hemorrhages, where the
production of red corpuscles has been vastly accelerated, one
would surely expect to see some undoubted sign of the deriva-
tion of the nucleated red corpuscle or its colorless predecessor
from the giant cell, if it is the function of this last cell to serve
as the origin of the new red corpuscles that are being formed.
My failure to find any perfectly clear examples of such a deri-
vation has compelled me to believe that the megakaryocytes
take no direct part in the production of new red corpuscles.

In a few cases I have obtained giant cells evidently belong-
ing to the class of megakaryocytes in which a smaller portion
of the nucleus seemed to be completely separated from the
main mass and was lying free in the cell, but always in such
cases there was some possibility that the appearance was de-

126 HOWELL. [Vot. IV.

ceptive. I was not able to obtain a sufficient number of clear
cases to convince me that this is a normal occurrence in the life
of these cells, though at the time I was convinced that their
most probable function was to form the erythroblasts or pro-
genitors of the nucleated red corpuscles; indeed, I abandoned
the theory reluctantly because the evidence seemed to be
opposed to it, or at least did not support it. If we adopt the
compromise view that the giant cells furnish some of the
erythroblasts while others, and probably most of the others,
arise in a different way, then we could understand why in rapid
regeneration of the blood after bleeding it is so rare to find
giant cells in the act of producing erythroblasts. But it does
not seem probable, to me at least, that these cells should be
produced in one organ by two different methods. Neverthe-
less, the constant presence of megakaryocytes in the blood-
forming organs induces me to believe that they have some
function to perform in connection with the formation of blood.
This is rendered more probable by the fact that in the embryo,
at least, the megakaryocytes can be found in the newly forming
blood-vessels surrounded by developing blood corpuscles. I
have found this in sections of the liver of an embryo cat where,
as has been described in another paper, the nucleated red cor-.
puscles and the erythroblasts lie in cords which are destined to
become the future blood-vessels of the liver. In some cases,
in fact, the cords may be seen to end in channels filled with
coagulated plasma and red corpuscles, with or without nuclei.
Now, in these cords of blood cells I have found the megakary-
ocytes, showing that they are connected in some way with the
blood (Fig. 5). In longitudinal sections of the hind leg of
the same embryo I have found developing blood-vessels lying
among the embryonic muscle fibres and in the blood-vessel
giant cells, — megakaryocyte, —as shown in Fig. 6. It is very
hard to understand what this cell is doing in such a place
if it is not connected with the production of either the formed
elements or of some of the chemical constituents of the blood.

As I have just said, I cannot find any corroborative evidence
for the first view, and am therefore inclined to look favorably
upon the second ; namely, that the function of the megakary-
ocyte is to manipulate, in some way, the material of the plasma
or lymph, forming some substance for the nourishment of the

No. 1.] GIANT CELLS OF THE MARROW. 127

<

developing blood cells. This view was suggested to me bya
curious phenomenon which I have occasionally found in con-
nection with these giant cells, and which, so far as I know, has
not been noticed before. In many sections of the marrow, but
especially in sections through the bone and marrow of the
femur of a foetal cat (9 cm. long), I have seen the megakary-
ocytes, either singly or in groups, with a delicate reticulum
radiating out from them on all sides, and enclosing within its
meshes the other elements of the marrow. A sketch of this
appearance is given in Fig. 7. I should add that Werner has
described but not figured what appears to be the same reticu-
lum. In the young developing marrow this appearance is so
common and so striking that I thought at first the megakary-
ocytes had for their function the formation of a supporting
reticulum for the marrow, secreting it, as it were, from the
cell substance. But when examinations were made of teased
specimens of the fresh marrow of young kittens to find if pos-
sible whether a giant cell with its reticulum could be teased
out from the other elements, I obtained the cells, surrounded
not by a reticulum, but by a very large envelope of exceedingly
fine and pale material (Fig. 8). Round the nucleus of the
cell was the ordinary granular protoplasm forming the body of
the cell, but outside of and surrounding this was a large enve-
lope of much more delicate and hyaline material, which did
not stain with methyl green. As this was watched under the
microscope, in a very dilute NaCl solution of methyl green, vac-
uoles began to form in it (Fig. 9), and becoming rapidly larger,
finally made a reticulum such as I had found in my sections
surrounding the cell. This convinced me that the reticulum
seen in the sections arose from the action of the fixing and
hardening reagents upon this secretion from the: cell.,. The
theory that the giant cells make a reticulum is rendered im-
probable, also, from the fact that they occur in the developing
blood-vessels of the embryo. In many cases in the teased
specimens the action of the reagent had gone so far that the
giant cells were found surrounded only by vesicular-like bodies
arising from the vacuolation of the secreted material, as shown
in Fig. 10. It seems to me that this broad envelope of mate-
rial surrounding the megakaryocyte, and evidently formed by it,
is very significant. As I found it, no nuclei were scattered

128 HOWELL. [Mou ivi

through it, and hence the most natural explanation is that it
is a material secreted by the cell which is finally dissolved in
the plasma, and is used, possibly, for the nutriment of the
blood-forming cells ; though this, of course, is mere speculation.
In sections nothing remains of the material except the reticu-
lum, and this does not stain with any of the reagents used, —
alum carmine, haematoxylin, eosin, saffranin, Ehrlich-Biondi’s
stain, indigo carmine (Shakspeare-Norris stain), —or at least
stains much more feebly than the protoplasmic cell substance.

SUMMARY.

The contents of the paper may be summed up briefly as
follows :

1. Giant cells fall into two classes: a. Polykaryocytes, or
multinucleated giant cells found in developing bone, in patho-
logical formations, or porous bodies kept in lymph cavities,
etc.; 4. Megakaryocytes, or large nucleated giant cells found
in the red marrow of the adult and in the blood-forming organs,
liver, spleen, etc., of the embryo.

2. The polykaryocytes have no special function, are not
related to the megakaryocytes, and are formed by the fusion
of smaller cells in consequence of too rapid growth.

3. The megakaryocytes form a peculiar class of cells. They
arise from the growth of small lymphoid cells, and afterwards
reproduce by direct division. During their life they form a
secreted material which can be seen for a time by the micro-
scope, but finally dissolves in the plasma.

They seem to take no direct part in the production of nucle-
ated red corpuscles or erythroblasts. After a certain period
the nucleus alters in such a way that it stains diffusely and
then fragments. This seems to be a degenerative change, and
probably ends in the total disintegration and dissolution of
thercell:

No. 1.] GIANT CELLS OF THE MARROW, 129

QW tom

REFERENCES.

Foa and Salvioli. Archiveo delle Scienze Mediche. Vol. IV", De 1
Bizzozero. Archives Ital. de Biologie. Vol. IV., Pp. 329.
Arnold. _Virchow’s Archiv f. path. Anat. u. Physiol. u. f. Klin. Med.
Bd. 93, S. 1; Bd. 7, S. 107. Archiv f. Mik. Anat. Bd. 30, S. 205.
Hamilton. A Text Book of Pathology. p. 291.
Werner. Ueber Theilungsvorgiinge in den Riesenzellen des Knocken-
marks. Inaugural Dissertation, Berlin, 1886.

6. Léwit. Sitzungsberichte d. K. Acad. d. Wiss. Bd. 92, Abth. III., 1885.

Foa. Archives Ital. de Biol. Vol. IV., p. 299. Griffini and Tizzoni.
Ibid., p. 303. .

130 HOWELL.

EXPLANATION OF PLATE.

Fic. 1. a, 4, ¢, 2. Drawings of megakaryocytes, to show some of the variations in
structure of the nucleus. e, / Megakaryocytes in which the nucleus stains diffusely
and fragments into smaller pieces, explained as degenerative changes.

Fic. 2. Three megakaryocytes; two in the act of dividing; from a camera lucida
sketch. . ;

Fic. 3. Two megakaryocytes; division complete, but the cells still connected;
from a camera lucida sketch.

Fic. 4. Four cells from the developing marrow in a section of the femur of a fcetal
cat 9 cms. long, stained with saffranin, and intended to illustrate the development
of a megakaryocyte from a marrow corpuscle.

Fic. 5. Section of the liver of a cat embryo 2.7 cms. long, showing a mega-
karyocyte lying in a developing blood vessel, and surrounded by erythroblasts. »

Fic. 6. Section of the hind leg of the same embryo, to show a megakaryocyte
lying in a developing blood-vessel of the muscular tissue. On one side there is still
a solid cord of erythroblasts, with some nucleated red corpuscles; on the other the
blood plasma and fully formed nucleated red corpuscles lie in contact with the giant
cell.

Fic. 7. Camera lucida sketch from the section of the femur of the foetal cat 9 cms.
to show the reticulum radiating from the megakaryocytes found in the marrow.

Fic. 8. A megakaryocyte surrounded by its broad envelope of secreted material
from a preparation of the marrow of a young kitten, teased in a weak solution of
methyl green in normal salt.

Fic. 9. The same cell, showing the vacuolation that takes place in the enveloping
substance. The vacuoles at first small, as on the under side of the cell, become larger,
until they form a structure resembling somewhat the reticulum shown in Fig. 7. The
sketches of the vacuolation were made at different times, and have been shown in
the same figure to indicate the gradual growth.

Fic. 10. A megakaryocyte from a similar preparation, in which the action of the
reagent had gone so far before the cell was examined, that a number of vesicles
adhering to the cell was all that remained of the original envelope.

Howell del.

ith. Ansty. Werner & Winter, Prandfirt

i;

Volume IV. October, 1890. Number 2,

JOURNAL

MORPHOLOGY,

CONTRIBUTIONS ON THE MORPHOLOGY OF
THE ACTINOZOA

I. THE STRUCTURE OF CERIANTHUS AMERICANUS.
J. PLAYFAIR McMURRICH.

THE genus Cerzanthus was established in 1829 by Delle
Chiaje for the Mediterranean form which we now know as
C. membranaceus, it having been originally described by Spallan-
zani as 7ubularia membranacea. Until 1854, however, no thor-
ough study of the internal structure was made, but in that year
appeared the excellent memoir of Haime (54). In this it is
shown that each “loge” has communicating with it two tenta-
cles, one belonging to the marginal, the other to the oral group.
Haime also described the arrangement of the mesenteries, show-
ing that two mesenteries, the cavity between them forming a
continuation of the “fossette gastrique” (siphonoglyphe), ex-
tend the entire length of the body to the terminal pore, while
the rest stop at a short distance below the internal opening of
the stomatodzum, and are unpaired, although they are alter-
nately slightly unequal in length and prominence. Haime de-
scribed, too, the hermaphroditism of this species, and gave an
incomplete account of some stages in its development. His
account of the-histology was, however, by no means exhaustive,
though admirable, when the facilities for such work at that time
are taken into consideration.

For twenty-five years nothing further was done towards the

132 MCMURRICH. iWon sive

elucidation of any members of the genus Certanthus, but in
1879 two papers of importance appeared. The brothers Hert-
wig ('79) in their studies on the nervous system of the Actini-
aria, examined histologically C. membranaceus and C. solttarius,
and added much to our knowledge of the minute anatomy of
these forms, discovering the nervous tissue, describing the
arrangement of the muscle-cells correctly, and showing the
similarity of all the tissues to those of the other groups of
Actiniaria. As regards the general structure, however, they
made no advance upon what had been done by Haime, not even
correcting some of the errors into which that author had fallen.

The other paper of 1879 was by von Heider, who treated
C. membranaceus in as thorough a manner as he had previously
done Sagartia troglodytes. Where the Hertwigs are lacking,
von Heider excels, giving a more correct account of the ana-
tomical features of the species than Haime had done, but,
his treatment of the histology is in some points not so com-
plete. As regards the anatomy, he showed that the pair of
elongated mesenteries are not the most ventral, but that be-
tween them is a pair reaching the wall of the siphonoglyphe,
but terminating a very short distance below the margin of the
groove. These are the ventral directives. He also extended
Haime’s discovery as to the alternating inequality in length of
the mesenteries, by showing that as a rule there is an alteration
of gonophoric and non-gonophoric mesenteries, and accordingly
divided the mesenteries into three groups ; namely, (1) Filament
septa, which are non-gonophoric ; (2) Genital septa; and (3) Con-
tinuous septa, which are represented only by the single pair which
reaches the terminal pore. Von Heider describes the Filament
septa as giving rise to the acontia, while the Genital septa are
provided with mesenterial filaments (craspeda, Gosse). The
Hertwigs, in a supplement to their description, after confirming
several of von Heider’s results, criticise this differentiation of
the filaments in the two groups of mesenteries, stating that ‘in
der Beschaffenheit der Mesenterialfilamente zwischen beiden
kein Unterschied vorhanden ist.’”’ It will be seen that, so far
as the structure of the filaments is concerned, this is true also
for C. Americanus, though there is a slight difference in the
arrangement of the different parts of the filament.

In 1880 a paper by Jourdan (80) appeared, written, however,

No. 2.] MORPHOLOGY OF THE ACTINOZOA. 133

before the publication of the contributions of the Hertwigs and
of von Heider. It adds nothing to Haime’s description of the
general structure, and falls much behind the Hertwigs’ contri-
bution in the treatment of the histology.

In 1888 a paper by C. Vogt (88) was published, in which
was confirmed the supposition of the Hertwigs that new mesen-
teries are formed in Certanthus solely at the dorsal surface, in
the region which corresponds in other orders of Actiniaria to
the intra-mesenterial space bounded by the dorsal directives.
Vogt also calls attention to the unpaired tentacle corresponding
to the ventral intra-mesenterial space, which had previously been
observed by Haime in Cerianthus and by A. Agassiz ('62) in
Arachnactis, and demonstrated the close relationship existing
between these two genera.

Later in the same year, H. V. Wilson (88) added to Vogt’s
observations by showing a similar method of formation of new
mesenteries in a free-swimming larva of an unidentified species
of Certanthus obtained at Nassau, Bahama Islands, W.I.

In the following year Fischer (89)? again called attention to
the unpaired ventral tentacle, and to the bilateral symmetry of
C. membranaceus.

Up to this time no accounts of the internal structure of any
other species of Cerianthus had been given. The Hertwigs
state that they studied C. solztarius, but make no special state-
ments concerning its general structure. In 1889, however,
Danielssen (89) published an account of the structure of a
Norwegian Cerianthus, which he termed C. borealis, and which
presents many important variations from the structure of C.
membranaceus. The principal anatomical peculiarities are the
small number of mesenteries, sixteen only, the occurrence of
either ova or spermatozoa only in any individual, and the differ-
ence in the arrangement of the mesenteries in the males and
females, all the mesenteries in the latter extending to the
terminal pore, except the ventral directives, which stop a short
distance above it, while in the males the arrangement is much
more similar to what is found in C. membranaceus.

1 The original paper I have not seen; my knowledge of its contents is derived
from the abstract in the Journal of the Royal Microscopical Society, December, 1889.

2 It seems probable that this form, which Danielssen holds to be distinct from
C. Lioydii, is not the same as Verrill’s C. dorealis, described in 1873. In this case

Verrill’s application of the name has the priority, as Danielssen’s description was not
published until 1877.

134 MCMURRICH. [Vou. IV.

CERIANTHUS AMERICANUS, L. AGASSIZ.

The earliest mention of this form is by L. Agassiz (’59), who
states that it was found by H. James Clark, in 1852, at Charles-
ton, S.C., where it lives in tubes sunk in the mud-flats of the
harbor. It is referred to the genus Cerzanthus, but no specific
name is given. Agassiz observed the terminal pore, which he
terms the anus, and states that the upper parts of the mesen-
teries bear female reproductive organs, and the lower parts
male organs.

The specific name is stated by Verrill (64) to have been be-
stowed in manuscript by Agassiz, and Verrill gives the first full
description of the species, from drawings made for Agassiz
by his artist, Burkhardt, and from alcoholic specimens in the
Museum of Comparative Zoology at Harvard College.

In a subsequent paper Verrill (’72) records its occurrence on
the coast of North Carolina, where it was collected by Dr. Yar-
row, and where it had previously been found by Stimpson, but
adds nothing to the description given in 1864.

Among the “ Challenger” material R. Hertwig (’82) found a
single specimen of a Cerzanthus obtained in thirteen fathoms in
the mouth of the Rio de la Plata. He identifies it with C. Amer
canus, but unwilling to mutilate the single specimen, did not
investigate it anatomically.

Finally I added (’87) a few points to the general description,
but gave no account of the internal anatomy and histology which
have never hitherto been examined.

1. EXTERNAL FEATURES.

The specimens of C. Americanus which I studied were found
at Beaufort, N.C., where the Summer Station of the Johns
Hopkins University was located in 1885. In the shallow sounds
there the bottom is largely very dark mud, sometimes with a
superficial coating of sand. Large areas of such mud are
uncovered at low tide, forming what are termed the mud-flats,
and it is on these flats that Cerianthus is found, usually just
below the average low-tide mark. It lives in cylindrical burrows
extending, usually at an angle, downwards for some distance,
how far I was not able to determine. Like other members of

No. 2.] MORPHOLOGY OF THE ACTINOZOA. 135

the genus it secretes a case, open at both ends, and composed of
hardened mucus and nematocysts. Animals removed from the
case and kept in an aquarium rapidly secrete for themselves a
new habitation, which, however, is naturally thinner than the
original one and of a lighter color, being almost white, while
normally the tube is dark gray, due to staining by the black
mud with which it is in contact. The inner surface is purplish
gray, being tinged by the pigment of the animal.

The largest specimen I obtained measured about 20 cm. in
length, with a diameter at the middle of 1.5-2 cm., and at the
disc of 1.8-2.5cm. The outer tentacles measured 3.4 cm., while
the oral series measured 1-1.2 cm. These measurements fall
very short of those given by Verrill, who states that the largest
specimens in expansion measure 60-70 cm. in length, with a
diameter at the disc of nearly 4 cm. and at the middle of the
column of 2.5 cm. My preserved specimens measure 5.5—6 cm.
in length, and about 1.5 cm. in diameter.

The color of the column is some shade of brown (Pl. VL,
Figs. 1 and 2), varying from pale chocolate-brown to deep pur-
plish brown. The upper part is always darker than the lower,
and in some cases the column is marked with longitudinal lines
of a lighter shade than the ground color. The marginal ten-
tacles are of a paler brown than the column, except the outer-
most, which are purplish blue. The oral tentacles in all the
specimens I observed were pure white; Verrill, on the other
hand, describes them as being darker than the marginal ones,
and marked with white longitudinal lines. In the Beaufort
specimens, however, the tentacles of both series are unmarked
by lines, spots, or annulations. The disc is yellow with white
lines crossing it radially.

The column is cylindrical and smooth, tapering gradually
towards the posterior extremity, which is rounded, and bears a
small terminal pore. The marginal tentacles vary somewhat in
number. Verrill states that they are 125 or more, but in the Beau-
fort specimens they did not amount to 100, varying, according to
the counts made, from 89 to 94 (95 ?). We know from the obser-
vations of Vogt and Fischer that there is always an odd number,
the unpaired tentacle corresponding to the space bounded by
the ventral directives. The absolute number of the tentacles
perhaps increases throughout the entire life of the animal, and

136 ' MCMURRICH. [VoL. IV.

the discrepancy between the number found in the Beaufort
specimens and that given by Verrill is probably in accord with
the smaller size of my specimens. A tentacle of the oral series
corresponds to and is opposite each marginal tentacle, and both
series seem to be arranged in three cycles, not four, as I stated in
my previous paper, but I was not able to ascertain the relations
of the various cycles in the two series. The accounts given by
von Heider (’79) and Fischer (’89) of these relations in C. mem-
branaceus differ materially. The number of cycles, however,
does not have the same significance here that it has in the
Hexactiniz, and the arrangement of the tentacles in cycles is
no doubt, as von Heider suggests, altogether mechanical, and
due to crowding, and accordingly their relation in the marginal
and oral series, and the number of the cycles in which they are
arranged, may vary.

2. INTERNAL STRUCTURE.

On laying open a specimen of C. Americanus by a longi-
tudinal incision, the appearance presented is that indicated in
Figure 1, Pl. VIL., in which, however, the disc and tentacles have
been omitted. In the upper part one sees the stomatodzeum
with a well-marked longitudinal groove —the siphonoglyphe.
By close examination it may be seen to be finely grooved lon-
gitudinally, each groove corresponding to a mesentery; a few
transverse grooves may usually be seen, but they are more or
less irregular, and are no doubt due to contraction. Its lower
margin is usually reflected, so that a transverse section in this
region gives the appearance represented in Figure 3.

The mesenteries are arranged on a very different plan for
what has been described for C. membranaceus and C. borealis,
Danl. On first laying open a specimen of C. Americanus, one
sees that more than one pair of mesenteries reach the posterior
extremity of the body, and yet all do not extend so far. In the
specimen figured twenty-three well-developed mesenteries are
present. The total number of mesenteries is really ninety-
two, as will be seen later, but of these only twenty-three
pass more than half-way down the column, and it is to the
arrangement of these that I wish first to direct the atten-
tion. Extending the entire length of the body from the
region of the siphonoglyphe is a pair of mesenteries corre-

No. 2.] MORPHOLOGY OF THE ACTINOZOA. 137

sponding to the similar pair in C. membranaceus, which von
Heider terms the Continuous septa. Between these, and in
reality constituting the ventral directives, is a pair, as in C. mem-
branaceus, which are very short, and hardly extend below the
level of the lower opening of the stomatodzum. Next to the
ventral continuous mesenteries on either side comes a mesentery
which only extends a short distance beyond the middle of the
column. Dorsal to it come three continuous mesenteries, the
middle one of which, however, hardly reaches the extremity of
the body. Then follow two extending slightly beyond the mid-
dle of the column, and similar in length therefore to the one
immediately succeeding the ventral continuous mesenteries :
and succeeding these is a single continuous mesentery. So far
the symmetry has been perfect, and I have found the arrange-
ment here described to hold in another specimen which I studied.
Unfortunately, the third specimen I had for investigation was
not favorable for the examination of the mesenteries.

The succeeding mesenteries passing dorsally on either side
vary from the regular arrangement. They are more recent in
date of formation than those towards the ventral line, and may
not yet have reached their final development, or may remain in
this somewhat immature condition. On one side, dorsal to the
continuous mesentery last mentioned, there is another similar to
it, but on the other side occurs one belonging to what may be
termed the second grade, reaching only to about the middle of
the column. Occupying the dorsal region are four mesenteries,
all of the second grade, two alternate ones, however, being
slightly longer than the other two. Upon what is the left side
of the figure a mesentery, also of the second grade, was detached
in making the longitudinal incision, and was omitted from the
drawing. The last mesentery of the second grade on the left
side is the youngest of those of the first two grades, being near-
est the median dorsal line.

I have denoted the mesenteries so far described according to
their length as the first and second grades, the latter being the
shorter ones which extend only about half-way down the
column. In the figure (Fig. 1) these mesenteries are the only
ones represented for the most part, but three (3) still smaller
than those of the second grade are indicated. In reality, the mes-
enteries of this third grade alternate with those of the first and

138 McMURRICH. (VoL. IV.

second grade, and there are consequently twenty-three of them.
These third-grade mesenteries extend only a short distance below
the internal opening of the stomatodzeum, not more than a centi-
metre, and usually less than that. Like the longer ones, they
are perfect in their upper part.

Figure 2, Pl. VIIL., is a semi-diagrammatic representation of the
mesenteries of the various grades, and it will be seen from this
that there is still a fourth grade of mesenteries, shorter than
any that have hitherto been described, and alternating with the
mesenteries of the other three grades. There are, therefore,
forty-six of them, and altogether in all the grades, accordingly,
there are ninety-two mesenteries, one more than the number of
tentacles, marginal or oral, of which I counted ninety-one. These
fourth-grade mesenteries hardly reach below the lower opening of
the stomatodzeum, and are not readily seen in a preserved speci-
men, being usually overlapped by the adjoining mesenteries, and
further concealed by the tangled mass of acontia which arise
from the edges of the mesenteries just below the stomatodzum.
The ventral directive mesenteries, as already mentioned, belong
to the fourth grade.

Figures 3, 4,and § show some interesting features in the rela-
tions of the mesenteries of the four grades. They are transverse
sections of the ventral region of the column in its upper part.
Figure 3 passes through the stomatodzum shortly above its
lower extremity, cutting its reflected portion. The ventral
siphonoglyphe (sz) is readily made out, the ectoderm lining it
not being thrown into folds as it is elsewhere. The ventral
directives (2) are still in connection with the stomatodzum,
but the other mesenteries have separated from it. Sections a
little higher up show that all the mesenteries are perfect; but
the ventral directives retain their connection with the sto-
matodzeum throughout a greater portion of its length than do the
others. The mesentery (1) immediately adjoining the direc-
tives is one of the ventral continuous mesenteries, which at
this level are narrow, as are all the mesenteries immediately
below the point where they lose connection with the sto-
matodeeum. Succeeding it comes a mesentery of the fourth
grade (4), and following this one of the third grade (3), both of
the same width as the ventral continuous mesentery. The mes-
enterial filaments of these three have the same structure, being

No. 2.] MORPHOLOGY OF THE ACTINOZOA. 139

somewhat bilobed, constricted by a well-marked “neck,” and
evidently comparable with the “ Flimmerstreifen” of the mes-
enterial filaments of the Hexactiniz. The section has not cut
all the mesenteries at the same relative level, so that those
furthest from the ventral median line show features which are
to be found in 1, 4, and 3, a little farther down. The mesentery
(4') which succeeds 3 is again of the fourth grade. It is much
wider than those nearer the middle line, and its mesenterial
filament is quite different, appearing simply as a small rounded
knob at the free edge of the mesentery not separated dis-
tinctly from the endoderm by a neck, and corresponding to the
“ Nesseldriisenstreif” of the Hexactiniz. A few sections
higher up this mesentery, and the other mesenteries of the
fourth grade represented (4'’ and 4''’), which resemble it in -
width and structure, are exactly similar in all points to 4. The
three mesenteries which alternate with 4’, 4’, and 4’’’ are of
the second (2), third (3’), and first (1’) grades, and exactly
resemble I.

Figure 4 is a section nearly 2 mm. below that just described.
The ventral directives, only one of which is figured (D), have
practically disappeared, being indicated simply by a slight
elevation of the mesoglaca. The mesenteries of the first, third,
and second grades nearest the middle line (1, 3, and 2) are
broader than they were higher up, but still retain the same kind
of filaments they possessed there. The more external (dorsal)
mesenteries are much wider, and, in fact, have now reached
their final width, and ova have begun to appear in their
mesogloea. Their mesenterial filaments have not been repre-
sented, but they are still of the same nature as they were
higher up. The mesenteries of the fourth grade (4, 4’, 4!’, and
4''') have lost all trace of their mesenterial filaments, and have
become very narrow.

Figure 5 is from a region about 1.5 mm. below the preceding
figure. The mesenteries of the fourth grade have now dis-
appeared, being represented, like the ventral directives, only by
slight projections of the mesogloea. The mesenteries of the
first three grades still persist ; all are gonophoric, but all have
lost their mesenterial filaments.

Still further down, in a section taken about 1 cm. lower, the
mesenteries of the third grade (3 and 3’) would have dis-

140 McMURRICH. [Vor. IV.

appeared, and in a section slightly below the middle of the
column only the mesenteries of the first grade would be found.

In a section a little higher up than the third (Fig. 5) of those
figured, on the gonophoric mesenteries, just before the mesen-
terial filaments die out, a very small portion of the ‘“ Nesseldrii-
senstreif’”’ can be seen. It is histologically like the same
portion in the mesenteries of the fourth grade, but does not
reach anything like the development it has upon these latter
mesenteries, being of very small extent, and somewhat apt to
be overlooked.

It will be seen from what has been said that the mesenteries of
the fourth grade are noticeably different from those of the other
three grades with which they alternate. They are much
shorter; they never bear reproductive elements; and they
possess both the ‘“ Flimmerstreifen”’ and the ‘ Nesseldriisen-
streif”’ of the mesenterial filaments well developed. On the
other hand, the mesenteries of the first three grades are all
gonophoric, and their filaments consist almost entirely of the
‘‘Flimmerstreifen,” the ‘ Nesseldriisenstreif”’ being very
short.

C. membranaceus has been found to be hermaphrodite by all
who make statements on this point, the ova and spermato-
zoa being both present upon all the gonophoric mesenteries.
C. borealis, Danl. is, according to its describer, bisexual, and
C. Americanus agrees with it. Agassiz, as already noted, states
that this last form is hermaphrodite, the ova occurring in the
upper part of the mesenteries, and the spermatozoa lower
down ; but this is certainly not the case in the three specimens
I had for study. All were females, ova only occurring in the
gonophoric mesenteries, and sections taken at varying distances
down the column to within 2 mm. of the posterior extremity
show no trace of spermatozoa.

Of course it is possible that C. Americanus may be dichog-
amous, as Lacaze-Duthiers (72) believes the Hexactinize to be.
In all the Actinians which I have examined, amounting to over
fifty species, with the exception of certain Zoanthez, which are
known to be hermaphrodite, I have never found any trace of
dichogamy or hermaphroditism. If dichogamy occurred as a
rule, one would expect to find occasionally, at any rate, some
traces of spermatozoa associated with ova, or of ova with sper-

No. 2.] MORPHOLOGY OF THE ACTINOZOA. I4I

matozoa ; but this, so far as I know, never occurs in any of the
Hexactinians. I believe, therefore, that C. Americanus is really
bisexual, and not dichogamous.

3. HISTOLOGY.

The maceration of fresh tissues gives the most satisfactory
results as to the structure of the histological elements in the
Actiniaria. This method I was unable to employ, and the
maceration of preserved specimens gave as usual unsatisfactory
results. Nearly all the facts I have to present have been
derived from the study of sections, and are therefore somewhat
fragmentary. They suffice, however, to show a very close
similarity in the histology of C. Americanus to that of C. mem-
branaceus, as described by the Hertwigs (’79), von Heider ('79),
and Jourdan (80), and, on the other hand, considerable differ-
ences from what Danielssen (’89) has described for C. borealis,
Danl.

(a) Tentacles and Disc.

The ectoderm in these parts is covered by a very distinct
cuticle, which shows a dotted appearance, produced probably
by the existence of perforations for the passage of the cilia.
The outer portion contains numerous nematocysts which stain
deeply with borax-carmine and are cylindrical or slightly curved
with the filament spirally coiled. They resemble those de-
scribed by von Heider and Haime in the same situations. Two
kinds of gland cells are present ; one resembles goblet cells, and
are by far the most abundant, the other kind occurring only
sparingly, and being of the structure figured by the Hertwigs
(Pi. VIII, Fig. 15,2) and: by Jourdan (Pl XII, Fig: 85; ¢).' 2
could not observe that the latter kind were more numerous in
the oral tentacles than in the marginal as Jourdan describes, the
histological structure of both series of tentacles being identical.
In the disc, however, they do seem to be more abundant than
in the tentacles. In sections which were slightly torn I could
perceive indications of the presence of “ Stiitzzellen’”’ and sen-
sory cells, but maceration preparations are necessary for their
proper study.

Below the epithelial layer to which these structures belong
comes the nerve layer. In the tentacles the nerve fibrils are

142 McMURRICH. [VoL. IV.

few and not readily distinguishable; but in the disc they are
much more distinct, and form a well-marked band in sections
(Pl. VIIL., Fig. 6, 2). This difference in the development of the
nerve tract is in correlation with the development of the lon-
gitudinal muscles in the two regions. Occasional nuclei can
be distinguished in the nerve region, which are probably the
nuclei of ganglion cells ; they are no larger, however, than the
nuclei of the cells of the epithelial layer. They appear to be
more numerous in the disc than in the tentacles.

The longitudinal muscles have the same development as in
C. membranaceus. In the tentacles they cover slight elevations
of the mesogloea, and are arranged in a single layer; in the
more contracted tentacles they appear to form two layers, the
fibrils of the upper layer alternating with those of the lower,
but they never show so extensive a development as that figured
by Jourdan (Pl. XII, Fig. 83). On the disc, however (Fig. 6,
/m.), they are arranged on both sides of delicate lamellz of the
mesogloea, which are arranged “like the leaves of a book,” the
entire muscle having a thickness of a 0.032 mm. I could find
in the oral tentacles no trace of the ectodermal circular muscles
described by Jourdan from maceration preparations.

The mesogloea is homogeneous and destitute of cells. Its
ectodermal surface in the disc is raised into thin lamellze for the
support of the longitudinal muscle fibres. These lamellze do
not terminate immediately below the nervous layer, however,
but branch, and send branching fibres up through this layer
(Fig. 6, pm.). This is very clearly seen in some of my prepara-
tions, especially some which were stained with eosin. One is
reminded by this arrangement of what R. Hertwig (88) has
described as occurring in //yanthopsis longifilis.

The endoderm is destitute of Zodxanthella. Its cells give
rise at their bases to muscle fibres, which, as usual, are arranged
circularly. Occasional gland cells are seen, but I did not find
them so numerous as the Hertwigs figure them in C. membra-

naceus ; they are of one kind only, namely, the granular club-
shaped kind.

(b) Zhe Column Wall.

The ectoderm of this portion of the body is characterized by
the great abundance of large nematocysts, with an irregularly

No. 2. } MORPHOLOGY OF THE ACTINOZOA. 143

coiled thread, similar to those originally described by Haime.
They are especially abundant in the upper part of the column,
and occur throughout the entire thickness of the epithelial
layer down to the nerve layer. In the lower part of the epi-
thelium, however, they are principally represented by highly
refractive globules of various sizes, some perfectly homogeneous,
others split with irregular portions of various sizes; these
globules I judge to be developing nematocysts on account of
their behavior to various staining reagents, which is exactly
like that shown by the fully developed cysts. They have been
described and similarly identified by Jourdan. A second form
of nematocyst is also present, chiefly in the outer portions of
epithelium. It is much smaller than the large Cnidz glomi-
ferze (Gosse), and is cylindrical, measuring 28 yw in length, and
5 » in breadth. It is well differentiated by both gold and
saffranin staining, taking with the former a faint pinkish tinge,
and with the latter a bright orange. It is clear, the spiral
portion of the filament not being visible, while the “ Axen-
korper”’ (Mobius) is very readily seen. A few nematocysts
resembling those found in the tentacles and disc also occur.
Undoubtedly too much stress has been laid upon slight dif-
ferences in shape, in distinguishing different forms of ne-
matocysts. Haime certainly erred in this respect, and von
Heider also, though to a less degree. The two forms described
by the latter from the tentacles differ only in size, and are
probably the same, and it does not appear to me to be neces-
sary to distinguish between the two forms of Cnide glomiferz
he describes from the column wall. Probably the second form
I have described above is identical with von Heider’s form d
from the column epithelium.

The epithelium is covered on its free surface by a cuticle.
Gland cells are very abundant, and are of the same kinds as
were found on the disc.

The nerve layer consists of a very strong band of fibres,
which lies immediately above the muscle band. It is traversed
at right angles by fine processes, both from the epithelial cells
and from the mesoglceal lamellae which support the muscle
fibres. A few nuclei are to be seen lying among the nerve
fibres, but they are very few and small, resembling those
found in the nerve layer of the disc. They are probably gan-

144 McCMURRICH. [Von. IV.

glion cells, but there is certainly no such development of ganglion
cells as Danielssen describes as occurring in his C. borealis.

This description agrees essentially with that of the Hertwigs.
It is well known, however, that von Heider’s observations
differ somewhat from those of the Hertwigs. He describes as
occurring in the lower part of the epithelial layer an “ Inter-
basalnetz”’ of fibres, formed by the anastomosis of fine branch-
ing processes of the epithelial cells. In the meshes of the
network are fine, sharply outlined points, which are supposed to
be cross-sections of delicate fibrils. These fibrils are nervous,
and send branches upwards to the epithelial cells, and down-
wards to the mesogloea. The Hertwigs, in discussing in an
appendix von Heider’s results, identify this ‘ Interbasalsub-
stanz’”’ with their nerve band. This, however, is a mistake. In
all sections taken from one of my specimens I get an appear-
ance similar to that shown in the Hertwigs’ Pl. VIII, Fig. 11;
in all the sections taken from another specimen, I get von
Heider’s ‘“Interbasalnetz.’””’ Why there is this difference in
the two specimens I cannot say. It may be due to a difference
in the amount of contraction. The nerve band in those prepa-
rations which show the “Interbasalnetz” is plainly visible,
lying between the muscle layer and the network, and corre-
sponding therefore with the fibrillar layer which von Heider
describes as appertaining to the “mesoderm,” which is shown
in his Pl. V., Fig. 35, f It is this fibrillar layer then, and
not the “ Interbasalnetz,’ which is the nerve band. It is
possible that the ‘‘ Interbasalnetz”’ appearance may be produced
by the great contraction of the mesogloea and of its processes,
which extend up into the epithelial layer. This idea is
strengthened by the fact that gland cells and nematocysts are
of very frequent occurrence in the network, —a fact that shows
that it belongs to the epithelial layer.

The longitudinal musculature of the column is as usual well
developed. For some distance below the margin it is no higher
than the musculature of the disc, but lower down it increases
in size, reaching its greatest height about the middle of the
column, where it is many times higher than on the disc. This
height it retains almost unaltered to within at least 2mm. of the
posterior extremity, except along the dorsal median line, where
it is throughout low as in C. membranaceus. It has essentially

INO; 25] MORPHOLOGY OF THE ACTINOZOA. 145

the same structure as in the disc, except that the fibres near
the base of the lamella are much smaller in diameter than
those which cover the greater portion of their surface. I have
not been able to discover the slightest trace of circular muscles
intermingled with the longitudinal, as Danielssen describes in
C. borealis, Danl.

The mesogloea presents the same structure here as on the
disc. The muscle lamellz are prolonged at their free edges
into numerous fine branching processes, which traverse the
nerve layer, and pass up into the epithelial region of the ec-
toderm. Continuations of the epithelial cells also traverse the
nerve layer, and pass down between the muscle Jamellz, and
are perhaps nervous, or partly nervous, and partly the basal
portions of the “Stiitzzellen.” A circular musculature is
present on the inner surface of the mesoglcea.

(c) The Stomatodeum.

As already stated, the surface of the stomatodzeum is raised
into numerous longitudinal folds, each of which corresponds to
an interval between two mesenteries (Fig. 7). The ectodermal
surface of the mesogloea is raised into slight ridges correspond-
ing to the folds of the ectoderm, and is provided with short
lamellz supporting the longitudinal muscle fibres, which have
a much smaller diameter in this region than elsewhere. The
delicate branching processes from the mesogloeal lamellz are
very evident (Fig. 7), especially those which arise from the
muscle processes covering the ridges. They can be traced for
some distance up into the ectodermal epithelium, forming
supports for its cells.

The epithelial and nerve layers have the same structure and
histological characters as in C. membranaceus. Circular muscles
occur in the endoderm.

(d) The Mesentertes.

The disc in Cerzanthus being funnel-shaped, a section made
transversely through the column wall in its uppermost part will
cut the disc tangentially (though slightly obliquely to its thick-
ness) and the marginal angles of the mesenteries obliquely ;
that is to say, the section of the mesenteries shows their actual!
thickness, but it is at an angle to both their length and breadth.

146 McMURRICH. [Vou. IV.

In such sections the mesenteries present a very different struc-
ture from what is found lower down.

The endoderm in such a section (Fig. 8) resembles very closely
in structure that of the column wall, but lower down, in sections
which pass through the column wall and the stomatodzeum, it
is much lower, very granular, and without any trace of cell out-
lines. In the gonophoric region (Fig. 9) it again becomes high,
higher even than in the uppermost region. The protoplasm of
the cells is crowded towards their free extremities, which take
the carmine stain, and in this region the nuclei are most abun-
dant ; towards the mesogloea, however, the cells form a network
(Fig. 10), the meshes of which are mainly occupied by a sub-
stance which does not stain. Slight traces of a granular sub-
stance are also present, and in some regions there are large
numbers of apparently homogeneous spherical bodies (Fig. 10),
which do stain somewhat deeply, and which vary considerably in
size. It is possible that they may be nuclei, but I am rather
inclined to think from their homogeneity and varying size that
they are food particles. They occur also in the endoderm of
other regions.

It is possible that the network is formed by branching and
anastomosing processes of mesogloea, but such an origin for it
could not be made out. If it should be of this nature, it would
be in accord to a certain extent with what Danielssen has
described in his C. borealis. Delicate lamellae having a wavy
outline project from the mesogloea of the mesenteries into the
epithelium: upon their surface are arranged both longitudinal
and transverse muscle fibres. No such arrangement occurs in
C. Americanus, nor apparently in C. membranaceus and solitarius,
but, as stated, the network, if mesogloeal, might be regarded as
representing it. The granular substance which lies along the
fibres composing the network no doubt corresponds to the mus-
cle fibres described by Danielssen, these, like the pinnate lamelle,
being “ausserst diinne.’” Maceration preparations of C. Amert-
canus failed to show the presence of muscle fibres in the meshes
of the network. In the endoderm of the mesenteries, as else-
where, no Zoéxanthelle are present.

In the sections which pass through the disc and column wall,
the endodermal musculature of the mesenteries is very clearly
seen (Fig. 8). The muscle fibres form a single flat layer on

No. 2.] MORPHOLOGY OF THE ACTINOZOA. 147

both surfaces of the mesogloea, and in the sections are cut more
or less obliquely, some being cut almost transversely, and others
more longitudinally. From the way they run, however, it is
clear that they are really transverse, and their apparent oblique,
or even longitudinal direction, is due to the manner in which the
mesentery is prolonged up into the angle formed by the column
wall and the funnel-shaped disc. Lower down they are not so
apparent, and in transverse sections of a mesentery in the gono-
phoric region they cannot be made out with certainty, although
maceration preparations show their presence. This arrange-
ment of the muscles agrees with what the Hertwigs have
described for C. membranaceus.

The mesogloea is differently developed in different regions.
It is thinnest in the stomatodzal region, and somewhat thicker
in the gonophoric region, being in both these parts almost homo-
geneous, and without any cells in its substance. In the upper-
most angle of the mesenteries it is much thicker than elsewhere
(Fig. 8), and contains cavities, reminding one of the cavities
found in the mesenteries of the Zoanthez, except that the con-
tents are not cellular as in that group, but consist of a granular
substance which does not stain at all with borax carmine.

As stated above, all my specimens were female. The ova are
large, and are imbedded in the mesogloea of the mesenteries, as
is well shown in the preparation figured (Fig. 9), where the
mesogloeal investment has separated from the ovum at one point.
The nucleus is large, and is always eccentric, usually projecting
very noticeably beyond the general surface of the ovum, which is
packed with densely staining yolk granules. One large nucleolus
is always present, but the rest of the nuclear substance in all
my preparations is apparently broken down, the nucleus appear-
ing as an irregularly shaped space with well-marked walls, con-
taining the large nucleolus and a few granules.

The histological details of the mesenterial filaments I hope to
describe fully in a future paper.

I have not been able to find any mesenterial stomata in C.
Americanus.

CLARK UNIVERSITY, WORCESTER.
March II, 1890.

148 McMURRICH.

BIBLIOGRAPHY.

(54) Harme, I.— Mémoire sur le Cérianthe (Cerianthus membranaceus). —
Ann. des Sct. Nat. 4me Série. T.1. 1854.

(59) AGassiz, L.— On some new Actinoid Polyps of the Coast of the United
States. — Proc. Boston Soc. Nat. Hist. VI. 1859-61.

(63) AGassiz, A. — On Arachnactis brachiolata, a species of Floating Actinia
found at Nahant, Massachusetts. — Four. Boston Soc. Nat. Hist. VII.
1863.

(64) VERRILL, A. E.— Revision of the Polypi of the Eastern Coast of the
United States. — Mem. Boston Soc. Nat. Hist. 1. 1866-69. [The paper
was published in 1864. ]

(72) VERRILL, A. E.— Brief contributions to Zodlogy from the Museum of
Yale College. No. XXII. On Radiata from the Coast of North Carolina.
— Amer. Four. Sct. and Arts. 3d Series. WII. 1872.

(72) LacazE-DuTHIERS, H. DE. — Développement des Coralliaires. — Arch.
de Zool. exp. et gén.. T.1. 1872.

(79) Hertwic, O. and R.— Die Actinien anatomisch und histologisch mit
besonderer Beriicksichtigung des Nervenmuskelsystems untersucht. —
Jena, 1879.

(79) VON HEIDER, A.— Cerianthus membranaceus Haime. Ein Beitrag zur
Anatomie der Actinien. — Sttzungsber. der kais. Akad. der Wissensch.
math-naturwiss. Classe. Wien. LXXIX. 1879.

(80) JouRDAN, E.— Recherches zoologiques et histologiques sur les Zoan-
thaires du Golfe de Marseilles. — Ann. des Sct. Nat. Ome Série. X. 1880.

(82) HERTWIG, R.— Report on the Actiniaria. — Reports of the Scientific
keesults of the Voyage of H. M.S. Challenger. Zodlogy. V1. 1882.

(87) McMurricu, J. P.— Notes on Actiniz obtained at Beaufort, N.C. —
Studies from the Biol. Labor. Fohns Hopkins University, Baltimore.
TVi 218872

(88) HERTWIG, R. — Supplement to the Report on the Actiniaria. — Reports
of the Scientific Results of the Voyage of H. M.S. Challenger. Zodlogy.
XXVI. 1888.

(88) Voct, C.— Des genres Arachnactis et Cerianthus.— Arch. de Biol.
VII. 1888.

(88) WiLson, H. V.—On the development of Manicina areolata. — Your.
of Morph. Il. 1888.

(89) FiscHER, P.— Sur la disposition des tentacules chez les Cérianthes. —
Bull. Soc. Zool. de France. XIV. 1889. [Abstract in Your. Roy. Micr.
Soc. Dec., 1889.]

(89) DANIELSSEN, D. C.—Cerianthus borealis. — Bergens Museums Aars-
beretning for 1888. Bergen, 1889.

150 McCMURRICH.

EXPLANATION OF PLATES VI. AND VII.

PLATE VI.

Fics. 1 and 2. Cerianthus Americanus, Ag. — Fig. 1, natural size; Fig. 2, reduced
one-third.

PLATE VII.

Fic. 1. View of specimen laid open by a longitudinal incision passing near the
mid-dorsal line. s? = siphonoglyphe, ac = acontia, s¢ = stomatodzum, 3 = mesentery
of third grade.

Fic. 2. Semi-diagrammatic, showing the relations of the mesenteries of the dif-
ferent grades. 3= mesentery of third grade, 4 = mesentery of fourth grade.

Fic. 3. Section through ventral portion of column, just above the lower end of the
stomatodzeum. J = ventral directive mesenteries, 1 and 1/= mesenteries of the first
grade, 2= mesentery of the second grade, 3 and 3'= mesenteries of the third grade,
4, 4/, 4/', and 4//’= mesenteries of the fourth grade.

Fic. 4. Section through ventral portion of column wall, about 2 mm. below Fig. 3.

Fic. 5. Section through ventral portion of column wall, about 1.5 mm. below Fig. 4.

Fic. 6. Portion of tangential section of disc. cm = circular muscles, mg = mes-
ogloea, 47 = longitudinal muscles, 2 = nerve layer, fz = process of mesogloea. (Zeiss.
obj. J, oc. 2.)

Fic. 7. Transverse section of stomatodzeum (Zeiss obj. J, oc. 2).

Fic. 8. Section of mesentery in the angle formed by the meeting of the disc and
column wall (Zeiss obj. J, oc. 2).

Fic. 9. Transverse section of mesentery in the gonophoric region, with ovum
(Zeiss obj. D, oc. 2).

Fic. 10. Portion of Fig. 9, more highly magnified to show the network. mg=
mesogloea, /= food particle (?). (Zeiss obj. J, oc. 2.)

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ON THE GUSTATORY ORGANS OF SOME OF THE
MAMMALIA.

FREDERICK TUCKERMAN,

CONTENTS.

PAGE

Entroductory Note 2c tr atesecrers c's nts. arsie cave. d sit atcha eer rte A pee ae 152
INDEX OF SPECIES

POLIMAYS ULV EUNEGHE, ye )50 hc ¥ valeieaiictiae ai ya ee ee 152
UI ELEAPIEAE SE LUISECHIUS Lis) Sie 0 000, Sipe vids we eg bois HE coe RMe Re A RTE TE 156
EA SEOLIMAYS \LUOMEOGE wrat b's,e/a' o's 5 #5410 ta, soe a ea mE US ee 157
eb RES MUAELOS CSILCT CLES 6.6, a\a) A/dm e vs eh cs 41% 3 0's) « Simla: crsldhepe te eee A 158
PPE DUS SEU It Karaoke oyna a0) « t's, asian d,o|tva,n asthe tke at ees OE 159
PD ESY PUSE UUNOSES 0) sta 01 3.7550) N53 2 4G; 0a !oigi a's 4 eal oe) SAR ee 160
MNES DVTULIBULUS 0) 55) 0 aa oko el nial o'nl viet siesslerak ance CaO eee ee 162
GREETUY DUCHESS LF UNCALIS 5. ohars shsici's iio) 9:4 veasdie ny coe oA Oe. 162
LEE PUSMUCAIE DESTIN Se. 3) a oleh ab 50's 2 hs \ala aig vitals Gee ae E ee 163
EON SA UMP SATERS a ALS) heh saan dics slots itis Seale tte Eee 166
MA PEPER VEY Si SPLCO DUS Ys \oloa'e vs 0\' a5 shie.o)e <isles 5) Sein Seon aie areas oe 167
MESURE AN: Soa ot ci'6s fase 3 toda 6:9! 53h Seal 4 Sia: 6 BUSTS SA CA Pe ee 169
CPOE SIUOCUICLANUG Rh iaisiatsie 364: 6 Sede Hak Cate RR ee 171
MOTELS PSU ECLLUS os 'e4c 50a i chah a, 2 shale’ exe, = 4) s'e'ia 3 ¥ RISEN ORR PETE 172
SYA NETL LE OM COTE TOO OO SCORER RTO OREO ER CCCs Ct ere ee be ewe 174
SD LAR GME OR ETECAUAR «0-2 sterale alainis o:s/alslaie pais’ aid ola sie gee eon eta ee 174
BUVCELO PSHE CLEP IEERELES ES NOs choca 2 arnt) dickens’ 3:5, Bia fat thdgal toy eee one RPT 175
LET OLUSUPSELEDIOIE Oe 9 eacnis vin gists das ode w on ences A Ie PTR 176
CHIL NITIES FEGSUATIES 31a. no bick ccs < olen njaiel bathed eae cll ARO Oe 178
PAVEELO CADE CHEF ECARA Ns in, 5 5 oi}, a1ale sisrsibieye) alle See Cathe oe q L79
TEAS TLDS CIE PEGLEPESES, 2383 ids td sata ay. 4, 350) 0 bia'6's ah apes Ma Nee Ge 180
GUIS IUPUS aia oss hos nied Sroye 2 t/a Ae leye) 4 ay shclo3 satis eld aration ep 181
CORUSTIAIR AIS ya) RT aida ole tens ate Terra afoa. 2 afeisi aia. we Me eNS e ee 183
GOATS JONPC ES ois ors ay ctnd as api hac ai oy aieledan Raa 184
LNOPRUS EBINFOFISLAIUES si oa: Sai solids ope ais sia") sho dee ETO 185
SE EDCBS ULEUIUIE TB ava fei wre Ss 4 28a se \0, 4[x Sata, She 4, Vaiw Wao Geer eT Ee 186
MTAPIE JOCCMMS 0 ora sy ase 8 5! sip dni sldear aes esis 4 shail a eR Aa ER es 188
MUACALUS: LYROM DIOS Scat Ds ciei Uis 4s alo d a, ais aris a shh gee GR RE TO 185
MAA EECUS FILOSUE iS ch'6' ste aaah ay ancresoiar otal ©: Sates erage a AEE aA ae 189

152 TUCKERMAN. [VoL. IV.

THE researches which are recorded in the following pages
were carried on in the Biological Laboratory of Clark Univer-
sity, Worcester, during the winter of 1889-90.

I am greatly indebted to the University for kindly obtaining
much of the material upon which this paper is based. I also
desire to express my thanks to Mr. Agassiz, Curator of the
Museum of Comparative Zodlogy at Cambridge, Dr. Whitman,
Professor of Animal Morphology in Clark University, Mr.
Bumpus, Associate Professor of Biology in Brown University,
and Dr. J. N. Hall, of Sterling, Colorado, for their kindness in
supplying me from time to time with valuable specimens. The
greater part of the material had been kept in dilute or ordinary
spirit. Of the remaining specimens, some were hardened in
absolute alcohol, others in osmic acid, and still others in
Miiller’s fluid, either alone or in a mixture of Miiller’s fluid and
alcohol in various proportions. The parts of the specimens
intended for microscopical examination were embedded in
celloidin, and for staining the tissues the usual reagents were
employed.

A table containing some comparative data respecting the
gustatory structures of the forms considered in the following
pages 1s appended to the paper.

THe Toncur or Didelphys virginiana.

I examined several tongues of Didelphys, most of them from
adult animals. All of the specimens had been hardened in
spirit before I received them.

General Description. — The organ measures 75 mm. in length,
18.5 mm. in breadth, and 13 mm. in thickness. Anteriorly it is
free from the fraenum linguze for 34 mm. The under surface is
smooth and marked by a longitudinal median ridge extending
from the franum to the tip. On either side of the ridge is a
wide but shallow groove. The tip, which is somewhat obtuse,
is bordered by a delicate fringe of simple filiform papillz, the
latter being probably tactile in function. The upper surface is
impressed by a number of transverse furrows, corresponding to
the roof of the palate. Anteriorly it is covered with closely
packed compound filiform papillz, to be presently described.
The fungiform papille are of fair size, and appear to be normal
in structure. They are not, however, very numerous, and, as

No. 2.] ON THE GUSTATORY ORGANS. 153

usual, are smallest about the tip. The circumvallate papillz
are three in number. They are arranged in an isosceles (or
more rarely an equilateral) triangle, the base of which measures
4.6 mm. The apex of the triangle is turned towards the epi-
glottis, and is distant some 12 mm. from the base of the tongue.
The posterior papilla is situated directly in the middle line of
the organ, and is much larger than the anterior papillae. The
region behind the triangle formed by the circumvallate papillae
lacks the usual fleshy elevations, and is quite smooth; the
immediate area around the papillae, however, is more or less
papillate. The lateral organs of taste (papillae foliatae) were so
effectually concealed that the superficial examination of the
tongue failed to reveal their presence. At each side of the
tongue, just above the line of union of the upper and lower
surfaces, is a fringe of rather coarse filiform papilla, curving
upwards, inwards, and backwards. The fringe terminates
before reaching the base of the glosso-palatine arch.

The tongue of a young Didelphys presented some points of
interest. This organ was 19 mm. in length and 7 mm. in
breadth. The dorsum was marked anteriorly by a longitudinal
mesial raphé. The raphé disappeared about 12 mm. from the
tip. The tip was rounded, and fringed with simple papilla as
in the adult organ. The circumvallate papilla were arranged in
the usual triangle, but were very closely set and very near the
base of the tongue’ The lateral fringe of filiform papillze at
the back of the tongue was well marked.

The Mechanical Papille. —The general surface of the tongue,
anterior to the triangle formed by the circumvallate papillz, is
covered with compound filiform papilla. Interspersed among
them are a lesser number of filiform papillz of a simple type.
About the tip, where the papillz are smaller and more closely
set, there appear to be about thirty to the square millimetre of
surface. Posteriorly there are only half that number covering
the same area. Each papilla rests upon one or more papillary
upgrowths of the mucosa, and, at a short distance from its base,
breaks up into a varying number of very long, slender, recurved
secondary papilla or processes. The number of these processes
varies greatly even in the same region. There are seldom less
than ten, and I have seen more than fifty. The average num-
ber appears to vary from sixteen to twenty. The free portion

154 TUCKERMAN. [Von. IV.

frequently exceeds a millimetre in length, the transverse diam-
eter being only about 0.021 mm. Anteriorly the papillz are
longer and more delicate than elsewhere. As they near the
tip, however, they decrease in number and are somewhat
shorter. Posteriorly they frequently bear a recurved spine
(doubtless a transitional form) in addition to their more slender
secondary processes. Still further back, in some papillz, the
secondary processes appear to have coalesced, forming a single
stout, sharp-pointed, hook-like spine, as in many of the higher
Mammalia. Papille thus modified are also sparingly inter-
spersed among the others at various parts of the dorsum. The
‘secondary papilla form one or more incomplete rings at the
summit of the main upgrowth, leaving within a_ horseshoe-
shaped cavity. Where two rings exist, the inner one is usually
less complete than the outer. More rarely, within the inner
ring a few secondary papille are irregularly scattered. The
secondary processes separate from the papillary body first in
front, those of the posterior side being given off at a higher
level. A few single filiform papilla, somewhat hair-like, are
scattered over the dorsum, particularly its anterior part.

When lowly magnified, the main body of the papilla (in verti-
cal section) appears to consist largely of a mass of cells in the
form of a thick column or inverted cone. This mass is com-
nosed of several distinct but not always sharply marked layers.
The basal layer is quite thin, and consists of small but clearly
defined, deeply staining columnar cells. Succeeding this is a
very thick layer, consisting below of nucleated polyhedral cells,
and above of fusiform cells. The cells of this layer are largely
granular and stain readily in haematoxylin, but less deeply than
those of the layer underlying. Resting upon this layer, and
also covering the lateral area of the papilla, is one consisting of
elongated cells, more or less granular. These cells are some-
what attenuated below, and large and swollen above and at the
sides. The nuclei are not always readily distinguishable, but
are of unusual size. The cells are not stained by haematoxylin.
Against this layer is a comparatively thin one, likewise com-
posed of cells fusiform in shape. The cells stain deeply, and
are apparently of the same general character as those consti-
tuting the bulk of the secondary papilla. The entire free por-
tion of the secondary processes in the anterior dorsal region

No. 2.]} ON THE GUSTATORY ORGANS. 155

stains quite deeply in hematoxylin. The secondary papilla of
the middle dorsal region, in which the cornification of the epi-
thelium is further advanced, are much less affected by staining
reagents. Externally the papillae are covered by a thin layer
of ordinary stratified epithelium.

This type of compound filiform papillz is characteristic of
the marsupial tongue, if not peculiar to it. The papillae, as
suggested by Poulton, who first described them,! have doubtless
been modified in a special manner for the capture of small
insects. The same observer has suggested the term “coro-
nate”? for those papilla which are surmounted by a ring of
recurved hair-like processes, and the term “fasciculate” for
those in which the appearance is more brush-like. For the
compound filiform papille of Dzdelphys virginiana the latter
term would be more closely descriptive than the former.

GUSTATORY STRUCTURES.

The Circumvallate Papille.— The posterior papilla follows
quite closely the type common to the higher animals. The
anterior pair resemble the corresponding papilla of Belideus
and Phalangista, but are further advanced. The posterior
papilla measures 0.75 mm. at its widest part, and is 0.70 mm.
in height. The upper surface is somewhat uneven, and over-
tops slightly the adjacent lingual area. The trench encircling
the papilla is narrow and fairly uniform in breadth. The an-
terior papillae are somewhat elongated, and average 0.60 mm. in
breadth and 0.90 mm. in height. They are deeply set, their
summits barely reaching the level of the general lingual surface.
Serous glands are abundant beneath and at the sides of the
papillae, and their ducts open into the trenches at their base
and lower part.

The taste-bulbs of the posterior papilla are mainly confined
to the inferior half of the lateral area. They are disposed in a
zone of twelve to fourteen closely packed tiers. The bulbs of
the anterior papille are similarly arranged, though with an
increased number of tiers. There are about sixty-five bulbs in
a tier. The bulbs of both regions are fairly uniform in size and
shape, and measure 0.054 mm. in length and 0.034 mm. in
breadth.

1 “On the Tongues of the Marsupialia,” Proc. Zool. Soc., 1883.

156 TUCKERMAN. [VoL. IV.

The Lateral Gustatory Organs. — These organs are somewhat
rudimentary, and consist of five or six irregular folds of the
mucous membrane. The intervening furrows are narrow, and
vary in depth from 0.5 mm. tol mm. Serous glands are fairly
abundant, and their ducts discharge at the bottom of the fur-
rows. Sections through some regions of the organ show the
sides of the folds filled with bulbs, there being twenty or more
tiers of them. As a rule, however, they are fewer in number
and restricted to the lower half of the lateral area. They meas-
ure 0.046 mm. in length and 0.030 mm. in breadth.

Bulbs were present at the upper part of the fungiform papillz.
In some sections as many as five could be counted, but they
were for the most part simple in structure, their apices failing
to reach the superficial layers of the epithelium. They are very
small, averaging only about 0.030 mm. in length and 0.016 mm.
in breadth.

In the young Dzdelphys the papille and trenches, save in a
few instances, were undifferentiated. The glands and their
ducts were likewise incomplete in their development. The
bulbs were mainly epithelial in position and, as usual in em-
bryos and the new-born, had only developed at the upper part
of the papillae, the lateral area being destitute of them. One of
the more advanced among them measured 0.027 mm. in length
and 0.018 mm. in breadth. I failed to identify bulbs in the
epiglottis of Dzdelphys.

THE TONGUE oF Bettongia cuniculus ?

This was the tongue of an embryo. It had been kept in
spirit.

General Description. — The organ is long and narrow, and is
free for nearly half its length. The under surface is marked by
a longitudinal median ridge extending from the fraenum to the
tip, and the papillate surface is impressed anteriorly by a mesial
raphe. Tungiform papillz are freely distributed over the dor-
sum. The circumvallate papille form a triangle near the base
of the organ. The anterior papilla could only be distinguished
with difficulty, their apices being barely visible within the nar-
row, slit-like openings of the trenches. The posterior papilla,
however, was clearly defined.

There are some indications of bulbs in the epithelium and

No. 2.] ON THE GUSTATORY ORGANS. 157

mucosa of the upper part and sides of the anterior papillz. In
the posterior papilla there are three or four tiers of bulbs. They
rest in depressions of the mucosa, and their apices traverse the
epithelium, but fail to penetrate its outer layers. They also
occur to some extent on the upper surface of the papilla.
Serous glands and ducts are present in the mucosa and sub-
mucosa, the latter being very plentiful. They open into the
trenches at their deeper part. No bulbs were detected either
in the fungiform papilla or epiglottis. No search was made for
the lateral gustatory organs.

Tue Toncue or Phascolomys wombat.

This tongue (that of a young animal) had been kept in dilute
spirit, and was not in a very favorable condition for minute
examination. The hardening was completed in absolute alcohol.

GUSTATORY STRUCTURES.

The Circumvallate Papille.— The papillae are arranged in
the usual triangle, the sides being a little shorter than the base.
Their summits are circular, or nearly so, and they are of nearly
equal size. They measure 0.75 mm. transversely, their height
being somewhat less. At their upper part they bear many sec-
ondary papilla; below they are attached to the tongue by a
narrow pedicel. The trenches are narrow and uniform in width.
Serous glands are abundant, and their ducts discharge at the
usual places.

The taste-bulbs are mainly restricted to the lower half of the
lateral walls of the papilla, although isolated ones not infre-
quently occur at higher levels. They are disposed in about ten
tiers, each tier containing on the average seventy closely packed
bulbs. They are also present in the epithelium of the outer
wall of the trench. Here they are disposed in five tiers. The
bulbs are long and narrow, and present considerable variation
in size. The mean length varies from 0.060 to 0.075 mm., and
the mean breadth from 0.024 to 0.035 mm. In a transverse
section through the centre of a bulb (0.030 mm. in diameter),
I counted some twenty-seven cells. In the subepithelial tissue
beneath the bulb region of the papillz is a rich nervous net-
work. From this network fibrils run to the bases of the bulbs

158 TUCKERMAN. [Vou. IV.

and also pass between them. Large ganglion cells are likewise
present in the axes of the papillae. For lack of sufficient
material no search was made for the lateral gustatory organs,
though they are doubtless present.

The fungiform papilla are quite numerous, especially over
the anterior dorsal surface. They are of the usual type and
probably contain bulbs, although none were found. No bulbs
were detected in the epiglottis.

THE TONGUE OF Phascolarctos cinereus.

This specimen was also from a young animal, and had like-
wise been kept in dilute spirit. It was subsequently placed in
absolute alcohol. For a careful study of this tongue, or that
of Phascolomys, fresh material, or material in a better state of
preservation than that which I had at my disposal, will be
necessary.

GUSTATORY STRUCTURES.

The Circumvallate Papille. —The papillz are very clearly
defined. They form a nearly equilateral triangle, the base
being a trifle less than the sides. The posterior papilla is
much the largest of the three, and is encircled by a narrow
trench. It is freely movable, and measures 0.55 mm. in diam-
eter and 0.50 mm. in height. Striated muscle-fibres terminate
at the base of the papilla. The anterior papillz are elongated,
and measure 0.50 mm. in height and 0.35 mm. transversely.
They are set in deep trenches, which open on the surface with
narrow, slit-like apertures. Anteriorly their summits terminate
in an apex. Further back they lose their apex, and become
more convex. At their extreme posterior limits they are com-
pletely roofed over. Their bases are somewhat constricted, and
their apices do not reach the level of the opening of the trench.
Serous glands are plentifully distributed to this gustatory area,
their ducts opening at the bottom of the trenches. Nerves and
large ganglion cells are present in the anterior papillz.

The bulbs are quite numerous. The arrangement of those
of the posterior papilla is much the same as in the circumvallate
papillae of Phascolomys. In the anterior papillae there appear
to be upwards of twenty-five tiers, a well-filled tier containing

No. 2.] ON THE GUSTATORY ORGANS. 159

about seventy-five bulbs. The apex of the papilla is free from
bulbs, but the ridge behind it bears them over the whole of its
circumference. The bulbs were too indistinct for their finer
details to be studied.

Papilla of the fungiform type are not very abundant, but
appear to be of normal structure. They are largest on the
dorsum and sides, just in front of the circumvallate papilla, and
contain bulbs. The usual fringe of filiform papillae at the side
of the base of the tongue, just above the lateral organ of taste,
was present. Below the fringe were a few small, mound-like
elevations, which doubtless marked the seat of the lateral taste
organ. Similar elevations are present in Ha/maturus and other
marsupials. Unfortunately, the material was not in a condition
for closer examination. The epiglottis was not examined.

THE TonGur oF Dasypus peba.

The material consisted of two spirit specimens, —a whole
tongue and the back part of a smaller one.

General Description. —The tongue is long and narrow, and
tapers gradually toa point. It measures 51 mm. in length and
near the base is 10mm. in diameter. It is free for 34 mm. from
the franum, two-thirds of its entire length, and is thus capable
of great prehensile power. The under surface is finely wrin-
kled, and marked by a median ridge extending from the fraenum
to the tip. The ridge is impressed longitudinally by two parallel
grooves. It is likewise transversely furrowed, the furrows being
parallel and 1.5 mm. apart. The anterior two-thirds of the pa-
pillate surface is sheathed with a thick layer of partially corni-
fied epithelium. The posterior third is transversely wrinkled,
the furrows being parallel and running across the tongue. The
basal end of the organ bends somewhat abruptly downwards,
forming a kind of pit. From this pit a deep groove extends
along the middle of the dorsum for6mm. Fungiform papilla
are not numerous, but are quite evenly distributed over the
middle and anterior dorsal surface and upon the sides. Some
of the papillae appear to be set in furrows, with their summits
below the level of the lingual surface. The circumvallate
papilla are two in number. They are 5 mm. apart, and 15 mm.
from the base of the organ. I failed to find the lateral organs

160 TUCKERMAN. [VoL. IV.

of taste in this species, but I think there can be but little doubt
of their existence.

GUSTATORY STRUCTURES.

The Circumvallate Papille.— The papilla are 0.40 mm. in
diameter and 0.95 mm. in height. Their upper surfaces are
more or less flattened, and their sides are vertical, or nearly so.
The trenches encircling them are narrow and very deep. Serous
glands are but sparingly present, and their ducts open at or near
the bottom of the trenches. At the lower part of the papillary
axis are a few isolated ganglion cells. The bulbs are disposed
on the lateral wall, often nearly filling it. They also occur to
some extent on the free surface of the papille. The average
number of tiers appears to be about eighteen (though there may
be twice that number), a well-filled tier containing from sixty
to seventy bulbs. The bulbs show some indications of a neck.
They measure 0.054 mm. in length, their greatest transverse
diameter being 0.030 mm. No bulbs were detected in the
fungiform papilla or in the epiglottis.

Tue ToncueE or Dasypus veillosus.

I received three specimens of this tongue. They had been
kept in spirit, and the tissues were in a fair state of preserva-
tion.

General Description.— The organ is long and narrow, and
tapers gradually to a point. It measures 81 mm. in length, and
is perfectly free for 50 mm. from the frenum. At its posterior
part it is 12 mm. in breadth and 14 mm. in thickness. The
under surface is marked by a distinct median ridge leading from
the fraenum to the tip. The extreme basal portion of the organ
is bent somewhat abruptly downwards, as in Dasypus peba. The
upper surface is quite densely papillate over nearly its entire
area. Papille of the fungiform type are sparingly scattered
over the dorsum and sides of the tongue. The two circum-
vallate papilla are on the same transverse line, and are set quite
close to the lateral margins of the organ. They are 7.3 mm.
apart, and 17mm. from the base of the tongue. There is a
lateral gustatory organ at each side of the base. The organs
are marked externally by three or four small, irregular openings,
running transversely to the long diameter of the tongue.

No. 2.] ON THE GUSTATORY ORGANS. 161

The Mechanical Papille.— These papilla differ to some ex-
tent from the compound filiform papillae of the marsupial tongue.
The secondary papillae are fewer in number, there being not
more than five or six to a papilla as a rule, and resemble some-
what the stout, hard spines of the Carnivora. Many of the
papillae bear at each lateral border a single recurved spine, the
space between being packed with epithelium. When viewed
in horizontal section they present a horseshoe-shaped cavity.
These papillae may be looked upon as representing an interme-
diate type between the “coronate” and “fasciculate”’ papillae of
the Marsupialia and the corresponding papillz of still higher
forms. Another and more simple form of papilla occurs on
this tongue near the lateral margins. It consists of a single
papillary upgrowth of the mucosa, overspreading which is a
layer of stratified epithelium. From the bed of epithelium
rises a single sharply pointed spine. The spine is cornified at
its upper part, and directed inwards and backwards.

GUSTATORY STRUCTURES.

The Circumvallate Papille.— The papillae were not devel-
oped alike in all the specimens. While some of them resemble
the papillae of higher animals, others approach more nearly the
marsupial type. The former, or more recent type, are I.3 mm.
in diameter and 1.1 mm. in height. They are flattened on top,
and barely reach the level of the lingual surface. The bulbs are
disposed around the lower part of the lateral area in eighteen
closely packed tiers. The circumvallate papilla of less recent
type are taller than the foregoing, and their sides converge as
they approach the opening of the trench. Their lateral area is
filled with bulbs to within a short distance of the top, there
being often thirty tiers of them. They measure 0.051 mm. in
length and 0.030 mm. in breadth. Serous glands are quite
abundant, the ducts opening into the trenches at their base and
sides.

The Lateral Gustatory Organs. — The lateral gustatory organ
of Dasypus villosus is not unlike that of Procyon lotor (described
by the writer in the “ Journal of Anatomy,” Vol. XXIV., 1890).
The superficial examination of this region showed several irreg-
ular slit-like openings, but I only succeeded in obtaining sec-
tions through one of them. This opening was 0.20 mm. in

162 TUCKERMAN. [ VoL. IV.

width, and led into a large, irregular shaped cavity or recess,
0.65 mm. in depth and 1 mm. in diameter. The walls of the
recess are not very thick and are lined with stratified epi-
thelium, resembling in the main that of the adjacent lingual
surface. From the floor of the recess rise two ridges, which
largely fill it. The larger of the two measures 0.40 mm. in
diameter and 0.45 mm. in height. Serous glands are fairly
abundant, and their ducts open into the spaces between the
ridges, and also at the sides of the recess towards its deeper
part. The ridges bear bulbs over their entire circumference.
Bulbs are also very irregularly scattered in the walls of the
cavity, and likewise occur in clusters near its mouth. They
are small, measuring 0.042 mm. in length and 0.024 mm. in
breadth.

The fungiform papillz appeared to be of the usual mammalian
type, but were destitute of bulbs. The latter were likewise
wanting in the epiglottis.

THE TONGUE OF Dasypus minutus.

This specimen had been kept in spirit, but was not in a favor-
able condition for minute study.

General Description. — The organ is 33 mm. in length, 5.5 mm.
in breadth, and is free from the frenum for 14 mm. The under
surface has the usual ridge, the upper anterior region being
grooved transversely. The tip was somewhat less pointed than
in the other Edentata examined. Although the entire tongue
was cut into sections, no circumvallate papillz or lateral gusta-
tory organs were found. It is quite probable, however, that they
were overlooked. The fungiform papillz are of good size, and
contained bulbs. Ina single section of one of them I counted
no less than six. They measured on the average 0.039 mm. in
length and 0.021 mm. in breadth. Simple and compound fili-
form papillze were present, the former being interspersed among
the latter. The epiglottis was not examined.

Tue Toncur oF Chlamyphorus truncatus.

I received only the anterior four-fifths of this tongue. The
piece measured 36 mm. in length. It had been kept in spirit,
and was sufficiently firm for cutting.

.

No. 2.] ON THE GUSTATORY ORGANS. 163

GUSTATORY STRUCTURES.

Lhe Circumvallate Papille. —The two papillz required the
aid of a powerful lens to reveal their presence. They are as
usual on the same transverse line, I.7 mm. apart, and lie com-
pletely concealed in deep and narrow trenches, their apices,
which are inclined inwards towards the median line, being
slightly below the openings of the latter. At a short distance
above their bases (which are constricted) the papilla: measure
0.23 mm. in diameter, their height being 0.6 mm., or nearly
three times the transverse diameter. I do not think it probable
that the mouths of the trenches can be closed. The arrange-
ment of the muscles beneath the papillz suggests a possible
drawing downwards of the entire region, but not of the papillz
alone. Glands of the serous type are sparingly scattered
through the connective tissue stroma underlying the papilla,
and their ducts open into the trenches at various levels.

The bulbs are restricted to the lower two-thirds of the lateral
area of the papilla. There may be seventeen or more tiers of
them. They were not clearly enough defined for me to deter-
mine the mean dimensions of the typical bulb of ‘this species.
One which I measured, and which was probably somewhat below
the mean, was 0.030 mm. in length and 0.018 mm. in breadth.
No lateral organs of taste were found on this piece of tongue.

The circumvallate papilla of Chlamyphorus approach quite
closely the marsupial type, the resemblance between them and
the anterior papillae of Belideus and Phalangista being very
marked.

THE Toncur oF Lepus campestris.

I received a fresh specimen of this tongue. The organ was
placed in a mixture of five parts Miiller’s fluid and one part
alcohol. After remaining in this mixture for fourteen days, it
was washed for a few hours in running water, and then trans-
ferred to ordinary spirit, where the hardening was completed.

General Description. —In many rodents the posterior portion
of the tongue rises somewhat abruptly above the level of the
anterior. This is a characteristic feature of the tongue of
Lepus. The organ shows two well-marked divisions, a more or
less flattened and expanded anterior portion, and a raised pos-

164 TUCKERMAN. (VoL. IV.

terior part. The two divisions are of nearly equal length, the
total length of the organ being 55 mm. The anterior division
is 15 mm. in breadth, 10 mm. in thickness, and is free from the
floor of the mouth for 14 mm. The upper surface and sides
of this division are covered with small, densely packed, cone-
shaped papilla, the apices of which are directed backwards.
The epithelium sheathing the papillae is dense and imbricated,
and in their upper half either partly or wholly cornified. The
papillate surface is impressed by a mesial furrow, extending
from the anterior limits of the posterior division nearly to the
tip. The tip is short, broad, and obtuse. The under surface is
somewhat wrinkled, and marked by a longitudinal median ridge.
Fungiform papillae are not especially numerous. They are
rather sparingly distributed over the anterior dorsal surface, and
the posterior division of the organ appears to be nearly destitute
of them. About the tip, however, particularly over its inferior
part, they are of good size and fairly abundant. The posterior
division, which rises somewhat abruptly above the level of the
preceding, is 13 mm. in breadth, and about the same in thick-
ness. The upper surface is somewhat convex, and, in front of
the circumvallate area, is covered with closely set mechanical
papilla, the points of which are directed backwards. The two
circumvallate papillz are placed one on either side of the median
line, and are6mm. apart. The lateral gustatory organs (papillee
foliatae) are situated obliquely on each side of the back of the
tongue, anterior to the glosso-palatine arch and circumvallate
papilla, their anterior extremity being directed downwards and
inwards. They are § mm. long, and, at their anterior end,
3 mm. in breadth. Viewed from above, they are small, convex
elevations, their lateral contours converging and forming an
apex at the posterior limits of the organ.

GUSTATORY STRUCTURES.

The Circumvallate Papille.— The general surface in front of
the papilla is covered with small papillary elevations. The
immediate area around them, however, is unpapillate. They
measure 0.60 mm. in diameter, and are a trifle less in height.
They are flattened on top, and the trenches which encircle them
are narrow and of uniform width. Mucous glands are very
plentiful, more so than those of the serous type. The ducts of

No. 2.] ON THE GUSTATORY ORGANS. 165

the former traverse the mucous membrane, and open, somewhat
obliquely, on the free lingual surface ; while those of the latter
discharge into the trenches at their lower part. The mucosa
forming the body of the circumvallate papillae consists of three
main portions or lamelle. The central lamella is much the
largest, and overtops the other two. At its upper part it is
cleft into a number of secondary papilla, the spaces between
which are filled to a common level with stratified epithelium.
The depressions between the lamellz not infrequently dip down
nearly to the base of the papilla, but they are for the most part
filled by epithelium. The average thickness of the stratified
pavement epithelium covering the papilla is about 0.045 mm.
This layer is thicker above than at the sides, but the difference
is only slight.

The bulbs of this gustatory area fill the middle third of the
papillary wall. Those of the outer wall of the trench are some-
what similarly placed. The bulbs, to all appearance, are in con-
tact by their edges, and, in the papilla, are disposed in a zone
of five tiers. Those embedded in the epithelium of the outer
wall are arranged in a girdle of four tiers. From horizontal
sections I estimated the mean number of bulbs in a tier of the
papilla at sixty, the mean number in a tier of the outer wall
being about seventy-five. The bulbs vary somewhat in size and
shape, but most of them have a fairly well-developed neck.
Their mean length is 0.049 mm. and their mean breadth
0.030 mm., they being a little smaller than in their eastern
congener, Lepus americanus. The papilla are well supplied
with nerves. Medullated fibres of the glosso-pharyngeus enter
the papilla at their base, and their finer (non-medullated)
branches form a delicate network in the mucosa directly be-
neath the bulb region. From the network fibrils enter the
bulbs, and also pass between them into the epithelium.

The Lateral Gustatory Organs. —The superficial dimensions
of these papillae have already been given. Each foliate struc-
ture consists of fourteen folds, nearly all of which bear bulbs
on their lateral area. The folds are separated by narrow fur-
rows, having an average depth of 0.30 mm. The mucosa com-
posing the body of each fold is divided, as is usual in Lepus,
into three secondary folds or lamellz, the primary or central
lamella being taller and slightly broader than the two lateral.

166 TUCKERMAN. [Vot. IV.

Spread over the lamelle is a thin layer of stratified pavement
epithelium, which usually fills up the depressions between them
to one level. Serous glands are plentiful in this region of the
tongue, and their ducts, which are exceedingly numerous, open
at the bottom of the furrows. The bulbs are disposed, midway
between the superior and inferior limits of the lateral wall of
the folds, in four to six closely set tiers. Each tier contains
about sixty bulbs in its entire length. I have counted as many
as eighty in a tier, but this number is largely in excess of the
mean. The dimensions of the bulbs are the same as in the cir-
cumvallate papilla. The fungiform papilla, particularly those
of the tip of the tongue, contain bulbs. I have not infrequently
seen six in a single section of a papilla of this type. The epi-
glottis was not examined, but bulbs have frequently been found
on the posterior surface of this organ in Leporide, not only by
the present writer, but by Krause, Davis, and others.

THE TONGUE OF Geomys bursartus.

The material consisted of a single spirit specimen.

General Description. — The organ is compressed laterally to
fit the narrow mouth cavity, and is somewhat humped up ante-
riorly and posteriorly. It measures 26 mm. in length, 6.6 mm.
in breadth, and 9.5 mm. in thickness, and is quite free for 13 mm.
from the frenum. The tip is drawn out into a rounded point.
The dorsal surface is covered with recurved filiform papilla. A
shallow median groove runs along the under surface. Fungi-
form papillae are not numerous, but occur upon the lower as well
as upper surface of the organ. There exists but one papilla of
the circumvallate type. It is set directly in the median line,
and very near the base of the tongue. The trench, both
anteriorly and posteriorly, is incomplete. It is anteriorly incom-
plete in Fiber zibethicus. The lateral gustatory organs lie below
the convex surface of the posterior elevated portion, and some-
what anterior to the circumvallate papilla.

GUSTATORY STRUCTURES.

The Circumvallate Papilla.— The papilla measures 0.60 mm.
transversely, and is 0.35 mm. in height. At its upper part it
bears many secondary papillz, the depressions between them
being filled by the epithelium. Serous glands are plentiful, and

No. 2.] ON THE GUSTATORY ORGANS. 167

their ducts open at the bottom of the trench. The bulbs are
more numerous than in /7der, but are similarly arranged. There
are eight tiers of them in the papilla and seven on the outer
wall. They form an unbroken chain around the bottom of the
trench, which is continued on to both walls to about the same
level. Isolated bulbs occur on the free surface of the papilla,
and occasionally extend along the outer wall of the trench nearly
to its upper angle. The mean length of the bulbs is 0.036 mm.
and the mean breadth 0.024 mm.

The Lateral Gustatory Organs. — The lateral taste organs are
simple in construction, and resemble those of /vber zibethicus.
They consist of four or five unequal folds of the mucosa, sepa-
rated by furrows of varying depth. The average depth is about
0.20mm. Serous glands are not abundant. There are three
or four tiers of bulbs, forming a chain around the bottom of the
furrow. Isolated bulbs also occur near the top of the lateral
wall. The bulbs of both the circumvallate and foliate taste
areas vary greatly in size and shape. Here they measure
0.036 mm. in length and 0.021 mm. in breadth.

The fungiform papillz are of normal structure, and bear a
few bulbs at their upper part. Those of the papillae of the mid-
dorsal region are only 0,020 mm. in length and 0.010 mm. in
breadth. In the papillae near the tip they are larger, and meas-
ure 0.039 mm. in length and 0.021 mm. in breadth. A careful
search of the epiglottis and other parts of the larynx failed to
reveal the presence of bulbs.

THE TonGuE or Hesperomys leucopus.

This tongue, like the last, was a spirit specimen.

General Description.— The shape of the organ suggests a
division into an anterior and a raised posterior part. The pos-
terior division is the longer of the two by one millimetre, the
total length of the organ being 13 mm. The anterior division
is free from the floor of the mouth for 4.5 mm. The tongue is
4 mm. in breadth and about the same in thickness. The upper
surface is covered with small conical papille, the apices of which
are directed backwards. Anteriorly it is marked by a deep
mesial groove which passes through the tip, and is continued
for a short distance on to the under surface. The under surface
is smooth, and somewhat hollowed out. Fungiform papillae are

168 TUCKERMAN. [VoL. IV.

not numerous, but are of the usual type. There is a single,
deeply set, circumvallate papilla situated in the median line,
2.5 mm. from the base of the organ. As in /%ber zibethicus, the
trench is anteriorly incomplete, but perfectly so posteriorly and
laterally.

GUSTATORY STRUCTURES.

The Circumvallate Papilla.— The general surface adjacent to
this gustatory area is more or less marked by papillary eleva-
tion of the mucous membrane. The papilla is quite simple in
structure, and measures 0.25 mm. in diameter and about the
same in height. It is flattened on top, and nearly encircled by
a wide trench. Mucous glands do not seem to be as abundant
as usual in this region of the tongue. Their ducts, however,
are very large, and pass through the mucous membrane to open
on the free lingual surface. Serous glands, on the contrary, are
quite plentiful, and even extend into the papilla itself. Their
ducts discharge into the trench at its deeper part. The bulbs
fill a portion of the lower part of the lateral area of the papilla,
but do not always extend toits base. The corresponding region
of the outer wall also contains bulbs. They are far from
numerous in this gustatory area, there being but three tiers in
the papilla and but two on the outer wall of the trench. They
traverse the epithelium obliquely, their bases being bent more
or less downwards. They have a short neck, and measure
0.048 mm. in length and 0.024 mm. in breadth.

The Lateral Gustatory Organs. — These organs were so incon-
spicuous as to be overlooked in the superficial examination of
the tongue. They are very simple and primitive in character,
and resemble in some degree a circumvallate papilla. They con-
sist merely of a single fold of the mucosa, 0.16 mm. in diam-
eter, on either side of which is a wide furrow, 0.18 mm. in depth.
In the underlying stroma serous glands are relatively plentiful.
There are two or three tiers of bulbs on the walls of the folds,
and one or two tiers on the opposed walls of the furrows.
They measure 0.042 mm. in length and 0.024 mm. in breadth.

The fungiform papilla are small, measuring only 0.14 mm. in
height and 0.05 mm. in diameter. They usually contain bulbs.
The latter lie partly in the epithelium and partly in the mucosa,
their apices, apparently, not piercing the outer layers of the

No. 2.] ON THE GUSTATORY ORGANS. 169

epithelium. They are much elongated, measuring 0.045 mm. in
length and 0.018 mm. in breadth. The filiform papillae follow
the usual type. The epiglottis was not examined.

Tue TONGUE oF Castor fiber.

I received a fresh specimen of this tongue. The organ
showed some indications of malformation, notably of the left
side. The hardening was conducted as in Lepus.

General Description. — The organ measures 74 mm. in length
and 25 mm. in breadth. The free portion is short, being only
about 18 mm. in length. The upper surface is soft and velvety
to the touch. Anteriorly it is flat and somewhat expanded, and
terminates in an obtuse apex. The fungiform papillz are of
fair size, and are quite numerous over the anterior fourth of the
dorsal surface and tip. Posteriorly they are less abundant, but
very conspicuous, projecting somewhat above the lingual sur-
face. Some of those just in front of the gustatory area are very
like circumvallate papille. At the posterior part of the dorsum,
and well towards the base, are three papillze of the circumvallate
type. They form a triangle, the apex of which looks towards
the epiglottis. The base of the triangle is 8 mm. in length, the
length of the sides being 5 mm. The posterior papilla is 11 mm.
distant from the base of the organ. The lateral gustatory
structures are situated a trifle anterior to the triangle formed
by the circumvallate papilla, the left lateral organ being much
further forward than the right.

GUSTATORY STRUCTURES.

The Civcumvallate Papille.—The papille are 0.26 mm. in
diameter and 0.16 mm. in height. Their summits are circular
or slightly oval, and are more or less marked by verrucose ele-
vations. Superimposed on one of the papilla was one of a
fungiform type. This is not a very unusual occurrence; I
have observed it in Lepus, and Schwalbe has called atten-
tion to it in Sws. A papilla thus placed does not always
bear bulbs, and in this instance I could not be sure of their
presence. The trenches encircling the papille are wide at the
mouth, but narrow and uniform at their lower part. Mucous
and serous glands are quite abundant in this region. The

170 TUCKERMAN. [Von. IV.

ducts of the former open as usual on the free lingual surface,
while those of the latter discharge into the trenches at their
base and deeper part. Bulbs are not very numerous in the
papillae of this taste area, and are chiefly limited to the lower
part of the lateral wall, where they are disposed in five or six
tiers. One of the bulbs divided transversely to its longitudinal
axis (but probably not exactly at right angles to the true axis)
consisted of at least thirty-six cells. The bulbs do not form a
continuous zone, but are somewhat irregularly scattered round
the edge of the papillary wall. They vary greatly in size and
shape, one of the largest measuring 0.060 mm. in length and
0.039 mm. in breadth. The mean length is probably 0.055 mm.,
and the mean breadth 0.031 mm. A few scattered bulbs are
present in the epithelium of the outer wall of the trench where
it curves beneath the papilla, and it is not unlikely that they
may also occur at higher levels, although none were detected.

The Lateral Gustatory Organs. —1 am not quite certain that
I had the foliate structure entire. The part in my possession
measured 5 mm. in length, and consisted of nine or ten folds,
the majority of which were bulb-bearing. The folds were not
separable into primary and secondary lamellz as in the Lepori-
dz, but were occasionally cleft for half their depth into two
quite equal portions, the interspace thus left being nearly free
from epithelium. The furrows separating the folds varied
greatly in width. Some of them were narrow above, but very
much dilated below, while others were narrow and of uniform
breadth throughout. The depth was fairly uniform, averaging
0.65 mm. Serous glands were exceedingly abundant, occurring
within the papillz as well as around them. The ducts were
plentiful, many of them being of small diameter but of unusual
length. They open at the bottom of the furrows and also at
the sides of the folds at different levels. A small bundle of
striated muscle-fibres terminated within the base of each fold.
The bulbs are disposed on the lateral area of the folds in seven
to nine tiers. They are likewise irregularly scattered in the
epithelium bordering their mid-furrow. In horizontal section
they are very closely packed. They measure 0.054 mm. in
length and 0.030 mm. in breadth, the gustatory pore having a
diameter of 0.0028 mm.

The fungiform papillae bear bulbs. Some of them possess

No. 2.] ON THE GUSTATORY ORGANS. 171

unusually long necks, and apparently reach the free surface.
The mucous glands of Nuhn are present in the tip of the
tongue, their ducts opening on its inferior surface. The epithe-
lium of the larynx was searched for bulbs, but none were
detected.

In many ways the gustatory structures of Castor fiber approach
closely those of Arctomys monax.

Tue ToNnGuE oF Cynomys ludovicianus.

The material consisted of two specimens. One of the tongues
had been kept in dilute spirit, and was gradually hardened in
. ordinary spirit and absolute alcohol. The other tongue, a fresh
specimen, was prepared for microscopical examination by the
method employed for Lepus and Castor.

General Description. — The organ is 41 mm. in length, 11 mm.
in breadth, and 8 mm. in thickness. The free portion measures
16 mm. in length. The papillate surface is wrinkled anteriorly,
and soft and velvety to the touch. The tip is obtuse, and
slightly cleft as in Avctomys, Fiber, and Hesperomys. The
fungiform papilla are small and inconspicuous, but are quite
evenly distributed over the anterior third and tip of the tongue.
The filiform papilla resemble those of #zder. The under sur-
face of the organ is impressed by a deep central groove. The
circumvallate papilla are three in number, and are situated
well back on the dorsum. They are arranged in an isosceles
triangle, the apex of the triangle being directed backwards.
The posterior papilla is 3.5 mm. from the base of the tongue.
The lateral gustatory organs are situated somewhat further
forward than the circumvallate papilla. When viewed from
above, they present a row of slit-like openings, below which
are a number of mound-like elevations. Each elevation is
pierced near its centre by a small aperture, which proved on
closer examination to be the mouth of a mucous duct. Above
the lateral taste organ is a fringe of filiform papillz, the points
of which are directed upwards, inwards, and backwards. The
fringe is continued on to the lower part of the glosso-palatine
arch.

GUSTATORY STRUCTURES.

The Circumvallate Papille. — The lingual surface adjacent to

the papillae is slightly papillate. The papillae are of good size

172 TUCKERMAN. [Vot. IV.

and quite movable. They measure I mm. in diameter and
0.55 mm. in height. Their free surface is flattened or slightly
convex, and they are occasionally cleft at their centre. The
trenches are narrow and uniform. Serous glands are not very
plentiful. Their ducts appear to open into the trenches at all
levels. The bulbs are disposed at the lower half of the lateral
area in six or seven tiers. I counted sixty bulbs in a tier, but
the circle was not quite complete. The bulbs are closely packed
in the tiers, and are fairly uniform in size and shape. They
measure 0.052 mm. in length and 0.030 mm. in breadth.

The Lateral Gustatory Organs.—These organs are about
2.40 mm. in length, and consist of five or six irregular folds of
the mucosa. The folds bear many secondary papille, and the
epithelium covering them is very much thicker on the upper
surface than at the sides. The furrows vary in depth, but
average about 0.30 mm. The serous glands and ducts offer
nothing unusual either in their position or structure. The bulbs
occupy a large portion of the lateral area of the folds, there
being from three to five tiers of them. They measure 0.051 mm.
in length and 0.032 mm. in breadth.

Most of the fungiform papillze contain bulbs. I found one
remarkably fine specimen in this region. It measured 0.056 mm.
in length and 0.036 mm. in breadth, and was almost wholly epi-
thelial in position.

Bulbs occur in the epithelium of the epiglottis and elsewhere
in the larynx. Those of the anterior surface of the epiglottis
are more or less spheroidal, and have a diameter of 0.042 mm.
Those of the posterior surface vary in length from 0.033 to
0.036 mm. Bulbs are also present on the inner surface of the
thyroid and arytenoid cartilages.

THE TONGUE OF ZJamuas striatus.

I received fresh specimens of this tongue. They were pre-
pared for histological examination by the method employed for
Lepus, Castor, etc.

General Description.— The tongue is 22 mm. long, 6 mm.
broad, and 5 mm. in thickness. The length of the free portion
isgmm. The papillate surface is marked anteriorly by a faint
mesial raphé. The dorsum in front of the circumvallate area
is covered with small recurved papilla, not unlike those of Fzder.

No. 2.] ON THE GUSTATORY ORGANS. 173

The apex of the tongue is obtuse, and the under surface smooth
and unmarked by ridge or groove. Papillae of the fungiform type
are thinly distributed over the dorsum. They appear to be of
normal structure. The circumvallate papilla, three in number,
are situated on the same transverse line. They are very near
together, and are 3.5 mm. distant from the base of the organ.
The lateral gustatory organs are quite far forward. A line
dividing the circumvallate papille at their centres, if continued
on to the under surface of the tongue, would pass through the
posterior limits of the lateral taste organs.

GUSTATORY STRUCTURES.

The Circumvallate Papille.—The papillae are 0.44 mm. in
diameter and 0.35 mm. in height. They are flattened on top,
and frequently cleft at the centre. Their sides are quite sym-
metrical, and at the lower part bend slightly inwards. The
trench encircling each papilla is deep and narrow. Serous
glands are not very plentiful in the underlying stroma. The
ducts open into the trenches at their deeper part. The bulbs
are disposed in five or six tiers, filling completely the overhang-
ing wall of the papilla. There are about fifty closely packed
bulbs in a tier. They are fairly uniform in size, measuring
0.051 mm. in length and 0.029 mm. in breadth.

The Lateral Gustatory Organs.— The organs measure 2.10
mm. in length, and consist of seven or eight bulb-bearing folds.
The folds are quite simple in construction, but vary somewhat
in size. They are separated by deep and narrow furrows, the
average depth being about 0.40 mm. Serous glands are not
abundant. Their ducts open at the usual places. The bulbs
are arranged in six to eight tiers, filling the lower half or lower
two-thirds of the folds. They measure 0.048 mm. in length
and 0.024 mm. in breadth.

All of the fungiform papilla examined contained bulbs. One
vertical section of a papilla showed two of equal size, measuring
0.039 mm. in length and 0.030 mm. in breadth. They were
placed obliquely near the summit, with their bases resting in
a depression of the mucosa, and their apices directed upwards
and slightly outwards.

Bulbs were present in the epiglottis and occurred elsewhere

174 TUCKERMAN. [Vou. 1V..

in the larynx. They measure in this region about 0.039 mm.
in length and 0.024 mm. in breadth.

THE TONGUE OF Sorex coopert ?

The material consisted of spirit specimens.

General Description. —The organ is 11 mm. long, and is free
from the frenum for 5 mm. The posterior portion is somewhat
raised above the level of the anterior, and the dorsum is trans-
versely ridged to fit the palatal grooves. The upper surface
and sides are beset with small, densely packed, filiform papillz.
The papillae are 0.15 mm. in height, and bear on their upper
part sharp-pointed, recurved, cornified spines. The two cir-
cumvallate papillze lie in the same plane, and are 0.5 mm. apart
and 3 mm. from the base of the organ. As seen from above
they are elliptical in form, and placed obliquely to the long axis
of the tongue, with their anterior extremity directed outwards.
The fungiform papilla are very small and scattered. Nothing
could be detected of the lateral gustatory organs. They are
doubtless present, though probably of a simple type.

GUSTATORY STRUCTURES.

The Circumvallate Papille.—The papillze have a transverse
diameter of 0.14 mm., and are 0.15 mm. in height. They are
flattened or concave on top, and project but slightly from the
opening of the trench. The sides converge somewhat sharply at
their lower part, giving the papillae a constricted base. The
trenches are narrow and quite uniform in breadth. Serous
glands are quite widely distributed through the mucosa and
submucosa, but are not very plentiful directly beneath the
papillae. Their ducts discharge at the usual places. The bulbs
resemble those of the Rodentia. They are arranged in three
or four tiers at the lower half of the lateral area, and measure
0.033 mm. in length and 0.020 mm. in breadth.

The fungiform papillz bear bulbs at their upper part. The
epiglottis was not examined.

Tue ToNGuE OF Alarina brevicauda.

This tongue, a spirit specimen, was not in a suitable condition
for minute study, and I am only able to give a very brief gen-
eral account.

No. 2.] ON THE GUSTATORY ORGANS. 175

General Description. — The organ measures 15 mm. in length,
4.5 mm. in breadth, and 3 mm. in thickness. It is free from
the fraenum for 5.5 mm. Papillz of the fungiform type are not
numerous. The two circumvallate papilla are on the same
transverse line, and are about 3 mm. from the base of the
tongue. They are clearly defined, and are 1.2 mm. from each
other and about the same distance from the lateral limits of the
organ. Their summits are circular, and measure 0.25 mm. in
diameter. Serous glands are not abundant. The epiglottis
was examined microscopically, but no bulbs were detected.

THe TONGUE OF Scalops argentatus.

The material consisted of spirit specimens.

General Description.— The organ is 25 mm. long, 7.5 mm.
wide, and 6 mm. in thickness. The free portion is 13 mm. in
length. The papillate surface is somewhat convex, and marked
by seven or eight transverse ridges corresponding to the palatal ,
grooves. The tip is rounded. The dorsum is covered with
thickly set, simple and compound filiform papillae of the ordinary
type. Fungiform papillz are not numerous, but are distributed
quite uniformly over the anterior two-thirds of the dorsum. At
about 5 mm. from the base of the organ are two elliptical-
shaped circumvallate papilla. They are set obliquely to the
long axis of the tongue, with their anterior extremity directed
outwards. The lateral gustatory structures were not easily de-
tectable. They are situated as usual at the sides of the base of
the organ, a little anterior to the circumvallate papilla, and con-
sist of a few simple, more or less irregular folds of the mucous

membrane.
GUSTATORY STRUCTURES.

The Circumvallate Papille.— The papillz are lobate. They
measure 0.90 mm. in diameter and 0.40 mm. in height. Both
serous and mucous glands appear to be plentiful around the
papilla. No regular arrangement of the bulbs could be deter-
mined from my sections. They measure 0.048 mm. in length
and 0.027 mm. in breadth.

The fungiform papillz are well supplied with bulbs. Some
of them are very small, being only about 0.021 mm. in length.
They measure on the average, however, 0.035 mm. in length and
0.024 mm. in breadth.

176 ZUCKERMAN. [Vor. IV.

The epiglottis and anterior surface of the larynx contain
bulb-like bodies. They are almost wholly epithelial in position,
and measure 0.036 mm. in length and 0.024 mm. in breadth.

Tue ToncuE oF Pteropus pselaphon.

I examined two tongues of this species, which is peculiar, I
think, to the Bonin Islands. They had been kept in spirit and
were well preserved.

General Description. —The organ measures 48 mm. in length,
14 mm. in breadth, and 10.5 mm. in thickness, and is free for
25 mm. from the frenum. The extreme posterior dorsal region
is more or less furrowed, and at the sides of the base are numer-
ous large filiform papilla, curving upwards, inwards, and back-
wards. The anterior dorsal surface is covered with closely
packed, recurved filiform papilla which, when stroked in the
opposite direction, convey the feeling of a fine-toothed rasp.
The tip of the organ is nearly pointed. The under surface is
smooth, and marked by a median groove extending from the
fraenum half-way to the tip. The fungiform papillz are of good
size, but not very abundant. They are thinly scattered over
the dorsum, and are also collected into a line at the sides, just
above the junction of the papillate and non-papillate surfaces.
The circumvallate papille, three in number, form a nearly equi-
lateral triangle. They are very close to the base of the tongue,
the papilla forming the apex of the triangle being but 3 mm.
distant from it. The anterior papilla are somewhat larger than ,
the posterior papilla, and are about I mm. apart.

The Mechanical Papille.—The compound filiform papilla
of Pteropus approach quite closely in their structure the corre-
sponding papilla: of the Marsupialia, of which they appear to be
a modified form. They are large and prominent along the mid-
dle of the dorsum, but anteriorly and laterally they are smaller
and more delicate, the transition from one form to the other
being somewhat abrupt. Anteriorly there are from eight to ten
papillae to the square millimetre of surface. Posteriorly they
are somewhat less thickly set. The larger ones measure at
their base 0.40 mm. in diameter. A little above the base the
diameter is very much less. Those near the tip have a trans-
verse diameter of 0.20 mm., and are about 0.45 mm. in height.
Fach papilla is seated upon a single main papillary upgrowth of

No. 2.] ON THE GUSTATORY ORGANS. 177

the mucosa. Covering the papilla is a dense mass of stratified
epithelium. No separation of the epithelium into distinct lay-
ers was possible in my sections. The secondary papillz or
processes are eight to twelve in number. At the lower part of
the papilla they are grouped near the centre of the main up-
growth. At higher levels they form an incomplete ring round
the edge, leaving a horseshoe-shaped space within. The free
ends of the secondary papilla are very short, and completely
cornified. They terminate in sharply pointed spines, the apices
of which are directed inwards and slightly backwards. The
secondary papilla of Pteropus separate from the main papillary
body at a very much higher level than in the Marsupialia, and
their free portion is consequently much shorter. The termi-
nal hooks are also relatively stouter in the former than in the
latter, and more completely cornified. The papille are largest
and fullest developed along the central portion of the dorsum.
Towards the lateral border and tip they lose the ring of second-
ary processes, the latter being replaced by one or more short
spines.

GUSTATORY STRUCTURES.

The Circumvallate Papille. — These papilla measure 1.25 mm.
transversely, and are 0.60 mm. in height. Their summits are
convex and slightly verrucose, and overtop the adjacent lingual
area. The trenches are rather narrow, but not deep. Serous
glands are very abundant within the papilla and beneath and
around them. The ducts are numerous and usually open at the
bottom of the trenches. Those of the intrapapillary glands
traverse the epithelium of the lateral wall, and open at a higher
level. The bulbs of this taste area occupy the overhanging wall
of the papilla, but are not entirely confined to it. They occur
in the epithelium of the outer wall of the trench, and isolated
ones are present on the lateral slopes of the upper surface of
the papilla. Those of the overhanging wall are quite closely
packed, and are disposed in a zone of eight to ten tiers. On
the outer wall their arrangement is less regular. In a well-
filled tier of the papilla there appear to be at least eighty bulbs.
In a transverse section through the lower part of the outer wall
I counted one hundred and seventy-five bulbs; the section,
however, included parts of several tiers. The bulbs are long

178 TUCKERMAN. (VoL. IV.

and slender, and measure 0.000 mm. in length and 0.029 mm. in
breadth.

The Lateral Gustatory Organs. — There is a somewhat strik-
ing resemblance noticeable between the gustatory folds of some
of the Chiroptera and a circumvallate papilla of the usual type.
In Pteropus this resemblance is less marked than in the genus
Vespertilio, the gustatory structures of which have already been
described in this Yournal by the present writer. The organs
consist of three or four very irrregular folds of the mucosa.
The intervening furrows are fairly uniform in width, but vary in
depth, the average depth being about 0.60 mm. Serous glands
are not very plentiful in this region. Their ducts open at the
usual places. The bulbs are restricted to the lateral walls of
the folds, and not infrequently nearly fill them. I counted
upwards of twenty tiers, but the mean number is somewhat
less. The bulbs are of the same shape and size as in the cir-
cumvallate papilla. The marginal fringe of filiform papillz,
peculiar to this region in many mammals, has already been
alluded to.

The fungiform papilla are well supplied with bulbs, and they
are also exceedingly numerous in the larynx, especially on the
inner surface of the cricoid and arytenoid cartilages.

THE TonGuE oF WVyctinomus nasutus.

The specimens had been kept in alcohol, and were in a fairly
good condition.

General Description.—The shape of the organ suggests a
division into an anterior and a raised posterior part. The
posterior division is 6 mm. in length, the total length of the
organ being 11 mm. The anterior division is 4.2 mm. in
breadth, 3 mm. in thickness, and is free from the floor of the
mouth for 3.5mm. The fungiform papillz are quite large on
the raised portion of the organ directly in front of the gustatory
area. They are also arranged ina line (for a part of the distance
in two rows) on each side of the tongue, at the junction of the
upper and lower surfaces. The circumvallate papilla, two in
number, are situated one on either side of the median line,
I mm. distant from the base. No search was made for the
lateral gustatory organs for want of sufficient material.

No. 2.] ON THE GUSTATORY ORGANS. 179

GUSTATORY STRUCTURES.

The Circumvallate Papille.— The papillz are 0.30 mm. in
diameter and 0.25 mm. in height, and project somewhat from
the opening of the trench. They are flattened on top with
symmetrically sloping sides curving downwards and inwards,
the bases of the papillz in consequence being somewhat con-
stricted. The trenches are uniform in breadth and fairly deep.
Serous glands are only sparingly present. There are five or
six tiers of bulbs, the uppermost tier sometimes being on a
level with the opening of the trench. In horizontal section I
counted fifty bulbs. They measure 0.050 mm. in length and
0.028 mm. in breadth.

All of the fungiform papillz examined bore bulbs, but they
were smaller than those of the circumvallate papillz. Bulbs
were also plentiful in the epithelium of the epiglottis and else-
where in the larynx

THE TonGuE oF Axntilocapra americana.

The material consisted of a single fresh specimen. It was
hardened by the method adopted with Lepus, etc.

General Description.— The organ measures 180 mm. in
length, and possesses the general characters of the ruminant
tongue. The fungiform papilla resemble black or dark-colored
beads, and are quite numerous, especially about the tip, includ-
ing its inferior portion. They are also arranged lineally on
each side of the dorsum from the tip to the gustatory area,
where they are not easily distinguished from those of the
circumvallate type. The circumvallate papilla are grouped in
two main portions, on each side of the median line, at the
posterior part of the dorsum. There appear to be about twenty-
six papilla on a side, those most posteriorly placed being
34 mm. from the base of the tongue.

GUSTATORY STRUCTURES.

The Circumvallate Papille.— The papillz are flattened or
slightly rounded on top, and their sides are vertical or nearly so.
The epithelium investing the lateral area is less thick than that
of the upper surface, the latter in turn being much thinner
than that of the adjacent lingual area. The papillae measure

180 TUCKERMAN. [Vor Ve

1.20 mm. transversely, and are 1.10 mm. in height. At their
upper part they bear many small secondary papille. The
trenches are narrow and of uniform width. Serous glands are
plentiful, and their ducts usually open at the bottom of the
trenches. (A few of the papilla show a marked reversion to an
earlier type. They consist merely of a simple semicircular
ridge, partially roofed over above, and bearing bulbs over its
circumference.) The bulbs are disposed in several tiers. In
some papillz the tiers are quite closely set, while in others
there is considerable space between them. When closely set
they are usually restricted to the middle portion of the lateral
wall, the space above and below being destitute of them. The
mean number of tiers is about twelve, and there are upwards of
eighty bulbs in a tier. They measure 0.069 mm. in length and
0.032 mm. in breadth.

No lateral gustatory organs were detected. Thus far in the
Ruminantia they have only been found, I think, in 7ragulus
javanicus, Camelopardalis giraffa, and Antilope mergens.

The fungiform papillz are of the usual type and bear bulbs.
The latter, however, appear to have relatively decreased in num-
ber, this perhaps being due to the unusual extent of bulb-
bearing surface offered in the gustatory area proper of the
tongue.

A few bulbs are present on the anterior surface of the epi-
glottis. Mucous glands are also abundant in this region.

THE ToNGuE oF Lutra canadensis.

I received several specimens of this tongue, all but one of
which were fresh. The fresh specimens were prepared by the
method employed for the preceding tongue. The spirit speci-
men was from a very young animal, and the hardening was
completed in ordinary alcohol.

General Description.— The organ is 71 mm. long, 23 mm.
wide, and 5 mm. in thickness. Anteriorly it is much tied down,
the free portion being only 15 mm. in length. The upper sur-
face is covered with filiform papilla, the apices of which are
directed backwards. When stroked in the opposite direction,
the papillae convey the feeling of a very fine rasp. A wide but
shallow median furrow leads from the area of the circumvallate
papilla to the tip. The circumvallate papilla, seven or eight

No. 2.] ON THE GUSTATORY ORGANS. 181

in number, are arranged in the form of an inverted V._ Fungi-
form papilla are grouped quite thickly in the space bounded
by the circumvallate papilla, and are also scattered elsewhere
on the dorsum. The tip of the organ is obtuse, and the under
surface smooth and marked by a narrow groove extending from
the fraenum to the apex.

In the young Lztra the dorsal surface, anterior to the cir-
cumvallate area, was thickly studded with fungiform papillze.
They were very evenly distributed, but were most thickly placed
along the median furrow, which in this tongue was well marked.
Far back on the dorsum were six or seven circumvallate papillze.

GUSTATORY STRUCTURES.

The Circumvallate Papille.—The papille are 0.55 mm. in
diameter and 0.75 mm. in height. The trenches encircling
them are narrow and deep. Glands of the serous type are not
very abundant. The ducts open into the trenches at different
levels. The papillze of the young Lztra were in better condition
for studying the arrangement of the bulbs than those of the adult
specimens. In the former the bulbs are disposed on the lateral
area in six to eight tiers. They are also present in the epithe-
lium of the outer wall, and even occur on the free upper surface
of the papilla. From horizontal sections there appear to be
about fifty bulbs in a tier. They measure 0.053 mm. in length
and 0.030 mm. in breadth.

The lateral gustatory organs appear to be rudimentary in
structure. They consist merely of a few simple folds of the
mucous membrane. The folds were destitute of bulbs, and no
glands of the serous type were detected in this region.

The fungiform papillz contained bulbs as usual. One from
a papilla of the mid-dorsal region measured 0.045 mm. in length
and 0.027 mm. in breadth. The base of the bulb rested against
the mucosa, and the apex penetrated the superficial layers of
the epithelium. No bulbs were detected in the epiglottis.

THE TONGUE OF Canis lupus.

This specimen and the following one were hardened in
Miiller’s fluid and alcohol, according to the method already
described.

182 TUCKERMAN. [ VoL. 1V-

General Description. — The organ is 156 mm. in length,
45 mm. in breadth, and 20 mm. in thickness. Anteriorly it is
free from the floor of the mouth for 73 mm., or nearly half its
length. The fore part of the papillate surface is impressed by
a well-marked mesial groove. Posteriorly the groove is con-
tinued as an indistinct raphé to the base of the organ. The
fungiform papilla are quite evenly distributed over the dorsum
and sides from the circumvallate area to the tip. They resem-
ble minute white beads, being largest posteriorly and gradually
decreasing in size as they approach the anterior limits of the
organ. The extreme posterior region and sides are covered as
usual with coarse, fleshy, recurved papillary elevations. The
under surface, except at the margins, which are somewhat
wrinkled, is quite smooth. The circumvallate papilla are two
in number. ‘They are on the same transverse line, 13 mm.
apart, and 26 mm. from the base of the tongue. At a short
distance from the base, near the line of union of the upper and
lower surfaces, the lateral gustatory organs are easily distin-
guishable. A line dividing the circumvallate papillae at their
centres, if continued on to the under surface of the tongue,
would pass through the posterior end of the lateral taste organs.

GUSTATORY STRUCTURES.

The Circumvallate Papille.— The papillae are laterally in-
voluted, and measure 1.25 mm. in diameter and 0.90 mm. in
height. Their summits are more or less verrucose, and project
somewhat from the trenches. The sides of the papilla are
quite symmetrical, and the trench encircling each is uniform in
width. Serous glands are sparingly present, and their ducts
open usually at the bottom of the trench. The bulbs are dis-
posed in sixteen closely packed tiers, filling the inferior half of
the papillary wall. A few bulbs are scattered at irregular inter-
vals on the outer wall of the trench, and, from some indications,
it is not improbable that isolated ones may also occur on the
upper surface of the papilla. Judging from horizontal sections,
there appear to be at least ninety bulbs in a tier. They meas-
ure 0,060 mm. in length and 0.031 mm. in breadth.

The Lateral Gustatory Organs. — These organs are 7 mm. in
length, and consist of nine or ten irregular folds. The furrows
vary in width and depth, the average depth being about 0.55 mm.

No. 2. | ON THE GUSTATORY ORGANS. 183

Serous glands are fairly abundant, and their ducts discharge as
usual. The bulbs are disposed at the sides of the folds in some
fourteen tiers. They vary but slightly from those of the cir-
cumvallate gustatory area, and measure 0.060 mm. in length
and 0.029 mm. in breadth.

The bulbs of the fungiform papillae are rather small and not
very numerous. They are placed as usual, The larynx was
not examined.

THE TonGurE oF Cants latrans.

General Description. —The organ measures 125 mm. in length,
30 mm. in breadth, and 20 mm. in thickness. It is free from
the fraenum linguze for 38 mm. The upper surface is marked
for nearly its entire length by a mesial groove or raphe. |» At
the extreme basal region are the usual fleshy elevations, and
the under surface presents a more or less wrinkled appearance.
The fungiform papilla are largest and most closely placed just
anterior to the circumvallate area, and are smallest along the
median groove and about the tip. The circumvallate papillee,
seven in number, are arranged in two rows, slightly converging
posteriorly, one on each side of the median line. The lateral
gustatory organs are placed nearly as in Canis lupus, their pos-
terior limits being on the same transverse line as the anterior
pair of circumvallate papillz.

GUSTATORY STRUCTURES.

The Circumvallate Papille. —The papille, like those of Cants
lupus, are laterally involuted. They vary greatly in size, but
average about 0.95 mm. in diameter and 0.80 mm. in height.
The adjacent lingual surface is papillate, and the papilla them-
selves are more or less verrucose and occasionally cleft. Serous
glands are not very plentiful. Their ducts discharge at various
levels. The bulbs of the papilla are limited to the lower third
of the lateral area, where they are disposed in ten to twelve.
closely packed tiers. They are also irregularly scattered in the
outer wall of the trench. There are about sixty-five bulbs in a
tier of the papilla. They measure 0.058 mm. in length and
0.033 mm. in breadth.

The Lateral Gustatory Organs. — These organs measure 5 mm.
in length. Each consists of seven or eight folds varying slightly

184 TUCKERMAN. PVOE- Ve

in size, but having the same general appearance. The furrows
have an average depth of 0.45 mm. Serous glands are fairly
plentiful, and their ducts open at all levels. The bulbs are dis-
posed at the sides of the folds in ten or more tiers, and also
occur sparingly on their upper surface. They are a trifle
smaller in this area than in the circumvallate papille.

The fungiform papilla are well supplied with bulbs, some-
times as many as eight being visible in a single section of a
papilla of this type. The epiglottis was not examined.

Tue TonGueE or Canis familaris.

General Description. — The organ possesses a considerable
extent of free portion. The under surface is smooth, and im-
pressed by a median groove extending from the fraenum half-
way to the tip. From this point it is continued as a slight
ridge to the apex. The tip, which is obtuse, is very slightly
bifid. The upper surface is marked anteriorly by a mesial
raphé. Papilla of the fungiform type are quite evenly distrib-
uted over the dorsum, and the usual fleshy elevations project
from the basal surface of the tongue. There are four to seven
circumvallate papilla, arranged very much as in Canzs latrans.
The lateral gustatory organs are clearly defined.

GUSTATORY STRUCTURES.

The Circumvallate Papille.— The papillz vary in size, but
their transverse diameter is almost always greater than their
height. Not infrequently they are cleft vertically. The trenches
are narrow and uniform in width. Serous glands are not very
abundant. The ducts open into the trenches at their base and
deeper part. The bulbs are disposed on the lateral area in ten
to fourteen closely packed tiers. They also occur irregularly
on the outer wall of the trench, near its upper part. They are
likewise present in the epithelium lining the vertical cleft of the
papilla, and, according to Ditlevsen, may sometimes be found
on their free upper surface. They are densely packed in the
tiers, the tiers of the larger papillae containing upwards of one
hundred and thirty bulbs. In the smaller papilla the number
in a tier is about eighty. The bulbs vary in length from 0.060
to 0.071 mm., and in breadth from 0.025 to 0.035 mm.

The Lateral Gustatory Organs. — These organs vary from

No. 2.] ON THE GUSTATORY ORGANS. 185

3.70 to 10 mm. in length. They consist of seven or eight folds,
most of which bear bulbs on one or both lateral areas. In some
folds the main papillary body is cleft into a few large secondary
papillae, the depressions between which are filled up to one level
with epithelium. The furrows are narrow, and average 0.50
mm. in depth. Serous glands are plentiful, the ducts opening
at the bottom of the furrows. The bulbs occasionally fill the
lateral area of the folds completely, and scattered bulbs are not
infrequent on their exposed surface. Usually there are from
ten to fourteen tiers on each lateral wall. I counted forty-six
bulbs in a tier, but the latter was not completely filled, nor did
it represent the entire width of the organ. The bulbs of this
region vary in size, and are a little smaller than in the circum-
vallate papilla. They measure 0.056 to 0.067 mm. in length
and 0.026 to 0.035 mm. in breadth.

Some of the bulbs of the fungiform papillz penetrate, and
even perforate, the outer layers of the epithelium. I found one
very beautiful flask-shaped specimen in this region. The basal
end rested against the mucosa, while a very delicate, but clearly
defined neck led to the exterior. The bulb measured 0.057 mm.
in length and 0.028 mm. in breadth.

Both Davis and Schofield have found bulbs at different parts
of the posterior surface of the epiglottis of Canzs famzliaris.
They have also been detected by Davis, and later by Sima-
nowsky, on the true vocal cords. The former investigator has
likewise observed them on the inner side of the arytenoid carti-
lages and on the aryepiglottic folds.

THE TonGuE oF Zalophus californianus.

The specimen came from a young individual, and had been
kept for some time in spirit.

General Description. —The organ is 58 mm. long, the length
of the free portion being 14mm. The tip is bifurcate. The
under surface is finely wrinkled, and impressed by a deep median
groove leading to the frenum. The anterior dorsal surface is
marked by a slight mesial raphé. Fungiform papilla are spar-
ingly scattered over the anterior dorsal region, and the anterior
margin and tip are fringed with filiform papilla. Near the base
of the organ are four or five papilla of the circumvallate type.
They are arranged in two unequal lines converging backwards.

186 TUCKERMAN. [VoL, I'v:

GUSTATORY STRUCTURES.

The Circumvallate Papille.—The papille are 0.90 mm. in
diameter and 0.45 mm. in height. The trenches are not deep,
and have a uniform breadth. Serous glands are very abundant,
and occur within the papillee themselves. The ducts open at or
near the bottom of the trenches. They are remarkably straight
and have a nearly uniform diameter. Some of them are quite
long, occasionally exceeding two millimetres in length. The
ducts of the intrapapillary glands pass through the epithelium
of the lateral wall and open about opposite the middle of the
trench. This being a young individual, the bulbs are present
on the upper surface as well as upon the sides of the papillz.
In one section I counted twelve on the upper area. The ar-
rangement of the tiers is very irregular. Isolated bulbs occur
on the outer wall of the trench, especially at its upper part.
The bulbs measure 0.054 mm. in length and 0.033 mm. in
breadth.

The lateral gustatory organs were represented by five or six
fairly symmetrical folds at the sides of the base of the tongue.
There were, however, no indications of bulbs, and serous glands
were likewise wanting in this region.

The fungiform papilla were very well supplied with bulbs.
The epiglottis was not examined.

THE TonGuE oF Phoca vitulina.

This tongue, of which I received a fresh specimen, was hard-
ened in Miiller’s fluid and alcohol.

General Description. — The tongue is 95 mm. in length, its
greatest transverse diameter is 51 mm., and it measures 30 mm.
in thickness. The organ possesses but little mobility, the free
portion being but 21 mm. in length, and there being but a small
extent of free margin. The tip is bifurcate, and fringed with
filiform papilla. There is a slight groove, 5 mm. long, on the
upper surface, and a narrower and much shallower one, 8 mm.
long, on the under surface. They both terminate at the tip.

The anterior and middle portions of the dorsum are covered
with large, closely set, cornified papilla, the apices of which are
directed inwards and backwards. The mucous membrane of
the remaining portion of the dorsal surface is thrown into numer-

No. 2.] ON THE GUSTATORY ORGANS. 187

ous, wavy, sub-parallel folds. Those of the lateral borders have
a more or less longitudinal direction, and are continued from
each side of the tongue on to its under surface, meeting in front
of the franum. I found some difficulty in identifying fungi-
form papilla. There appear, however, to be a few of this type
scattered here and there between the folds. The circumvallate .
papilla are ten to twelve in number. They are in the form of
an inverted V, the apex of the V being about 21 mm. from the
base of the organ. No lateral gustatory structures were de-
tected.

The folds of the dorsum measure on the average 0.60 mm. in
height and 0.70 mm. in breadth. They are flattened or slightly
convex on top, and are covered with a fairly uniform layer of
epithelium. The epithelium is a trifle thicker on the upper sur-
face than at the sides and, in places, appears to be somewhat
cornified. The mechanical papille covering the anterior portion
of the dorsum are wedge- or cone-shaped, and measure 0.50 mm.
in height and 0.40 mm. in breadth. They bear at their upper
part from one to three recurved spinules. The epithelium
sheathing the papillze is partly, and that composing the spinules
wholly, cornified. Underlying the papille, and only separated
from them by a thin layer of mucosa, is a large amount of fat
in the form of lobules and groups of fat-cells.

GUSTATORY STRUCTURES.

The Circumvallate Papille.— The papillze vary greatly in size.
One of the larger ones measured 2.30 mm. transversely, and
was Imm. in height. At their upper part they break up into
many secondary papilla. In some papilla the upper portion is
cleft and furrowed. The trenches vary in width and depth.
Nerve-fibres enter the papilla at their base, and, towards their
upper part, divide and form a rather coarse plexus. Serous
glands are not very abundant. Their ducts, which are often
very large, open at the bottom of the trenches. There is a
great abundance of fat in the mucosa and submucosa, and
groups of fat-cells also occur within the papilla. The bulbs are
far from numerous. They differ in size and shape, and are very
irregular in their distribution. One of them measured 0.060 mm.
in length and 0.036 mm. in breadth.

188 TUCKERM AN. [ Vox. IV.

Ture Toncur oF Hapale jacchus.

This specimen and the two following had been preserved in
spirit. They were none of them fully adult.

General Description. —The anterior dorsal surface of the
organ is impressed on each side of the median line by four or
five transverse furrows. The fungiform papillee are quite large,
though not very numerous. The under surface presents a wide
and deep wedge-shaped groove extending from the franum to
the tip. The tip is obtuse. In Quadrumana the circumvallate
papilla: are usually three in number. In this specimen, unfort-
unately, the tongue had been divided just behind the anterior
pair, so that I had no opportunity of examining either the pos-
terior papilla or the lateral gustatory organs. The two anterior
papillz are 3 mm. apart, and 1.5 mm. from the lateral limits of
the organ.

GUSTATORY STRUCTURES.

The Circumvallate Papille.—The papille are 0.33 mm. in
diameter and 0.28 mm. in height. They are flattened on top,
and their sides incline inwards at the base. Serous glands are
not plentiful. Their ducts open as usual into the trenches at
their deeper part. The bulbs are irregularly scattered over the
upper surface and sides of the papilla. From horizontal sec-
tions there appear to be about thirty-five in a tier. They are
quite small, measuring only 0.039 mm. in length and 0.025 mm.
in breadth.

The bulbs of the fungiform papilla present the usual appear-
ance. The epiglottis was not examined.

Tue TonGue oF Macacus cynomolgus.

General Description. —The organ measures 50 mm. in length,
16 mm. in breadth, and is free from the frenum for 15 mm.
Fungiform papillz of normal structure are thickly distributed
over the dorsum, and also extend on to the under surface. Those
about the tips are quite closely set. The circumvallate papilla,
of which there are two pairs, —an anterior and a posterior, —
are situated well back on the dorsum. The posterior pair are
Imm. apart, and 11 mm. from the base of the organ. The
anterior pair are 6 mm. from the posterior, and are 10 mm.
apart. The lateral gustatory organs are placed about as usual.

No. 2.] ON THE GUSTATORY ORGANS. 189

GUSTATORY STRUCTURES. ,,

The Circumvallate Papille. —The papilla are of nearly equal
size. Their summits are more or less flattened, and their sides
symmetrical and nearly vertical. The trenches are narrow and
deep. Serous glands are abundant, and their ducts open at or
near the bottom of the trenches. The bulbs are numerous,
and are disposed in a varying number of tiers. In some sec-
tions there appear to be not less than twenty tiers, but the mean
is probably ten. The number of bulbs in a well-filled tier is about
a hundred. They measure 0.058 mm. in length and 0.032 mm.
in breadth.

The Lateral Gustatory Organs. — The lateral taste organs are
6 mm. in length, and consist of seven or eight bulb-bearing
folds. The furrows are quite uniform in breadth, and average
0.70 mm. in depth. Serous glands and ducts are quite plenti-
ful. The bulbs are disposed at the sides of the folds in nine or
ten tiers, and measure 0.057 mm. in length and 0.031 mm. in
breadth.

The fungiform papillz contain many bulbs, those about the
tip being particularly well supplied. In a section of a papilla
of this region I have seen as many as twelve. No true bulbs
were detected in the epiglottis.

THE TONGUE OF Macacus rhesus.

General Description. —The organ is 41 mm. in length, 15 mm.
in breadth, and is free from the frenum for 13 mm. The tip
is rounded, and covered with closely set fungiform papilla.
Papillze of this type are also thickly distributed over the dor-
sum, and upon the sides and under surface to the lateral line of
union of the papillate with the non-papillate surface. The cir-
cumvallate papilla, three in number, are arranged in an isosceles
triangle, the apex of the triangle being directed backwards.
The posterior papilla is 11 mm. from the base of the organ.
The papillae viewed from above present a smooth, slightly oval
surface. The lateral gustatory organs are placed more obliquely
than in Macacus cynomolgus, their posterior end being nearly on
a level with the upper surface of the tongue.

190 TUCKERMAN. PVOE ViVi:

GUSTATORY STRUCTURES.

The Circumvallate Papille. —The summits of the papillz are
smooth, but more convex than in A/acacus cynomolgus. The
trenches are narrow and of uniform breadth. Serous glands
are not very abundant. The ducts open into the trenches at
their deeper part. The taste bulbs are disposed at the sides of
the papillee in ten to twelve tiers. A few bulbs are also scat-
tered over their upper surface. They are not very closely
packed, there being only about fifty bulbs in a tier. They vary
greatly in size and shape, but the mean length is 0.068 mm. and
the mean breadth 0.036 mm.

The Lateral Gustatory Organs. —These organs consist of a few
folds of the mucous membrane. The intervening furrows are
narrow, and 0.60 mm. in depth. Serous glands are not abun-
dant. The bulbs, of which there are ten or more tiers, measure
0.066 mm. in length and 0.036 mm. in breadth.

The fungiform papillz are of normal structure, and are richly
supplied with bulbs. One deeply placed bulb, lying directly in
the long axis of a papilla, measured 0.062 mm. in length and
0.045 mm. in breadth, the canal leading from it to the exterior
having a length of 0.016 mm. and diameter of 0.003 mm.
Bulbs were also present on the anterior surface of the epiglottis.

CONCLUDING REMARKS.

Far-reaching generalizations, based upon the study of the
gustatory organs of the widely separated forms (some of them
scarcely typical of their kind) which have been considered in
this paper, would be of very doubtful value. It is therefore my
intention to say but little at this time, reserving further remarks
for a future occasion.

In the Marsupialia the circumvallate papilla are three in
number. They are arranged in the form of an equilateral or
isosceles triangle, the apex of which is directed backwards! In
some genera, as Halmaturus, Macropus, Petrogale, and Dasyu-
rus, these papillae might very justly be called gustatory ridges,
from their general shape, extreme protection, and concealed
position. In these respects, as well as in the arrangement of
the bulbs, they resemble the ridges of Orutthorhynchus ana-

1In Dendrolagus, according to Owen (Comp. Anat. and Phys. of Verts., vol. iii.,
1869, p. 191), the apex of the triangle is turned forwards.

No. 2.] ON THE GUSTATORY ORGANS. IOI

tinus. In other genera, as Phalangista, Belideus, Acrobates,
Bettongia, Phascolarctos, and Didelphys, the posterior papilla
follows more closely the type characteristic of higher animals,
while the anterior pair are still in a transitional stage of develop-
ment. In Phascolomys, Perameles, and some species of Didel-
phys, all three papillz are closely allied to the type common to
higher animals.

Although the lateral gustatory organs have not been detected
in all marsupials in which a search for them has been made,
they have been found in a sufficient number of species to render
their existence in all scarcely open to doubt. In Ha/maturus,
according to Poulton, the organ consists of a row of gland ducts,
in the walls of which scattered bulbs are developed. In Macro-
pus, Petrogale, and Phascolarctos they are more advanced, the
ducts opening at the bottom of depressions. In Perameles the
organ consists of a single epithelial-lined furrow, in the walls of
which are several tiers of bulbs. In Phalangista, Belideus,
Acrobates, and Didelphys the organs are less simple in struc-
ture, and the ducts open at the bottom of the furrows as in the
higher mammals.

Edentata have but two circumvallate papilla. The posterior
papilla has disappeared, to reappear in the Castoridze, Sciuride,
Pteropodidz, and Quadrumana. They are on the same trans-
verse line, and may be considered as representing the anterior
papillae of the Marsupialia. While some species show a very
decided advance in all, or nearly all, the points which the papillae
of the two orders have in common, other species appear to fol-
low closely the marsupial type. In Dasypus peba they resemble
in the main those of higher animals. In Chlamyphorus truncatus
they approach quite closely the marsupial type, the resemblance
between them and the anterior papille of Belideus and Phalan-
gista being very marked. The papille of Dasypus villosus
appear to hold an intermediate position, both types being repre-
sented, although more or less modified. This species was the
only one in which I detected lateral gustatory organs, although
they are doubtless present in the others. The organs were
wholly unlike any yet described except those of Procyon Jotor.

Bulbs appear to be wanting in the larynx of both the Marsu-
pialia and Edentata. Further research, however, may show
their existence in this region, although it is not unlikely that
their appearance here is more recent.

hVorEs EV

TUCKERMAN.

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THE GRIGIN OF THE TEST_CELLS OF ‘ASCIDIANS.
t. Hy MORGAN, Pa: D:

A worb of explanation and apology seems necessary on add-
ing another account to the long list of descriptions of the origin
of the test-cells. While studying the embryology of Clavellina
in the spring of 1888, I became interested in the origin of the
test-cells, and the work was continued during the summer of
the same year. A preliminary note was written in October, 88,
and published in the Johns Hopkins University Circular, Vol.
VIIL;. No..72:

At the same time I obtained the paper of Van Beneden and
Julin in the Archives de Biologie, Tome, VI., 87, in which I
found conclusions almost exactly similar to those to which I
had come. It seemed then unnecessary to publish a full account
of the work, and the figures and descriptions were laid aside.

In the spring of 89, Dr. M. v. Davidoff published a new
account of the origin of the test-cells (Mittheilungen aus der Zool-
ogischen Station zu Neapel), in which he differs essentially from
Van Beneden and Julin. Thus the whole question became once
more unsettled by the conflicting accounts of Van Beneden and
Davidoff, and seemed worth working over again.

During the summer of ’89 I carefully prepared ovaries of
several Ascidians by the methods described in detail by David-
off, hoping in this way to meet him to some extent on his own
grounds, and to test the value of the new methods of prepara-
tion. During the winter of 89-90 this material was examined,
and gave not the slightest evidence of such an origin of the
test-cells as described by Davidoff. Here again I was irresist-
ably led to the same conclusions as those reached by Van
Beneden.

As the results obtained by the methods described by Davidoff
differed in no essential points from those obtained by other
methods, it seemed unnecessa y to draw all the figures from
such preparations. Several genera were examined, including

196 MORGAN. [VoL. IV.

Cynthia ocellata, Cynthia partita, Ascidia amorpha, Molgula
manhattensis, Perophora viridis, Amarectum stellatum, and
Clavellina sp. ?

Finally, during the summer of ’g90, a new method of preparation
was obtained, which confirmed, from another point of view, the
previous results, and helped to make clear the exact origin of
the test-cells.

Cynthia ocellata. — The ova are arranged around a central
cavity, which is the body cavity according to Van Beneden.
This cavity, which communicates by a duct with the atrial
cavity, is lined by a germinal epithelium, within which the ova
originate.

In sections through the ovary, nuclei are seen lying along the
wall of the cavity of the ovary; and here and there one is seen
to have enlarged, and the protoplasm about it to have increased
in quantity. This nucleus and protoplasm form the commence-
ment of a new egg. Such a condition is shown in Fig. 1,
Pl. VIII. At the periphery of this young egg another nucleus
is seen. This nucleus is one of those peripherally lying nuclei
which go to form the follicular nuclei of the egg. In Fig. 2
we see an older stage: at f is a nucleus of the forming follicle
with its protoplasm stretching over the surface of the egg.
Other and similar nuclei lie around the egg in other sections of
the series. The follicular protoplasm surrounding the egg is
not a continuous mass, but is split up into cells corresponding
in number to the follicular nuclei. The cell walls are so
extremely thin that at this stage they cannot be seen in such
sections.

Passing to a later stage, as shown in Fig. 3, the egg is
seen while enlarging to have pushed inwards from the germi-
nal membrane, which is still, however, attached to one side of
the egg.

The peripheral zone of protoplasm of the follicle is much
wider than in the last figure, and now is seen clearly to cover
the whole surface of the egg. Four follicular nuclei are seen
in this section. The nucleus of the ovum has also enlarged,
and contains in the figure a single large nucleolus.

Fig. 4 is part of an egg of Cynthia partita (hardened in picro-
acetic acid). The follicular zone is wider than in the last
figure, and the follicular nuclei are a little larger. The egg

No. 2.] TEST-CELLS OF ASCIDIANS. 197

has passed into the general stroma of the ovary. This is the
stage in development just before the formation of the test-
cells.

In the next figure (5) the test-cells have begun to appear.
At ¢c the follicular zone is seen pushing into the substance of
the egg, and a careful examination shows faint indications of
cell outlines at this place. In other words, one of the follicular
cells has changed somewhat its position, and has come to lie a
little interior to the cells of the follicular zone. The cell con-
tains a nucleus which agrees in all details with those of the
follicle. At ¢c’ another such cell is seen; and at ¢c’’ a cell of
the follicular zone is seen pushing into the interior of the egg.

These three cells, which take a more internal position, are
the follicular cells, which are in process of conversion into test-
cells. Two main sources of error may arise in interpreting
the sections at this stage and are carefully to be avoided.

Inner nuclei may be seen if the section passes — not through
the centre, but — near one end of the egg, where the convexity
of the surface is so great relatively to the plane of the section,
that two or more layers of the nuclei of the follicle may appear
in the same section. <A careful count was made in all cases of
the sections and the central ones chosen for study. Again, an
error may arise when the microtome knife does not cut the egg
cleanly, but turns over part of the follicular zone.

The stage in development in which the test-cells appear is
very constant, and I have never succeeded in finding any nuclei
or cells inside of the follicular zone before such a stage of
development is reached. In Fig. 6 we see a cross-section of
an egg of C. ocellata, in which the migration of test-cells is
completed. The follicular zone is seen to be divided up into
distinct cells, which are sharply separated from the egg. At
the periphery of the yolk, and touching the follicular zone,
lie the test-cells. These do not stain so deeply as the proto-
plasm of the egg, and in all respects resemble the cells of the
follicie.

After a stage represented by Fig. 6 has been reached, the num-
ber of test-cells does not seem to increase; although between
Figs. 5 and 6 it is probable that the test-cells divide at the
periphery of the yolk.

In Fig. 6 there is seen at the outer periphery of the follicle

198 MORGAN. [VoL. IV.

a number of small nuclei lying in close contact with the follicle
cells. These seem to belong to the stroma of the ovary, and
to be added secondarily to the growing ovum.

In later stages the test-cells do not seem materially to change
either in number, or size, or structure, but the follicular cells
continue to increase in size and become much vacuolated.

By the methods of preparation ordinarily employed, the boun-
daries of the cells of the follicle do not appear in sections of young
ova. In order to determine the shape and arrangement of the
follicular cells, I experimented with other methods and found one
which made clear the structure of the cells, and indirectly con-
firmed the conclusions reached by previous methods of prepara-
tions. The fresh ovaries were teased apart in very dilute osmic
acid, washed in distilled water, and placed in a one per cent
solution of silver nitrate, where they remained for half an hour;
then put into a two per cent solution of acetic acid for the
same length of time, and placed in the sunlight. They were
then examined under the microscope, and the cell boundaries
were distinctly seen. The eggs were then carried through the
usual processes, imbedded in paraffin, and cut into thin sections.

In young ova, the follicular cells were found from surface views
to have irregular outlines, and in general appearance to resemble
peritoneal epithelial cells. A bit of the periphery of the egg is
shown in Fig. 7, which corresponds approximately to an eg¢
at an age represented in Fig. 3.

As the egg increases in size, the outer surface area of each
follicular cell diminishes ; but it will be remembered they are
increasing in thickness during this period. At the time when
the test-cells are forming, corresponding to Fig. 5, the follic-
ular cells have reached a size shown in surface view by Fig. 8.
In Figs. 9 and 10 are drawn sections of the periphery of eggs
at this stage, showing the origin of the test-cells. Here it
is seen that one of the follicular cells pushes itself inside of
the follicular zone and becomes a test-cell. Comparing Fig. 8
with 9 and 10, the size of the test-cells is seen to exactly corre-
spond to that of the follicular cells; so that we find at the time
when the test-cells are formed that the follicular cells have
reached exactly the same size as the test-cells, and this by itself
would lend probability to the view that the inner cells are
derived from the outer. Taken in connection with what we learn

No. 2.] TEST-CELLS OF ASCIDIANS. 199

from sections, it seems to me to leave not the slightest doubt as
to the origin of the test-cells. The protoplasm of the egg of
Cynthia stains deeply, and the color cannot easily be extracted.
In this respect it is unfavorable material to determine the truth
of Davidoff’s position; but in the next form, C/aved/ina, the pro-
toplasm (and yolk) stains scarcely at all, and wandering nuclei
could be easily observed were there any such in the egg between
the nucleus and the follicle.

Clavellina sp.?—The ascidian from which the material for
work was obtained was collected at Green Turtle Cay, Ba-
hamas, but I have not identified the species. The eggs are
extremely large and very favorable for study, both on ac-
count of their size and for other peculiarities which will be
seen in the following description. On account of the large
amount of food yolk in this American C/avellina, no better
material could be obtained to test the correctness of Davidoff’s
theory. The test-cells are not formed till the egg is quite large,
and has a good deal of yolk; so that if the nucleus of the test-
cells came from the nucleus of the ovum, these migrating pieces
would have to travel a long distance from the centre to the
periphery of the egg, and the chances of seeing such a migra-
tion would be very great.

The young nuclei of the ovum arise in the germinal epi-
thelium of the ovary, and are at first like the ordinary nuclei of
the membrane. The earliest stage is shown in Fig. 11. A
somewhat older ovum is shown in Fig. 12, in which the proto-
plasm about the nucleus has increased, and there is a sharper
distinction between the egg proper and the more peripherally
lying follicular nuclei. The protoplasm of the follicular nuclei
is seen to be thickening over the surface of the ovum, beginning
at that part of the periphery of the egg which is in contact with
the germinal membrane. In Fig. 13 the ovum has left the
surface of the cavity of the ovary and passed into its substance.
The peripheral zone of follicular protoplasm has spread over the
whole surface of the ovum, and the follicular nuclei, which are
now more numerous than in the last figure, lie within this proto-
plasm. At this stage in the history of the ovum of Clavellina
the yolk commences to form, and appears as a zone around the
nucleus. It is seen in Fig. 12 as an irregular mass, which in
the figure is colored a lighter shade than the surrounding

200 MORGAN. [Vou. IV.

protoplasm. There is no sharp line of demarcation in the
section between yolk and surrounding protoplasm, but the
latter sends processes into the former.

Passing to a stage represented by Fig. 14, we have essen-
tially the same conditions as in the last figure. The ovum has
increased very much in size, and the increase is largely due to
the accumulation of yolk. Around the periphery of the ovum
is a zone of protoplasm which does not contain yolk, and outside
of this is the follicular protoplasm with its nuclei. As a rule,
the nucleus of the ovum lies near to the centre of the egg, but
in the section from which the figure was drawn it was near one
end of the egg. It was also irregular in outline, but this is
exceptional at this stage. In Figs. 15 and 16 we have the
first indication of an origin of the test-cells. In Fig. 15 only
a part of the egg is shown, but it will be easily seen that there
has been a very great increase in size as compared with Fig.
14. At the periphery of the yolk lies a narrow zone of proto-
plasm, and the follicular protoplasm around the egg is wider
than in the last figures. Here and there a nucleus lies nearer
to the inner part of the zone, and it is generally accompanied
by an ingrowth of protoplasm from the follicular zone. These
ingrowths are the first trace of the formation of the test-
cells. Where they project into the ovum, they are clearly seen
to push through the peripheral zone of protoplasm of the egg.

The follicular zone stains differently from the protoplasm of
the ovum, and one thus distinctly sees that the ingrowth of pro-
toplasm with its contained nucleus belongs in each case to the
follicular zone and represents migrating cells into the proto-
plasm of the egg. In Fig. 16 we see three stages in the in-
growth of follicular cells. The earliest is shown at ¢c, where an
ordinary follicular nucleus has moved a little interior to the
general row, and at the same place the follicular protoplasm has
become thickened on the inner side. At ¢c’ the follicular nu-
cleus has passed still further within, and the surrounding proto-
plasm projects further into the egg, passing through the zone
of protoplasm. At ¢c!’ we see a somewhat exceptional condi-
tion, owing to the large amount of follicular protoplasm which
has pushed into the ovum. It has passed the zone of proto-
plasm of the egg, and projects into the yolk substance. Within
it and at its base lies a follicular nucleus. In later stages all of

No. 2.] TEST-CELLS OF ASCIDIANS. 201

these ingrowths become constricted off to form the test-cells.
After separation from the follicular zone, the test-cells multiply
at the periphery of the egg, and form each clusters of four or
five cells closely connected.

In Fig. 17 is shown a section through the periphery of the
egg, taken at a time when the test-cells were entirely within
the follicular zone. Three such cells are seen in the figure, and
it will be also noticed that at the same time the peripheral zone
of protoplasm of the egg becomes extremely narrow, and at the
time of its disappearance a membrane appears around the egg
between the follicular cells and the yolk, and enclosed within
this membrane are the test-cells. Most probably this peripheral
protoplasm either secretes the membrane before its disappear-
ance, or becomes converted into it.

In Fig. 18 is drawn a part of the periphery of an egg of
Clavellina when the egg is nearly mature. The test-cells are
seen lying in clusters of from one to a dozen at the periphery of
the yolk and just within the egg membrane. Outside of the
membrane the columnar follicular cells form a layer completely
investing the egg, and beyond the follicular cells is an imper-
fect layer formed of scattered cells which seem to be derived
from the inner substance of the ovary, although I have not care-
fully traced their origin.

The account I gave of the formation of the test-cells of Clavel-
/ina in my preliminary note is not quite clear nor entirely free
from error. This is due to my not distinctly recognizing that
the peripheral zone of protoplasm of the egg belonged entirely
to the ovum; but I hope the preceding account will make clear
the true position of the egg membrane.

It seems useless to give an extended criticism of the results
and theory of Davidoff. Our views are diametrically opposed,
and it seems to me irreconcilable.

My own results agree essentially with those of Van Beneden
and Julin, and must stand or fall with theirs. Those who care
for a fuller knowledge of Davidoff’s position can refer to his
paper (in the Mittheilungen aus der Zoologischen Station zu
Neapel. Neunter Band. I. Heft. 1889). I will give here very
briefly an outline of his views, so that I may contrast them with
the preceding account.

In very young eggs (much younger than those in which I

202 MORGAN. [Von. IV.

have found the test-cells forming) Davidoff believes the nucleus
to constrict off portions of itself which he calls buds (Knospen
Nucleogemmz, Kernknospen). These nucleogemme migrate to
the periphery of the egg, and divide in many cases into smaller
nuclei, although xo mitotic, nuclear figures were found, and the
more peripheral nuclei stained more deeply than the more
central bodies. At a later stage of development (which corre-
sponds to a stage in which I have found the test-cells constricted
off from the follicular zone) Davidoff finds these peripheral
nuclei — nucleogemmze — ¢o multiply by mitotic division. At
the stage when these karyokinetic figures are found, the pe-
riphery of the egg is colored more intensely than elsewhere.
Then the peripheral protoplasm of the egg divides itself into
cells with a nucleogemma in each such cell.

Next follows a short account of the formation of the ova of
Fritillaria. The young ovary contains a few cells which he
calls odblasts. They subsequently fuse, forming a syncytium.
From the nuclei of this syncytium—the karyoblasts — there
are budded off nucleogemmz which, after cutting off some of
the protoplasm of the syncytium, form the true ova. Comparing
the Ascidian with the Appendicularian developments, Davidoff
concludes, ‘ Bevor wir indessen diesen Vergleich mit Erfolg
durchfiihren k6nnen, mussen wir uns die Frage vorlegen, was
denn das Ei der Ascidien selbst ist. Nach allem Gesagten
kann dasselbe nicht mit einem Ei der Fritallaria homologisirt
werden. Wir mussen viel mehr zuruckgreifen und sehen, ob
wir die Ascidien-Eier mit einem der Ooblasten des Fritallaria
—Ovarium vergleichen konnen. ... Wir konnen also jetzt
mit Recht den Schluss ziehen, dass das Et der Ascidien ergent-
lich kein solches in gewolhnilichen Sinne tst. Es ist vielmehr ein
Ooblast, welcher erst Eier producirt.”” These eggs are the
test-cells, or, as Davidoff concludes to call them, abortive eggs.
By some such complicated explanation with its complicated
terminology, he attempts to homologize the formation of the
ova in the Appendicularian and Ascidian. Such a process of
formation of the Ascidian egg seems so highly improbable in
itself, and the method by which the odblast divides into abor-
tive eggs and egg proper is so out of the usual course of events,
that it must take exceedingly strong evidence to support such
a position. On the other hand, the increase in area of the follic-

No. 2.] TEST-CELLS OF ASCIDIANS. 203

ular cells by a process in which some of the cells are pushed
interior to the others is easily understood. We see in the ovo-
genesis of the squid a parallel case, where the process is carried
to even a greater extent, the substance of the ovum being per-
meated by the trabeculae of the follicular zone. In the squid
there being many cells pushed in at several points, and in the
Ascidian a single cell here and there over the surface.

The origin of the ova in vertebrates at the surface of the
ovary, and their migration into its stroma, accompanied by
follicular cells, themselves derived from the peripheral cells of
the ovary, and their subsequent arrangement around the young
egg into two or more layers, furnishes a most striking resem-
blance to the origin of ova, and follicular and test-cells, of the
Ascidians.

In Davidoff’s paper he says that Van Beneden, who examined
his first series of slides, concluded that in Dzsta/pa the test-cells,
after originating from the follicular cells, migrated into the yolk.
If this be true, then the difficulties of determination of the ori-
gin of the test-cells would be greatly increased, from his own
point of view. And as in the origin of the test-cells it is not
conceivable that in one Ascidian we have true ova, and in an-
other odblasts to replace them, when all the other processes are
identical, no explanation of our work being done in different
genera seems possible. After having gone over again and again
sections of ovaries prepared by many methods, I have been as
often forced to accept the account I have given in the first
part of this paper, both because no traces of nucleogemmz have
ever been observed, and also because I believe I have been able
to trace step by step the origin of the test-cells from the sur-
rounding follicular zone.

THE MARINE BIOLOGICAL LABORATORY, Woop’s HOoLt,
July 15, 1890.

DESCRIPTION OF PLATE VIII.

[All the figures are camera drawings. Figs. 1, 2, 3, and 6 inclusive are from prep-
arations hardened in Kleinenberg’s picro-sulphuric and stained in borax-carmine.
Identical preparations were obtained with Davidoff’s acetic-sublimate and acetic-
picric. Figs. 4 and 5 were hardened in acetic-picric and stained in borax-carmine.
Figs. 11-17 were prepared in picro-nitric acid, and I must express my obligation to
Dr. H. V. Wilson, who supplemented my now imperfect material from some that
he had carefully preserved in the Bahamas. Figs. 7, 8, 9 and 10 are from prepara-
tions killed in weak osmic, stained in silver nitrate, etc., and cut in paraffin.]

Figs. 1, 2, 3, and 6 are sections of ova of Cynthia ocellata. Fig. 1, Zeiss 4 f.;
Figs. 2, 3, 6, Zeiss 2 f.

Figs. 4 and 5 are sections of ova of Cynthia partita. Zeiss 2 f.

Figs. 7, 8, 9, 10 are from Cynthia partita ; Figs. 7 and 8, surface views of follicle
cells; Figs. 9 and 10, cross-section of egg. Zeiss 2 f.

Figs, 11-17 inclusive are sections of ova of Clavellina. Zeiss 2 f.

;
_ = :
Margan,del, —
A AMeisel.tith Boston.

THE ORIGIN OF THE MESOBLAST-BANDS IN
ANNELIDS.

EDMUND B. WILSON.

le

SINCE the publication of Salensky’s memoirs on the formation
of the germ-layers in various Polycheta (Psygmobranchus,
Nereis, Pileolaria, Terebella, Aricia)! it has been generally
accepted that the mesoblast-bands (“secondary mesoblast”’)
of annelids arise in some cases by direct proliferation from
the ventral ectoblast, and often without the agency of pole-cells,
or teloblasts (“primary mesoblasts, or pro-mesoblasts’’). “ Par-
tout,” he says, “ou j’airéussi a observer les jeunes stades de
l’evolution du mésoderme somatique, ce feuillet, a4 son début,
consistait en un épaississement ectodermique qui, sous forme de
deux bandelettes, regne suivant l’axe longitudinal du corps; ce
n'est que dans le cours du développement qu'il se sépare de
Vectoderme.”2 This result was in harmony with Kleinenberg’s
éa. ier studies upon Lumbricus ;* it received, apparently, a com-
plete confirmation through the same author’s splendid study of
Lopadorhynchus,* a work that seemed at the time to place the
ectoblastic origin of the entire mesoblast in a number of the
marine annelids beyond the possibility of doubt.

Yet this conclusion in one sense only served to render the
general subject of mesoblast-formation in annelids less intel-
ligible than ever. It had been demonstrated by Kowalevsky,°®
Hatschek,® and Whitman,’ that in some cases the mesoblast
first appears in the form of a pair of large cells (teloblasts), by

1 Arch. de Biol., Iil., IV., VI.

2 Arch. de Biol., V1., p. 618.

3 Quart. Four. Mic. Sci., X1X., 1879.

* Zettsch. f. wiss. Zool., XLIV., 1886.

5 Mim. Acad. Imp. St. Petersburg, XV1., 187%.
© Arb. Zool. Inst. Wien., \., 1878.

7 Quart. Four. Mic. Sct., XVIUII., 1878.

206 WILSON. [Vou. IV.

the proliferation of which the paired mesoblastic bands are pro-
duced. The teloblasts are often differentiated at a very early
period, — sometimes even prior to the gastrulation, — arising
near the region corresponding to the posterior lip of the blasto-
pore. In no case do they arise from the ectoblast ; in some
cases they seem to arise from, or at least to be closely associated
with, the cells of the archenteron. In still another class of cases
the mesoblast appears to arise neither by delamination from
the ectoblast, nor from teloblasts, but from a central mass of
‘“‘mes-entoblast,” the lateral portions of which give rise to the
mesoblast-bands, and the central portion to the entoblast. This
would seem to be the case, for instance, in the development of
Enchytreoides as described by Roule;! and it is apparently the
case also in Sfzvorbis, according to the investigations of my
former pupil, Miss H. Randolph, whose preparations I have
carefully examined.”

How are these various modes of mesoblast-formation to be
reconciled? It is impossible to doubt the homology of the mes-
oblastic bands in Polygordius, Eupomatus, Lumbricus, Clepsine,
Lopadorhynchus, and Enchytreoides —a series that includes rep-
resentatives of the three modes of mesoblast-formation I have
mentioned. It must, therefore, be possible to reduce these
modes of development to a common type, and it is a remarkable
illustration of the elementary state of our knowledge of annelid
development that no one, as far as I am aware, has made even
a suggestion as to how this is to be done. The only exception
known to me is an hypothesis put forward in my recent paper

1 4nn. a. Sci. Nat., VIL, 1889.

2 Gédtte has described the mesoblast of Spzrorbis nautiloides as arising from a pair
of large teloblasts, derived from the archenteron. This, however, is certainly a
blunder. As Salensky has suggested, and Miss Randolph has conclusively proved,
the rounded body, mistaken by Gitte for a pair of primary mesoblasts, is simply the
rounded, hollow posterior part of the mesenteron. The mesoblast first appears in
the form of two lateral masses which join each other posteriorly, and in some cases.
seem to fuse with the walls of the mesenteron. Salensky describes these masses
as delaminating from the ectoblast in P2/eolaria, a form nearly related to Sfzrordzs.
In the latter form, however, the mesoblast-cells are quite distinct from the ectoblast
from the earliest period at which they are distinguishable, while they are so intimately
related with the entoblast, as to form with it, apparently, a common mass of mes-
entoblast, as in xchytreoides. Miss Randolph’s researches are, however, still

incomplete, and a more thorough study of the earlier stages may lead to a different
interpretation.

No. 2.] THE MESOBLAST-BANDS IN ANNELIDS. 207

on Lumbricus !|—a somewhat unsatisfactory suggestion, which,
however, had the merit of emphasizing the importance of a care-
ful study of the relations between the germ-bands and the blas-
topore in the Polycheta. During the past two years I have
investigated several forms, with this end in view, in the hope of
finding facts that might bring into some relation the teloblastic
origin of the mesoblast (represented by Clepszue, etc.) and the
delaminate mode of origin (Lopadorhynchus), but until recently
the puzzle remained as greatas ever. During the past summer,
however, I was fortunate enough, through the kindness of. Dr.
E. A. Andrews, to procure very abundant material for the study
of the early stages of two species of Merezs (V. imbata, Ehlers,
and JV. megalops, Verrill), and the facts thus brought to light
point the way, as I believe, to a solution of the problem. The
interest of the results depends largely upon the completeness
with which the early stages can be followed in detail; and this
in turn is owing to the extraordinarily favorable character of the
eges for observation. They are transparent, of comparatively
large size, and they may be procured in abundance. The four
primary entoblast cells (which are the remnants of the four
macromeres of the typical eight-cell stage) remain undivided
until a very late stage; z.e. until the trochophore form is at-
tained and the eyes and mouth have appeared. Thus the axes
of the embryo may be located with the greatest precision
throughout the entire process of cleavage and gastrulation ; and
the certainty of the orientation is increased by the following
circumstance. The transparent macromeres contain large oil-
drops, which run together during the development, until, in the
great majority of cases, only four are left (one in each macro-
mere), viz.: a pair of smaller and a pair of larger drops, in corre-
spondence with the size of the macromeres in which they are
respectively contained. The smaller pair mark the anterior ex-
tremity, the larger the posterior, and they are bilaterally ar-
ranged on either side the middle line (f Fig. 6). There are
other advantages, equally great, that facilitate the precise study
of the early stages, but these will be described hereafter.

Aided by these favorable conditions, I have been able to
trace the origin of the mesoblast-bands from the beginning of
development. As in Lopadorhynchus and similar types, the

1 Your. Morphology, I11., 1889.

208 WILSON. [Vou. IV.

trochophore seems to consist at first of two layers only. The
mesoblast, like the neural foundations and those of the seta-sacs,
arises directly from a thickened bilobed ventral plate; ze. it
seems to arise from the ectoblast. But a further examination
of the matter gives it a different aspect. The cells of the ven-
tral plate differ markedly from the remaining cells of the outer
layer. They are larger, differently granulated, and upon treat-
ment with certain reagents (combinations of acetic acid, etc.),
assume a brownish color that differentiates them very sharply
from those of the upper pole. This renders it possible to trace

Tt

II

Fic. 1.— Eight-cell stage of Nereis viewed from the upper pole. I.-I., the first
cleavage-plane; II.-II., the second; 4, B, C, D, the four macromeres; a, 4, c, d, the
corresponding micromeres.!

their origin, cell by cell, from the beginning of development,
and this origin is such as to show that the mesoblast is com-
pletely segregated in the anterior part of the plate, while the
posterior part alone gives rise to ectoblastic structures (neural
plates, seta-sacs). Moreover, each of the two divisions of the
ventral plate may be traced back to a single cell (pro-teloblast),
which zs obviously homologous to a corresponding cell in the early
embryo of Clepsine. A detailed account of the cleavage will be
given in a forthcoming paper, but the following synopsis will
suffice for the present purpose.

1 All of the cuts are from camera drawings, and are not schematized in outline,
though slightly simplified.

No. 2] THE MESOBLAST-BANDS IN ANNELIDS. 209

I have followed the cleavage process many times and find it
to be extremely constant, the variations being so insignificant
as to have no effect on its general character. The first cleavage
divides the ovum into a larger (AB) and a smaller (CD) segment ;
the second divides CD into equal parts, Cand D, and AB into
unequal parts, of which 4 is the largest of the four, and B is
intermediate in size between it and the two smaller segments,
Cand D. The third or equatorial cleavage separates four
micromeres (@, 6, c, d@) at the upper pole from four macromeres
(A, B, C, D, respectively, Fig. 1). The later development shows

dif

Fic, 2.— View from the upper pole of the sixteen-cell stage of Wereis. _X, the
first pro-teloblast which has been separated from A.

that the second cleavage-plane (II.-II.) corresponds precisely to
the future median vertical plane of the adult body; C and D are
the anterior macromeres; A and B, the posterior. The embryo
is, therefore, not bilaterally symmetrical, from any point of view.
Bilateral symmetry is, however, soon to be established.

At the fourth cleavage (Fig. 2) the four micromeres divide,
not quite equally, and three new micromeres are formed from
B, C, and D, respectively. A, at the same time, separates off
a large characteristically granulated cell (X), which I shall call
the first pro-teloblast. The regularity of the cleavage now
ceases. A little later A again divides, separating off a second
cell (Y), somewhat smaller than .X, but with a similar granu-
lation of the protoplasm. This I shall call the second pro-

210 WILSON. [Vou. IV.

teloblast. It lies between A and X, anterior, ventral, and
somewhat to the left hand of the latter (Fig. 3). A is now
approximately of the same size as B, and the embryo is bilater-
ally symmetrical, though not absolutely so, since both the pro-
teloblasts are slightly displaced to the left, Y somewhat more
so than X.

The gastrulation is strictly epibolic, the posterior lip of the
blastopore is formed by the progeny of Y, the second pro-

pS

Fic. 3. — View of a later stage from the lower pole, showing the two pro-teloblasts,
X and Y, the four macromeres, and the edge of the cap of micromeres.

teloblast, and the stomodzeum is developed from the micromeres
that close in the blastopore.

The principal interest of the development, from our present
point of view, centres in the fate of the pro-teloblasts. From
these two cells the entire ventral plate arises, its anterior cells
From Y, tts posterior cells from X. From Y arise the mesoblast-
bands, from X the neural plates, the seta-sacs, and other structures
still undetermined. Y first divides into two equal cells (the
primary mesoblasts), and X soon does the same, first, however,
invariably separating two smaller cells (Fig. 4). The bilateral
symmetry of the embryo is now conspicuous, though the slight
displacement of the teloblasts to the left still remains. Each of
the primary mesoblasts now separates off a somewhat smaller
cell at its outer side, thus forming a transverse series of four

No. 2.] THE MESOBLAST-BANDS IN ANNELIDS. 2th.

cells which lie at the surface and form the posterior lip of the
blastopore. Each of the X’s now separates off a smaller cell

Fic. 4. — View corresponding to that given in Fig. 3, after the division of the pro-
teloblasts. Y, Y,the primary mesoblasts; , X, the two primary posterior teloblasts.

in front, then a small cell at its outer side, and finally divides
longitudinally into two equal parts. At this period (Fig. 5) the

II

Fic. 5.— View from the ventral side of a still later Veve’s embryo. The ventral
plate consists of fourteen cells (two small lateral cells are not shown). 4, the blas-
topore; Pr, micromeres from which the prototroch is almost immediately afterwards
developed. :

212 WILSON. [VoL. IV.

ventral plate consists of twelve principal cells, arranged in two
longitudinal rows of three each on each side of the median line.
Anteriorly are the two primary mesoblasts with their first-formed
derivatives (Y—Y) ; posteriorly are four teloblasts (X—X), and
four smaller cells derived from them, as shown in the figure.

In the next stage the four smaller ’s divide obliquely, while
four smaller cells are produced anteriorly from the four larger
Y’s. The four Y’s are now rapidly pushed into the interior,
being overgrown by the progeny of .Y, which advance upon them
from behind (Fig. 6), and by the micromeres advancing from the

Fic. 6.— View of a still later stage in optical longitudinal section from the right-
hand side. I.-I., the first cleavage-plane; Zc, the ectoblast derived from the micro-
meres; /7, the prototroch; OJ, the oil-globules; Y, primary mesoblast in process
of pushing in; X—X, progeny of the first pro-teloblast. The blastopore, which was
situated just anterior to Y, has been closed in by the micromeres.

sides and in front. As they are pushed in, the smaller Y’s
divide, and a second pair of smaller cells are separated from the
two large Y’s (primary mesoblasts), which are now scarcely larger
than the cells to which they have given rise, or than the super-
ficial cells (descendants of ) among which they are imbedded.
After the next division the teloblasts become indistinguishable,
and the mesoblast-bands seem to fuse with the ventral plate ;
so that an examination of the embryo at this period, without
a knowledge of its earlier history, would certainly lead to the
conclusion that the mesoblast-bands arise by proliferation from

No. 2.] THE MESOBLAST-BANDS [N ANNELIDS. 213

the ventral ectoblast, as Salensky describes it. From their
point of origin the bands, each now represented by a group
of six or seven cells, extend upwards between the outer layer
and the four entoblast-cells, along the line of contact of the
two anterior cells (C, D) and the two posterior (4, 4). Their
later history will be described elsewhere.

Let us now return to the progeny of X. Increasing rapidly
in number, both by their own divisions and by the addition of
cells formed from the four posterior teloblasts, they give rise
to a broad, bilobed plate, consisting throughout of a single layer
of granular cells, and occupying the greater part of the lower
half of the embryo. The prototroch is developed from a series
of micromeres, at first single, that encircles the equatorial belt
of the embryo, and lies immediately behind the four posterior
teloblasts. The latter persist for a considerable period, but
ultimately disappear. The two outer ones first break up into
smaller cells, and as this takes place, the remaining two separate
from each other along the median line. Thus the ventral plate
becomes bilobed behind, with a V-shaped area between the two
lobes, and a single teloblast at the tip of each. This teloblast
remains until each half of the ventral plate contains fifty or
more cells, still lying quite at the surface. Ultimately it dis-
appears, and the proctodzeum is formed in the anterior part
of the V-shaped area. At a still later period the ventral plate
thickens, becoming several layers deep on each side the median
line, and gives rise to the neural plates and the seta-sacs. Its
relation to the nephridia and the circular muscles is still under
investigation.

Let us now compare these facts with the development of
Clepsine and Lopadorhynchus. In Clepsine the large posterior
macromere first separates off a single micromere (as in /Vere7s),
and then divides into two large cells. The upper right-hand
cell (“‘neuro-nephroblast ” of Whitman) has precisely the same
relation to the rest of the embryo as the first pro-teloblast, ,
of Nereis. In Clepsine this cell breaks up into eight teloblasts,
viz. (still using Whitman’s terminology) : two neuroblasts, four
nephroblasts, and two lateral teloblasts ; and these give rise to
all the structures of the ventral body-wall excepting those aris-
ing from the mesoblast-bands ; z.e. the neural foundations, a part
of the nephridia, some of the ventral ectoblast (probably), and

214 WILSON. [VoL. IV.

perhaps to other structures! In JVerezs the corresponding cell
breaks up into four instead of eight teloblasts, which give rise
likewise to those parts of the ventral body-wall not derived from
the mesoblast-bands; z.e. the neural plates, the ventral ecto-
blast, the seta-sacs, and probably other structures (for the pres-
ent I leave the nephridia and the circular muscles out of consid-
eration). The foregoing considerations render it practically cer-
tain that the first pro-teloblast of Nerets (X) ts the homologue of
the “neuro-nephroblast” of Clepsine. In Clepsine the second and
larger cell produced by the division of the large posterior ma-
cromere (which I shall call the common primary mesoblast)
divides into equal parts, which become the primary mesoblasts,
and give rise to the mesoblast-bands, in the usual manner. The
second pro-teloblast (Y) of Nereis ts therefore the homologue of the
common primary mesoblast of Clepsine. It therefore appears
that Clepsine and WVerets agree in every essential feature of de-
velopment. They differ only in secondary details, —in the ulti-
mate number and arrangement of the teloblasts, and in the fact
that they and their products are at first superficial and form a
part of the outer layer, — falsely called the ectoblast. It is in-
teresting to recall in this connection the fact that in Lumbricus
eight of the ten teloblasts likewise remain for a long time at the
surface.

Turning now to Lopadorhynchus, we find here also a bilobed
ventral plate, as in JVerezs, but no teloblasts, 2% the earliest stages
thus far observed. From it arise, as in /Verezs, the mesoblast-
bands, the neural plates, and the seta-sacs ; and I think the ven-
tral plate must be regarded as completely homologous in the
two forms. They differ only in the earlier segregation and dif-
ferentiation of the mesoblastic material in Verezs which leads to
the formation of a pair of transitory teloblasts, which, however,
form part of the ventral plate. Meyer makes the interesting
statement in a recent paper? that he has satisfied himself that
the neural elements and the mesoblast-bands do not arise in
Lopadorhynchus from a common foundation, but from “ Zwei
verschiedene, nur dicht zusammengedrangte Bildungsheerde.”

1 Bergh asserts that the lateral teloblasts and the nephroblasts give rise, in Lum-
éricus, to circular muscles, and are therefore myoblasts. I shall return to this question
in a later paper.

2 Biol. Centralblatt, 1 Juli, 1890.

No. 2.] THE MESOBLAST-BANDS IN ANNELIDS. 215

This is precisely what occurs in Merezs. The term “ectoblast”’
as applied to the ventral plate as a whole is, however, a misno-
mer. Its cells form rather a still undifferentiated tissue which
is nowise to be regarded in the same light as the ectoblast of
the upper pole.

HH,

It now becomes an interesting question whether the second-
ary mesoblast of annelids can be shown in all cases to arise
from a single pair of teloblasts; for the case of MWerezs shows
that they may be present only in very early stages, so as easily
to be overlooked. The entire subject demands re-investigation
from this point of view. I have carefully studied the develop-
ment of Hydrotdes dianthus (a form nearly allied to Expomatus),
by following the cleavage of the living ovum, by examination of
stained and cleared embryos, and by actual sections. The
cleavage is in every detail identical with that of Ewpomatus, the
gastrulation takes place in essentially the same manner, and the
trochophore is of quite the same type. Yet I have been unable
to identify the teloblasts at any period. They are certainly not
present at a stage when the mesoblast-bands consist of not more
than five or six cells each «(f Hatschek’s Figs. 43 to 46).! At
this period each band ends posteriorly in a group of about three
cells, two of which, not perceptibly larger than the others, are
joined by a narrow bridge of protoplasm stretching across in the
angle between the proctodzeum and the wall of the anal vesicle.
The head-kidney lies outside the mesoblast-band, and is only
connected with it at its anterior end.?

In its earliest recognizable condition (cf. Hatschek’s Fig. 33)
the mesoblast-band consists of a group of three or four cells

1 Arb. a. d. Zool. Inst. Wien., VI., 1885.

2 J have been able, by the study of these embryos, to establish the interesting fact
that the head-kidney opens posteriorly into the proctodeum. Under a high power
(Zeiss Apochromatic Homogeneous, 3 mm.) the canal can easily be followed from
its beginning near the front end of the organ, along the outer dorsal border of the
latter, into the antero-lateral part of the proctodeum. This can be done most
readily during the activity of the cilia (which is intermittent) ; but the canal can
easily be followed in the quiescent state, in both the side and ventral views of the
larva. This fact seems to remove all doubt of the homology of the trochophoran
head-kidney with the nephridia of the Rotifera. I shall return to this point at
another time.

216 WILSON. [Vot. IV.

wedged into the angle between the posterior part of the archen-
teron and the ectoblast : no one of them can be identified as the
teloblast. Besides this ‘secondary mesoblast”’ there are at this
period a number of scattered cells, lying in the narrow cleavage-
space. Some of these are applied to the ectoblast, some to the
entoblast, where they form a contractile network surrounding
the archenteron ; some stretch across the cleavage-cavity. These
cells obviously represent a part of the “primary mesoblast”’ or
“mesenchyme,” the relation of which to the secondary meso-
blast of the germ-bands has become one of the most interesting
questions of annelid embryology. My observations on Hydvroides
indicate that the primary mesoblast arises in a manner similar
to the process in Polygordius, as described by Repiachoff !
and Metschnikoff,? though I am not yet able to give decisive
evidence, in spite of repeated examination of the question.
According to these authors the primary mesoblast arises in the
form of isolated cells that take their origin “ héchst wahrschein-
lich,” in the entoblast, at or just before the time of gastrulation.
Metschnikoff makes no special mention of the formation of the
secondary mesoblast-bands; but his general account implies,
and his figures bear out this interpretation as far as they go,
that there is no real distinction between the primary and sec-
ondary mesoblast, the latter arising in exactly the same manner
as the former. For the present I am obliged to believe that
the primary and secondary mesoblast have the same relation
in fydrotdes. The mesenchyme cells are of all shapes, —
branching, elongate, rounded,— and appear to graduate both
in form and in position into those of the germ-bands. The
latter appear about the time of gastrulation, as two bilateral
masses of cells that are pushed into the cleavage-cavity near
the blastopore. Some of these cells appear to pass forwards
and give rise to the “mesenchyme”; the remainder form the
‘“‘secondary”’ mesoblast-bands, which at first are posteriorly at
the sides of the proctodazum, but afterwards come into con-
nection by a bridge that is developed on the ventral (anterior)
side of that structure.

Through the courtesy of Mr. Agassiz I have had an oppor-
tunity to investigate Polygordius, the larve of which are very

1 Zool. Anzeiger, 1881.
42. fe We. L001, SAND 1OS2:

No. 2.] THE MESOBLAST-BANDS /N ANNELIDS. 217

abundant at Newport. In the very large series of larve in my
possession all stages are represented from the adult condition!
down to the youngest described by Hatschek and Fraipont.
The species is closely similar to P. neapolitanum as described
by Fraipont. An examination of the mesoblast-bands in these
larvee showed, to my extreme surprise, that xo ¢elodblasts of any
kind are present, even in the youngest stages. The mesoblast-
bands end behind, as in Hydvozdes, in a group of two or three
cells, which are somewhat larger than those in front, it is true,
but have none of the characteristics of teloblasts. Only in a
single case have I found at the tip of the band a cell distinctly
larger than the others, and this was on one side only. Is this
absence of the pole-cells peculiar to the American species of
Polygordius? Possibly; yet there is a fact that forces me to
suspect the possibility of error on the part of Hatschek and
Fraipont, improbable as such a suspicion may appear. If the
germ-bands of a young larva be viewed ex face from the ventral
side in suitably prepared specimens, a very clear picture of the
germ-bands will be seen, closely similar to that represented
in Hatschek’s Fig. 57. At the tip of each germ-band isa large,
rounded, clear cell, meeting its fellow in the median line just
in front of the anus, and to all appearances a “ primary meso-
blast.” For such I at first mistook these cells, but, to my great
surprise, sections in the various planes all agreed in showing
that they formed part of the ectoblast, lay at the surface of the
body, and had no connection whatever with the mesoblast-
bands, though the latter end just below them. These cells
are clear, vacuolated, with minute nuclei, and are undoubtedly
homologous with the anal vesicles or anal glands of other
annelid trochophores; though as far as I am aware they are
here described for the first time. They have no connection
with the paratroch.

1 T have repeatedly observed the sudden metamorphosis of the last larval stage into
the adult, which has been briefly mentioned by Kleinenberg. The cells of both the
prototroch and paratroch are suddenly thrown off, and continue to swim for some
time after their complete separation from the body of the young worm. The proto-
trochal cells are often thrown off, over the head, in the form of a girdle, which in
some cases—TI have observed it at least four or five times—is swallowed and
digested by the animal. This recalls the curious habit of Actinotrocha, which at its
metamorphosis throws off and swallows the greater part of the prae-oral lobe (Cald-

well). This metamorphosis certainly throws an interesting light on the origin
of such a mode of development as that of Pididium.

218 WILSON. [Vou. IV.

I hesitate to suggest that two such experienced observers as
Hatschek and Fraipont can have fallen into error on this point,
yet it is a singular fact that neither of them figures or refers to
the conspicuous prz-anal gland-cells, while both describe a large
mesoblast-cell in exactly the same position, but lying below the
surface. Each gives a single figure of the ‘‘ primary mesoblasts”’
in cross-section, lying in the cleavage-cavity. But Hatschek
gives also, in the same series of figures, exceedingly definite
representations of “die fotalen Langskandlen,” which, as Meyer
and Fraipont have shown, and as I have convinced myself from
the study of many series of sections, have no existence in
nature. Fraipont’s figure is scarcely more satisfactory. The
minute nuclei and clear protoplasm of his figure have no resem-
blance to the large, conspicuous nuclei and granular protoplasm
of teloblasts; they at once suggest the small nuclei and vacu-
olated cell-body of the ‘anal gland-cells.”” A closely similar
view might easily be had of a rather thick, slightly oblique
section, the “ gland-cells’’ being tangentially cut, and appearing
to lie inside the ectoblast on account of the sharp curvature of
the surface where they lie. And in view of these facts I venture
to suggest the desirability of a re-examination of the mesoblast-
bands in the European species of Polygordzus.

The non-existence of the teloblasts in later stages, which is
apparently the rule among the Polychzta and (?) Archiannelids,
by no means, however, affords any presumption that they are
not present in earlier stages. The case of Mevets shows that
it will not be safe to assume the absence of teloblasts without
following the development, cell by cell, from the very beginning,
and that wherever it is possible to make such a detailed study,
we may pretty confidently expect to find teloblasts. There is,
I believe, every reason to believe that in Hydrotdes, for example,
the two groups of mesoblast-cells take origin in two cells, though
they may appear at so early a period and differ so little in
appearance from the adjacent cells as to elude any but the most
searching examination. And it does not seem very rash to
predict that the secondary mesoblast-bands, even of Lopado-
rhynchus, will yet be shown to arise by teloblastic development.

MARINE BIOLOGICAL LABORATORY, Woop’s HOLL, MASss.,
September, 1890.

No. 2.] LHE MESOBLAST-BANDS IN ANNELIDS. 219

While the foregoing article was in process of publication I received R. S.
Bergh’s full paper on the germ-bands of Lumbricus [Zeitschr. J. wiss. Zool.,
L. 3, 1890]. This admirable work contains a complete confirmation of my
discovery of the teloblasts and cell-rows of the middle stratum of the germ-
bands in Oligochata—a confirmation which has especial significance on
account of the curiously exaggerated and, to me, quite inexplicable, hostility
with which its author saw fit to receive the original announcement of that
discovery. Dr. Bergh’s paper, as a matter of course, exhibits in some degree
the well-known and characteristic skill of its author in belittling the work of
other investigators, but I observe with interest many signs of progress, both
in knowledge of the germ-bands and in acquaintance with the usual forms of
courtesy in scientific discussion. The author, for instance, no longer seeks to
discredit my work by innuendoes directed against my scientific good faith ;
indeed, the existence of the teloblasts and cell-rows has even become «< sehr
leicht zu konstatiren” —an evidence of progress on which I congratulate
my learned and courteous critic. It is not improbable that Dr. Bergh may
in time be able to observe with equal ease the teloblasts and other structures
I have described in WVerezs.

Apart from the development of the glandular portions of the nephridia, my
general account of the structure, mode of growth, and relations of the various
parts of the germ-bands in Luméricus is confirmed in every respect. He
adds, however, the very interesting discovery that the products of the neuro-
blasts are reinforced by a median nerve-plexus that is taken up into the
ventral nerve-chain between the two neural cords. His account of the
‘‘nephroblasts ” and ‘lateral teloblasts” differs entirely from that given by
‘Whitman and myself. Their connection with the nephridia is denied, and
they are asserted to be ‘‘ myoblasts” which give rise to the circular muscles.
This result, if well founded, is of the greatest interest, and marks an im-
portant advance in our knowledge of annelid embryology. It is to be hoped,
however, that the question may be carefully re-examined, by some fair-minded
observer, in Clepsne or some other favorable form.

BRYN MAwe, PA.

CONCERNING VERTEBRATE CEPHALOGENESIS.
HOWARD AYERS.

In this short resumé of some of the results of my investiga-
tions in vertebrate cephalogenesis, I shall not give a complete
account of the morphological data upon which the conclusions
are based ; but shall introduce only such facts as seem necessary
for this presentation.

A more detailed account with a critical consideration of the
literature bearing upon the subject is reserved for a more
extended and illustrated publication which I have in prepara-
tion.

The problem of the origin of the vertebrate head, more espe-
cially the brain, from the invertebrate type is, so far as our
knowledge yet reaches, an insoluble one; but given the verte-
brate type of body and central nervous system, we are in posi-
tion to clearly demonstrate its phylogenetic outcome as repre-
sented in the mammalian head and brain.

Starting with the central nervous system of Amphioxus, we
have to deal with an organ which affords many points of contrast
with the axial nervous system of higher vertebrates and which
serves, as some authorities think, to bridge over the chasm
between the invertebrate type (arthropod and annelid) and the
vertebrate.

With the exception of an undetermined small number of
anterior segments, the nerve cord of Amphioxus is divided up
into a series of physiologically equal segments as Steiner has
shown ; but I feel confident from anatomical facts that further
and more detailed experimentation will show that there are some
modifications to be introduced into Steiner’s results, and that
there are differentiations physiologically which the crude meth-
ods of experiment used by him excluded from the physiological
reaction. Steiner’s results on other vertebrates would lead to a
conclusion differing from that which he has published with

regard to Amphioxys. For in his experiments on Amphioxus
225

222 HOWARD AYERS. [VioL. IV.

he was unable to take into account any of the higher sense
organs. Of course their relation to locomotion and the central
mechanism effecting this relation entirely escaped him.

I hope to show that there are certain ganglionic centres in
the anterior end of the nerve cord of Amphioxus, which compel
us to call this region a brain strictly comparable with that of
other vertebrates; for in this region we have the centres of
special sense brought into relation with locomotion.

In my final paper I shall give an historical review of the ideas
that have been held concerning vertebrate cephalogenesis. For
it is in the gradual evolution of these ideas that we have the
firmest support, and completest precedent, for leaving the pre-
vailing ideas for those that can be shown to come nearer the
truth in nature. The idea that the human skull (as typical of
the highest vertebrates and hence erroneously thought to most
certainly present genuine vertebrate conditions) was formed of
relatively slightly modified vertebral bodies and their processess,
had its eminent exponents and ranits course. It was succeeded
by the theory that it was only in the cartilaginous cranium that
remnants of primitive segments were to be sought. This form
of solution was modified by the statement that only a portion of
the primordial cranium could possibly show traces of the primi-
tive segmented character.

This brings me to the expression of the view, which has firm
support in facts, that the primordial cranium is never influenced
by vertebral segmentation, as it appears in, and belongs to, a
stage antecedent to the formation of vertebra; further, that it
is not primarily influenced by the mesomeric segmentation of
the body, since it arose at a later period phylogenetically and
after the mesomery ontogenetically. The primordial cranium
has been gradually acquired by vertebrates, and its rudiments
were developed first in or near the horizontal plane in which the
chorda lies. Extending in a direction more or less parallel to
the chorda, it does not necessarily show traces of the primitive
segmentation of that part of the body in which it is developed,
since many, at least, of the features of segmentation, had long
vanished before a protecting cranium formed about the nervous
axis. In such a structure we would expect ccenogenetic (onto-
genetic) variations to be of frequent occurrence.

What I have said above does not exclude the possibility of

No. 2. ] VERTEBRATE CEPHALOGENESSS. 223

the inclusion of vertebral remains within the skull, nor is this
view influenced by the fact that such inclusions are known to
exist, e.g. in the cartilaginous fishes, etc., and for this reason; the
skull is formed about the brain and includes various organs, not
always the same, within its substance. As illustrations of the
variations met with, we have in some cases the aorta included
in the cartilaginous basis cranii, though usually it is left out,
the notochord usually for the whole of its cranial length, though
it may be left out in part, as for example its anterior end may lie
below the cartilage or above, z.e. inside the cranial cavity either
projecting beyond the pituitary prominence or running out on
the inner face of the skull behind it. There are other facts of
this class which have not received enough attention.

A. The anterior end of the neural axis of Amphioxus ts a
brain, and corresponds with a certain definite portion of the
brains of other vertebrates. Its anterior wall ts the homologue
of the lamina terminalis of other vertebrate brains, and the ante-
rior portion of tts unpaired ventricle 1s the thalamocele. There
7s a posterior portion of the ventricle intimately associated with
a ganglionic tract, which corresponds to the mesocele, while the
myelocele remains, more or less widened, varying much im
different individuals, but always in an undifferentiated condition.

Although the nervous system of Amphioxus has been often-
times studied, many features of importance remain to be de-
scribed, and the well-established relations deserve reconsideration
in connection with the new ones. With the improvements in
methods of preparation, results unobtainable otherwise are
reached, which show that we may still hope for more light on
important questions from even well-worked fields. The prepa-
rations I have studied were made by the celloidin method of
imbedding and nitric acid maceration process, and I can recom-
mend both as giving excellent results.

I may say, to begin with, that I do not consider Amphioxus
to be a degraded form, in the usual sense of that word as
employed in vertebrate phylogeny. Its larval modifications
cannot be called degradations of structure, nor are its adult
peculiarities to be looked upon in the light of degraded or
bizarre modifications of zorma/l vertebrate conditions; on the
contrary, they are to be regarded as not only xorma/, but also

224 HOWARD AYERS. [Vor Ve

as primitive, and in a certain degree ancestral, conditions of
structure.

The derivation of the vertebrate phylum from some simple
type closely approaching Amphioxus, in detail of structure, in
almost! all its organs, as we know them at the present time,
becomes a morphological necessity. Instead of precluding the
possibility of establishing the claims of Amphioxus to such
position in the phylum, every advance in our knowledge of the
life-history of Amphioxus serves only to strengthen such claims.

It is to be regretted that certain zodlogists should have so
dulled their morphological sense as to be found denying to
Amphioxus any position whatsoever among vertebrates. As
an offset to such rash opinions, I will only quote the words of
Professor Huxley, giving his conclusions after a careful study
of the anatomy of Amphioxus, and a detailed comparison with
other fish forms: ‘In all other respects, however, it conforms
(except in the absence of auditory organs) to the vertebrate
type; and considering its resemblance to the early stages of
Petromyzon, . . . I can see no reason for removing it from the
class of Pisces.” I might quote other eminent authority in
favor of the strictly vertebrate nature of Amphioxus, but I think
this should suffice. Surely we may never hope to know more
of the mystery of the origin of vertebrates by ignoring the most
important of the still accessible forms that lead us back to the
beginning of our type.

It has become necessary, in any discussion of the homologies
of the vertebrate brain, to define more accurately what is to be
understood by the term “vertebrate brain.” Evidently the
definition of the word must include the names and relations,
as far as lies within the province of a definition, of all those
parts common to all vertebrate forms, excluding from the prin-
cipal sentence of the definition all exceptions however important.
We have not lacked for definitions more or less specific, but
until quite recently I do not know of any investigator who has
attempted to define the organ in terms harmonious with the

1 July 27. I have just seen Boveri’s paper, “Uber die Niere des Amphioxus ” ;
and since this investigator has shown in most conclusive manner by his important
discovery, that the kidneys of Amphioxus are ancestral structures, I am ready to
withdraw the limiting word “almost,” since it was mainly with reference to the
kidney system that the reservation was made.

INO: 2: VERTEBRATE CEPHALOGENESIS. 225

present condition of morphology and physiology. Steiner has
shown, by a beautiful series of experiments on numerous forms,
that we may only speak of a brain from the physiologist’s stand-
point when we have associated with the general centre of loco-
motion one or more of the organs of the higher senses, and this
definition is entirely admissible from a morphological standpoint.
While Steiner's experiments do not show the presence of a
brain in Amphioxus, they do not, I think, in any way demon-
strate or even render probable its absence, and it can be shown
that the morphological or physical basis required by Steiner’s
definition is present.

I would define the vertebrate brain as follows : The “vertebrate
brain” ts that portion of the anterior part of the axial nerve cord,
associated with organs of special sense, containing an enlargement
of the central canal which is carried out into all structures formed
by the outgrowth of the brain wall. Its walls contain the principal
centres for the co-ordination of sensations and movements. All
further additions to this simple brain (Amphioxus) are made in
response to the demands of the organs of special sense, with which
1s associated extension of the co-ordination apparatus. With such
additions we have the compound brain of all other known verte-
brates up to man inclusive.

Reasons why the anterior end of the nerve cord of Amphi-
oxus is a brain. It is a brain because

1. It forms the anterior termination of the neural axis.

2. It stands in intimate relation to the sense organs eye and
nose.

3. It gives off at least two pairs of sensory nerves provided
with peripheral ganglia.

4. It possesses large groups of ganglion cells forming centres
of co-ordination.

5. It possesses an enlarged section of the central canal in the
form of an unpaired ventricle with three well marked divertic-
ula, two optic, one olfactory.

6. It is the largest part of the nervous system, at a time when
the massive musculature and branchial apparatus of the anterior
middle fourth of the body has not reached the stage requiring
much enlarged central accommodations.!

‘ When, however, the innervation of the locomotive apparatus (which in the
adult animal more than equals the remaining organs in bulk) is fully developed, the

226 HOWARD AYERS. [VoL. IV.

7. It shows in young larva growth to such an extent as to
cause a ventral flexure of the chorda, the brain itself bending
downwards, thus producing a “cranial flexure.”

8. It shows in all other details of structure that z¢ zs ot sim-
ply the anterior end of the spinal chord, but a brain.

g. It shows in a larval stage soon after the differentiation
of fibres in the neural axis (larve with one gill slit), a marked
differentiation into ganglionic and fibrous regions, and the
boundaries of the unpaired ventricle as well as the lamina ter-
minalis are distinctly marked out. There is then a ventricular
segment of the brain reserved for the special sense organs. The
fibres appear simultaneously with the formation of the pigment
spot, and are in all probability the ways by means of which the
sensations from this special sense organ are conveyed backwards
to the motor centres.

10. Szuce Amphioxus ts a vertebrate, these relations MUST have
direct and tmportant bearings on the phylogeny of the vertebrate
brain and head, and will afford us invaluable aid in clearing up
these intricate problems.

B. The large collections of ganglion cells gust posterior to the
thalamocele are homologous with the medullary nuclet of other
vertebrates, since thetr connections show them to be centres for the
control of the branchial apparatus, and the sensory and motor
structures lying in the territory of the gill basket, — e.g. centres
of respiration, deglutition, etc.

These groups of large ganglion cells do not reach quite to the
thalamoceele. A portion of the wall bounding the ventricle
posteriorly is free from them, and is made up of such cells as
are strictly comparable with the cellular elements of the mid-
brain of higher forms. From this narrow territory and the
central canal contained in it is derived the mesencephalon and

brain is relatively smaller, but it still has its peculiar structure. We have other in-
stances of the actual preponderance in size of circumscribed tracts of the nervous
axis over the brain. In those ancient forms, ¢.g. Stegosaurus, whose remains have
been described by Marsh and others, where the sacral canal is shown to greatly
exceed the cranial cavity in its cubic dimensions. But here there is a doubt as to
whether the nervous axis in the sacral canal really filled the space, or whether it bore
proportionately the same relations to its cavity that the brain did to its cranial space.
There is reason to seriously doubt that the sacral enlargement of the cord was actually
larger and more important than the brain in relation to the locomotive mechanism of
the animal.

No. 2.] VERTEBRATE CEPHALOGENESYIS. 227

mesoccele of higher forms. The cerebellum is simply a dorsal
commissure of the medullary region, as has been shown by
Osborne and others from studies on higher vertebrates. I have
found no trace of a cerebellar commissure in Amphioxus. The
open ventricle of the medulla is produced by the great increase
of ganglion cells, and especially of the fibre tracts which must
of necessity pass through this region in order to reach the cen-
tres of co-ordination in the parts anterior to it. The basal and
lateral portions of its walls are thus greatly thickened, the gan-
glion cells of the nerve nuclei arrange themselves in harmony
with these changed conditions, and the dorsal wall is greatly
extended without at the same time being structurally compli-
cated. The medulla of higher vertebrates is not co-extensive in
all forms and includes a varying number of the segments repre-
sented in Amphioxus by simple spinal segments.

The brain of Amphioxus possesses all those functions which
in higher vertebrates are possessed by thalamencephalon mid-
brain, and medulla, except an undetermined number of the pos-
terior segments of the medulla of the higher forms. Of course
these functions are all of a milder nature than in the higher
animals.

The reason for Steiner’s failure to find a medullary centre of
co-ordination lies in the intense nature of the stimuli applied to
the nervous apparatus (resection, etc.) and to the small size of
the region to which the stimuli must be applied. The nervous
apparatus is of such a delicate nature that such strong stimuli
serve to call forth the activities of each segmental centre of
locomotion, and prevent the observation of any other results of
stimulation.

Much more delicate methods must be used to obtain reaction
of the organs of special sense. Of the possible means of ex-
perimentation, which appear to me likely to give good results in
the case of the pigment spot and the olfactive pit, are circum-
scribed application of bundles of light rays of varying intensity and
wave lengths, and the application of olfactive stimuli by means
of fine capillary tubes to the resting animals in sea-water.

C. The ontogenetic changes of the neural axis in other verte-
brates carries the brain through the condition which in Amphi-
oxus remains permanent as the adult brain.

228 HOWARD AYERS. [VoL. IV.

As has been demonstrated by several recent investigators,
the fore-brain and olfactory lobes are simple outgrowths of the
dorsal wall of the thalamencephalon. They appear early in the
development, it is true, but such early appearance is undoubtedly
a coenogenetic phenomenon. That there is a substantial agree-
ment between the Amphioxus brain, and the higher vertebrate
brain of this stage, is perfectly evident. There is always a thala-
moccele bounded anteriorly by a lamina terminalis to mark the
boundary of the primitive end of the neural axis, and above, be-
hind and on either side of this primitive lamina terminalis, the
optic diverticula are given off from the brain.

D. All the sense organs developed in connection with the anterior
end of the Amphioxus body are probably paired; some of them
certainly are, e.g. the eye-spot.

As I shall show in the next paragraph, the eye of Amphioxus
exhibits unmistakable traces of bilateral symmetry and a ten-
dency to develop into two pigmented areas in connection with
diverticula of the thalamoccele, so that here I need only mention
the fact, that I have been able to trace fibres from the olfactory
organ through the walls of the olfactive diverticulum or bulbus
olfactorius to both sides of the brain. Whatever this organ
may prove to be, on further investigation of its ontogeny it is
supplied from dzlateral brain centres and affects both right and
left co-ordinating tracts. The organs of special sense developed
in the course of the two anterior pairs of nerves are certainly
bilaterally symmetrical structures.

E. The eye-spot or eye of Amphioxus ts the forerunner of the
vertebrate eye, and shows traces of several stages in the develop-
ment of the retina of higher forms. In itself it 1s not an organ
of sight, but a light-perceiving organ.

After acareful study of the Amphioxus eye-spot, and related
structures, I have become convinced that this animal presents us
with the earliest stage in the phylogenetic development of the
vertebrate eye.

As is well known, this eye-spot, which is considered a pigment
spot, lies across the anterior end of the neural axis in the ante-
rior end or wall of the brain, z.e. in the lamina terminalis, in an
extended sense. It is not widely known, however, that this

No. 2.]} VERTEBRATE CEPHALOGENESYSS. 229

pigment spot assumes a large variety of shapes as well as posi-
tions, with respect to the anterior wall of the ventricle and to
the first pair of cranial nerves. The most usual form is that of
a slightly bilobed mass ; the lobes being placed to the right and
left of the median line, so as to cover the roots of the first pair
of cranial nerves more or less completely.

Other forms have been described by the various authors who
have written upon the subject, and they are easily observable
in any series of individuals. As already stated, the spot is very
variable in size and shape, and its extremes may be set, as the
convex or concave (forwards) lens shape, and the close group-
ing of most of the pigment cells in the wall of the lamina ter-
minalis on the one hand, and the separation of the mass into
three portions (two ventral and lateral, and one dorsal and me-
dian) with outlying scattered pigment cells on the other hand.
These various forms grade insensibly into each other, and
evidently depend in large degree, if not entirely, upon the migra-
tory capabilities of the pigment cells in a state of nature.

These variations, then, are caused by the shifting of the pig-
ment cells. It may be frequently observed that the two pig-
ment masses pass out into the two nerves of the first pair.
Where this modification occurs, it will be found associated with
a pair of diverticula of the median ventricle which make their
way out into the nerve roots. These diverticula are lined by
the same cells that form the inner lining of the median ven-
tricle, and the pigment cells lie among the cells of the second
layer, or layer of percipient elements.

In this manner the light-perceiving organs are carried toward
the surface at the ends of diverticula of the primitive brain
cavity, reproducing in the phylogeny of Amphioxus the first
steps of the series known for the remaining vertebrates.

We have only to think of these two pigmented optic struc-
tures brought into relation with the ectodermic structures —
lens, cornea, etc., — of the higher vertebrate eye, in order to
have all the steps of eye production carried out in Amphioxus.
We know of no such relation of the ectoderm in the Amphi-
oxus, and consequently conclude that, all facts considered, Am-
phioxus is descended from some ancestral form along with other
vertebrates, but that, owing to its simple habits of life, has
never required a lens or other focusing apparatus for the pur-

230 HOWARD AYERS. [VoL. IV.

pose of image perception, — simple light-perception sufficing for
all its needs.

As Hatschek has shown, the pigment spot appears in the
nervous layer long before the closure of the anterior neuropore.
One of the causes operating to transfer the pigment from the
brain wall to the periphery is the constant growth from the
larva to the adult, and the consequent thickening and increas-
ing opacity of the superjacent tissue.

After these diverticula have formed, there is left of the an-
terior brain wall between them a median portion which, as we
shall see, is the homologue of the lamina terminalis of the
remaining vertebrates.

The lamina terminalis of this stage contains the basis of the
cerebral lobes, and the olfactive'centres of all the higher forms.

The relation of the parts here described corresponds with the
facts as given in the latest and most accurate studies of verte-
brate ontogeny from all classes of vertebrates, which form a
valuable basis for the examination of this proposition from the
comparative standpoint.

An examination of Hatschek’s figures, 64, 65, 67, and 60, for
an explanation of the relation of the parts, shows that the most
anterior portion of the neural plate of the young larva remains
for a long time in its primitive flattened, uninclosed condition.
Here the dorsal surface of the plate is still exposed to the direct
action of external stimuli; its face is in or near the horizontal
plane, and hence looks vertically, or nearly vertically, upwards.
The eye-spot is developed in this area, and it clearly occupies
the most favorable position for an important sense organ that
the body of Amphioxus affords.

As the anterior neuropore closes up more and more, the an-
terior lip grows upward curving backwards, the pigment spot
comes to lie in the vertical portion of the anterior end of the
now completed canal; consequently it is strictly terminal in its
adult relations, but may, and frequently does, assume a position
at the side and behind the end of the axis.

For greater functional power, the central (median) portion of
the pigment spot has grown upwards (dorsad), and carrying with
it a portion of the ventricular wall has produced the pineal eye.

Let us follow through the development of the nerve cord of
the larval Amphioxus. Formed as a thickened plate of cells on

No. 2.] VERTEBRATE CEPHALOGENESIS. 231

the dorsal surface of the embryo, for a long time it lies open
and exposed to the sea-water; its cells bear cilia which are
doubtless sensitive to various stimuli. Long before the neural
plate is converted into a cylinder (but only after the plate has
been overgrown by the lip of the blastopore), pigment makes its
appearance in some of the cells of the plate. At present we
have no complete account of the appearance or early relations
of this pigment matter; but the mass called the eye-spot ap-
pears shortly after the first mass which arises in the middle of
the length of the plate, which at this stage falls in the fifth
somite. The eye-spot soon becomes much more important
than any of the posterior pigment-cell groups, and remains so
throughout life. As already stated, it at first faces upward,
but later acquires a facing at right angles to this direction on
account of the closure of the neuropore, the development of
the head fin, and the gradual thickening of the tissue directly
above the spot. Of course with this thickening of the body
walls, more and more light is cut off from the spot, and the
direction from which light reaches the spot in greatest abun-
dance is from the front and sides of the head. The head fin
fold divides the light falling upon the spot into two lateral bun-
dles of rays, which, owing to less thickness of tissue through
which they are compelled to pass, fall upon the sides of the
spot with greater energy than light from any other source.

It is, then, owing to the relations of the external features
of the head that the primitively median pigment body (not
necessarily from an unpaired source) divides into two lateral
portions, each of which strives to get nearer the surface, and
in accomplishing the transposition cause an outgrowth of the
brain wall, producing the two antero-lateral diverticula spoken
of above.

The relation of the two diverticula to the bases of the first
pair of nerves is of great interest in this connection, for, as we
know, the surface of the head end of the body of Amphioxus —
especially of the young —is pigmented along certain lines, over
certain areas; and it appears highly probable that the pigment
cells of the surface have still some intimate relation to the light
and heat perception, — one or both. The pigment of the cen-
tral canal is derived from the pigment of the superficial ecto-
derm, which in the adult has nearly disappeared.

232 HOWARD AYERS. [Vor. IV.

The primitive sense cells of the ectoderm transmitted through
the nerve fibres of the first pair of nerves general impressions,
from which those percipient elements of the brain associated
with pigment cells selected vibrations of those wave lengths
productive of the sensations of light and heat. The percip-
ient elements not associated with pigment cells selected and
were affected by only such stimuli as gave rise to other sensa-
tions, taste, smell, touch, etc.

Now that we have seen how most of the structural details of
the brain form presented to us by all vertebrates higher than
Amphioxus may have arisen out of the simple Amphioxus type,
there remains for consideration a structure whose significance,
both morphological and physiological, is still a matter of dis-
cussion, viz., the hypophysis. So far as I have been able to
discover, there does not exist in Amphioxus the slightest trace
of such an organ; but it is not difficult to see how such a struc-
ture could arise from the ventral portion of the thalamoceele, in
connection with the further development of the mouth and its
sense organs. For the present I am not in position to say
more of this structure, than that the hypophysis in Ammoccetes
is intimately related with the olfactive area, and was probably an
organ of taste.

We have then, in Amphioxus, all the stages in the develop-
ment of the paired eyes of vertebrates (as well as of the pineal
or unpaired eye) demanded by the theoretical considerations,
save the final ones of the formation of the completed optic vesi-
cles and lenses. The completion of these stages occurred
somewhere in the phyletic series between the Amphioxus condi-
tion and the Cyclostome condition. In the Cyclostomata
(Ammoccetes) the optic vesicles grow out only slowly, and after
the animal has passed the Amphioxus stage.

It should not be forgotten that the optic vesicle, as it pushes
out from the brain, presents its anterior hemisphere to the exte-
rior; and that in most vertebrates this half remains unpigmented,
while it is this anterior portion which is pigmented in Amphi-
oxus. Such must have been the primitive condition of the optic
vesicle however developed.

From what has been said, it follows that there is no posterior
hemisphere developed, because the process never goes far enough
to form a spherical bulb, and an optic stalk. .

No. 2.] VERTEBRATE CEPHALOGENES!S. 233

That these differences are due to primitiveness of the organs
in Amphioxus, and not to any inherent fundamental differ-
ences between the eyes of Amphioxus and the corresponding
parts in other vertebrates, will be apparent to any one who will
analyze and compare the earliest stages in the development of
the eye in the several vertebrate groups.

We cannot believe that the paired eyes of vertebrates, as
they are known from the Cyclostomes upward, sprang into
existence with complete optic vesicles, optic stalks and nerves.
Much less, that they were provided from the first with lens and
cornea; on the contrary, the facts of ontogeny, the existence of
such an idea as that of evolution in the domain of biological
science, compel us to assume that the process was a gradual
one and lasted through a long period of time, progressing only
by minute increments, beginning with such a rudiment as I have
shown to exist in Amphioxus. With the increase in complexity
of organization of the type and specialization of the functions of
the sense organs, came the added increments, which have re-
sulted in the very complex, and in many ways still unknown,
structure and relations of the paired and median eyes.

It is further unavoidable, that we conclude that the rudiments
of eyes were functional from the very first or incipient stages
through each modification up to the completed form, and that
the physiological function of the organs in question have under-
gone as extensive, as important, and in every way a parallel
growth, by slight increments acquired with the added morpho-
logical increments.

To those morphologists who object to the view I have thus
briefly stated and endeavored to support, that such simple diver-
ticula as are found in Amphioxus associated with pigment spots
are entirely inadequate to give rise to such complicated struct-
ures as the eyes, I wish to give answer here: that we have in
the case of the extremely complicated organ of the mammalian
ear, an organ which has originated in a very similar manner,
and whose every stage of development may be traced in existing
vertebrates and about which morphologists are agreed that it is
formed by simple involution of primitively superficial sense
organs. The development of the cochlea, and its contained
structures, is another instance of the production of an extremely
complicated organ from a simple diverticulum pushed out from a

234 HOWARD AYERS. [Vou. IV.

previously existing cavity, — every stage of whose phylogeny lies
before us in existing vertebrates.

The chief merits of the theory I have here attempted to
establish is, that no demand is made upon the organism to sup-
ply any new structures, but simply to develop organs and struct-
ures already existing, in connection with a function of which we
have evidence enough that all animals endeavor to acquire in
ever increasing perfection, —z.e. light-perception and the possi-
bilities which grow out of it.

F. The pigment of the eye-spot of Amphioxus 1s contained in
cells which normally le inside the bounds of the nerve mass, and
whenever found outside in microscopical preparations, tt 1s to be
considered as misplaced by chemical or mechanical means.

Sections through the Amphioxus brain, prepared without con-
traction, or tearing, from well-preserved material, never show, so
far as my experience goes, the free pigment granules lying in
the interspace between the anterior end of brain and its sheath.
This point is worthy of mention, since the presence of such
free pigment particles has given rise to erroneous ideas as to
the structure of the eye-spot, and the nature of the pigment
deposit.

G. The pigment bodies of the central nervous system of Am-
phioxus are connected with, and form a part of, segmental sensory
structures.

These structures are doubtless derived from the superficial
sense organs of that ancestral form in which the neural plate
had not yet been converted into a tube, or had not received a
protective covering.

As they exist in Amphioxus to-day, the regularity of their
disposition is somewhat interfered with by the rearrangement
of the cellular elements forming the bases of these structures,
which are now transformed into a part of the central nervous
apparatus. They consequently retain only indirect connection
with the periphery.

After the formation of the neural plate as an embryonic or-
gan, the cells retain their embryonic condition for a varying
length of time. The transformation of these cells into the
elongated and greatly enlarged spherical, functionally active

No. 2.] VERTEBRATE CEPHALOGENESIS. 235

cells, occurs concomitantly with the development of the meso-
dermic somites and the peripheral sense organs.

This resting stage into which the cells of the medullary plate
as a whole pass, as soon as the plate has been fully formed, illus-
trates the retardation of the development of the structure, and
consequently function, of the apparatus, and this fully serves to
explain why the superficial sensory organs and their pigment
bodies remain undeveloped until the nervous system is entirely
inclosed. For, although the medullary plate enters on a resting
phase, the remaining organs of the body continue their develop-
ment, especially the mesodermic structures, which by rapid
growth inclose the nervous plate long before the resting phase
has passed, and the differentiation of the permanent fibres and
ganglion cells of the nervous cord begins.

H. Each one of the pigment bodies ts connected with, forms a
deposit in, an ameboid cell. All these cells retain their ameboid
nature throughout life, the pigment cells of the eye-spot not
excluded.

The pigment-bearing cells are relatively large, being in the
majority of cases equal to the middle size ganglion cells. The
pigment makes its appearance in the cells of the growing larva
in the form of particles of melanin, which may or may not fuse
into a single mass. The particles are related in some unknown
way to the protoplasmic structure of the cell, and are controlled
by the cell, massed together in a more or less irregular lump in
the contracted condition, or spread out in the form of threads,
sheets, rows of particles, etc., in the expanded condition, of the
cell.

The pigment cells arise bilaterally near the centre of the
somites, and multiply in such a way that they take in more or
less of the segment longitudinally. In the contracted condition
the pigment frequently appears in the form of crescentic bodies,
hemispherical cups, either simple or with rays streaming out
from the periphery.

The nucleus of the cell is frequently visible, though the cell
wall is usually hidden by the pigment.

The nucleus is eccentric in position and in carmine stains,
it colors, with its cell, much like the ganglion cells with which
it is associated.

236 HOWARD AVERS. [VoL. IV.

According to Stieda, the general shape of the cells during
life is that of an irregular star.

It is not at all difficult to find, in the preserved Amphioxus,
cells with several pigment processes, and often the length of
the processes exceeds twice the diameter of the central mass.

I. The pigment of the axial nervous system of Amphioxus is
in process of migration towards the anterior end of the body —
towards the eye.

Where the eye is large, the interspace between it and the first
pigment group is long, and wece versa. With the increasing
opacity of the body walls, of the sheaths, and of the central
nervous system itself, correlated with an increase in the degree
of complexity and heightening of the function of the light-
perceiving organs, the pigment cells scattered throughout the
entire length of the neural axis migrate to the eye, and become
associated with the percipient layer.

The interspace between the eye-spot and the first group of
pigment bodies is dependent upon the size of the eye-spot —
z.e. upon the sufficiency of its function for the body.

Stieda describes the pigment bodies as lying mostly ina plane
passing horizontally through the ventral wall of the central
canal; I find however that the cells oftentimes make their way
dorsad along the walls of the canal, and may send processes
outward from the canal reaching quite to the periphery of the
cord.

J. Rhode’s conclusion that the giant ganglion cells of the ante-
vior portion of the spinal cord of Amphioxus send out axis cylin-
ders only caudad 7s erroneous, and as my preparations show,
Stieda’s observations are correct both in figure and text.

Notwithstanding Rhode’s positive assertion to the contrary,
I am fully convinced that the giant ganglion cells show connec-
tion by means of a plurality of axis-cylinders, with structures
lying both caudad and cephalad of them.

K. The vertebrate ear has developed within the phylum above
Amphioxus, and arose from one of the primary sense organs of the
lateral line system, at a period phylogenetically later than the
Sormation of the canal system of these sense organs. The ear

No. 2.] VERTEBRATE CEPHALOGENESYS. 237

organ has retained in its inclosed state the tendencies of growth
possessed by the surface organs, and in the vertebrates above the
Ichthyopsida offers the only remnant of the perfected primitive
canal system of sense organs.

The position of the ear capsule does not mark a divide betweei
two morphologically different portions of the brain, nor has this
capsule played any part in the formation of the brain contours.

No explanation has ever been offered of the origin of those
peculiar structures, the semicircular canals, of which Foster
says, “ But the peculiar features of the semicircular canals sug-
gest almost irresistibly that they are special agents” in the
equilibration of the body.

The solution here offered makes clear why these canals are
semicircular, and why they, as canals, have such sfeczal and
morphologically significant relations to the sense organs in the
ampulle, and it further helps us materially in forming a judg-
ment as to the function of these organs which have so long
proved a fruitful source of speculation, experiment, and differ-
ence of opinion.

Our knowledge of the development of the sense organs of
the lateral line, and of the canal system, so intimately associated
with them, we owe to several writers, but E. P. Allis has given
us the most detailed and accurate account of these organs as
they occur in Amia. Briefly stated, the steps of the develop-
mental process are as follows: 1. The layers of the ectoderm
thicken over certain areas and sink below the general level of
the surface of the body. These thickened bodies lie thus in
the bottom of relatively extensive surface depressions of the
general body surface. The ultimate sense organs are meanwhile
forming, and as they do so each sinks below the bottom of its
depression and lies in a pit, the lips of which soon grow upwards
and inwards towards each other. By their coalescence they
form an arch over the organ, which is soon converted into a
canal, by the rapid growth of the edges of the arch away from
the organ, and their fusion along the median line of the depres-
sion over which they arch as they grow.

Canals originating thus, continue to grow until they meet
some other canal opening, or until the exhaustion of the impulse.
In the former case the two terminal pores of the canal fuse into
one; in the latter the primitive canal possesses a strictly terminal

238 HOWARD AYERS. [Vo. IV.

opening. The sense organs, in the meantime, have become
differentiated out of the indifferent epithelium, and present the
appearance of a rounded elevation covered with hair or rod-
bearing sensory, columnar, epithelial cells.

The key to the solution of the whole problem lies in explain-
ing the development of the internal ear on the basis of the
development of the canal organs of the surface of the body.

When we examine the development of the canals and cochlea
from the primary auditory vesicle, in the light of Allis’ investi-
gations, we find that the whole process consists in the further
development within the head of one of the depressed areas and
its sense organs, that are found in numbers on the body (head
region) of all embryo vertebrates, and just as the primary sense
organs of the surface of the body may (and usually do) produce
several or many organs, so does the organ inclosed within the
vesicular involution of the ear continue to multiply, until it has
produced the constituent parts of the adult ear of a given verte-
brate form.

Some of the deductions from these premises are as fol-
lows : —

a. Each natural group of vertebrates at the present day has
a type of internal ear closely adhered to by all its members.

The so-called single semicircular canal of Myxine being, in
reality, a double structure possessing two ampullz and ampullar
sense organs, the homology of parts between Myxine and
Petromyzon is essentially complete.

6. The semicircular canals, among the Elsamobranchs, many
times show a condition of canal development, indicative of the
fusion of the primary pores at the two ends of the same canal,
with an incipient separation of the primary pore thus formed
from the rest of the ear.

c. In any study of the functions of the internal ear, we can-
not lose sight of primitive function of the sense organ giving
rise to sensory apparatus of the ear. For the functions have
not suffered greater alterations than the structures, and there is
no difficulty whatever in making out the structural relations of
the primary and finished organs.

d. If the function of equilibration, as supposed, belongs in large
part, or entirely, to the semicircular canals and their ampullar
organs on account of their spatial relations, we cannot overlook

No. 2.] VERTEBRATE CEPHALOGENESYSS. 239

the fact that, among the lower vertebrate forms, canal organs are
placed in much more favorable position (on the surface of the
body) for the reception of stimuli than are the deeply buried ear
canals ; besides which, the former lie further from the centre of
motion, and would consequently execute greater excursions than
the corresponding ear canals during any given motion of the
body.

These surface canals occupy positions in the three planes of
space just as the ear canals do.

e. Since from an extended consideration of the experimental
evidence, derived from investigations of the function of brain
and ear, with special reference to the function of equilibration,
we find that the semicircular canals, and their organs, are not
more directly connected with the centre of equilibration than
many other sense organs, and other parts of the body not sen-
sory in function, and since we know, from their history of devel-
opment, that they were purely protective structures originally,
and have only had the semicircular form and spatial relations
impressed upon them, as it seems, by the purely mechanical cir-
cumstances of involution, independently of any special function
more than they possessed on the surface of the body, we must
conclude that the semicircular canals have never, and do not
now, possess any peculiar or special relation to the function of
equilibration.

f. The system of canal organs is a very ancient, and, so far as
the existing vertebrates are concerned, a very primitive, system.
Amphioxus apparently has no trace of it, but all other verte-
brates show, by the possession of an internal ear with semicir-
cular canals, etc., that their ancestors must have possessed a
well-developed system of canal organs in the head region.

From this primitive condition the existing variety of forms
of canal organs and other sensory structures of this class have
been derived.

L. The higher sense organs of the Cyclostomata are all patred,
since the nose (i.e. the nasal or olfactive epithelium) exists in the
embryo as well as the adult in the form of two circumscribed areas
lying on either side of the median line, each of which receives the
entire nerve supply afforded by the olfactory nerve of its side.

It is evidently not consistent with our morphological ideas, to

240 HOWARD AYERS. [VoL. IV.

consider an organ unpaired and median when its essential
structures are distinctly paired and bilaterally symmetrical, —
even though some accessory portion has been so modified as to
have lost all trace of its double origin. So far as I have dis-
covered, the general acceptation has been that the Cyclostomes
possessed only a single nasal organ, a single median olfactive
area to which both of the olfactory nerves gave up their fibres.
It is true, this supposed relation of two olfactory nerves to a
single olfactive area has caused comment, and the explanation has
been offered that we had to do with a modified condition, —a
degradation of high sense organs. But as my sections show,
the proximal portion of the nasal pit is divided by a median, non-
olfactive raphe, into two lateral pockets, or right and left nasal
pits, to which alone the olfactory nerve of the right and left
sides, respectively, are distributed. The median territory is sup-
plied by branches from another nerve, which one I have not
been able to determine specifically.

I wish to call attention to the fact that, among the lower
vertebrates there is a connection of the two olfactory pits across
the median line by way of the mouth, here especially distinct, but
among the higher forms it is also evident. This connection is
usually brought about by means of grooves, placing the olfactive
pit in communication with the mouth. Even in the mammalia
the formation of a median partition to the extent of the forma-
tion of two zasal canals is a secondary process. The primitive
or embryonic condition shows two simple pits, separated by a
more or less distinct partition, which is usually a broad one.
Of course in this stage the pits are not deep, and by the time
their boundary walls become extensive the median portion is
also well developed. In Petromyzon the partition remains
rudimentary.

I have, from my studies on Ammocortes and Petromyzon,
ventured to make my statement a general one for the Cyclosto-
tomata ; and I feel sure there is no reason to doubt that Myxine
and Bdellostoma will be found to have their nasal apparatus,
bilaterally symmetrical, with as great distinctness as Petromyzon
shows this very important condition. The embryonic rudiment
of the nasal pits in the earliest stages of epithelial differentia-
tion, needs further study, as none of my stages are young
cnough to prove that the olfactive areas arise entirely independ-

No. 2.] VERTEBRATE CEPHALOGENES/S. 241

ently of each other, though I have no hesitation in venturing
the prediction that this will be found to be the case.

M. Zhe parietal-pineal eye of the Cyclostomata and other
vertebrates has been developed from a median portion of the pig-
mented eye of Amphioxus. The rudiments of this eye were de-
rived from (segmental) sense organs, but the eye ttself is never
developed from two right and left halves, in so far as the closure
of the medullary folds would necessitate thts.

No absolute demonstration of this view is at present possi-
ble, but I wish to offer the following considerations in sup-
port of it.

1. The parietal eye of vertebrates is formed as a hollow out-
growth of the anterior portion of the roof of the primitive
thalamoccele, and its cells contain pigment.

2. The pigment contained within the cells of the bulbar, or
functional portion of the pineal eye, is derived phylogenetically
from the median portion of the pigment body of the anterior
end of the primitive thalamoccele of an Amphioxus-like form.

3. The genetic connection of these three organs, for light-
perception and sight, viz., the two lateral eyes and the median
unpaired eye, is furthermore rendered probable by the central
connection of all three organs, which is found to be in the optic
thalami.

4. The same essential elements are present in eacn, — pigment
cells and percipient rods and ‘cones.

5. All three organs were formed to supply the demand for
the restoration of the more perfect conditions for light-percep-
tion, destroyed by the folding in of the medullary canal.

6. The two kinds of eyes were primarily alike in structure
and lensless; both formed lenses, the paired eyes first, —and this
condition has been retained by all descendants, the pineal eye
only in certain forms, — ancestors of the groups still showing
traces of the lens body, and their descendants. The double
nature of this organ, recently described in Leydig and Selenka,
does not affect the above views, for, so far as we yet know (and
the matter needs further investigation), both of the tubular out-
growths, although intimately related, are materially different in
one respect, viz., only ove, the epiphysis, contains pigment in its
distal portion.

242 HOWARD AYERS. [VOELVE

N. Zhe neural axis of all vertebrates ts co-extenstve with that
of the chorda, or vice versa, since the neural axis ts phylogenetically
as well as ontogenetically the older structure.

In this I can confirm Keibel’s observation on mammals, by
my own on Sharks. The same is true of the early larval Cyclos-
tomata and Amphioxus. My own results, the outcome of a
study of Acanthias vulgaris and Galeus canis, at the Maine Bio-
logical Laboratory, Wood’s Holl, Mass., were obtained several
months before the publication of Dr. Keibel’s paper. This rela-
tion of chorda and neural axis exists, not only in all the higher
vertebrates, but in Amphioxus as well, during the stage in which
the organs in question arrive at their complete separation from
the surrounding tissues; but while in higher vertebrates the
notochord suffers a shortening of its anterior end, and not infre-
quently of its posterior end also, in Amphioxus the anterior
end secondarily grows out into a process for the support of the
pointed anterior end of the head.

A cephalic flexure occurs in both, and is more or less com-
pletely obliterated in both in the later stages, though from dif-
ferent causes.

In no craniate vertebrate does the notochord extend the
entire length of the embryo when first formed, but ends in
an undifferentiated mass of cells placed below and in front
of the neural axis, and later grows out in front of it. This out-
growth is soon overcome by the much greater and more rapid
growth of the brain. The conditions in Amphioxus are simpler
in that the notochord is, from the first, distinct quite to the end
of the neural axis, the anterior termination of the body being
relatively much larger than in embryo of the higher vertebrata
of corresponding stages.

O. The pituitary prominence of the skulls of vertebrates does
not mark a fixed point, as the relation of the anterior end of the
chorda and of the hypophystal organs clearly proves.

I reserve a detailed consideration of this proposition for my
illustrated publication.

P. In the discussion of the segmentation of the head it has
become necessary to deny any segmental value whatsoever to any
portion of the chondro, — or ossicranium. They have no greater
segmental value than the intestine. And all apparent segmental

No. 2.] VERTEBRATE CEPHALOGENESIS. 243

characters have been impressed upon them by other organs of seg-
mental nature. Not only does ontology force this concluston, but
the historical development of our anatomical knowledge alike com-
pels tts admission.

It is taken for granted that the head region has been formed
by a gradual process of modification of the anterior end of some
ancestral type, whose body, with the exception of the first and
last segment, was composed of nearly, if not quite, homodyna-
mous segments. The problems are to determine what was the
value of these primitive segments, and how they have been so
modified as to produce the head. Obviously the only way open
to the morphologist is to determine what existing vertebrates
show the steps of this process, and the extent to which the
primitive segmentation persists in the highly differentiated ver-
tebrates of this age.

Few if any topics of vertebrate anatomy have received the
attention of so many morphologists, or have been discussed
with such interest, as the nature and development of the head.

It has long been recognized that within the vertebrate phylum
the cephalon undergoes a most wonderful degree of differen-
tiation, unequalled by any other portion of the body. As the
lowest step in this series we have Amphioxus with the head less
developed, both morphologically and physiologically, than many
annelids and arthropods, — with this reservation of course, that
in the Lancelet, as we say, the type is higher. From Amphioxus
to the next stage, as represented by the Cyclostomes, the transi-
tional forms are unknown, but the larval development of Petromy-
zon gives some interesting hints as to the manner in which
these transitional stages have been passed through. From the
Cyclostomes up to man, the series is practically unbroken, and
allows us to trace, with a great degree of accuracy, the course
of development, and to formulate some ideas as to the causes
which have been at work effecting the progress.

Morphological conclusions, in the realm of neurology, have
undergone great modification within the last decade, and as
our knowledge increases we have found it necessary to widen
our views as to what is normal and essential for a neural seg-
ment. From a general survey of the field, it is clear that a
classification of the cephalic structures of modern vertebrates
must be made, separating those which are genetically connected

244 HOWARD AYERS. [Vot. IV.

with the primitive organs, persisting with no change of function
from those which have appeared much later and in a modified
condition of the body. The brain is of the former class, the
cranium of the latter. As an illustration, we may take the
cases found in nature of a completely segmented vertebral
column and its separation from the skull by an articulation,
and the opposite condition of a continuous cartilaginous skele-
ton, including vertebral column and cranium. The explanation
current is, that in the latter case we have to do with secondary
fusion, and that we have here a modified condition derived from
the typically segmented condition by a lack of development
of the articulation. I consider it the true view, that a continu-
ous cartilaginous skeleton has been, and is in some living cases,
the primitive and normal condition, though of course I do not
deny the production of more or less continuous portions of the
skeleton by a process of secondary fusion.

The segmentation of the contained or enveloping organs does
not predicate the segmentation of the containing or enveloping
structure, any more than the formation of an ear capsule of a
mammal, out of separate bones, predicates the segmentation of
the sense organ contained.

Q. The head cavities, or spaces included within the mesoblastic
somites occurring in the head region, possess relatively the great-
est importance in an acraniate stage before a skull or anything
comparable with a primordial cranium has made us appearance.
This is true from the ontogenetic as well as phylogenetic stand-
point.

R. The hypophysis is a structure which arose tn the vertebrate
phylum long after the chorda was established, as Amphioxus
proves, and was connected in an important way with the infundib-
ulum. It arose as an organ of taste, and the infundibulum was
its nerve.

S. The optic chiasm (the trochlear chiasm as well) has arisen
within the vertebrate group above the Amphioxus condition and in
the following manner : —

The fibres supplying the pigment spots (or muscle) arose from
ganglion cells of the multipolar kind, and as we know in Amphi-
oxus and Cyclostomes, these fibres cross the middle line not

No. 2. ] VERTEBRATE CEPHALOGENESYIS. 245

unfrequently, or the cells lie in the axial line of the nerve cord
or brain. By a gradual process of development, it came about
that the majority of fibres for the right side of the body found
their central ends in the left side of the brain, for some as yet
unknown cause, although this transposition was probably inti-
mately connected with the development of the system of asso-
ciation fibres.

T. The explanation of the increased number of gill slits of
Amphioxus over those of other vertebrates (which certainly show
traces of considerable reduction in number) is to be found in the
habits of the Amphioxus, which ts not a free swimming animal,
and cannot be a predatory one. It depends, for tis food, upon the
size and power of its branchial apparatus to create currents and
keep moving a sufficient volume of water to supply tt with the
requisite amount of food, which 1s contained tn only limited quan-
tities therein.

As the animal grows larger its needs are greater; the bran-
chial chamber and apparatus must increase to allow a larger
volume of water to pass.

U. The branchial apparatus of Amphioxus is then not merely a
respiratory apparatus, but more an apparatus for the collection of
food and for the transfer of such collected store to the pharyngeal
opening for deglutition. A much smaller organ than the bran-
chial basket of the adult animal would suffice for the adequate
respiration.

We have only to call to mind the peculiarly inactive life that
the animal leads, to become satisfied that its so-called respiratory
apparatus is much more important as a food collector and
strainer than as a respiration organ.

This modification of the branchial apparatus, for food collec-
tion, is paralleled in higher vertebrate by the production of
mandibulo-maxillary region for the same purpose.

THE LAKE LABORATORY, MILWAUKEE, WIS.
April 21, 1890.

\ ; se} 40 itt ; 4 hie Pee bi yes é (lit chi Be

{ ott

Volume IV. Fanuary, 1891. Number 3.

JOURNAL

OF

MORPHOLR@GY:

STUDIES ON CEPHALOPODS.

il

CLEAVAGE OF THE OVUM.

S. WATASE,

CiLarK UNIVERSITY.

CONTENTS.

PAGE
Ts (Untroductiomicytetre sorters eetokarets ever eusichelcotaiesereta store creer lelevevelel otal ala tenebaerstevele 247
Id: (Remarks sonithe "Amma @ varmgigey aster eyeeuejelie er exel sional let chey helen otais aleueia)<isiskenetaire 250

III. Relation of the External Phenomena of Cleavage of the Ovum to the Inter-
Mali Phenomena ox) Cary OKIMesis aelaerchelere ater) telelicteleyeieledalate ster atelele retest 256
IV. Cleavage of the Ovum of the Cephalopod, Zo/igo Pealei, Lesueur......... 278
V. Remarks on the Variation of Cleavage in the Cephalopod........... 289-294.

bt

In the following pages, I will first attempt to treat the general
morphology of the animal ovum from the standpoint of some
embryological and morphological facts and theories. In the
next place, the relation of the external phenomena of cleavage,
as shown by the behavior of the cytoplasm, to the internal
phenomena of nuclear cleavage or caryokinesis will be dis-
cussed. In this connection, some theories on caryokinesis will
be examined, my interpretation of the cleavage phenomena
being that they are essentially the azalysis of the potential
tissues contained in the cleavage nucleus, and that caryokinesis

247

248 WATASE. [Vor TVs

is the method of such analysis, and the achromatic spindle the
instrument used in the analysis. The cleavage of the squid
ovum will then be described, and finally variations in the cleav-
age of the same animal will be discussed.

The observations were made during the summers of 1888 and
1889, while I was a student at the Johns Hopkins University,
at the Woods Holl Laboratory of the U. S. Fish Commisson,
through the kindness of Professor M. MacDonald.

The pressure of other studies at the time and the difficulty of
obtaining a constant supply of the most desirable stages have
left my observations incomplete in many respects. Although the
squid eggs are abundant during the summer months at Woods
Holl, it is difficult to get the early stages, since the bunches
of egg-capsules caught in a dredge or in a trawl are usually too
far advanced for the study of cleavage. The following observa-
tions were made on material obtained from two female squids.
On the night of September 3, 1888, one squid, Loligo Pealei,
kept in an aquarium, laid four capsules of eggs. The eggs were
examined immediately, and the cleavage stages were followed.
All the figures on Pls. IX and XII, except Fig. 29, were drawn
from specimens obtained from this source. Subsequently I
killed the same squid and fertilized the remaining ova artifi-
cially. A few better drawings of the surface views of the divid-
ing eggs were thus obtained.

One day in August, 1889, another specimen of the female
squid with ripe eggs was kindly given to me by Professor F.
M. McFarland of the Marine Biological Laboratory. The eggs
were taken out and artificially fertilized with spermatozoa which
were found in the spermatophores in the inside of the external
buccal membrane of the same animal. All the figures in Pls. X
and XI, and Fig. 29 in Pl. XII, were drawn from this material.
Judging from my experience, I believe that the study of the
early stages of cleavage is best accomplished with artificially
fertilized ova. Such ova have many advantages over those laid
naturally. In the first place, the eggs can be studied free from
the jelly covering, which interferes in no small measure with the
process of successful manipulation. In the second place, the
ova may be obtained at any stage of their cleavage at the time
when it is most convenient to work.

If one succeed in catching a gravid female, it is an easy mat-

No. 3-] STUDIES ON CEPHALOPODS. 249

ter to get hundreds of ripe ova from the ege-reservoir. The eggs,
when ripe, shine through the thin wall of the body underneath the
mantle, and easily roll out through an incision in the wall, as the
shot would do through a similar opening in the sac which con-
tained them. A search for the spermatophores may then be made
in the adult male, which, in the reproductive season, carries Sev-
eral bunches in the inside of the mantle chamber. If the search
in the mantle chamber fail, we can usually get quantities of them
in the reservoir, through the thin walls of which can be seen, regu-
larly arranged, white, elongated, spindle-shaped spermatophores.
Search for a male is often rendered unnecessary, for a number of
spermatophores are usually carried by the females on the inside
of the arms, and may usually be found on the little horseshoe-
shaped prominence on the inner surface of the outer buccal mem-
brane of the same animal from which the eggs were taken. This
is a matter of great convenience to the investigator, for a single
female with well-matured eggs will usually have spermatozoa
enough to fertilize all the eggs. Taking hold of the bunch of
spermatophores with the forceps and shaking them into a dish
with a little water in it, the spermatozoa can be liberated easily.
The process can be facilitated by cutting the spermatophores
with scissors into a few fragments. By pouring the water, frag-
ments of the spermatophores and all, over the eggs, and mixing
them thoroughly, the artificial part of the fertilization may be
said to have been accomplished. If one take an egg and a few
drops of the water after this process and examine them under
the microscope, one will see a multitude of spermatozoa covering
the surface of the ovum, and with little perseverance one can
follow the penetration of the sperm-cell into the ovum through
the micropyle. Between one and two hours after the mixture
of the sperm and the ova, the first furrow of the cleavage was
observed, and thence at intervals of five to ten minutes, the
succeeding furrows of cleavage were introduced, the interval
between the two successive furrows becoming shorter and
shorter as the stage advances.

After the majority of ova have commenced to divide, it is
necessary to change the water frequently, particularly when a
large number of eggs are kept in the same vessel.

In order to separate the blastoderm, the egg at any given
stage of division was killed by a mixture of sea-water, acetic

250 WATASE. [VoL. IV.

acid, and osmic acid, in the proportion recommended by the
Hertwigs for their well-known macerating fluid. The propor-
tion of the osmic acid may be reduced, or may be altogether
excluded from the reagent, as it affects the eyes of the observer
while he is closely watching the effect of the reagent upon the
egg and teasing away the blastoderm with needles. I found it
most satisfactory to remove the ovum from the action of the
reagent as soon as the translucent protoplasmic germ-disc turns
whitish opaque, which takes place very quickly, and complete
the operation of separating the blastoderm in dilute glycerine.

The blastoderm thus separated may afterwards be stained with
dilute Schneider’s acetic carmine, or with Erlich’s hematoxylin,
and then mounted in glycerine; or may be left unstained, as
most things can be satisfactorily made out without any staining,
and by avoiding this unnecessary process of technique in this
case, the risk of dislocating or injuring the delicate blastoderm
in other ways is thus greatly lessened — the risk all the more to
be avoided as the blastoderm is not firmly fixed to the slide,
and a little movement of the cover-glass is often enough to
destroy the whole specimen.

Treatment of living eggs with Perenyi’s fluid for a few seconds
is excellent for the surface study of cleavage. It turns the
protoplasmic portion of the ovum opaque yellow, and brings
out the cleavage furrows distinctly, while the rest of the egg
remains translucent as before.

iT:

It was a disputed question at the time when the cell-theory
was first promulgated, how much of the animal ovum is to be
homologized with Schwann’s scheme of an organized cell —
whether the germinal vesicle alone is to be taken as such, or
the whole substance of the ovum, including both the cytoplasm
and the germinal vesicle.

This failure to recognize the true cellular nature of the ovum
and of the cleavage segments, coupled with the false doctrine of
the Cytoblastema of Schwann, stood in the way of a true under-
standing of the cleavage process, and obscured the significance
of the cell-theory itself.

Our understanding of the cell-theory was made possible (1) dy

No. 3.] STUDIES ON CEPHALOPODS. 251

the accurate determination of the ovum as a single cell; (2) and
by the identification of cleavage with cell-division.

In regard to the first question, although Schwann, after con-
siderable hesitation, inclined to the conclusion that the ovum is
morphologically a cell, and that the germinal vesicle is its
nucleus, still he felt compelled by the evidences available in his
time to admit that “it was impossible to decide the question
whether the germinal vesicle be cell or nucleus.”

In regard to the second question, he was totally unaware of
its true significance, as his theory of the Cytoblastema very well
shows. Within a few years of the publication of Schwann’s
great work, however, it was definitely determined that the
cleavage of the ovum is a process of cell-multiplication ; and
that in the whole domain of developmental phenomena of animal
tissues no cell-formation ever takes place independently of those
cells already existing in the organism.

Following the identification of the germinal vesicle of the
ovum as the true homologue of a typical cell-nucleus, and
the cleavage of the ovum asa process of cell-formation, came the
problem of the promorphological character of the ovum. This
is an entirely distinct problem from that of the germ-layers, and
is essentially the outcome of a more careful investigation into
the early phenomena of egg-development. The existence of the
germ-layer theory did not depend on morphological views of the
ovum. The germ-layers were recognized and studied before
naturalists knew anything about cleavage and the morphology
of the ovum. The cytological method deals with facts before
the cells arrange themselves into definite layers. It deals with
a certain cell or a certain group of cells in an early stage of
cleavage, from which different embryonic organs and germ-
layers themselves are derived. For example, it traces the
mesoderm to a pair of cells, or the future ectoderm and endo-
derm to the two-cell stage of development. It seeks to explain
the differentiation of the axes of the adult or larval organism by
tracing them to certain recognizable axes in the extremely early
stages of development, or even in the unsegmented ovum itself.

It seeks to find out the significance of the different planes of
cleavage in their relation to the origin of tissues in the larva or
the adult. Thus, instead of starting with the formation of the
germ-layers in the study of organogeny, it goes a step further

252 WATASE. [VoL. IV.

and attempts to derive those germ-layers themselves from a
simpler cleavage segment or segments; and instead of compar-
ing well-arranged layers in the establishment of homologies of
structures in different organisms, it seeks to reduce them toa
still simpler aggregation of cells. The teloblasts of Whitman
and Wilson, in which an early differentiation of the tissue-germs
is recognized, furnish admirable examples of this class of phe-
nomena. May not the well-known fact of the early separation
of sexual cells in the body of a metazoan embryo belong to the
same category of facts?

While the teloblasts represent only a part of the entire organ-
ism, as, for example, the nervous system or the excretory organ,
the sexual cells contain the “ Anlage”’ of the complete organism.
The difference appears to me to be more one of degree than of
kind. While the one completes its development during the
ontogeny of the organism, the other completes its development
at the next generation.

Although this tendency to recognize early differentiation of
parts in an early stage of the dividing ovum is essentially the
outcome of more recent studies, it was foreshadowed in the
works of several earlier investigators. Von Baer, as far back as
1834, recognized the animal and vegetable poles in the frog’s
ovum. Newport, as far back as 1854, identified by an ingen-
ious device the first cleavage plane of the ovum with the
median axis of the embryo in the frog, and pointed out the
origin of the head and tail end of the embryo from definite
parts of the ovum. Pfliiger and Roux have more recently
shown the coincidence of the median axis of the embryo with
the plane of the first cleavage furrow in the ovum of the frog.
The striking series of results brought out by the latter in
experimental embryology are the most important contributions
in this connection. Mark has discussed the “ primitive axis”
of the ovum in Zzmax ; Goette has fully treated the relation of
the promorphological axes of the ovum in the annelid, and their
relation to the cleavage and early differentiation of tissue-cells ;
Hatschek has pointed out the early exhibition of the bilaterality
in the ova of Zeredo and Pedicellina ; Brooks, in the ova of the
American oyster. E. van Beneden, in various papers on Ascaris,
Tunicates, etc., has called particular attention to the importance
of the study of the promorphological relation of the ovum.

No. 3.] STUDIES ON CEPHALOPODS. 253

Agassiz and Whitman have most fully discussed the impor-
tance of this question, while Hallez was led to the formulation
of an important law in regard to the orientation of insect ova.
Blochmann and Wheeler have described the early exhibition of
the bilateral arrangement in insect ova, extending even to the
distribution of yolk-granules.

Several others may be mentioned whose work bears more or
less on this important problem. Some of these we will examine
more in detail later. It may be pointed out, however, at present
that in the study of segmentation of the ovum, a mere quanti-
tative enumeration of blastomeres at the successive stages of
egg-cleavage alone, has ceased to be a matter of any great im-
portance.

Van Beneden and Julin! have demonstrated in the egg of
Clavellina the existence of a bilateral axis before it begins to
segment. The anterior and posterior extremities, the left and
right sides, the dorsal and ventral aspects of the larva can be
definitely pointed out in the unsegmented ovum. The first
furrow of cleavage coincides with the median plane of the
bilateral larva, the left half of the larval organization being
derived from the first primary blastomere which lies on the left
side of the first furrow of cleavage, and the right half of the
body, from the right cleavage sphere. Properly speaking, the
body of the larval Clavellina consists of two identical halves
which meet in the median line. All structures in the body are
paired, or have double origin corresponding to the bilateral
symmetry of the ovum. The median structures, such as the
notochord, digestive tube, are derived from two sources, — the
right and left halves of the body, — although later, one side of
the body may be invaded by the cells from the other side.

Chabry,? who worked on Asczdia aspersa, has confirmed the
results of the above-mentioned authors, and has established
with certainty the primitively double character of such median
structures as the eye, otolith, notochord, the atrium, and the
organ of fixation. He thinks that the eye in the adult is de-
rived from the cells situated in the anterior end of the right
half of the body of the larva.

1 Ed. van Beneden et C. Julin: Za segmentation chez les Ascidiens et ses rapports
avec Vorganisation dela larve. Archives de Biologie, Tome V, 1884.
* La segmentation chez les Ascidies simples. Jour. Anat. et Physiol., T. 20, 1885.

254 WATASE. [Vot. IV.

Van Beneden!in his recent work with Neyt, on Ascarts, is
inclined to carry his former conclusion still further, and believes
that the ovum as well as the other cells of the body probably
have the inherent bilateral structure, and traces the _ bilat-
eral symmetry of all Metazoa, either in the larval stages or
retained through their life histories, to the bilateral structure of
the unsegmented ova themselves. “Les premiers blastomeres
ont, comme |’ceuf fécondé, non seulement une symeétrie mono-
axone, mais une structure bilatérale. J] est probable que c’est
la un caractére comme a toute cellule et l’on doit concevoir
un organisme cellulaire, non comme formé de couches con-
centriques, mais comme présentant essentiellement un axe a
extrémités différentes et un plan unique de symétrie. Cette
symeétrie bilatéral de la cellule est probablement la cause de la
symétrie bilaterale des organismes plus complexes, des animaux
en particulier. Il est bien prouvé maintenant que la symeétrie
bilaterale est primordiale chez les Radiaires, les Echinodermes,
et les Zoophytes, comme chez les Mollusques, les Vers, les
Arthropodes et les Chordés: la symétrie radiée n’apparait que
secondairment chez les Echinodermes et les Coelentérés. Nous
pensons que la méme démonstration sera faite une jour pour les
protozoaires et pour les végétaux.”

Hallez,? who paid especial attention to the promorphology of
the ova of insects, finds that there is a definite law of orienta-
tion in the ova, a law which he restricts to insects at present.
The law runs as follows: “ La cellule-cuf possede la méme orten-
tation que l’organisme maternel quit l’a produtte: elle a un pole
cephalique et un pole caudal, wn coté arotte et un coté gauche, une
Jace dorsal et une face ventral; et ces différentes faces de la cellule-
awuf coincident aux faces correspondantes de l’embryon.” Hallez
observes elsewhere ® that, “ L’ceuf, pendant une période de son
histoire, faite partie de l’organisme maternelle a titre de simple
élément histologique. Or, les expériences de sections et de
régénérations, faites sur les Planaires et autres animaux, mont-
rent que chaque trongon, si petit qu'il soit, conserve la méme
orientation, c’est-a-dire les deux polarités céphalique et caudal,

1 Nouvelles recherches sur la fecondation et la division mitosigue chez l’ Ascaris
mégalocéphale, 1887.

2 Hallez: Comptes Rendus, 103, 1886.

8 Hallez: Comptes Rendus, 101, 188s.

No. 3.] STUDIES ON CEPHALOPODS. 266

qu'il avait dans l’animal entier. C’est quelque chose de com-
parable a l’expérience de l’aimant brisé. Ne peut-on en conclure
que chaque élément histologique posséde, ‘ui aussi, ces deux
polarités de l’animal, polarités qui persistraient dans la cellule-
ceuf, aprés qu’elle a cessé de faire partie des tissus maternels?”’

Mark? has recently called attention to the fact that in the
ovarian ovum of Lepidosteus, the polar differentiation of the
ovum can already be pointed out by the existence of a peculiar
secretory activity of the egg-protoplasm at one point, which cor-
responds to the future micropyle region. According to Mark,
the egg-membranes of the ovum — the villous layer and the zona
radiata — are produced by the secretion from the surface of the
ovum, and the change in the egg-membranes corresponding to
the region of the future micropyle is due to the diminished se-
cretory activity of that particular part of the ovum.

Mention must be made in this connection of the important
fact established by Rabl,? Carnoy,? and Gehuchten,* that the
resting nuclei in the tissue-cells of certain animals, such as the
epithelial cells of the Salamander, the testis cells of a certain
Arachnid and of Crustacea, the intestinal cells of a larval
Dipteron, there are either two poles or an axis, around which
chromosomes arrange themselves in a definite way.

The preceding, taken somewhat at random, will show one
essential fact ; viz. that, however diverse the examples, they all
point to one and the same conclusion, namely, that the metazoan
ovum and its derivatives, the tissue-cells, are more than a homo-
geneous, isotropic mass of protoplasm, devoid of definite sym-
metry. The study of caryokinetic figure shows, van Beneden
points out, that the cell is not only uniaxial, but also bilateral.
In several forms of ova carefully studied, the axes of the caryo-
kinetic figure correspond in a definite way with the recognizable
axes of a given ovum, the external shape of which is chiefly
determined by the quantity and distribution of the food-yolk.
The axes thus determined are maintained through the differ-

1E.L. Mark: Studies on Lepidosteus. Bull. Mus. Comp. Zool. Harvard College.
Vol. XIX, No. 1, 1890.

2 Rabl: Ueber Zelltheilung, Morph. Jahrb., Bd. X, 1885; Ueber Zellthetlung,
Anat. Anzeiger, IV, Jahrg. No. 1, 1889.

3 Carnoy: La Cytodiéredse chez la Arthropodes. La Cellule, Tome I, 1885.

4 Gehuchten: L’Axe organique du noyau. La Cellule, Tome V, 1889.

256 WATASE. [Vor. IV.

ent stages of growth and give rise to definite axes of the iarval
or of the adult organism. If these facts be more firmly estab-
lished by the further investigation of the subject, we may say
with van Beneden:! “L’ancienne théorie de 1’évolution ne
serait pas aussi dénuée de tout fondement qu’on le croit
aujourd’hui.”’

1

It has been pointed out that the cytological study of the
animal ovum is different from the study of germ-layers. It
has also been pointed out that the morphology of a bilateral
organism, at least in those well-established cases, has its begin-
ning in the morphology of its bilateral ovum.

The next important point to be borne in mind, it appears to
me, in the study of the cleavage of the ovum, is the phenomena
of tissue-differentiation, which become more and more manifest
with the growth of the ovum and the progress of its cleavage.
Now what is meant by the differentiation of tissues? What is
the relation of the tissues thus differentiated to the original
ovum from which they sprung? How does one tissue-cell
differ from another, which belongs to another tissue ?

Strictly speaking, the differentiation of tissues in the devel-
oping organism means the formation of two or more different
kinds of protoplasm out of one. For the sake of clearness we
may begin our inquiry by asking, How does one egg which is a
single nucleated cell, and which gives rise to one animal, differ
from another which gives rise to an entirely different organism?
Both are simple nucleated cells, and as such they are morpho-
logically alike, but one egg will develop into one organism and
the other into another after a repeated division and subdivision,
exactly under the same circumstances, and often in the same
space of time. The difference in result cannot, therefore, be
attributed to difference of conditions under which they develop,
but to something inherent in the ova themselves. In other
words, the egg-cell of a jelly-fish must have had from the begin-
ning the potentiality of becoming a jelly-fish and nothing else ;
and similarly, the starfish ovum must have been a potential star-
fish from the beginning. To imagine, therefore, that all proto-

1 Van Beneden: Recherches sur la maturation de Peuf, la fecondation et la divi-
ston cellulaire. Archives de Biologie, Tome IV, 1883.

Nodi.3)] STUDIES ON CEPHALOPODS. 257

plasm is identical, because no difference is recognizable by any
means at our disposal, would be an error. Deep within the two
particles of protoplasm which give rise to two different organ-
isms, there must be a corresponding difference which lies at the
bottom of all differences. In short, the eggs of two different
animals must be supposed to differ in their elementary consti-
tution, as much as their adult organisms differ in anatomical
structure. ‘‘ From general scientific principles,” says Professor
Sachs,! “we must assume ¢hat for each visible external difference
of organ, there ts a corresponding difference tn tts material sub-
stance, exactly as we regard the form of a crystal as an expres-
sion of the material properties of the crystallizing substance.”
And again, says the distinguished German botanist, “ Even the
different shapes of the two sexual cells —of an antherozoid or a
pollen grain compared with the odsphere — indicate plainly that
both are constituted differently as to material, since the external
form as well as the internal structure of any body is the neces-
sary expression of its material constitution. Difference of form
always indicates difference of material substance.”’ This doc-
trine of Form and Matter, or of Mechanism and Function as
expressed in the language of physiology, is the basis of our bio-
logical inquiries. As is clearly expressed in the words of Pro-
fessor Burdon Sanderson,? we must assume “ that every apprect-
able difference of structure corresponds to a difference of function ;
and conversely, each endowment of a living organ must be
explained, if explained at all, as springing from its structure” ;
or, in short, we must hold to the principle “ that Aiving material
acts by virtue of its structure, provided we allow the term struc-
ture to be used in a sense which carries tt beyond the limits of
anatomical investigation, t.e. beyond the knowledge which can
be attained either by the scalpel or the microscope.” Given
protoplasm of definite structure, and we have its definite func-
tion or property. Or conversely, we observe a certain property
in a given mass of protoplasm, and we regard it as springing
from a definite structure. When structure varies, the function
must vary also; and when we observe certain peculiar proper-
ties we must ascribe them to peculiarities in structure.
1 Sachs: Lectures on the Physiology of Plants. 1887.

2 Burdon Sanderson: Presidential Address to the Section of Biology, British Asso-
ctation for the Advancement of Science, 1889. Nature, Vol. 40, No. 22.

258 WATASE. [VoL. IV.

One rational answer to our inquiry is possible; viz. the proto-
plasmic structure of the egg which gives rise to one organism,
must differ from that of the egg which gives rise to another dif-
ferent organism, the differences between the two being relatively
as great as those which the two adult organisms display in their
anatomical relationships.

If the similarities of two organisms must be attributed to the
corresponding similarities of the protoplasmic structure of the
fertilized ova from which they respectively arise, the source of
similarities in the latter must be sought for in the community
of their hereditary antecedents. Hence, one way to place the
doctrine of phyletic kinship of two or more organisms upon a
scientific basis, would be, if such a thing were possible, to demon-
strate the molecular or structural affinity of their germ-cells.
The embryological phenomena of a developing organism may
be expressed in the terms of protoplasmic metamorphosis. Two
organisms at the same stage of development would represent
the same stage of protoplasmic structure. The budding of a
new cell or the formation of a new organ would correspond to
the birth of a new phase in the course of the metamorphosis of
the original protoplasm of the egg.

To turn to our main point, namely, the development of the
organism as first indicated by the cleavage of its protoplasmic
material.

What is the cleavage of the ovum? What is accomplished
by it? Is it the mere sundering of material which has no more
reference to the future organization of the embryo than the
snowflakes bear to the size and shape of a future avalanche? Or
is it a “ histogenetic sundering” of material in which every step
in the process has a definite relation to the building up of the
future embryo? That each step of cleavage has some definite
significance in relation to the organization of the adult or the
larva, at least in certain forms which have been most carefully
studied, there can be no question. Thus, in a certain Tunicate
already referred to, it has been observed that the nuclear sub-
stance of the ovum is divided, during the first cleavage, in such
a manner that one of the new nuclei, by its division, gives rise
to the right, and the other to the left side of the adult organism.
In another case, as in some worms, it has been maintained, that
the first division of the nucleus distributes the nuclear substance

NOs STUDIES ON CEPHALOPODS. 259

into future ectoderm and entoderm. And again, the formation
of a certain organ, or a system of tissues, has been traced in a
most definite manner to a particular cell or group of cells in an
early stage of cleavage, as is shown by the well-known researches
of Whitman on Clepsine, or more recently in the admirable work
of Wilson on Lumbricus and Nereis. The more carefully the
phenomena are studied, the more astonishing is the regularity
and precision with which the cleavage process is conducted and
the differentiation of tissues is accomplished.

The occurrence of variations or irregularities in the mode of
cleavage in a certain animal — irregularities as judged by the
arrangement of superficial cytoplasmic furrows — does not in-
validate the importance of the conclusion which can be derived
from the study of forms where a great regularity prevails. For
the essential feature of the cleavage process is the division and
distribution of the nuclear substance of the ovum, and in so far
as the nuclear substance is distributed in such a manner as to
produce a symmetry of growth in the developing organism, it is
immaterial whether its total quantity be divided exactly in two
equal halves and distributed into right and left at the first cleay-
age, or whether it be divided into dissimilar portions and the
equilibrium of growth be gradually secured during the subse-
quent stages of cleavage. The distribution of the nuclear sub-
stance may have been just as accurate and precise in one case
as in the other.

A comparative study of cleavage of different ova affords
another example illustrating this point. For instance, as my
friend Dr. C. Ishikawa tells me, the summer and winter eggs
of a certain form of Daphnidze undergo different “types” of
cleavage, one being holoblastic and the other being meroblastic,
the difference being probably produced by the amount of food-
yolk. Dr. Ishikawa has kindly placed at my disposal several
interesting drawings which illustwate this point clearly. The
accompanying figures show the difference of the modes of
cleavage between the yolkless summer egg and the winter egg,
in which food-yolk is abundant, from one and the same species of
animal. The summer egg belongs to the regular, holoblastic
type of cleavage, and the winter egg, to the meroblastic type,
showing a close resemblance to the ova of some insects.

260 WATASE. [VoL. IV.

Polyphemus oculus +

Figures Iand // show the holoblastic cleavage of the summer eggs.
Figures I/I and ZV show the meroblastic cleavage of the winter eggs. All from
unpublished drawings of Dr. C. Ishikawa.

From Dr. Ishikawa’s private communication, as well as from
the published account by Grobben,! Weismann and Ishikawa,”

1 Grobben: Die Embryonalentwicklung von Moina rectirostris. Wiener Arbeiten,
Bd. II, 1879.

? Weismann and Ishikawa: Ueber die Bildung der Richtungskirper bet thierischen,
Eiern. Ber. d. naturf. Gesellsch. zu Freiburg, Bd. III, 1887, Heft 1, Taf. I, II,
and Ill. Sbid.: Ueber die Paracopulationim Daphnidenei. Zoologische Jahrbiicher,
Bd. IV, 1889, Heft 1, Taf. X, XI, XII, XIII.

No. 3.] STUDIES ON CEPHALOPODS. 261

on the early development phenomena of the various forms of
Daphnide, I constructed the following table, which shows at a
glance the intimate connection existing between the so-called
“types” of cleavage and the relative amount of deutoplasm
contained in the protoplasm of the egg, and this conclusively,
because the difference in cleavage is seen in the ova of one and
the same species of animal.

Broods of Lges. Types of Quantity of

Cleavage. food-yolk.

Polyphemus oculus...... Summer OIA Enis holoblastie sched Storevets scanty.
VATE I Rapes talehavevonntelens METODIASEG!|tye)s)sletstel plenty.

Bythotrephes longimanus. pee dope we Hoi holoblastic ap aepajodia tye scanty.
RAMUS Gnoop oct adc meroblastic......... plenty.

Miia vee Oe Summer Hidsindigomubc Heloblaste noddhao gen scanty.
NNAIDESE AOS Gb golaec tc MELODLASELCHys!= state) tere plenty.

Leptodora hyalina’ sik +: Summer Bg PHI ENS Oro meroblastic Sas acon plenty.
Write So bblocow ooo MELO DAstic:. sis) 2! ore\a1s plenty.

Diep ina tees ane Summer SIC AUO Sw OBL meroblastic Meier toror sere plenty.
\NAWIUISTE G Game goomoor meroblasticey. 1st plenty.

In Leptodora and Daphnia, both the winter and summer eggs
contain plenty of food-yolk, so that the cleavage is not com-
plete ; while in Polyphemus, Bythotrephes, and Mozna, the dif-
ference between the cleavage of winter and summer eggs are
so sharply marked that the two broods of eggs may be taken
for two entirely different organisms, if the affinity of the
“types” of cleavage of the ova were in any way indicative of
the systematic affinity of the adult organisms. The same may
be said in regard to the cleavages in different species of Perzpatus,
as the studies of Sedgwick and others have shown. The same
is true in the case of Renz/la,as was shown by Wilson. In
short, if we classify animals by the “types” of cleavage, or
differences of cleavage, rather than with reference to the poten-
tial qualities of the nuclear substance, we fall into an error of
placing nearly related species of organisms in different cate-
gories ; nay, we even fall into the absurdity of separating the
individuals of one and the same species into different groups.

Sachs ! gives an instance illustrating the same fact in plants;
viz. six different forms of cleavage taking place in the pollen
mother-cells from one and the same orchid, the difference in
cleavage being produced by the slight individual variation in

1 Joc. cit.

262 WATASE. [Vou. IV.

the outline of the original mother-cells. He points out that the
mode of cytoplasmic cleavage of cells depends chiefly upon the
configuration of a growing organ, and I may add, of an organ-
ism, at the time when it begins to grow, and not upon its phy-
letic significance. As an example, he cites the cleavage of
cells in a glandular hair of the gourd-plant and the embryo of a
Phanerogam, both of which undergo essentially the same ‘“‘type”’
of cleavage in every detail so far as we can judge from the re-
semblances in the external appearances of their cleavage furrows.

Balfour pointed out long ago that the similarity or dissimi-
larity of the cleavage process in the ova of different animals as
indicated by the external phenomena alone, has no value what-
ever in estimating the systematic affinities of different organisms
which develop from them. Given two animal ova with their
external configurations alike, owing to the similar distribution
of food-yolk and of the germ-protoplasm, then the early phe-
nomena of cleavage will be also alike, whatever may be the
difference of animals which later develop from them.

That the argument based on the arrangement of superficial
furrows alone is not entitled to any weight, is further shown by
their total absence in several forms of ova, which nevertheless
develop into perfect organisms. It has been shown that in a
certain plant, the cytoplasm becomes divided without a corre-
sponding division of its nucleus. Such facts seem to point to
_ the conclusion that the division of the cytoplasm and that of
the nucleus are two independent phenomena, and that one pro-
cess can occur without the other, and that when they do occur
in close succession, as in ordinary cell-division, it is to be looked
upon as a case of coincidence.2_ At any rate, the following con-
clusion seems to be a valid one; viz. that the division of the
nucleus and that of the cytoplasm are due to different causes.
The cleavage furrows are devoid of significance, taken by
themselves. The formation of a cleavage furrow is a negative
phenomenon, a reflex manifestation of some other activities
going on elsewhere.

It is now quite generally conceded that the nucleus of the

1 For different examples in which the caryokinetic division of the nucleus is not
followed by the division of the cytoplasm, see KGlliker: Handbuch der Gewebelehre.
6th edition, p. 61, 1889.

2 Sachs: Lectures on the Physiology of Plants. 1887.

No. 3.] STUDIES ON CEPHALOPODS. 263

fertilized ovum contains all the hereditary characteristics of
the parent organism. It is this structure in the ovum which
stamps the particular characteristics upon an organism of a
given species. The study of fertilization has clearly demon-
strated the metamorphosis of the sperm-nucleus into a constit-
uent part of the cleavage-nucleus, and thence it is distributed
to all nuclei formed in the subsequent cleavages. Morphologi-
cally, all the hereditary characteristics which the infant organ-
isms inherit from the par-
ents, must be traced back
to a certain number of
chromosomes which come
from the sperm and egg-
nuclei of the ~ fertilized
ovum. By cleavage, the
potential characteristics ,
become gradually analyzed
into their special attributes -
— the attributes which we
assign to different tissues
of the larval or the adult
organism. If, therefore, I
may use one word to charac-
terize the whole process of
cleavage of the ovum, the
term Axzalysis will perhaps
best express our interpreta-
tion of the phenomenon.
It is true that we know
very little as to the essen-
tial respects in which the nuclear substance in the entodermic
cleavage sphere differs from the similar substance in the ecto-
dermic sphere. In the present state of our knowledge on this
subject, we can only infer a structural difference of the pro-
toplasm from the careful study of the fate of the respective seg-
ments. If, for instance, one cell gives rise to a sense-organ, the
fundamental molecular structure of that cell must be different
from another which contains all the germs of an excretory
organ, just as we are forced to conclude that the ova of differ-
ent organisms are of necessity different, even if they appear

Figure V.— Loligo.

264 WATASE. [VoL. IV.

identical by the means of observation at our disposal. Thus,
instead of inferring function from structure, we infer structure
from function, and conclude that wherever we detect a differ-
ence in function the protoplasmic structure must be different
also. When, therefore, we speak of the analysis of nuclear
substances we do not speak from actual knowledge of the sub-
stances thus analyzed, but from purely scientific reasoning.

A. Centre of Archoplasm.

8. Extra-nuclear archoplas-
mic filaments.

C. Cytoplasm.

D. Interzonal archoplasmic
filaments.

£. Intra-nuclear archoplas-
mic filaments.

#, Remnant of nuclear cav-
ity filled with nuclear
fluid.

M. Limit of the original
nuclear cavity, sharply
separated from the
surrounding cytoplasm.

n, n'. Two daughter chromo-
some bands.

figure VT, — Asterias.

It is probable that during cleavage, the original nuclear sub-
stance may undergo a series of molecular changes, and split up
into a number of protoplasmic substances, each of a different
molecular structure, and that as a final result of this chain of
metamorphoses different kinds of tissue-cells come into exist-
ence.!' In short, different morphological stages of the develop-
ing ovum may be considered as so many different molecular
conditions of the protoplasm. And perhaps the molecular
constitution of a dividing ovum in its earlier stage may differ
more from that of the later larval stage, than two organisms

1 See in this connection, Roux: Bedeutung der Kerntheilungsfiguren. 1883.

Now 3.4 STUDIES ON CEPHALOPODS. 265

belonging to different species would differ from each other in
their adult condition. Professor Weismann’s phrase — “ onto-
genetic stages of idioplasm’’ —aptly expresses our meaning on
this subject. For the metamorphoses of structures and of em-
bryonic tissues must of necessity correspond to the change in
the constituent protoplasm. Without change in the nuclear
substance, development is impossible ; the egg must remain an
egg forever.

If all the determining elements of future tissues are contained
in the nucleus of the ovum, and if cleavage is the process by
which these elements are analyzed into more tangible tissues,
the question naturally arises as to the method of analysis em-
ployed in such a process.

Such a method we find in Caryokinesis.

I will, therefore, describe the process which may be termed
the mechanics of nuclear division, as based on my observations
on Cephalopods and Echinoderms.!

It is now agreed by many foremost investigators of the sub-
ject that the essential feature of caryokinesis lies in the
division of the chromatic substance of the nucleus among the
daughter cells, and that the complicated system of spindle rays
is the mechanism to effect such a division. The development
of a spindle clearly shows this, and the following is an attempt
towards a further confirmation of the current view on the sub-
ject, as held especially by E. van Beneden and T. Boveri. In
one important respect my view is just opposite to that of these
authors, but this difference lies more in the interpretation of
phenomena than in the facts themselves.

First of all, I will endeavor to describe the anatomy of a
well-developed caryokinetic figure in the Cephalopod egg, upon
which my observations have been chiefly carried on. The ques-
tion of nomenclature presents some difficulty. I will use here
a set of terms of a simple descriptive character, descriptive of
function, of origin, or of topographic relationship of different
parts.

The accompanying illustration, Fig. V, shows a caryokinetic

1 The substance of this portion has been given in my two previous notes on the
subject: Karyokinesis and the Cleavage of the Ovum, Johns Hopkins Univ. Circulars,
April, 1890; On Caryokinesis, Biological Lectures delivered at the Marine Biological
Station, Wood’s Holl, 1891, pp. 168-187.

266 WATASE. [Vou. IV.

figure in the blastoderm of the squid. Fig. VI shows the same
in a more advanced condition, as seen in the developing ovum
of a starfish (Asterias vulgaris) common at Wood’s Holl. The
latter was brought out by the application of the method recom-
mended by Boveri in his recent paper.! The figure shows a
striking resemblance to those given by Agassiz and Whitman 2
in their recent work on the development of the osseous fishes.

The figure consists of two essential anatomical features, (1)
the central, elliptical body, and (2) the two star-like, radiating
structures. The former corresponds to the outline of the origi-
nal nucleus, as will be shown later, and the latter constitute the
asters of Fol, spheres attractives of van Beneden, or, to use a
more recent nomenclature, the archoplasmic spheres of Boveri.
The central area of archoplasm (A) is situated in the substance
of the cytoplasm (C). From the granular archoplasmic substance
as a centre, there radiate out in all directions a large number of
fibre-like rays, the avchoplasmic filaments (B) and (£). A por-
tion of these ray-fibres penetrate into the elliptical part of the
figure, and constitute the zxtva-nuclear archoplasmic filaments
(Z); while those lying outside of the elliptical body are the
extra-nuclear archoplasmic filaments (BP).

The elliptical portion of the figure consists of three parts, two
terminal and one intermediate. The terminal portion, which
presents different optical properties from the intermediate part,
consists of a hemispherical mass of a slightly stainable, semi-
liquid substance, which I believe to be the nuclear sap of the
original nucleus. Into this part the archoplasmic rays extend,
as has already been mentioned. The two terminal masses of
stainable substance are separated from the intermediate non-
stainable bundle of filaments by parallel chromatic “plates” (z'),
(2), —the chromosomes (Waldeyer) of the original nucleus. The
non-stainable intermediate filaments above referred to are the
enterzonal archoplasmic filaments (D), —“ interzonal filaments”
of Mark, ‘filaments réunissants”’ of van Beneden, “ gubernacu-
lum” of Maupas, ‘“ Verbindungschlauch”’ of Strasburger, “ con-
nective filaments,” “ Verbindungsfaden,” etc., of authors.

One plate of chromosomes goes to one daughter nucleus, and

1 Zellen Studien, III, pp. 6, 7, 1890.
2 Agassiz and Whitman: JZemoirs of the Museum of Comparative Zoblogy at
Harvard College, Vol. XIV, No. 1, 1889.

No. 3.] STUDIES ON CEPHALOPODS. 267

the other to another. The cytoplasm accumulates around each,
and there follows a separation into two cells, each with its dis-
tinct nucleus. da eben 2

If one examine a nucleus at ea Ls
a tolerably early stage of caryo-
kinesis, one will see a phe-
nomenon such as is shown in
Fig. VII. The nucleus with
a network of chromosomes is
intercepted between two archo-
plasmic spheres. More than
this, however. That portion
of the archoplasmic rays which
falls on the surface of the
nucleus presses that part in-
ward and so flattens that side
of the nucleus. This polar

flattening of the nucleus goes ide Lp Sa)
on until the nucleus presents of
the appearance shown in Fig. Figure VII. — Loligo.

— Nucleus, x, nucleolus (?).
VIII N—Nucl leolus (?)

Space only forbids the illustration of the further changes,
but it may be easily imagined that when this flattening of the
nucleus is continued, the whole Oe ee ens
solid contents of the nucleus Toes He oy
are reduced to a single flat A
sheet, as it were, as shown in
Fig. 1X, forming the equatorial Fee en aE
chromatic “ plate.” The spin- oe i] WN E
dle, then, as its history clearly TRS FAA NS:
indicates, consists of two cones ree 4 Dos CUES iy ‘ seet-M
with their bases turned toward = N=77 Qed fo Sf€
each other, and with their 9-05 VS
apices in the archoplasmic SSS a
centres, as was first pointed ge bas: Sey
out by van Beneden.

This stage of caryokinesis
with its single chromatic AY
“plate’’ leads to another with
two daughter “plates,’——a phase which has been called by
Flemming, metakinesis.

as
Saat NS
~

Figure VII.

268 WATASE. FVOLE. TV:

The question naturally arises, How is this separation of a
single “plate” into two “ plates’ effected? With the separa-
tion of the two daughter “ plates” of chromosomes, there comes
into existence a series of parallel interzonal filaments which lie
between the two separating “plates.’’ The separation of the
daughter ‘“‘plates”’ of chromosomes, and the formation of the
interzogal filaments, are so intimately connected with one
another that we naturally look for a causal connection which

eu ae underlies the parallel se-
NN ries of phenomena. Any
a theory which explains the
= one must also explain the
as esas other.

EE iy, Rae Before I venture a sug-

gestion in regard to this
important point, it will
ae UU blah iN aes be important to find out
BSS Vi whether the phenomenon
BS \ WW pe of polar flattening of the
SS ale ie nucleus, such as I have
“ Wa oot described in the previous
pages, is one of normal
character or not. That the
said phenomenon is of a
normal character is shown
by its constant occurrence
in the blastoderm of the
squid, where all the differ-
ent stages of caryokinetic division can be observed without
any difficulty. My recent studies on the eggs of a starfish (As¢e-
rias vulgaris), with this particular point in mind, convinced me
that there, as in the squid, the polar invasion of the nucleus by
the archoplasmic laments is the matter of normal occurrence.
This phase, however, is extremely transient, and quickly passes
into the equatorial plate stage.
In connection with the caryokinetic phenomena in Pagurus

striatus, Carnoy! observed the formation of the equatorial chro-
matic plate by the “gradual retraction of the chromatic loops

Figure IX,

1 Carnoy: Cytodiérése chez les Arthropodes, Pl. VII, Fig. 44, a, 4, c, d, f, p. 316.

NG. 3s] STUDIES ON CEPHALOPODS. 269

towards the equator” of the nucleus. In the first stage, the
nucleus retains the elliptical outline, with a complete nuclear
membrane, and two archoplasmic asters at the two antipodal
ends of the nucleus. In the second stage, the retraction of the
nuclear filaments towards the equator has commenced, and with
it, the rudiment of the archoplasmic spindle appears inside of
the nuclear membrane, presenting very much the same appear-
ance as in Fig. VII, p. 267, previously described. This retrac-
tion towards the equator goes still further in the third stage,
and with it the shape of the spindle becomes more perfect. In
the fourth stage, the nuclear filaments are reduced into a plate-
like structure, and the original cavity of the nucleus, still sharply
bounded by the nuclear membrane, contains the complete spin-
dle; and in the succeeding figure (f) the metakinesis in its
advanced stage is represented. The resemblance of this series
of figures to the series I have already given is perfect.

These observations, according to Carnoy, show two important
facts : —

(1) The persistence of the nuclear membrane through the
stages of caryokinesis.

(2) The developmental history of the spindle. After all this,
Carnoy,! however, concludes, “Ils prouvent d’abord que le
fuseau peut dériver exclusivement du noyau.”

It appears to me to say that the spindle is formed inside of
the nuclear membrane is one thing, but it is quite another to
infer from it that the spindle is derived from the nucleus. Why
may it not be formed by the invasion of the archoplasmic fila-
ments from the outside of the nucleus, through the porous
nuclear membrane, as was pointed out by Strasburger and
Guignard ?

I am strongly inclined to believe that the interesting series
of stages in the formation of the spindle and of the chromatic
equatorial “plate,” given by Carnoy in the place above referred
to, belongs to the same order of facts presented in the previous
pages in connection with the origin of the spindle in the Cephalo-
pod blastoderm. May not the beautifui series of figures given
by Rabl? and Schewiakoff,? showing the gradual formation of

1 Joc. cit. p. 317: 2 Rabl: Ueber Zelitheilung. Morph. Jahrbuch., Bd. X.

8 Schewiakoff: Ueber die Karyokinetische Kernthetlung der Euglypha alveolata.
fbid., Bd. XIII.

270 WATASE. [Vous ive

the spindle with the corresponding retraction of the chromatic
filaments toward the equator of the nucleus, show the same
thing, and that the spindle material does not originate inside
of the nucleus, properly speaking, but comes from the outside?

The most convincing facts in regard to the truth of the polar
invasion of the archoplasmic filaments, in the nucleus with the
firm nuclear membrane around it, are afforded by Biitschli’s?
observation in the living egg of Bvanchipus, by Mark? in
Limax, by Platner® in Aulostoma, and by Garnault* in Heder.
Further, I believe that the facts given by these naturalists have
the same significance in the course of caryokinetic phenomena,
which I have ascribed to them in the preceding pages, in the
case of Cephalopods.

To van Beneden’s works references have already been made
in the preceding pages. The following is the summary of his
more recent results in his studies on the mechanics of caryo-
kinesis in Ascaris megalocephala, which he carried out in con-
junction with A. Neyt:—®

“Dans notre opinion,’ van Beneden and Neyt observe, “tous
les mouvements internes qui accompagnent la division cellulaire
ont leur cause immédiate dans la contractilité des fibrilles du
protoplasme cellulaire [archoplasmic fibrils] et dans leur arrange-
ment en une sorte de systeme musculaire radiaire, [archoplasmic
sphere] composé de groupes antagonistes ; le corpuscule central
joue dans le systeme le réle d’un organe d’insertion. Des divers
organes de la cellule c’est lui qui se divise en premier lieu, et
son dédoublements amene le groupement des éléments con-
tractils de la cellule en deux syst¢mes ayant chacun leur centre.
La présence de ces deux systemes entraine la division cellulaire
et détermine activement le cheminement des étoiles chromatiques
secondaires dans des directions opposées. Une partie impor-
tante des phénomenes qui constituent la cinése a donc sa cause
efficiente, zon dans le noyau, mais dans le corps protoplasmique
de la cellule.

10, Biitschli: Studien iiber die ersten Entwicklungsvorgiinge der Eizelle, die
Zelitheilung und die Conjugation der Infusorien. 1875. Taf. XIII, Fig. 14.

2 Mark: Maturation, Fertilization, Segmentation of Limax

3 Platner: Arch. f. Mik. Anat., Bd. 33.

*Garnault: Bull. Scientifique de la France et de la Belgique, Tome XXII.

° E. van Beneden and A. Neyt: Mouvelle recherches, etc., p. 280. (Italics are my
own.)

Now.) STUDIES ON CEPHALOPODS. 271

“D’ou vient l’impulsion qui détermine le dédoublement des
corpuscules centraux, la formation des cordons pelotonnés et la
division longitudinale des anses? Réside-t-elle dans le noyau,
ou dans le corps cellulaire? Aucune donnée positive ne permet
de résoudre cette question. Nous n’avons réussi a établir que
deux choses: cest /’existence dans la cellule d'un appareil ou
aun mécanisme qui préside a la division cellulaire, comme notre
systéme musculatre & la locomotion, et le dédoublement de ce
mécanisme préalablement a la division nucléaire.”

To this must be added his account of the origin of interzonal
fibrils. As van Beneden! observes, with the division of the
equatorial chromosomes and their respective migrations towards
opposite poles, there comes into existence, intercepted by two
daughter ‘“ plates,” a lamina, which he calls the “dame intermé-
diatre,’ and the substance which composes it, the “ swdstance
entermédtare”’ (p. 543).

This substance is described as being “relativement sombre
et chromophile, mais se colorant de moins en moins au fur et a
mesure que l’écartement [of daughter chromatic stars] progresse”’
(p. 556).

As the separation of the chromatic loops progresses, one can
positively ascertain the existence of achromatic fibrils in this
intermediary substance. To these achromatic fibrils van Bene-
den gave the name of the “‘/ilaments réunissants,” to distinguish
them from the achromatic spindle fibrils directly arising from
the sphéres attractives (pp. 557, 558).

As to the origin of the entire achromatic spindle, van Beneden
considers it as highly probable that it is derived from two sources,
a part from the sphéres attractives (cytoplasmic in their origin),
and a part from the achromatic part of the nucleus.

The achromatic constituent of the nucleus contributes to the
formation of the swbstance intermédiare with its fibrils, while
the ends of the spindle are derived from a portion of the attrac-
tive spheres. The gradual lengthening of the achromatic fibrils
in the interzonal space is due to the antagonistic pulling forces
emanating from the attractive spheres which act through the
contractile fibrils whose distal ends are attached to separating

1E. van Beneden: Recherches sur la maturation de l’euf et la fécondation.
Archives des Biologie, Tome IV, 1883.

272 WATASE. [Vor IVs

daughter stars. For the graphic illustrations of this view, the
reader is referred to the diagrams constructed by Boveri.

Boveri's? view on the origin of the interzonal filaments in
Ascaris is a remarkable one. He agrees with van Beneden in
regarding the interzonal fibrils as essentially different from the
archoplasmic spindle fibres. In fact, Boveri does not regard the
interzonal filaments as filaments at all, but simply as the optical
expression of longitudinal folds of the “lame tntermédiatre”’ of
van Beneden, brought about by the contraction of the archo-
plasmic fibrils, whose distal extremities are fastened to the chro-
mosomes of the nucleus, and the consequent stretching of the
intermediary lamina in the longitudinal direction of the spindle.
The folds thus produced run parallel with the longitudinal axis
of the caryokinetic figure, and give rise to the filamentous ap-
pearance. Boveri admits that the interzonal filaments in the
caryokinetic figures in other cells, demand a different explana-
tion from that given above.

It would be entirely out of place here to enter into the exami-
nation of the details of the phenomena so admirably brought out
by Boveri. But before leaving Boveri I would like to call atten-
tion to his Fig. 41,3 Taf. XX, in the above-mentioned memoir,
in which a group of archoplasmic threads from the upper centre
fasten themselves to two chromatic loops in common with some-
what longer archoplasmic threads arising from the opposite
centre.

The figure suggests the idea that the upper bundle of archo-
plasmic threads are yielding to the greater pushing force of the
lower. If the archoplasmic fibrils are contractile in movement
during the stages of metakinesis, and are pulling the chromo-
somes towards their own archoplasmic centres, how does it
happen that this bundle shows such a peculiar curvature, its
convex surface being turned away from the centre which is sup-
posed to be the centre of attraction ?

Another point I would like to observe in connection with the
theory of van Beneden and Boveri on the function of the archo-

1 Boveri: Zellen-Studien, II. Jenaische Zeitschrift, Bd. XXII, Taf. XXI, Figs.
64a, 64%. See, also, Geddes and Thompson: Zhe Evolution of Sex, p. 222. Lon-
don, 1889.

2 Boveri: loc. cit,

3 Reproduced in Geddes and Thompson: The Evolution of Sex, p. 146, Fig. 3.

No. 3-] STUDIES ON CEPHALOPODS. 273

plasmic fibrils, and that is the duplex activity of the archo-
plasmic threads at two different periods of caryokinesis. In
the first half of the process, the fibrils are distensible, stretching
away from the archoplasmic centres, and in the latter half, the
activity becomes just reversed and then become contractile,
travelling towards the respective centres from which they arise.

Strasburger has always held that the archoplasmic spindle
threads have a cytoplasmic origin, the threads penetrating the
nucleus from two poles, through the porous nuclear membrane.
In his more recent studies on Spirogyra," he conclusively supports
his former conclusion.

As to the origin and significance of the interzonal fibrils,
Strasburger says :—*

« Augenscheinlich sammelt sich zwischen den beiden Kern-
plattenhalften ein osmotisch wirksamer Stoff an, der Zellsaft aus
der Umgebung an sich zieht und der die angrenzende Plasma-
hiille nach aussen treibt. Es lasst sich annehmen, das dieser
Stoff zuvor zwischen den Spindelfasern angesammelt war; ja ich
méchte noch weiter gehen und die Vermuthung aussprechen, das
derselbe dem urspriinglichen Kernsaft entstammt.” Through-
out the process of nuclear division the nucleus is enclosed by a
cytoplasmic mantle. As the two daughter nuclei separate
wider and wider, the equatorial achromatic space lying between
them assumes the swollen barrel shape, which is enveloped by
an extremely thin cytoplasmic bag. This interzonal structure is
designated as the Verbindungsschlauch, in which the equatorial
suspensory threads appear prominent as pronounced ribs. This
“ Verbindungsschlauch ” constantly increases in length and
breadth by osmotic absorption of fluid through its walls. Stras-
burger offers the following suggestion in regard to the signifi-
cance of this structure: “Es unterleigt kaum einem Zweifel,
das wir in dem Verbindungsschlauche eine Einrichtung vor
uns haben, die mit Zuhiilfenahme osmotischer Druckkrafte bei
Spirogyra dazu dient, die Zellkerne auseinander zu treiben,
sie in bestimmter gegenseitiger Lage zu erhalten, endlich den
Auschluss der gesammten Theilungssfigur an die vordringende
Scheidewande herzustellen” (pp. 21, 22).

Strasburger, as has been seen, regards the interzonal filaments

1 Ueber Kern- und Zelltheilung in Pflanzenreiche, etc. 1888.
2 Joc. cit. p. 17.

274 WATASE. [Vior. TV2

as first arising from the achromatic portion of the original
nucleus, to which, by osmotic action, is added the substance
from the surrounding cytoplasm.

While Strasburger regards the “ Verbindungsschlauch”’ as a
mechanism for separating the two daughter chromatic “plates”
apart, he also ascribes to the latter a certain automatic capacity
for movement. The chromatic threads may probably use the
spindle filaments as supports in the course of their migration.
Strasburger also supposes that the poles of the spindle exert a
certain influence upon the behavior of the chromatic threads.
“In welcher Art man sich die Wirkung der Pole denken mag,
kommt dabei zunachst nicht in Betracht, es ware ja moglich,
dass es sich dabei um einen chemischen Reiz handelt, ahnlich
demjenigen, der die Plasmodien, Bacterien oder Oscillaria-
Faden veranlasst, bestimmte Bewegungsrichtungen einzu-
schlagen’”’ (p. 153).

Strasburger then recognizes four possible factors which de-
termine the movement of the chromatic threads: (1) Spindle
fibres, cytoplasmic in origin, penetrating the nucleus, through
the nuclear membrane; (2) the ‘ Verbindungsschlauch,” partly
nuclear, partly cytoplasmic in origin, driving the two daughter
chromatic plates wider apart towards the poles of the spindle;
(3) the chromatic granules forming the nuclear threads having
an automatic power of movement, and travelling themselves
towards the poles, using the achromatic spindle fibres as sup-
ports; and (4) the poles may exert a certain influence upon the
migrating chromatic threads, whose nature is difficult to imagine,
but may possibly be chemical in its nature.

There are a few more theories recently published, attempting
to explain the phenomena of caryokinesis on a mechanical
ground. The main object of the present paper not being to
discuss caryokinesis, I cannot enter into a fuller examination of
different views on the subject at present.

To turn again to the description of the caryokinetic process
in Cephalopod. As to the filamentous nature of the interzonal
substance, there can be no question, as several observers have
abundantly shown. My own studies on Cephalopods and Echi-
noderms have convinced me of the truth of this conclusion.
Further, no optical difference could be observed between the
archoplasmic fibrils at the poles of the spindle and the filamen-

ING. Sc] STUDIES ON CEPHALOPODS. 275

tous bodies in the intermediate zone, which fact has already been
pointed out by several investigators.

Observing, then, (1) that the interzonal portion of the caryo-
kinetic figure consists of the bundle of filamentous substance,
(2) that this filamentous substance is essentially the same as
the archoplasmic filaments of the spindle, (3) that the length of
these filaments is exactly the same as the space between the
parallel bands of chromosomes in all stages, (4) that the archo-
plasmic filaments have been growing in length from the poles
toward the equator of the nucleus, and (5) that the interzonal
filaments came into existence exactly at the moment when the
single equatorial “ plate” was dividing into two parallel daughter
“plates,” the following view becomes probable, viz. that after
the archoplasmic filaments from the two centres have reduced
the chromatic contents of the nucleus into a flat “plate” by
gradual lengthening, they continue to grow in the same manner,
and push through between each other, just as two brushes
would do if their ends were pushed together. Their free
ends dovetail with each other. The distal extremity of each
archoplasmic filament being fastened to the chromosome, the
latter will be carried by the former at its tip, and will be pushed
forward as long as the filament continues to grow. Two op-
posing systems of the archoplasmic filaments behaving in a
similar way, and lengthening in a contrary direction, would
reduce the spherical nucleus first to a biconcave disc, then to
a flat “plate,” and finally, into two parallel “plates,” each
“plate” travelling in an opposite direction. The interzonal
filaments then, according to this view, are the actual continua-
tions of the archoplasmic filaments; but, instead of consisting
of a single system, as at either end of the spindle, they are
composed of two systems, each dovetailing with the other, and
growing in contrary directions. Jnterzonal filaments are, there-
fore, the prolongations of the zntranuclear filaments. 1 am
further inclined to believe that a certain optical peculiarity of
the interzonal region, as, for instance, its aversion to take stains,
is due to the existence in it of a proportionally large number of
non-stainable archoplasmic filaments.

Having briefly sketched the general outline of the process by
which the characteristic shape of a caryokinetic figure originates,
it would be appropriate to devote a few words to the obscure

276 WATASE. [Vou. IV.

point as to the origin and movement of the archoplasm itself.
But as a matter of fact, we know as yet very little in regard to
the origin of the archoplasm, which has sometimes a definite
body in its centre —the centrosome. A great authority like
van Beneden looks upon it as a permanent organ of the cell,
equal in value to the nucleus itself; but the whole question of
its origin and its apparent homologues, which pass by different
names in different cells, is too complicated and obscure to be
discussed in this place.

The later history of the archoplasm is, however, better known.
When we examine a cell at the close of caryokinetic division,
we see a small nucleus with the archoplasmic sphere at one side
of it, appearing somewhat like a satellite of a planet. This
small nucleus is one of the daughter nuclei of the previous gen-
eration, and is destined to become the mother nucleus of the
next. Just as new nuclei arise by the division of the old one,
so the new archoplasmic spheres also arise by the division of
the previous one. In the Cephalopod blastoderm the division
of the mother archoplasmic sphere into two daughter spheres
could be observed with sufficient clearness. In Ascarts, its
division has been most carefully studied by several investiga-
tors. At first the two daughter spheres lie in close opposition ;
later, they separate more and more widely. As each sphere has
a system of radiating filaments, there is formed a little spindle
where they come into contact. This spindle lies outside of the
nucleus, and has nothing to do with the larger one which has
been described already. The daughter archoplasmic spheres
migrate further apart, and finally settle themselves on the
opposite sides of the nucleus. Their effect on the latter is soon
seen. That surface of the nucleus upon which the archoplasm
rests soon shows signs of flattening, as was shown in Fig. VII.
This polar flattening of the nucleus has been interpreted as due
to the pressure exerted by the growing archoplasmic filaments.
The growth of the filaments continues, and the effects it pro-
duces upon the nucleus, in the arrangement and distribution of
the chromosomes, have already been described. Compare in
this connection the series of stages shown in Figs. VII, VIII,
IX, and V.

The above is a brief sketch of the mechanics of nuclear
division, as I interpret them from the study of Cephalopod

No. 3.] STUDIES ON CEPHALOPODS. 277

blastoderm. The descriptions of the different stages, which are
but summarily given in connection with the few diagrams, will
be suplemented in a future paper on the caryokinesis in Cephal-
opods and Echinoderms.

The preceding descriptions of the phenomena of caryokinesis
in Cephalopods refer to four important topics : (1) the origin of
the spindle ; (2) its behavior towards the nucleus; (3) the forma-
tion of the equatorial chromatic “ plate”; and (4) the separa-
tion of the daughter plates and the formation of the interzonal
filaments.

I have attempted to show that these series of phenomena are
the continuation of one and the same process, with no reversal
of activity in the middle, nor with the introduction of several
hypothetical factors.

The spindle, however, plays only a part in the production of
the caryokinetic phenomena. The whole behavior of the chro-
mosomes preparatory to division, such as the transformation
from a resting condition to a coil stage, followed by the longi-
tudinal splitting of each filament, — phenomena which take
place independently of the influence of the spindle, — has
received no consideration, and, so far as I can see, has no
causal connection with the behavior of the archoplasm, although
both tend to accomplish the same end, viz. the formation of two
nuclei out of one. It is conceivable that one mother coil may
sometimes split into two different kinds of substances, and the
archoplasmic filaments play simply the part of a distributing
agent in carrying these into opposite halves of the dividing cell.
In view of the general theoretical conclusion regarding the inti-
mate correlation between form and matter, and mechanism and
function, such a view does not appear improbable; for, as has
already been stated, the differences of two cells lie in their
structure, and the structure being the expression of the chemi-
cal substance of the protoplasm which composes them, wher-
ever we find the difference of structure we find difference of
substance or substances, and wherever we find difference in the
substance we find difference in property or function. It is
probable, as has been mentioned already, that the nuclear sub-
stance, by its constant metamorphoses, gives rise to a series of
substances, to isolate and distribute which is the function of the
spindle, a number of differently constituted cells being thus
produced,

278 WATASE. [Vor. IV<

If, in conclusion, I recapitulate what has been said in a few
words, the cleavage of the ovum may be characterized as Azaly-
sis of different protoplasmic substances which form the bases
of different tissues, caryokinesis as the J/ethod, and the archo-
plasmic spindle as the /wstrument.

EV:

Since the work of Kolliker was published in 1844, there have
appeared a number of valuable observations on the development
of Cephalopods, touching more or less on the cleavage of the
ovum, such as those of Bobretzky, Lankester, Ussow, Brooks,
and Bruce, and quite recently an important contribution on the
cleavage in Sepza by Vialleton.

So far as the observation of facts are concerned, there is very
little chance for error in forms like the Cephalopods. Differences
of methods, so far as they come within the limits of approved
histological practice, can make but a little difference, particu-
larly in the study of such a subject as this, where everything
comes out with a diagrammatic clearness. In regard to the
illustrations of the different stages in Lolzgo Pealet which ac-
company this paper, I feel confident of their general correctness,
as far as they go. Most of the preparations from which the
illustrations on my plates were drawn are still in existence, and
can be verified at any time. I will not, therefore, enter into a
critical comparison of the facts presented by different authors.
In regard to the interpretation of the phenomena, differences
aré naturally expected. Whatever may be the value of the pre-
ceding remarks concerning the cleavage of the ovum in general,
I will attempt to treat the cleavage phenomena in Lo/zgo from
that standpoint. In this point my paper differs from that of
any previous worker on the Cephalopod embryology.

Before we enter into the descriptive details of the present
subject, a few points of a more general nature occur to me,
which may be introduced here.

The idea of ascribing a comparatively high, complex structure
to the ovum, such as the differentiation of the axes, etc., in the
unsegmented egg is attended with scepticism, or accepted with
a certain amount of reserve by more cautious embryologists.
It appears to me, this hesitation arises chiefly from the prevalent

No. 3.] STUDIES ON CEPHALOPODS. 279

idea that the ovum of a metazoan represents phylogenetically
a protozoan which is supposed to have possessed a radial sym-
metry. We must, however, bear in mind that the ovum at
the moment of its first separation as a single cell in the then
embryonic body of the parent organism, is different from the
ovum at the stage ready for cleavage. If by the growth of an
organism is meant an increase in the volume of the original
protoplasm and the development of the external configuration,
and the cleavage of its material is regarded as a secondary feat-
ure which may be dispensed with in the strict definition of the
term as applied in the organic kingdom, then the history of the
ovarian ovum is just as much a growth in all of its essential
features as the conversion of the segmented ovum into an
embryo, and the embryo into an adult organism.

The ovarian ovum increases in size; it acquires protective
apparatus around it; it develops a particular external configura-
tion; and, above all, it accumulates a greater or less amount
of yolk-substance for future consumption. The ovum at the
time it is ready for cleavage is not the same organism as when
it was first formed inside of the maternal tissues, any more than
the two segment stage of the same ovum is the same organism
as it is at an advanced stage of its ontogeny. There is a
growth and development in the unicellular phases of the one,
as there are growth and development in the polycellular phases
of the other. When, therefore, one says that the ovum of a
metazoan is the phylogenetic representation of its protozoan
ancestry, we may ask whether one refers to the primitive ovum
first found in the embryo, or to one advanced to the stage ready
to divide, between which, we must remember, there exist various
stages of growth and development.

From the developmental standpoint the origin of the ovum is
just as old as many of the functional tissues of the parent
organism. It is true that the growth of the ovarian ovum is
an extremely slow process compared with other tissue-cells, but,
nevertheless, it is a growth, and a continuous one, and directly
depends on the nourishment and protection afforded by the
tissues of the parent organism. Even if we admit that the uni-
cellular ovum, irrespective of its stages of growth, represents
actually the condition of the ancestral protozoan, a highly dif-
ferentiated axial symmetry of a certain metazoan ovum cannot

280 WATASE. [Vot. IV.

be said to be an aberrant feature unrepresented in the ancestral
protozoa, so long as the existing forms of the protozoa often
show such a high degree of differentiation in that particular
respect.

It appears to me admissible to say at present that the ovum,
which may start out without any definite axis at first, may
acquire it later, and at the moment ready for its cleavage the
distribution of its protoplasmic substances may be such as to
exhibit a perfect symmetry, and the furrows of cleavage may
have a certain definite relation to the inherent arrangement of
the protoplasmic substances which constitute the ovum. Hence,
in a certain case, the plane of the first cleavage furrow may
coincide with the plane of the median axis of the embryo, and
the sundering of the protoplasmic material may take place into
right and left, according to the pre-existing organization of the
egg at the time of cleavage; and in another case the first cleav-
age may roughly correspond to the differentiation of the ecto-
derm and the endoderm, also according to the pre-organized
constitution of the protoplasmic materials of the ovum.

It does not appear strange, therefore, that we may detect a
certain structural differentiation in the unsegmented ovum, with
all the axes of the future organism already foreshadowed in it,
and the axial symmetry of the embryonic organism identical
with that of the adult.

The general shape of the ovum of the squid at the time it is
ready for cleavage is oblong, having the shape of a hen’s egg,
with one end more pointed than the other (Fig. 1, Pl. IX). On
the pointed end is situated the germ-protoplasm, spreading its
thin pellicle over the huge mass of the food-yolk and completely
enveloping it. The germ-protoplasm at the pointed pole shows
such a thickening, that, when viewed from the side, it presents
a lenticular outline, sharply distinguished from the underlying
mass of food-yolk (Fig. X). The polar globules, three in num-
ber, one a little larger than the rest, are seen floating in the
perivitelline fluid inside the chorion.

By keeping the exact position of the segmentation nucleus
and the outline of the whole germ-disc as shown by the optical
section in view, and rolling the egg in its longitudinal axis, we
observe two important facts. At one time we observe a slight
inequality in the amount of cytoplasm on both sides of the

STUDIES ON CEPHALOPODS.

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282 WATASE. [Vou. IV.

polar extremity (Fig. XI), and at the next moment the wings,
as it were, of the cytoplasm on both sides of the segmentation
nucleus become perfectly identical in outline (Fig. X). By
rolling the egg, we are constantly brought to view the same
alternation of facts with perfect regularity.

Coupled with these phenomena is another presented by the
general outline of the ovum. When the wings of the cytoplasm
on both sides of the segmentation nucleus appear unequal
(Fig. XI), then the whole outline of the ovum corresponding
to the above is also unequal, the longer arm of the germ-wing
being accompanied by the broader, convex outline (A), while
the side corresponding to the shorter wing of the cytoplasm
descends rather abruptly (P). By a further study of the phe-
nomena through successive stages of development, it can be
demonstrated that this more convex border corresponds to the
anterior, and the less convex border corresponds to the posterior,
side of the egg (Figs. XII, XIII). The view (Fig. X) in which
the wings of the cytoplasm are equal is the one in which the
optical axis of observation coincides with the antero-posterior
axis of the ovum; hence the two wings correspond to the right
and the left sides of the organism (R,L, Fig. X). The view
in which the distribution of the cytoplasm around the nucleus
is unequal is the one in which the optical axis of observation
coincides with the transverse axis of the ovum (Fig. XI).
The longer wing of the cytoplasm corresponds to the anterior
border of the ovum (A), and the shorter wing corresponds to
the posterior border of the same (P). Thus in the unsegmented
ovum of the Lo/igo, the surface study alone indicates that there
is an anterior border (A), different from the posterior (P), and
with it there is a right (R) and /eft stde (L) of the organism. As
the later embryological studies abundantly show, the end where
the germ-protoplasm is situated corresponds to the dorsal stde
(D), and the opposite pole, where the food-yolk predominates,
is the ventral side (V) of the organism.

This surface observation, when combined with the study of
the blastoderm completely separated from the bulk of food-yolk,
makes the point still more clear. Such a specimen is shown
in Fig. 16, Pl. X. The germ shows four well-marked concen-
tric zones. The segmentation nucleus occupies the innermost
circle. Next around it comes a zone of clear substance in

No. 3.] STUDIES ON CEPHALOPODS. 283

which the achromatic radiating fibrils abound. Next to this is
the zone of granular cytoplasm which passes rather suddenly
‘nto the zone of the thinner protoplasmic pellicle, a portion of
which is shown in the figure. The whole mass of the yolk-
substance is enveloped by the extension of this same pellicle.

A further study of the same blastoderm will show that the
nucleus is not situated exactly in the centre of the germ-disc,
but it is slightly eccentric. Taking the outermost limit of the
granular zone as the contour of the essential part of the devel-
oping egg, the cleavage nucleus is found situated nearer to one
side than to the other. I have made a series of careful meas-
urements in regard to this point, and found in a large majority
of cases, this eccentric position of the nucleus in the blastodisc
is well pronounced, and even discernible without any aid of a
micrometer. This fact was already noticed by Vialleton in
Sepia. Comparing this observation on the isolated blastoderm
with the surface study, we notice an interesting coincidence
of the two. For if we draw a straight line through the cleav-
age nucleus, transverse to the germ-disc, as it is represented
in the plate, it will divide the latter into two unequal halves,
the one lying in front of the line being larger than the one lying
behind it, while if we draw a straight line at right angles to
the first and through the same nucleus, it will divide the disc
into two identical bilateral halves. The larger segment lying
wn front of the transverse line corresponds to the anterior
half, and the smaller segment behind the line corresponds to
the posterior half of the ovum. The two bilateral identical
segments falling on both sides of our second imaginary line
correspond to the right and the left half of the ovum.

Bearing these points in mind, we will proceed to the examina-
tion of the different stages of cleavage, with special reference
to the development of symmetry as manifested by the cleavage
furrows.

Fig. 17 shows a stage in which the caryokinetic figure of the
cleavage nucleus has just reached a stage where the chromatic
contents of the nucleus have been reduced into a “ plate,” and
the general outline of the spindle has become completed. The
spindle is formed entirely within the original nuclear cavity,
which is sharply separated from the cytoplasmic surrounding.
The archoplasmic rays of the aster run in all directions, and

284 WATASE. [Vou. IV.

come of them meet with the rays of the opposite side in the
plane of the equatorial chromatin “plate.” This plane corre-
sponds to the median axis of the ovum.

Fig. 18 represents the blastoderm with the first furrow of
cleavage completed. The furrow is deepest in the area where
the layer of cytoplasm is thickest, becoming gradually shallower
and shallower until it finally disappears in the peripheral zone of
the blastoderm, or in the zone of the protoplasmic pellicle.
Each daughter nucleus has a characteristic bean-shaped outline,
with the broader convex borders turned toward each other.
The outline of the original interzonal substance is not distinct.

The direction of the furrow of cleavage corresponds exactly
with the plane of the median axis of the adult organism. The
two segments of the blastoderm correspond to the right and the
left half of the adult organism respectively. The “median”
or the “unpaired” structure as such, therefore, strictly speak-
ing, has no existence in the squid. All parts are paired histologi-
cally, and derive their material from two bilateral sources. The
median structure, the siphon, arises in that way, as is well known.
Organs like the digestive tube, must also be considered as arising
by the meeting of some descendant of cells derived from two
halves of the original blastoderm such as I have been describing.

Fig. 19 represents a stage in which each of the two daughter
nuclei of the previous figure is dividing. In the nature of the
archoplasmic spindle and the appearance of the equatorial
chromosomes, they do not offer any recognizable difference
from those seen in a preceding stage. One important point to
be noticed in this stage is the direction of the longitudinal axis
of the caryokinetic figure in reference to the plane of the first
cleavage furrow. The axis of the spindle does not run exactly
in the same direction with the first cleavage furrow, but slightly
diverges from it anteriorly. If the axes of the two spindles on
both sides of the first cleavage furrow be prolonged both ante-
riorly and posteriorly, they will soon meet in the prolongation
of the median plane of the blastoderm in the posterior part,
while they will diverge more and more as they are prolonged
in the anterior part.

This stage introduces us into the next one (Fig. 20), where
the slight antero-posterior differentiations shown in the disposi-
tion of the caryokinetic figure in relation to a fixed plane in the

NOW S| STUDIES ON CEPHALOPODS. 285

previous stage, becomes still more emphasized in the arrange-
ment of the resulting nuclei. For the sake of convenience, I
will call the segments in front of the second furrow of cleavage
the anterior, and those behind it the posterior, segments of the
blastoderm. The nuclei of the anterior two segments take dif-
ferent positions from those of the posterior ones. This is shown
by the attitude they assume in reference to the median plane, as
will be seen in the figure.

The antero-posterior differentiation of the blastoderm is made
still more manifest in the succeeding stage, Fig. 21. The longi-
tudinal axis of the caryokinetic figure in each of the posterior
segments is almost parallel with the second furrow of cleavage,
while the same axis in the anterior segment makes an angle of
about 45° with it.

The antero-posterior differentiation indicated by the disposi-
tion of the caryokinetic figure in the previous stage is made
still more apparent by the appearance of the cleavage furrows,
Fig. 22. While the third furrows in the posterior segments run
nearly parallel to the first and nearly at right angles to the sec-
ond cleavage plane, those in the anterior segments take quite a
different direction, as will be seen in the figure. They run
parallel to, or even converge towards the median axis in the
posterior half, while in the anterior half of the blastoderm they
run away from the median plane.

In Fig. 23 the twelve segment stage of the blastoderm is
shown. No two cells on one side of the blastoderm are alike, in
size, in shape, or perhaps in their destiny. Just where the suc-
ceeding cleavage furrows come in, is indicated by the equa-
torial plane of the caryokinetic figure.

Fig. 24 represents the sixteen segment stage of the blasto-
derm. The bilateral arrangement of the segments is perfect.
One important fact which will be discussed later may here be
pointed out ; viz. all the segments in front of the first cleavage
furrow are in a more advanced stage of caryokinesis than those
situated behind. This difference in the rate of growth in the
anterior and posterior halves of the blastoderm is most pro-
nounced in the behavior of four central cells, surrounded by
twelve marginal cells. Two central segments which are situated
anteriorly are considerably larger than the other two central
segments situated behind. The former belong to the original

286 WATASE. [Vou. IV.

anterior half of the blastoderm, and the latter are derived from
the original posterior segments.

The nuclei in the central segments are decidedly in different
stages of growth, Those in the anterior segments are far more
advanced than those in the posterior segments. The outlines
of the anterior central segments present interesting points for
study.

In the first place, we see that the shape of a given cell is
determined by the condition of the environment ; that is, it con-
forms to the space determined by the meeting of several other
segments. The lateral horn on the outer border of each cell
fits into the space between the heads of two marginal segments.

On the other hand, the same cell appears to me to offer
another example of how the outline of a given cell may arise
from another cause. For instance, the conical process at the
anterior border of the same cell, penetrating into the substance
of the marginal cell is produced by a different cause from that
producing lateral horns already referred to. The anterior horn
came into existence in connection with the production of the
caryokinetic phenomena going inside of its cell boundary, and
has some intimate connections with the archoplasmic sphere
which les nearest to it.

Fig. 25 represents the blastoderm at the twenty-two cell stage.
The number of the marginal cells is twelve—the number we
had in the preceding stage. The inner cells have been increased
to ten from the four previously present. The history of the
two pairs of inner cells of the previous stage (Fig. 24) is again
repeated in the present stage. The two anterior segments
which were further advanced than the posterior pair in Fig. 24,
have nearly completed the division, and the posterior pair which
were in a resting condition are in pretty well advanced stages
of caryokinesis.

The right segment is, however, a little further advanced than
the left segment. Two posterior marginal segments in direct
contact with the two inner cells above described also show that
the right segment is further advanced than the left one. The
segments between second and fourth furrows of cleavage in the
posterior half of the blastoderm, lying on the right and left side
respectively of the two inner segments above described, show
also difference in the degrees of division, the right half being in
advance to that of the left.

No. 3.] STUDIES ON CEPHALOPODS. 287

Fig. 26 represents a blastoderm with 30 segments, with 12
inner cells with 18 marginal cells. Two of the inner cells have
nearly completed division, but each has been counted as one.
Most of the cells are in a resting condition, and the rate of
growth is uniform on both sides of the blastoderm.

Fig. 27 represents the 32 cell stage, with 14 inner cells and 18
marginal cells — 16 cells on each side of the first or the second
cleavage furrow.

If one, however, divide the blastoderm into front and behind
by the furrow of the first cleavage, as is clearly indicated by the
state of nuclear changes, we find an interesting contrast in
the division of the inner and marginal segments. For while we
find 10 marginal segments out of the entire 16 of the blastoderm
in the front half alone, we find only 8 in the posterior half. In
the quantity of the inner segments, however, the posterior halt
is ahead of the anterior; for the former contains 8, and the
latter, only 6. Hence, while the anterior half is ahead of the
posterior in the number of marginal cells, the latter is ahead of
the former in the number of the inner cells; and hence the
numerical superiority of the anterior half of the blastoderm is
produced by cells of larger dimension than the cells in the
posterior half, the anterior half of the blastoderm occupying a
larger area than the posterior half. Besides the 6 inner cells in
the anterior half occupy a larger area than the area covered by
8 inner cells in the posterior half, altogether making the devel-
opment of the front half more conspicuous than that of the
posterior half.

Fig. 28, Pl. XII, shows a 32 cell stage —16 in front and 16
behind the second cleavage furrow (2'-2). The first cleavage
furrow (1'-1) divides the blastoderm into two bilateral halves, of
which the left side is more advanced than the right side, as the
conditions of their nuclei clearly show. The numerical propor-
tions of the marginal and the inner cells in the posterior and
anterior halves are exactly the same as in Fig. 27 (Pl. XI),
described already. The development of a pair of marginal
segments in Fig. 28, the segments lying between 2! and 4, and
2 and 4 respectively, on each side of the blastoderm, is con-
siderably different from the development of the corresponding
segments in Fig. 27 or Fig. 26. In Fig. 28 the marginal seg-
ment (4-2') does not reach the margin of the blastodisc at all,

288 WATASE. [VOL S1Vs

as in (4-2') Fig. 27. The corresponding segment on the right
side (4-2), Fig. 28, tapers gradually to a point as it comes
nearer to the periphery of the disc, and does not expand as in
(4-2) marginal segment in Fig. 27. In my former communica-
tion! I failed to trace the true homology of that diminutive
marginal segment in Fig. 28 (4-2) and (4-2), and a certain
confusion has resulted in the naming of the marginal cleavage
furrows.

Fig. 29 shows the blastoderm in the same stage as Fig. 28.
The arrangement of segments is pretty much like that in Fig.
27. An interesting point in this specimen is that there exists
a pair of triangular areas in the anterior half of the blastoderm,
where the cytoplasmic cleavage of the segments is not complete.
I cannot tell whether this is due to the incomplete division or
due to the refusion of the original segments once completely
divided. At any rate, it is interesting to notice that the dis-
tribution of these areas of incomplete division is perfectly
bilateral and can be traced originally to a pair of single seg-
ments in the anterior half of the blastoderm in the eight cell
stage (Fig..22; Pl. -X1).

The faint cell-boundaries, such as are represented in the
figure, were all that could be seen soon after the blastoderm
was prepared fourteen months ago. The specimen is still in
existence, but fusion of the segments is almost complete and
the original faint boundary lines are hardly discernible. The
area looks, in fact, like an unusually large marginal segment
with four caryokinetic figures in it.

The remaining three stages, Figs. 30, 31, and 32, do not need
any special comment. The condition of the nucleus in every
cell has been faithfully represented, showing at a glance which
segment is further advanced than the others.

Fig. 30 represents a stage with 60 segments— 30 on each side
of the median axis or the plane of the first cleavage furrow.
Counted with reference to the plane of the second cleavage
furrow, we find 32 segments in the front half of the original
blastoderm, and 28 in the posterior, showing that growth in
the anterior half is more vigorous than in the posterior. In
other words, each of two segments in the anterior half of
the blastoderm in the 4 cell stage, such as are shown in Figs.

1 Johns Hopkins University Circulars. March, 1889.

No. 3.] STUDIES ON CEPHALOPODS. 289

20 and 21, has divided into 16 segments, while each segment
in the posterior half has divided into 14. We must also bear in
mind that the size of the inner cells in the posterior half is gen-
erally smaller than that of the anterior ones, showing that the
original anterior half of the blastoderm is ahead of the poste-
rior in the number of segments as well as in the actual bulk
of the protoplasm. An examination of the caryokinetic figures
will show that the segments on the left side are further advanced
than those on the right side.

It is a difficult matter to decide of just how many segments a
given stage of the blastoderm consists, when the division of
cells is such a gradual process, and when there exist stages
showing perfect transition from one condition to another. Such
a stage is shown in Fig. 31. The number of segments differs
according to the kind of criterion one takes in deciding what
constitutes a completion of cell-division. One thing is, however,
certain, —that the growth of the blastoderm, as indicated by
the conditions of the nuclei in different parts, is not uniform.
The right side of the figure is decidedly ahead of the left half,
and the left quadrant in the anterior half of the blastoderm is
decidedly behind the right quadrant in the same half.

Fig. 32 represents the blastoderm at the 116 cell stage. The
left half of the blastoderm is in advance of the right half,
although their line of demarcation does not exactly coincide
with the plane of the first cleavage. The cells situated in the
central portion of the blastoderm are backward in growth and
division, as in all preceding cases.

The numbers around the marginal cells indicate the order of
succession of cleavage furrows in the marginal zone alone, in
this case, as well as in all of the preceding specimens we have
described.

V.

By examining the stages of division more carefully, we find
that in many cases the divisions of the different segments in
the same ovum do not take place at the same time and with the
same velocity. We further find a curious fact, that when one
segment or a few segments on one side of the blastoderm show
a tendency to vary in a certain particular direction, the corre-
sponding segment or segments on the opposite side show the
same tendency.

290 WATASE. [VoL. IV.

Figs. 24 and 27, Pl. XI, show two stages of cleavage. All
the blastomeres in front of the second cleavage furrow (2'—2)
are more advanced than those lying behind it, as shown by the |
stages of the caryokinetic figures. The nuclei in the posterior
half of the blastoderm shown in Fig. 27 are all in the “resting”
condition, while all of those in the anterior half have advanced
as far as the equatorial plate stage. The same fact has been
observed by Vialleton, in Sepza. If I represent the condition
in a diagram, and reduce back all the segments to the original
blastomeres from which they have sprung, it will be something
like Fig. XVI.

saneenean—| D>

Wy [--—~-------

Fig. XIV. —The unsegmented blastoderm of the squid, showing the eccentric
position of the nucleus 7; A, anterior; P, posterior; L, left; R, right.

Fig. XV.— A diagram showing the one-sided development of the blastoderm.
The segment / gave rise to all the segments in the left half of the blastoderm, such
as are shown in Figs. 28, 30, 32. ‘

Fig. XVI.—A diagram showing the unequal cleavage in the ‘anterior and the
posterior halves of the blastoderm. All the segments showing the more advanced
stages in caryokinesis such as are shown in Figs. 24 and 27 were descended from the
two anterior segments a./. and a.7.

Fig. XVII.—A diagram showing precocious cleavage among the descendants of
one segment, 2.7.

Fig. XVIII. — A diagram showing the seats of abnormal (triple) caryokinesis, on
the corresponding sides of the ovun, £.2.’, 6.7!

Fig. XIX.—A diagram showing the original segments, @.2’, @.7.', from which
a pair of fused areas on both sides of the blastoderm (Fig. 29, Pl. XII) were
descended.

No. 3.] STUDIES ON CEPHALOPODS. 201

The first furrow of cleavage is indicated by (1/-1); and the
second furrow by (2’-2); a./, a.r. represent the anterior blasto-
meres. All the cleavage segments in front of the line (2'—2) in
Figs. 24 and 27, showing the more advanced condition of caryo-
kinesis than those lying behind, were descended from these two
blastomeres a./. and a.7., as represented in the diagram (XVI) ;
and all those segments showing the backward conditions in their
nuclear changes were derived from the two segments which lie
behind them.

Fig. 25, Pl. XI, shows another interesting variation. All the
four segments, which descended from one quadrant (p.7, Fig.
XVII) are further advanced than their homologues on the
opposite half, which have descended from the corresponding
quadrants on the opposite half.

Figs. 28, 30, 31, 32, were all taken from the eggs of one and
the same squid. As the examination of caryokinetic figures in
each segment of the blastoderm will show, those segments lying
on the one side of the first cleavage furrow are more advanced
than those on the other, making exceptions of those few segments
which are found along the posterior half of the median axis.

I cannot well describe the extremely slight differences shown
by different blastomeres in different parts of the blastoderm.
A glance over the figures and the comparison of different seg-
ments will show them far more clearly than pages of verbal
description.

As I have stated already, Fig. 31 was taken from the same
lot of eggs as Figs. 28, 30, and 32. But it is the right side of
the blastoderm which shows a more advanced condition, and not
the left side, as in the others. I am inclined to believe now
that in this case, the blastoderm was mounted with the wrong
side up, and what appears as the right side in the figure belongs
really to the left side of the animal. If such was the case, the
left-handed variation, as shown in the series of four stages, Figs.
28, 30, 31, and 33, becomes interesting, since all the eggs come
from one and the same animal. It is probable that this ten-
dency to vary in the same direction may be due to the same
hereditary characteristics inherited from the parent organism.

Fig. 29 is an interesting example of what may be designated
as the analogous variation of both sides of the germ. Four seg-
ments on each side of the blastoderm show a curious phenom-

292 WATASE. [Vor. IV.

enon of fusion. It may have been an imperfect division at first,
however. At any rate, the behavior of these four corresponding
segments on each side of the median axis is interesting. The
specimen as it now stands has lost all the faint cleavage lines
which existed at first, and the four blastomeres have completely
fused with each other. The origin of this mass of fused seg-
ments can be traced to a single blastomere at the eight cell
stage. In the diagram, Fig. XIX, the segments a./.' and ar!
on both sides of the first cleavage furrow (1'-1) represent the
original segments which gave rise to the four imperfectly divided
segments described above.

Another curious example which I have met is one in which
the abnormal caryokinesis in one segment on one side of the
body is shared by the corresponding segment on the other (7.2
and p.r.', Fig. XVIII). In both segments mentioned above, the
nuclei were seen in the state of being divided into three nuclei,
as indicated by the triasters.

The fact that the groups of cells which vary simultaneously
on both sides of the bilateral ovum can be traced back to a
single segment in an earlier stage appears to me to be a matter
of considerable importance. Although I was unable to trace the
whole history of such segment or segments, it is probable they
give rise to or constitute a corresponding part of some future
bilateral structure. Another fact, namely, the unusual mode of
caryokinesis occurring in the corresponding segments of the
bilateral blastoderm when all other cells in the body are dividing
normally, is a point worthy of consideration.

And finally, we must bear in mind that the precocious cleav-
age may occur in the descendants of a single blastomere alone
and not in pairs, as in Fig. 24.

This phenomenon of uzegual growth of different parts, as 1
may call it in an empirical way, has been observed by several
naturalists.

Brooks ! figured unequal cleavage in the bilateral blastoderm
of Batrachus.

His? developed this fact of unequal growth of different parts
into a principle, although his principle more especially refers

1 Alternation of Periods of Rest with Periods of Activity in the Segmenting Eggs of
Vertebrates, Studies from Biol. Laborat., Johns Hopkins Univ., Vol. II, 1882.
2 Unsere Korperform, etc., 1874.

Na. 3: ] STUDIES ON CEPHALOPODS. 293

to a much later stage of embryonic development with special
application to the facts of organogeny. Newport! noticed long
ago, in the frog, that ‘‘about two hours after the completion of
the crucial cleft [the second furrow of cleavage] a new series of
changes is set up in the egg. The clefts no longer include the
whole circumference of the egg, but are confined to the splitting
of the larger into smaller pieces, after a binary plan; and this
process does not begin at once over the whole surface, but
appears first in a given spot, and then pursues a definite course ;
thus each of the two pieces seen from above on one side (behind)
of the crucial cleft becomes subdivided, producing four segments
on one side of that line, whilst there are only two on the other.
When this subdivision is nearly completed, and not till then, a
corresponding change takes place in the two segments on the
other side (in front) of the sulcus.”

What are the effective causes which produce these differences
in different parts of one and the same blastoderm ?

Unless we study different forms more extensively, discussion
in this field is not likely to be of any particular value. The
following remarks are therefore only provisional ones, with
special reference to the results of my Cephalopod study.

Growth of any two given parts in an organism may be similar
or different, according as the inherent nature of the material
which constitutes the given parts are similar or different. Two
parts fundamentally alike, on the other hand, may grow at dif-
ferent times and at different rates, according to the conditions
of environment which affect them. In other words, unequal
growth may take place in two parts intrinsically similar, one of
the parts, however, being more favorably situated than the
other.

By which of these categories of causes may the unequal growths
we have seen in the blastoderms of the squid be accounted for ?
To characterize this whole series of variation simply as patho-
logical is not an explanation. On the other hand, it has been
suggested that the unequal growth such as is manifested in the
alternate cleavage of the squid ovum may be explained as due
solely to the influence of external conditions. I am, however,
disposed to believe that external conditions have nothing to do

1 Researches on the Impregnation of the Ovum in the Amphibia; and on the Early
Stages of Development of the Embryo, 3a Series. Philosophical Transactions, 1854,
Pp: 241.

204, WATASE. [Vou. IV.

<

with this particular case. My reasons are as follows: I have
shown that the area which is the seat of a particular variation
either in the behavior of the nucleus or of the cytoplasm, such
as the peculiar fusion or the incomplete division of the cyto-
plasm, or in the velocity of the caryokinetic division, can always
be traced to one single segment ina still earlier stage. In other
words, when an area consisting of a small number of cells shows
a peculiar modification, that area can always be traced toa single
cell in a still earlier stage, from which it has descended. When
analogous phenomena of variation occur on both sides of the
bilateral axis of the blastoderm, the area where such phenomena
occur can be traced to one corresponding cell on each side of
the axis, which came into existence at the same time on both
sides. Or when the variation occurs only on one side, or at one
spot, this area of variability is made up of the descendants of a
single cell.

It is difficult to suppose that the environment affects one
quadrant of the blastoderm at the four cell stage different from
another; or how the external conditions can affect the corre-
sponding segments on each side of the blastoderm and divide
their nuclei with three asters, when all the rest of the nuclei
are divided with two, it is difficult to imagine.

The cause of unequal cleavage in the various cases we have
examined appears to me to be an internal one, due to the pecu-
liarities of the particular protoplasmic structure which composes
the segment or segments. When, therefore, the right and left
halves of the bilateral blastoderms show a difference of velocity
in their cleavage, I believe it is due to the slight qualitative
inequalities induced by the first division, — inequalities which
appear more and more exaggerated as the cleavage process
advances.

These facts seem to point to two conclusions : —

1) That the earlier cleavage processes are more fundamental,
and, from the morphological standpoint, more significant than
the later ones.

2) That, as I have already mentioned, since the eggs from
the same animal show similar variations in cleavage, such a
tendency to vary may become hereditary. This conclusion is,
however, a provisional one.

CLARK UNIVERSITY, WORCESTER, MASS.,
November 19, 1890.

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206 WATASE.

EXPLANATION OF PLATE IX.
Loligo Pealet. -

All the figures in this plate have been magnified 45 diameters.

Fic. 1. Unsegmented ovum, with three polar globules, one of which has not been
made distinct by the engraver. The whitish cap at the pointed pole of the egg is the
germ-disc.

Fics. 2-8. Different stages of cleavage. The eggs have been slightly tipped over
so as to show the cleavage field better.

FIGS. 9, 10, 11. Side views of more advanced stages of cleavage, showing the
relation of the marginal elongated cells to the yolk-mass and to the central smaller
cells.

Fics. 12, 13, 14. Views from the cleavage-poles.

Fic. 15. An abnormal specimen, with the germ-protoplasm at the side of the
ovum, and not at the top as is usual. Cleavage is, however, normal.

Journal of Morphology. Vol. Ne

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298 WATASE.

EXPLANATION OF PLATE X.
Loligo Pealet.

All the figures in this plate have been magnified 90 diameters. Polar globules
have not been drawn in the figures.

Fic. 16. Unsegmented stage of the germ-disc. There are considerable variations
in the size of the germ-disc in different ova. This is an unusually large one. Notice
that the anterior half of the germ-disc is larger than the posterior half.

Fic. 17. A germ-disc at the midst of the first cleavage.

Fic. 18. A germ-disc at the completion of the first cleavage furrow.

Fic. 19. A germ-disc at the midst of the second cleavage.

Fic, 20. A germ-disc at the conclusion of the second cleavage. Gaping of the
first cleavage furrow at the anterior and posterior margin of the blastoderm in this
figure as well as in the next, is produced by the pressure of the cover-glass on the
convex germ-disc.

Fic, 21, A germ-disc at the beginning of the third cleavage. Notice the differ-
ence in the directions of cleavage in the anterior and the posterior half of the disc,
as indicated by the directions of the caryokinetic axes.

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300 WATASE.

EXPLANATION OF PLATE XI.
Loligo Pealet.

All figures in this plate have been magnified 65 diameters.

Fic. 22. A blastoderm consisting of 8 cells, 4 in front, and 4 behind the second
furrow of cleavage. All the cells in front are larger than those behind.

Fic. 23. A blastoderm consisting of 12 cells.

Fic. 24. A blastoderm consisting of 18 cells. All the segments in front of the
second furrow of cleavage are more advanced than those behind it.

Fic. 25. A blastoderm consisting of 22 cells. :

Fic. 26. A blastoderm which has just entered into the 32-cell stage. A wide cleft
in the left side of the blastoderm, 2’, corresponding to the second furrow of cleavage,
was produced by the separation of the adjacent segment due to the pressure by cover-
glass.

Fic. 27. A blastoderm consisting of 32 segments, with all the segments in the
front half of the blastoderm in the midst of division, while the nuclei in the posterior
half are in a “resting” condition. A group of 6 inner cells, and a pair of marginal
cells along the first cleavage furrow, in the posterior half of the blastoderm, present
the characteristic appearance of this stage. As the later stages will show, these
groups of cells make the slowest growth in the whole blastoderm.

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302 WATASE.

EXPLANATION OF PLATE XII.
Loligo Pealet.

All the figures in this plate have been magnified 65 diameters.

Fic. 28. A blastoderm with 32 cells. The left half is ahead of the right half in
the progress of division.

Fic. 29. A blastoderm consisting of 32 cells. This specimen shows a pair of
peculiarly fused areas which are symmetrically arranged on both sides of the median
furrow of cleavage.

Fic. 30. A blastoderm consisting of 60 cells. The left-hand side of the blasto-
derm is further advanced than the right side.

Fic. 31. A blastoderm consisting of 90 cells. In this specimen, the right-hand
side is further advanced than the left half.

Fic. 32. A blastoderm consisting of 116 cells. The left half ahead of the right.

All the specimens represented in Figs. 28, 30, 31, and 32 have been obtained from
the eggs of the same squid.

Lith Ansty Werner A Winter, Frankia tH,

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Journal of Mornhology. Vol.

1°

CONTRIBUTIONS ON THE MORPHOLOGY OF THE
ACTINOZOA.

Il. ON THE DEVELOPMENT OF THE HEXACTINIA.
J. PLAYFAIR McMURRICH.

For the past few years I have been endeavoring, as occasion
offered, to obtain the material necessary for the study of the
development of some Hexactinian, but my efforts have been only
partially successful. In 1887 I obtained at Nassau, Bahama
Islands, W.I., a few embryos of two different Actiniaria, —
Aulactinia stelloides, McM. and Rhodactis Sancti Thome (Duch.
and Mich.). Both these forms, however, retain the embryos in
the interior of the body until the mesenteries have formed, the
embryos from Aw/actinia possessing, when extruded, from eight
to twelve perfect mesenteries, while those from Rhodactis are
extruded somewhat earlier, while they are furnished with only
two or four perfect mesenteries. These forms, therefore, agree,
as regards the retention of the embryos, with the majority of
forms whose embryology has been studied.

There are some forms, however, in which the ova are extruded
unfertilized. Adamsia parasitica is one of these according to
Kowalewsky (75), and I have found that Metridium margt-
natum, so abundant on the New England coast, does so like-
wise. During the summer of 1889, aided by the generous
enthusiasm and energy of two assistants, I was able through
the facilities offered by the Marine Biological Laboratory at
Woods Holl, Mass., to collect and keep large numbers of this
Actinian in aquaria, and so obtained some ova which were
artificially fertilized by sperm extruded by other individuals at
the same time. Unfortunately spermatozoa were not always
obtainable when required, and consequently I was obliged to
allow a large number of ova, obtained at various times, to decay,
and was only successful in rearing embryos in a very limited
number of cases. There are consequently many gaps in my
observations, which might readily have been filled in by the

304 McMURRICH. [Vou. IV.

study of more abundant material. Those ova which were fertil-
ized I did not succeed in rearing up to the time of formation of
the mesenteries, owing probably to ignorance of the proper food
which they require at this period. I had hoped to be more suc-
cessful during the summer just past, but my endeavors to obtain
ova were entirely fruitless, though spermatozoa were plentiful.

1. THE SEGMENTATION AND FORMATION OF THE GERM-LAYERS
IN METRIDIUM.

In his classic monograph on the development of the Actinians
Lacaze-Duthiers (72) advances the suggestion that these forms
are hermaphrodite. Later observers have not succeeded in
confirming this opinion in so far as the Hexactinians are con-
cerned, though the coexistence of ova and spermatozoa has
been found in certain Zoantheze and Cerianthez. I have never
found in Metridium, or in any of the numerous other Hexac-
tinia I have examined, any trace of hermaphroditism, and
believe that it may be accepted as a rule that the Hexactinians
are bi-sexual. Dichogamy may possibly occur, but the evidence
seems strongly against even this.

The immature ova of JZetridium lie closely packed together
in the mesogloea of the gonophoric mesenteries, mutual pres-
sure compelling them to assume irregular shapes. When set
free by teasing, the younger ova are almost spherical, while the
older ones (Pl. XIII, Fig. 1) are somewhat irregular, the majority
possessing a more or less elongated process extending out from
the circumference at one point, sometimes in the neighborhood
of the nucleus, sometimes distant from it. The nucleus is large,
with a single large nucleolus, and is always situated eccentri-
cally, lying as a rule nearest that pole of the ovum which is
adjacent to the free surface of the mesentery. The peculiar
filamental apparatus which the Hertwigs have described in con-
nection with the immature ova of Adamsia parasitica (79) and
other forms (’82), I have never observed in Metridium.

The mature eggs are perfectly spherical, pinkish in color,
and opaque with small granules of food-yolk. They measure
0.124 mm. to 0.159 mm. in diameter, and each ovum is surrounded
by a delicate membrane in which no perforations are visible.
No nucleus can be detected in the fresh ova after their extru-

No. 3.] MORPHOLOGY OF THE ACTINOZOA. 305

sion, though they are evidently thoroughly mature, and ready
for fertilization, after which segmentation begins without any
intermediate formation of polar globules. It seems almost cer-
tain that these bodies are formed before the extrusion of the
ova, possibly while they are still in the mesenteries. I have
endeavored in vain to observe their formation, but do not attrib-
ute my want of success to their non-existence. Haddon (’89)
has figured (Pl. XXXVI, Fig. 10) a condition which he suggests
as representing the formation of a polar globule while the ovum
is still in the mesentery, but I cannot consider the formation of
polar globules before the setting free of the ovum, to be proved
by what he has figured, there being no evidence, such as the
existence of karyokinesis, to show that the body being extruded
from the ovum really represents a polar globule. It seems
more probable that the figure represents an artefact.

The first indications of segmentation occurred in the ova of
Metridium about three-quarters of an hour after adding the
sperm to the water containing the ova, and consisted in the for-
mation of two slight elevations at one pole of the egg (Fig. 2),
reminding one very much of what occurs in the ova of Cteno-
phores. A slight groove appears later between the elevations,
and, gradually deepening (Fig. 3), finally cuts the ovum into
two spherules. I did not observe the formation of the eleva-
tions in all cases, but it seems certain that the segmentation
furrow always starts at one pole of the egg in the typical Co-
lenterate manner,—a method of segmentation, by the way,
which does not seem by any means to be confined to that
group, but to be of more widespread occurrence.

The two spherules are usually slightly unequal in size (Fig. 4),
but the amount of the inequality may vary considerably, being
well marked in some cases, but hardly noticeable in others.

When the segmentation furrow is completed, the spherules
are rounded, and in very slight contact with each other. Soon,
however, the contiguous faces flatten down (Fig. 4), and a re-
fusion of the spherules occurs. This phenomenon is very
marked in the four-celled stage. I have seen the spherules in
this stage become reduced by refusion to two, which, on their
part, underwent refusion, so that there was a return to an appar-
ently unsegmented ovum. I was not able, however, to watch
the further development of this egg, so cannot say that it was

306 McCMURRICH. [VoL. IV.

perfectly normal, but it is certain that the refusion phenomena
are well marked.

At the close of the two-celled stage the ovum is slightly
oval, and shows no external trace of having consisted of two
distinct spherules. It then divides at once into four spherules,
the cleavage furrows arising at the exterior and passing towards
the centre, the segmentation belonging to that variety which
Metschnikoff distinguishes as centripetal (86). Two of the
spherules are usually smaller than the other two, and two lie
upon a different plane than the other two (Fig. 5). How this
is brought about I cannot say; it may be due either to the
nuclei of the original spherules dividing obliquely to the plane
which originally separated them, or else to the nucleus of one
of the original spherules dividing in a plane at right angles
to that in which the other divides, as Ludwig has described in
Asterina (82).

Subsequent divisions lead to the formation of eight (Fig. 6),
sixteen, thirty-two, and sixty-four cells, although occasional irreg-
ularities were seen, such as stages in which there were seven
or eleven cells. The irregularity of the spherules evident in
the earliest stages becomes reduced as development progresses,
so that it becomes impossible to orient the embryos in later
stages, and so determine the relation of the axes of the ovum
and embryo. Inequalities are to be seen in the cells of young
blastulas (Fig. 7), but they are irregular, and do not serve to
mark out a pole of the embryo. The result of segmentation is
the formation of a hollow blastula, with walls composed of elon-
gated columnar cells richly packed with granules of food-yolk,
with a few clear vacuoles, and with their small nuclei situated
peripherally. This blastula is somewhat pyriform in shape (Fig.
8), and is ciliated, swimming about at the bottom of the vessel
in which it is contained, the narrower end being anterior, and
progression being accompanied by rotation about the long axis.
The cilia cover the entire body, and are equal in length except
at the pole which is anterior in swimming, where there is a tuft
of longer cilia, similar to what has been found in other Actinian
larvee. In sections of embryos in this stage the interior appeared
to be quite empty, but in others again (Fig. 11) it was filled
with what seemed to be a coagulable fluid, in which were scat-
tered granules of food-yolk evidently derived from the partial

No. 3.] MORPHOLOGY OF THE ACTINOZOA. 307

disintegration of certain of the blastula cells. As will be seen
later, this disintegration is a normal occurrence, but the time
when it makes its appearance seems to vary in different cases.
Viewed externally, the larva seems to persist in this condi-
tion for some time (Fig. 9), but sections show that very impor-
tant changes are going on during this apparent rest. These
changes consist of the formation of the endoderm by delamina-
tion. I was not able to observe the nuclear phenomena accom-
panying this process, but there seems little room for doubt that
it really is a delamination. It is multipolar in its distribution,
and cuts off the peripheral third from the inner two-thirds of
each cell (Fig. 12). The outer cells so formed become some-
what spherical, are somewhat granular, and show a distinct
nucleus. A peculiar feature which characterizes them, and
which seems to be invariably present after delamination is
completed, is a clear vacuole, larger than the nucleus, occu-
pying the central end of each cell; somewhat similar vacuoles
are figured by E. B. Wilson (83) in Renzl/a, but what may be
their significance is not apparent. The inner or endoderm cells
are much larger than those belonging to the ectoderm, and like
these are granular with food-yolk, and here and there show a
clear vacuole similar to those of the ectoderm cells, but having
no definite position in the cell. One peculiar feature of the
endoderm cells is the difficulty which exists in observing their
nuclei. In specimens in which the delamination is just begin-
ning, only a few cells having separated into the endodermal and
ectodermal moities, bodies which stain somewhat more deeply,
and are as a rule somewhat larger than the yolk-granules, can
be observed scattered about at different levels in the inner por-
tions of the wall of the blastula. Only one of these bodies
is to be seen in each cell, there being in addition, of course,
the nucleus near the periphery, which will eventually belong to
the ectodermal moiety. They do not stain nearly as deeply as the
peripheral nuclei, but nevertheless I am inclined to believe them
to be the nuclei of the endodermal cells, it being evident from
the presence of the large vacuole at the junction of the outer
and middle thirds of the cells in which they occur that they are
quite ready for division (Fig. 11). When the delamination is
tomplete, however, these bodies cannot be satisfactorily made
put, and the endoderm cells are apparently without nuclei,

308 McMURRICH. [Vou. IV.

though it seems more probable that they are actually present,
though undiscoverable. H. V. Wilson (’88), it is to be noticed,
found the same difficulty in observing the nuclei of the delami-
nated endodermal cells of Manzcina.

As already remarked, the cavity of the blastula becomes more
or less filled by a mass of yolk-spherules. Occasionally the
appearance of these spherules may occur quite early, but at
other times, and perhaps more normally, it is delayed until the
beginning of delamination. The granules come from the break-
ing down more or less completely of the endoderm cells. That
they have this origin is, I think, clearly shown in Figs. 11 and
12. In the former the disintegration seems to have affected
some cells to a very great extent, but in the latter it seems for
the most part to be only the central ends of certain cells that
are affected. The central mass is certainly not cellular, the cell
outline having become obscured, as in certain other Actinozoa,
but is composed simply and solely of yolk-granules, sometimes
imbedded in a homogeneous coagulable matrix. In some cases
where the central matter made its appearance during the blas-
tula stage, the inner ends of certain cells seemed to fall off into
the central cavity, and then to undergo disintegration, but in
no case could there be said to be actual cellular elements in the
central mass. This is a point of considerable importance, hav-
ing very decided bearing upon what has been described for other
Actinians, and allowing of inferences as to the actual occurrence
in these superficially studied cases.

At the conclusion of the delamination a slight depression is
to be observed at the posterior pole of the larva (Fig. 9), and
soon after this breaks through, a communication of the interior
cavity with the exterior being thus established. The endo-
dermal cells have by this time arranged themselves in a definite
layer, separated by a slight space from the ectoderm, and the
larva has the appearance of a typical invaginate gastrula (Fig.
10). In fact, before making my sections, I was persuaded that
the formation of the germ-layers was by invagination. It is not
easy to observe in optical sections the endoderm cells soon after
their delamination, and larvz with the slight depression at the
posterior end appear to be single-layered (Fig. 9), the depression
seeming to be the commencement of an invagination. The fact
that I was not able to observe any intermediate stages between

No. 3.] MORPHOLOGY OF THE ACTINOZOA. 309

embryos with the depression and the fully formed gastrula sur-
prised me somewhat, but still the appearances presented by
optical sections seemed so clear, and the gastrula so similar
to an invaginate gastrula, that I believed that I had missed
or overlooked the intermediate stages. Sections, however,
explained the matter at once, and demonstrated that instead
of with invagination we have to do with delamination. I make
special mention of this mistake, since, as I shall shortly endeavor
to show, other observers have probably fallen into the same
error.

As to what becomes of the yolk-spherules occupying the cen-
tral cavity, I have no evidence. It is possible that they may be
absorbed by the endoderm cells as soon as they have reached
their final differentiation, or they may pass to the exterior
through the blastopore and be lost to the larva.

Beyond this stage I was unable to rear the embryos. A few
Actinozoan larve obtained by skimming are almost certainly
those of Metridium, but I prefer to leave them undescribed for
the present, partly on account of the uncertainty of the identifi-
cation and partly because they belong to later stages which may
be more satisfactorily studied in the larger embryos of Rhodactis
and Auwlactinia.

Between the latest stage of Metridium and the earliest which
I possess of Rhodactis, there is an hiatus, during which there
occurs the formation of a very important and characteristic
structure, the stomatodzum. Still, from what occurs in other
forms, we may readily imagine how its formation takes place in
Metridium. Certanthus apparently has a stage almost if not
quite identical with the last stage of Metridium described, and
in this form the lips of the “gastrula”’ mouth bend in to form
the two-layered stomatodzum. It seems probable that the
same thing occurs in Metridium.

A comparison of the methods of formation of the germ-layers
in the Actinozoa, so far as they are known, is of considerable
interest. Our knowledge is derived from the study of only a
relatively small number of forms ; nevertheless, it suggests some
important ideas. The amount of food-yolk seems to have a very
important influence on the details of the development, but the
general mode of formation of the germ-layers is the same in the
majority of forms. Amongst the Alcyonaria we have accounts

310 MCMURRICH. [Vou. IV.

of the early development of at least four different forms. In
Alcyonium digitatum, according to Kowalewsky ('73), the finely
granular protoplasm separates in the form of spherules from a
central coarsely granular mass, which thus becomes enclosed by
protoplasmic spherules, and later on divides up into a number
of cells. In Clavularia (Kowalewsky and Marion, ’83) the yolk
does not exert so great an opposing influence to the division
forces, and we see the planes extending much further towards
the centre of the egg than in Alcyonzum, although there is at
first a small portion of yolk which does not share in the divis-
ion. It gradually disappears, however, with the later divisions,
and an embryo similar to that of Adcyontum is formed. In
Renilla (E. B. Wilson, 83) the phenomenon is essentially the
same as in Clavularia, but in Sympodium (Kowalewsky and
Marion, ’83) the segmentation seems to be total. The early
phases of segmentation have been thoroughly worked out only
in Renzlla, and we know that in it considerable variation may
occur in the manner of division, the ovum sometimes dividing
at once into eight, sixteen, or even thirty-two cells, besides show-
ing other peculiarities. This did not depend on the sudden
division of the nucleus into such a number of parts, but the
nuclear division went on, no doubt, in the usual manner, the
cytoplasmic division being inhibited to a greater or less extent
by the amount of food-yolk present. In the segmentation of
Alcyontum the same process is apparently carried still farther.
In all these forms, and in Gorgonta Cavolini described by
Von Koch (87), the segmentation leads to the formation of a
solid structure consisting of an external layer of cells (ectoderm)
surrounding a solid central mass of cells. This is the organism
for which Metschnikoff (86) has proposed the term Parenchy-
mella or Phagocytella, —rather cumbersome names, which may
perhaps be more conveniently replaced by Sterrula. In only one
of the forms studied have we definite evidence as to the manner
of formation of the central mass, and that is in Rezz//a, in which
Wilson has shown that a Sterroblastula (Goette) results from
the segmentation, and that the inner ends of its cells delaminate
off to form the central solid mass. The similarity of the Ster-
rulas of the other forms to that of Renilla seems to indicate
that the same process of delamination occurs in them likewise.
It certainly cannot be a process of invagination which gives rise

No. 3.] MORPHOLOGY OF THE ACTINOZOA. 311

to the Sterrula, and there are physical difficulties in the way of
immigration.

There has been described, however, among the Alcyonarians
a single case of invagination following the formation of an
archiblastula. Haeckel (’75) states that he has found such an
occurrence in Monoxenia. I have not been able to consult his
original account (75 a) of this phenomenon, and cannot accord-
ingly determine the value of the statement ; but in view of what
is known to occur in other Alcyonaria, and since he also states
that he has found the same thing in an Actinian, — which, as I
shall later endeavor to show, seems doubtful, —I think it is not
safe to rely on the accuracy of the statement until it is con-
firmed by further observations.

Up to the present, we know nothing as to the formation of
the germ-layers in the Edwardsiz, the recent representatives
of the ancestors of the Hexactiniz ; but the Alcyonarians must
be regarded as standing with the Edwardsiz at the bottom of
the Actinozoan stem, since the two groups agree in the number
of their mesenteries, —a feature of fundamental importance in
the present state of our ideas on the subject of the evolution of
the Actinozoa, the difference in the mode of arrangement of the
longitudinal muscles being of secondary value. The occurrence
of a Sterrula, and its formation by delamination in this some-
what primitive group of Actinozoa, is accordingly of importance
as affording a basis whereon to build an explanation of what is
found in the Hexactiniz.

The most definite statements made hitherto as to the early
development of the Hexactiniz are by H. V. Wilson (’88), for
Mancina. Segmentation results in the formation of a hollow
blastula, and by the delamination of the blastula cells, the blas-
toccel becomes filled up with a mass of yolk-bearing cells whose
outlines and nuclei cannot readily be made out. The embryo is
then a Sterrula, and agrees in the manner of its development
with the Alcyonaria. Jourdan’s description (80) of the devel-
opment of Balanophyllia does not extend to stages sufficiently
early to enable one to state what the method of formation of
the germ-layers is ; but it is probable that it is closely similar
to what occurs in Manzcina, and a Sterrula (see his Fig. 126,
Pl. XVII) is the result.

In A. parasitica (Adamsia Rondeleti of Andres), Kowalew-

212 McMURRICH. [VoL. IV.

sky (75) found the method to be as follows: “This phenome-
non (the segmentation) is quite regular, —z.e. the egg divides
into two spherules, then into four, and so on. There results
from this segmentation a mass of cells without the formation of
that central cavity which is termed the segmentation cavity.
After the segmentation is completed, the embryo covers itself
with vibratile cilia, and begins to swim.” A Sterrula is conse-
quently formed in this case likewise, though from the descrip-
tion quoted it is impossible to state the exact manner in which
it arises.

Before proceeding to compare these forms with Metridzum, it
will be necessary to mention certain cases in which an invagi-
nate gastrula has been stated to occur. These are a form
related to A. mesembryanthemum and Certanthus, according
to Kowalewsky ('75 and ’73),; and A. egutna, according to
Jourdan (80). I regret exceedingly that Kowalewsky’s impor-
tant paper is inaccessible to me, and I have been obliged to rely
on the abstract given in Hoffman and Schwalbe’s Jahresbe-
richt, and on the translation of a portion of the original paper
by Giard (Kowalewsky, ’75). As for the figures, I have been
able to see only those reproduced by Mark (’84), in Pls. XI and
XII of the Selections from Embryological Monographs. Fig.
27 of Pl. XI, representing the gastrula of A. mesembryanthe-
mum (°), is very similar to that given by Jourdan from A. eguzna,
and it certainly looks like an early stage of invagination.
Nevertheless, it seems to me, in view of what I have described
for Metridium, that there is a very great chance that both
Kowalewsky and Jourdan have been mistaken in their interpre-
tation of what actually occurs. I have found in Metridium
what I considered malformations, which approach very closely
to what these authors describe and figure. I find in my notes
the following statements: ‘In many (z.e. swimming blastulas)
a depression could be observed upon one side, giving rise to
a slight projection into the blastoccel. The invagination (ze.
depression) appears to occur mostly at the side. Never saw
one at end.” I have drawings of optical sections of this which
present an appearance not very unlike what Kowalewsky and
Jourdan have figured. It must be noticed, too, that neither of
these authors were able to trace directly one of these stages
into the fully formed gastrula stage. Jourdan gives as the suc-

No. 3.] MORPHOLOGY OF THE ACTINOZOA. 313

ceeding stage a form in which the ectoderm and endoderm are
well differentiated histologically, the endoderm cavity is filled
up with food-yolk, and the stomatodzeum has already formed.
There is evidently a great hiatus between the two stages.
Kowalewsky’s description (75), likewise, leads one to infer that
he observed only isolated stages, and did not actually witness
the conversion of the early invagination stage (Mark, ’84, PI.
XI, Fig. 27) into the fully formed gastrula (Fig. 28), which, it is
to be noticed, is exactly similar to my figure of the gastrula of
Metridium (Pl. XIII, Fig. 10).

These facts, taken into consideration with the difficulty of
observing the delamination by optical sections, render it not
improbable that the interpretation of the figures referred to is
erroneous, and that what really occurs is what I have described
for Metridiuwm, and there is a further point which renders this
probability almost a certainty. Jourdan’s Fig. 117 shows the
endodermic cavity to be filled with food-yolk, which is not pres-
ent in the stage represented in Fig. 116. Whence does this
food-yolk come? Kowalewsky’s figures of the gastrula of Cevz-
anthus (Mark, ’84, Pl. XII, Figs. 2 and 3) show essentially the
same thing, though the amount of food-yolk present in the
endodermal cavity is much smaller. Kowalewsky believes (73)
that the food-yolk is secreted by the endoderm cells after invagi-
nation, and is later on reabsorbed by them —a very remarkable
proceeding. In the gastrula of AZetridium food-yolk is to be
found in the endoderm cavity, since it occurs in the blastoccel,
and I think it exceedingly probable that future observations
will show that, instead of invagination occurring in these forms,
the process of endoderm formation is essentially what I have
described above for MWetridium. The cases of supposed gastru-
lation by invagination which have been described for the Hex-
actiniz must be denoted, along with Haeckel’s account of
Monoxenta, as not authenticated.

In all the Actinozoa, then, concerning which we have reli-
able information, the endoderm is formed by delamination. It
remains now to be considered whether what we find in MWetri-
dium or in Renilla is the more primitive. This inquiry leads to
another; namely, Was delamination the primitive method of
endoderm formation, or 1s tt a secondary modification of a still
earlier phenomenon ?

314 MCMURRICH. [VOES TV.

An affirmative answer to the first part of this question formed
the basis of Lankester’s planula theory (77), while the second
part of the interrogation is considered a correct statement of
affairs by the supporters of the Gastraea theory, invagination
being according to them the original phenomenon. It is diffi-
cult to understand how one process could have been converted
into the other, as is required by either theory. Lankester’s
theory, too, finds little support from the facts of development in
the higher forms, whereas the Gastrzea theory is strengthened
by them. On the other hand, the Gastraea theory weakens when
we come to study the developmental phenomena of the lower
Metazoa.

I have already shown the improbability of the occurrence
of an invaginate gastrula in the Actinozoa. In the Scypho-
medusz, it is certain apparently that it occurs in Pe/agza and
Nausithoe, but these are certainly exceptions. Goette (87) has
shown that a gastrula-like structure may be produced in Aurelia
aurita without invagination, by the hollowing out of a Sterrula;
and I have found this same structure to be formed by the
immigration of certain of the blastula cells in Cyanea artica.
Lucernarta, which is probably a very primitive Scyphomedusan,
likewise forms a Sterrula (Kowalewsky, ’84), and, so far as is
known, an invaginate gastrula occurs in the Scyphomedusz
only in two isolated cases. The formation of a Sterrula is to
be regarded as typical for the Scyphomeduse.

In the Hydrozoa not a single case of invagination is known!
See then what a showing we have among the Cnidaria, only
two authentic cases of invagination for the formation of the
endoderm are known !

How then is the endoderm formed in this group? Delami-
nation occurs in the Actinozoa and in certain Hydrozoa, but
there is a process back of this, from which it has been derived:
I mean immigration. The most suggestive and admirable work
of Metschnikoff (’86), has placed the occurrence of this process
beyond a doubt; and that author has been led to regard the

Parenchymella (Sterrula) as an ancestral form of the Ccelen-
_terates, from which, as he shows, a gastrula by invagination
might readily be derived. With this opinion I desire to express
my full agreement, and I have elsewhere (’91) endeavored to
extend the idea somewhat. It will be unnecessary, therefore,

No. 3-] MORPHOLOGY OF THE ACTINOZOA. ane

to enter into a lengthened discussion of the various subordinate
and, so far as the essentials of the theory are concerned, unim-
portant details, which I have there discussed.

There is one point, however, which bears directly upon the
question before us at present, and that is the origin of delami-
nation from immigration. Metschnikoff’s explanation of this
is not, I think, quite satisfactory. He supposes, in fact, that
the two phenomena are contemporaneous, and that both existed
in the ancestral Metazoa, the endoderm being formed by “mixed
delamination ”’ ; z.e. partly by true delamination and partly by
immigration. Where, therefore, in the embryos of higher forms
we find immigration alone occurring, there has been a secondary
specialization, the delamination having been suppressed ; simi-
larly with forms in which delamination alone occurs. It seems
to me, however, that, taking into consideration the occurrence
of immigration alone in such forms as Volvox and Protospongia,
the absence of delamination so far as is known among the
Sponges, and its comparatively rare occurrence among the
Hydrozoa, since the formation of a solid morula instead of
a blastula, such as occurs for instance in Aydractinia, is to
be regarded as precocious immigration rather than precocious
delamination, — taking these facts into consideration, it would
seem that immigration was a more primitive phenomenon than
delamination.

I think a clew to the transformation of the one process into
the other is to be found among the Actinozoa, in which, as
I have shown, we have variations from such a delamination as
occurs in Alcyonium, to what we find in Metridium. In
Renilla the protoplasm is so weighted down that the comple-
tion of the division plane is difficult. The yolk is segregated
towards the central part of the ovum, the periphery being more
purely protoplasmic, and in the later stages the nuclei of the
cells are in the peripheral portion. By segmentation a Sterro-
blastula is formed. There are, consequently, physical difficul-
ties in the way of immigration, and, in addition, the food-yolk
renders each cell as a whole inert, and deprives it of the energy
necessary for immigration. The peripheral protoplasm, how-
ever, is unhampered by yolk, and cuts loose from the central
yolk-containing portion, a differentiation of the Sterroblastula
into a Sterrula thus occurring by delamination. There seems

316 McMURRICH. [VornDVe

to be here a difficulty, in the fact that immigration is not accom-
panied by a division of the cell. Immigration, however, must
necessitate a considerable expenditure of energy, by the cell in
general, which energy, it seems quite conceivable, might be
turned, when immigration became unnecessary or impossible,
to the production of a division.

Supposing the delamination to have arisen in this way in the
lower Actinozoa, it is probable that on a subsequent loss of
food-yolk by the more recent forms, delamination would persist
as the mode of formation of the central mass or endoderm, even
though a large blastoccel was present in the blastula.

I believe then that the phenomena occurring in Wetridinm
are to be explained on the supposition that the Hexactiniz are
descended from forms whose ova possessed a relatively large
amount of food-yolk. What we find in the Alcyonaria is the
original condition, and the more typical delamination of the
Hexactiniz has been derived from this. In Revzz//a the central
cells, heavily laden with food-yolk, are separated from the pro-
portionately more protoplasmic ectoderm ; later, a certain num-
ber of the central cells become transformed into the endoderm
layer, while the rest degenerate and their contents serve as food
for the developing embryo, being absorbed by its endoderm
cells. So too, though to a less extent, in Manzcina; the yolk is
here much less in quantity, but still the same processes occur as
in Renilla. In Metridium the yolk is much more reduced, so
that the blastula cavity never becomes filled up, the delami-
nated cells becoming almost entirely converted into the defini-
tive endoderm, a few only disintegrating, and giving rise to the
granules of food-yolk which lie in the endodermal cavity.

2. THE FORMATION OF THE First EIGHT MESENTERIES IN
RHODACTIS.

The embryos of Rhodactis Sancti-Thome, when extruded by
the parent, have, as stated above, from two to four perfect mes-
enteries. In shape they are pyriform, the mouth being situated
at the narrower pole. The ectoderm is high, and shows a differ-
entiation into the various kinds of cells usually to be found in
that layer in the Actinians, but does not yet present that pecul-
iar vacuolated appearance, due to an excessive development

No. 3.] MORPHOLOGY OF THE ACTINOZOA. 317

of glandular elements, which characterizes the adult (McMur-
rich, ‘89 a). At the aboral pole there is a peculiar differentiation
of the ectoderm which is not found elsewhere on the body.
Immediately external to the mesogloea, which is well developed
at this stage, there is to be seen in longitudinal sections of the
embryo, a layer (Fig. 13, 7) much more faintly stained with
hematoxylin than either the mesogloea (mg) or the portion of
ectoderm (ec) which lies superficial to it ; it is crossed by numer-
ous fine fibrils, in the meshes of which lies a clear, faintly gran-
ular substance, which does not stain at all, or but very faintly.
The fibres are readily seen to be delicate prolongations of the
mesogloea extending up into the ectoderm, and appearing in
cross-section as deeply stained, round, homogeneous dots, and
the matrix in which they lie is apparently composed of nerve-
fibrils. It occupies the relative position held by the nerve-layer
in adults, and it is probable that it is of that nature, although I
have not been able to distinguish ganglion cells in it. In swim-
ming, the aboral end is anterior, and the special development
of sensory cells and a nerve-plexus at that pole is not surprising.
The layer is distinguishable only for a short distance up the
sides of the body, though probably it extends in a very much
less developed condition all over the body. The special devel-
opment at the aboral end must be considered a larval adapta-
tion.

The endoderm consists of large, vacuolated cells, of the same
nature as those found in the adult. They contain large num-
bers of Zooxanthellz, which occur, however, also in the ecto-
derm, and the large nematocysts, which I described as occurring
in the endoderm of the adults, are present in the embryos only
in the ectoderm. The endoderm cells in the younger stages
completely fill up the central cavity, and show little or no
arrangement into a definite layer. It seems probable from this
that the formation of the germ-layers in this form differs some-
what from that described for MMJetridium, and more nearly ap-
proaches what occurs in the Alcyonaria and in Manicina.

In the youngest specimen obtained, the first pair of mesen-
teries is in process of formation. The number of embryos at
my disposal is not sufficient to trace out fully all the processes
concerned in the formation of these mesenteries, but I can
merely deduce what is presumably taking place from conditions

318 MCMURRICH. (VOLE. DVE.

which I find in a limited number of preparations; a more com-
plete series is necessary to determine accurately the actual
occurrences. We have, however, a description of a more ample
series of embryos of JMJanzcina, by H. V. Wilson (88), and this
furnishes a basis for understanding more disconnected stages. In
a transverse section a little above the one figured (PI. xiii, Fig. 14),
the stomatodzum is seen to lie in close apposition to the column
wall, there being no endoderm between its mesogloea and that
of the column. A little lower, as in the section figured, it has
separated, and the mesentery is to be seen extending from it to
the column. The axis of the stomatodzeum is oblique to the
long axis of the embryo, and therefore the section does not cut
the stomatodzum transversely. The other mesentery of the
first pair is seen also in this section, and it is noticeable that it
has made its appearance independently of any apposition of the
stomatodzeum to its point of origin. In another specimen
(Fig. 15), however, I find that such an apposition does occur
in the manner described by H. V. Wilson for Manicina (’88),
the stomatodzum passing over from the point of origin of the
first formed mesentery, drawing this with it, to apply itself to
the column wall at the region where the second mesentery is to
be formed. It is certain, however, that this apposition of the
stomatodzum is not necessary for the formation of the second
mesentery, which may rise while the stomatodzum is still in its
upper part in contact with the place of origin of the first mes-
entery.

There is as yet no development of mesenterial filaments.
The stomatodzeal ectoderm can be traced downwards in a series
of transverse sections somewhat further on one side than on the
other, but this is a necessary consequence of the obliquity of
the stomatodzum to the long axis of the embryo. It is possible
that there may be a slight elongation, but it is doubtful, since
in the specimen from which Fig. 14 is taken, the stomatodzal
ectoderm is cut in the sections lower down on the side on which
the stomatodzeum is in contact with the column wall than on
the other, and if we consider this as indicating the formation of
a filament, we will have the filament of the second mesentery
more fully developed than that of the first, which is possible,
but not to be expected.

The stomatodzum is now slung by two mesenteries. It lies

No. 3.-] MORPHOLOGY OF THE ACTINOZOA. 319

embedded in the endoderm, which, in the upper part of the body,
does not yet show any signs of forming a definite layer, although
below this, differentiation is accomplished, and a well-marked
cavity exists. There are consequently in the upper part of the
body as yet no intermesenterial cavities. In its upper part the
stomatodzum is circular in section, but below it is elongated
in the plane of the two mesenteries, which are situated nearly
opposite each other.

The mesenteries of the second pair make their appearance
simultaneously and without any migration and apposition of the
stomatodzum to their line of origin. They are formed in the
larger space, cut off by the two previously formed mesenteries,
and gradually increase in size until they reach the stomatodzeum,
with which they unite.

The third and fourth pairs of mesenteries also appear simul-
taneously (Fig. 16), the third pair being in the interval between
the mesenteries of the first pair; z.e. at the ventral surface of
the embryo, and the fourth pair in the interval between the
mesenteries of the second pair, or at the dorsal surface of
the embryo. In the oldest embryo of Rodactis which I possess
these mesenteries are not yet perfect, and the longitudinal mus-
cles have not developed, so that it is impossible to get definite
evidence as to which pairs of mesenteries are really the direc-
tives. The manner of flattening of the stomatodzeum in its lower
part would suggest that each pair of directives consists of one
mesentery of the first pair and one of the second; but it is to be
noticed that the upper part of the stomatodzum does not take
part in this flattening, which is probably to be considered as a
temporary condition. It seems more probable that the future
and permanent flattening of the stomatodzeum is at right angles
to the one existing in these early embryos of Rhodactzs, and
that the third and fourth pair of mesenteries will form the direc-
tives of the adult.

H. V. Wilson has described in Maniczna a peculiar reflection
of the ectoderm of the stomatodzum upon the external (z.e.
normally endodermal) surface of that organ, and considers it to
be concerned with the formation of the mesenterial filaments,
with the exception of those of the first pair of mesenteries. In
my specimens of Rhodactis the formation of the mesenterial
filaments differs considerably from what has been described for

320 MCMURRICH. [Mors DVi

Manicina, the filaments of the first pair not appearing until a
very much later stage of the development. In Manzcina a down-
growth of ectoderm pushing away the endoderm occurs while the
stomatodzeum is in contact with the column wall; in other words,
the filament makes its appearance before the mesentery. In
Rhodactis this is not the case, but the mesenteries are well
developed, and the second pair well formed and even perfect,
before the formation of the filaments begins. They appear to
be down-growths of the stomatodzeal ectoderm, extending down
some distance below the lower end of the stomatodzeum. It
must be noticed, however, that the histological characteristics
of the down-growths are not quite identical with those of the
stomatodzeum, and I cannot be certain that there is histological
continuity.

In one specimen only was there any trace of the filaments of
the second pair. Eight mesenteries were formed, two pairs
only being perfect. Here I found not the slightest trace of any
reflection of the ectoderm, but the filament, which was devel-
oped only in one of the pairs, was apparently simply a slight
down-growth from the stomatodzeal ectoderm. It developed
in exactly the same manner as the filaments of the first pair,
and there is not the slightest indication in this specimen of a
reflection of the stomatodzeal ectoderm. In a younger speci-
men, however, in which only the filaments of the first pair have
begun to form, there is a peculiar formation of the lower end of
the stomatodzum (Fig. 17), which is somewhat different from
what Wilson describes. The stomatodzum on one side is split
from below upwards for a short distance, and the ectoderm of
the edge of the slit is reflected for a short distance. The slit
figured is between the mesenteries of the second pair, and the
ectoderm is reflected so as to enclose their extremities, but is
not continued down at all to form mesenterial filaments. On
the side of the stomatodzeum, opposite the slit and between the
mesenteries of the first pair, is a piece of reflected ectoderm,
which is the upper edge of a slit similar to, but less in extent
than, that of the other side. The reflected ectoderm of this
side seems to be continuous with the mesenterial filaments of
the first pair of mesenteries. In estimating the significance of
these ectodermal reflections it must be remembered that even
in adult individuals of various forms reflections of the lower

No: 3: ] MORPHOLOGY OF THE ACTINOZOA. 321

edge of the stomatodzeum, similar to what I have just described,
occur, and apparently have had something to do with the forma-
tion of the ‘“ Flimmerstreifen”’ of the filaments. These struc-
tures, however, have not made their appearance in any of the
Rhodactis embryos I have had for study.

In another specimen, in which the second pair of mesenteries
was just forming, I found some distance up upon the endoder-
mal surface of the stomatodzeum a patch of ectoderm (Fig. 18).
It was not situated symmetrically, but was close to one of
the mesenteries of the first pair. I thought at first that this
might be a piece of reflected ectoderm which had migrated up-
wards some distance. It had evidently, however, completely
lost its continuity with the stomatodzeal ectoderm below, while
on the other hand the mesogloea, which ought to have occurred
below it, separating it from the stomatodzal ectoderm, was not
present, so that it was in direct continuity with the stomato-
dzeal ectoderm, and it seems as if there had been an escape of
ectoderm upon the endodermal surface of the stomatodzeum
through a perforation of the mesoglcea. I am strongly inclined
to take this to be a malformation, especially as we have already
seen that the filaments of the second pair of mesenteries develop
only after the mesenteries have become perfect, there being
therefore no necessity for an ectodermal reflection, even though
they are developed from that layer.

The evidence, so far as it goes, indicates that in Rhodactis
there is no reflection of the stomatodzal ectoderm in connec-
tion with the formation of the mesenterial filaments, and in
Aulactinia the evidence points to the same direction. The
question arises as to whether the arrangement in MJantczina is
the more primitive, or whether the formation of the filaments
is typically postponed until the mesenteries have become per-
fect. The latter seems to me to be the more primitive method,
since in all probability the mesenteries are phylogenetically
older structures than the filaments. In embryos of Edwardsza,
too, I find that the filaments, with the exception perhaps of
those of the first pair of mesenteries, do not appear till the eight
mesenteries have become perfect. This has an important bear-
ing upon the question, since Edwardsza must be regarded as
representing an ancestral condition of the Hexactinians. I
think, then, that the peculiarities of Mancina are secondary,

322 McMURRICH. [Vou. IV.

and are perhaps to be referred to the early appearance of the
mesenterial filaments.

It must be remembered, however, that there are different
opinions as to the nature of the mesenterial filaments in the
Actinozoa, some authors considering them to be endodermal and
others ectodermal. As stated above, they appear to be ectoder-
mal in Rhodactis, at least the median portion, which is all that
is developed, does. At the same time I must refrain from ex-
pressing any certainty on this point, (1) on account of the slight
difference in histology between the ectoderm of the stomato-
dzum and of the filaments, and (2) because it seemed, in the
case of a very young filament of the second pair of mesenteries,
that, in a series of transverse sections, there was a very short
streak of indifferent cells intervening between the stomatodzum
and the filament, interrupting the continuity of these struc-
tures. If this be the case, it would seem that the filaments, or
at least their median portion, were endodermal, which seems
to be the case with those of Azlactinza, to be described later.

3. LATER STAGES IN AULACTINIA.

In the youngest embryo of Aw/actinza which I possess, the
endoderm presents certain peculiarities which allow us to infer
the mode of development in the earlier stages to a certain ex-
tent. There are already eight perfect mesenteries, only two of
which — those of the first pair —are provided with mesenterial
filaments. The endoderm has arranged itself into a somewhat
definite layer, but lying scattered about in the body cavity of
the embryo are numerous somewhat large cellular elements and
yolk-granules. The endoderm is in a stage of differentiation
which corresponds fairly well with that represented in Renzlla
by E. B. Wilson (83) in his Fig: 125; Pl. 57. From the occur-
rence of these cellular elements, it seems probable that the
formation of the germ-layers took place in a manner similar
to what is found in the Alcyonarians, rather than like what I
have described for Metridium.

The stomatodeum in these youngest larve is elongated
throughout its entire length in a direction at right angles to the
elongation noticed in its lower part in Rhodactis, so that a line
drawn in the axis of the elongation passes through the inter-

No. 3.] MORPHOLOGY OF THE ACTINOZOA. 323

mesenterial spaces between the third and fourth pairs of mesen-
teries. Upon the mesenteries the longitudinal muscle processes
have begun to appear. The manner of their arrangement may
be better seen, however, from an older specimen, a figure of which
is given (Pl. XIII, Fig. 19). It will here be seen that the em-
bryo is bilaterally symmetrical, the line separating the antimeres
passing between the mesenteries of the third and fourth pairs,
and therefore in the direction of the elongation of the stomato-
dzeum. The mesenteries of the third and fourth pairs are seen
to have their longitudinal muscles on the faces which are turned
away from each other, and accordingly correspond to the direc-
tives of the adult. The mesenteries of the first and second
pairs do not form a pair in the same manner as the directives,
but each mesentery of these pairs has its longitudinal muscles
upon the face which is directed towards the third pair of mesen-
teries, which we may term the ventral directives.

We have then in this stage an arrangement which exactly
reproduces the condition permanent in the Edwardsi@, and
which may therefore be termed the Edwardsza stage. The sig-
nificance of this from a phylogenetic standpoint I have already
elsewhere pointed out (’89), and do not intend to enter into this
question here, hoping to return to it with a discussion of other
investigations on the subject in a future number of these
“Contributions.”

The next stage witnesses the formation of two additional
pairs of mesenteries, which make their appearance simulta-
neously upon either side of the mesenteries of the first pair
(Fig. 19). These grow rapidly, finally reaching the stomato-
dzeum, and developing their longitudinal muscles. Twelve per-
fect mesenteries are then present. The mesenteries of the fifth
pair have their longitudinal muscles on the face which is turned
towards the mesenteries of the first pair, and those of the sixth
pair have them on the face which is turned towards the mesen-
teries of the second pair, z.e. upon the dorsal surface in each
case. When these youngest mesenteries have become perfect
we have a stage which exactly resembles the condition perma-
nent in Halcampa, and which, therefore, may be termed the
Halcampa stage.

The mesenteries are arranged in six pairs. Two of these,
occupying the dorsal and ventral surfaces, are formed each of

324 MCMURRICH. [Vor. 1Ve

mesenteries which have arisen simultaneously, or nearly so, in
corresponding regions of each antimere of the bilaterally sym-
metrical embryo, and have their longitudinal muscles upon the
faces turned away from each other. The other four pairs have,
however, a very different constitution. Each consists of one
mesentery formed previously to the directives, and one formed
after their completion. Whereas in the case of the directives
the mesenteries forming each pair are homochromous in their
formation; in the lateral pairs, the mesenteries of each pair
are heterochromous. These heterochromous pairs differ from
the directives in the position of the longitudinal muscles which
are on the adjacent faces of each pair.

In succeeding stages as Lacaze-Duthiers pointed out (72)
the appearance of new mesenteries is governed by a law different
from that which operated up to the conclusion of the Halcampa
stage. Up to this period the mesenteries made their appear-
ance bilaterally, one on each side of the dorso-ventral line, the
tendency towards bilateral symmetry being the governing force
which determined the manner of their development. In the
stages which now supervene, the new pairs of mesenteries make
their appearance as if governed by a radial symmetry, develop-
ing simultaneously in the intervals between each of the six
Halcampa pairs, the mesenteries of each pair forming contigu-
ously. In this way a stage with twelve pairs of mesenteries is
formed, having all the characteristics of a Hexactinian. Em-
bryos showing the formation of this second cycle of mesenteries
are the oldest that I possess.

As regards the formation of the mesenterial filamentals my
observations on Azlactinta are somewhat more definite than
those on Rhodactis, though at the same time I do not regard
them as perfectly conclusive. Taken, however, into consider-
ation with what I have seen in adult Actinians, and with the
very definite observations of E. B. Wilson on the development
of the filaments in the Alcyonaria (’83 and ’84), I am inclined
to believe that the median streak of the mesenterial filaments
of Audlactinia are developed from the endoderm.

As already stated, in the youngest Am/actinia embryo mesen-
terial filaments are present only on the mesenteries of the first
pair, so that I have ample material illustrating their formation
on the other mesenteries. In all cases it is the median streak

Nox 3] MORPHOLOGY OF THE ACTINOZOA. 325

(Nesseldriisenstreif) which is first formed, and for some time it
is the only portion of the filament which is present. I shall
consider its formation first, reserving a description of the forma-
tion of the lateral streaks (Fdémmerstreifen) until later.

In an embryo in which the second cycle of mesenterial pairs
is making its appearance, the first four pairs of mesenteries are
already provided with fully formed filaments. The fifth and
sixth pairs are just forming them, while in the mesenteries of
the second cycle, which are yet quite small, there is no trace of
them. It is certain that the reflection of stomatodzeal ectoderm
described by H. V. Wilson does not exist. The mesenteries of
the fifth and sixth pairs have reached the stomatodzeum and
have fused with it for about the upper half of its length; below,
however, they are still separate from it. In a section which
passes through the stomatodzum immediately below the point
of separation of the mesentery from it (Fig. 21), it can be seen
that no ectoderm exists upon its outer surface, while in sections
lower down (Fig. 22), the mesenterial filament is seen. There
is therefore no connection between the stomatodzeal ectoderm
and the cells forming the free edge of the mesentery. The con-
clusion is evident that the median streak of the mesenterial fila-
ment is a product of the endoderm.

I have verified this result in the case of the third mesenteries
in younger larvae, and have no reason to doubt its accuracy.
The statements of H. V. Wilson (88) and E. B. Wilson (’84)
are both so definite, and at the same time so absolutely opposed
to one another that it is of importance to determine, by the
study of a number of forms, what actually takes place. It does
not seem probable that structures having the same morphologi-
cal and physiological characteristics should be derived in such
nearly related forms as the Alcyonaria and Hexactinians from
two different germ-layers, and the question seems to be as to
the correctness of the interpretation of the observations made
by the authors mentioned. What I have found inclines me to
accept the observations of E. B. Wilson.

The filaments, when first formed, consist, as stated above,
solely of the median streak. This is small at first, and differs
considerably from the structure of older filaments, as can be
seen from a comparison of Figs. 20 and 23. In the younger
filament (Fig. 20) the cells are elongated, differing markedly

326 McMURRICH. [Vot. IV.

from the endoderm cells in the vicinity, but do not show any
very decided differentiation into gland and nettle cells. The
older filament (Fig. 23), on the other hand, has numerous nettle
cells which stand out prominently in specimens stained with
haematoxylin, and gland cells are quite numerous. Upon each
side of the filament the endoderm cells are heightened so as to
form a very decided wing upon either side, and there is no trace
of any extension of the filament cells out upon this wing. The
appearance which is seen in Fig. 21 is due to the section having
passed through a bend of the filament. Owing to the contrac-
tion of the embryos the filaments are much contorted, and sec-
tions frequently give an appearance as if several filaments
branched out from the margin of the mesentery. A reconstruc-
tion in wax of a mesentery and its filament showed, however,
that this appearance was deceptive, and was due to the bending
and twisting of the filament.

There is one bend, however, which is constant in all the
embryos, and occurs a short distance below the point where
the mesentery leaves the stomatodeum. The edge of the
mesentery is here bent upon itself, so that transverse sections
show a portion of the mesentery attached to the stomatodzeum,
its edge being turned towards the edge of the rest of the mesen-
tery (Fig. 24). This bend is important since it is upon the por-
tion that lies morphologically above it, that the lateral streaks
of the filaments are developed.

In the embryo from which Figs. 21 and 22 were taken, the
lateral streaks had developed upon the first and second pairs of
mesenteries. In these in a section taken just below the point
where the bend mentioned above occurs (Fig. 24), the median
streak (ms) of the filament is well differentiated, and the two
endodermic wings may be seen on that portion of the filament
which is below (morphologically speaking) the bend. On the
upper part of the mesentery, that which in the section is at-
tached to the stomatodzum, quite a different structure is found.
The mesogloea of the mesentery here branches into three pro-
cesses at its free edge, and these support the three lobes of the
mesenterial filament. The two lateral lobes (Flimmerstreifen)
(/s) are readily to be made out from the dark stain which they
take, and on tracing them onwards towards the lower edge of
the stomatodzeum the epithelium which forms them can be seen

No. 3.] MORPHOLOGY OF THE ACTINOZOA. 327

to pass, without any solution of continuity, directly into the
stomatodzal ectoderm. It would seem, then, that the lateral
streaks of the filaments are ectodermal in their origin, and
consist of two down-growths of the stomatodzum along the
sides of the mesenteries close to their free edge.

The third or median process of this portion of the filament
has a quite different structure. It does not at all resemble the
lateral streaks of the filament, neither is it similar to the median
streak (Nesseldriisenstreif), which is found lower down. It
appears to be more or less undifferentiated, and resembles the
endoderm in its structure rather than the ectoderm, although
the similarity is rather obscured by the presence in the general
endoderm of large numbers of Zooxanthellz, which are absent
in the median process. I believe it to be endodermal in its
nature, and to be the persisting endoderm which covered the
free edge of the filament before the lateral streaks made their
appearance, the endoderm at the sides of the free edge only
being pushed down or replaced by the ectodermal Flimmer-
streifen.

I have not considered it necessary to review the literature
upon the formation of the mesenterial filaments; this has been
sufficiently done in the papers of E. B. Wilson and H. V. Wil-
son, already referred to. It may be seen that my results as
stated above agree perfectly with those obtained by E. B.
Wilson (84), and fully bear out the suggestion advanced by
him that the Flimmerstreifen of the Hexactinian filament is
equivalent to the dorsal mesenteries of the Alcyonaria, and that
the median streak (Nesseldriisenstreif) is equivalent to the six
ventral filaments of members of that group. On the other
hand, I am utterly at variance with H. V. Wilson on the ques-
tion of the origin of both portions of the filament. Wilson
homologizes the simple unlobed filament of M/anzczua with the
entire trilobed filament of such a form as Aw/lactinia. He sup-
poses that in primitive forms the filament was simple and
unlobed, and that by a process of physiological differentiation
became divided into a central glandular and lateral respiratory
portions. This theory I cannot accept. The mere fact that J
believe in the origin of the two portions of the filament from
different germ-layers precludes its acceptance; but in addition
the manner of formation of the lateral streaks shows them to

328 McMURRICH. [Vou. IV.

have had an independent origin from the median streak. If
we may judge from the individual development, the median
streak was the first to make its appearance in the ancestors of
the Actinozoa, and they remained with such simple glandular
filaments, formed by a differentiation of the endodermal cells
along the free edges of the mesenteries, for some time. Later,
the ciliated respiratory portions of the mesentery developed by
a down-growth of the stomatodzeal ectoderm, and were at first
confined to the upper portion of the mesentery where the gland-
ular streak was not developed, but later, extending their limits,
they came to overlap the glandular portion, and thus the fila-
ment became trilobed throughout a portion of its length.

CLARK UNIVERSITY, WORCESTER, MAss.,
October 14, 1890.

BIBLIOGRAPHY.

(72) H. DE LacazE-DUTHIERS. — Développement des Coralliaires. — Arch.
de Zool. exp. et gin. 1., I]. 1872-3.

(73) A. KowALEwsky. — Untersuchungen tiber die Entwicklung der Coelen-
teraten. — Wachr. Gesellsch. Freunde Naturerkenntniss, Anthrop.,
und Ethnol. Moskow. X. 1873. [Abstract in Hoffman und Schwalbe’s
Fahresbericht for 1873. ]

(75) A. KowaLewsky.— Contributions a histoire du développement des
Actinies. — Fournal de Zoologie. (Gervais.) IV. 1875. [Transl. of
a portion of preceding paper by A. Giard.]

(75) E. HAEcKEL. — Die Gastrula und die Eifurchung der Thiere. — Fe-
naische Zeitschr. IX. 1875.

(75a) E. HAEcKEL. — Arabische Korallen. — Berlin, 1875.

(77) E. Ray LANKESTER. — Notes on the Embryology and Classification
of the Animal Kingdom. — Quart. Fourn. Micr. Sci. XVII. 1877.
(79) O. and R. Hertwic.— Die Actinien. — Fenatsche Zettschr. XIV.

1879.

(80) E. JouRDAN. — Recherches zoologiques et histologiques sur les Zoan-
thaires du Golfe de Marseille. — Ann. des Sci. Nat. Ome Série. X.
1880.

(82) R. HERtTwIG. — Actiniaria of Challenger Expedition. — Reports on the
Scientific Results of the Voyage of H.M.S. Challenger. Zodlogy. VI.
1882.

(83) A. KOWALEwsky and A. F. Marion. — Documents pour V’histoire em-
bryogénique des Alcyonaires.— Ann. Museum d’Hist. Nat. de Marseille.
I. 1883.

(83) E. B. Witson.— The Development of Renilla. — Phzlosoph. Trans.
Roy. Soc. London, 1883.

No. 3.] MORPHOLOGY OF THE ACTINOZOA. 329

(84) A. KowaLewsky. — Entwicklungsgeschichte der Lucernaria. — Zoolog.
Anzeiger. VII. 1884.

(84) E. L. Marx.—Selections from Embryological Monographs. _ III.
Polyps. — Mem. Museum Comp. Zool. Cambridge. IX. 1884.

(84) E. B. WiLson. — The mesenterial filaments of the Alcyonaria. — MZ7tth.
a. a. Zool. Station zu Neapel. V. 1884.

(86) E. METSCHNIKOFF.— Embryologische Studien an Medusen. — Wien,
1886.

(87) A. GoETTE. — Entwicklungsgeschichte der Aurelia aurita und Coty-
lorhiza tuberculata. — Adhandl. zur Entwicklungsgesch. der Thiere. 1V
Heft. 1887.

(87) G. von Kocu. — Die Gorgoniden. — Fauna u. Flora des Golfes von
Neapel. Monogr. XV. 1887.

(88) H. V. Witson.—On the Development of Manicina. — Fourn. of
Morphology. V1. 1888.

(89) A. C. Happon.—A Revision of the British Actinia. Part I.—
Scient. Trans. Roy. Dublin Soc. Series Il. IV. 1889.

(89) J. PLAyFaAIR McMurricu. — On the occurrence of an Edwardsia stage
in the free-swimming embryos of Hexactinie.— Fohnus Hopkins Univ.
Circulars. VIII. 1889.

(89@2) J. PLAYFAIR McMurricu. — The Actiniaria of the Bahama Islands,
W.1.— Fourn. of Morphology. Wl. 1889.

(91) J. PLAyFAIR McMurricu. — The Gastea-theory and its Successors. —
Biological Lectures. Boston, 1891.

330 MCMURRICH.

EXPLANATION OF PLATE XIII.

Fics, 1-12 are of Metridium marginatum.

Fic. 1. Immature ovum taken from the ovary.

Fic, 2. Commencement of the first cleavage.

Fic. 3. First cleavage about half completed.

Fic. 4. Flattening out of spherules at conclusion of the first cleavage.
Fic. 5. Four-celled stage.

Fic. 6. Eight-celled stage.

Fic. 7. Early blastula; figure drawn from preserved ovum.

Fic. 8, Optical section of free-swimming blastula.

Fic. 9. Optical section of blastula, just before the breaking through of the mouth,
showing the depression at the posterior pole. Drawing made from preserved speci-
men.

Fic. 10, Optical section of gastrula stage ; drawn from a preserved and cleared
specimen.

Fic. 11. Section through blastula, 48 hours old. Camera. Zeiss, D, 2.

Fic. 12, Section through embryo, showing the formation of endoderm by delami-
nation. Camera. Zeiss, D, 2.

Fics. 13-18 are from Rhodactis Sancti- Thome.

Fic. 13. Portion of longitudinal section through anterior (aboral) pole of young
larva. ex=endoderm, mg=mesoglea, n= nerve-layer, ec=ectoderm. Camera.
Zeiss;, |, 2:

Fic. 14. Transverse section through larva, showing formation of second mesentery.
Camera. Zeiss, A, 4.

Fic. 15. Transverse section through larva, showing formation of second mesentery
by apposition of stomatodzeum to its line of formation. Camera. Zeiss, A, 4. The
details of the endoderm in this and the three succeeding figures are not completed.

Fic. 16. Transverse section, showing the formation of the third and fourth pairs
of mesenteries. Camera. Zeiss, A, 4.

Fic. 17. Transverse section through larva, showing reflection of stomatodzal ecto-
derm. Camera. Zeiss, A, 4.

Fic. 18. Transverse section through larva, showing ectoderm on outer surface of
stomatodatum, apparently having broken through the mesogloea. Camera. Zeiss, A, 4.

Fics. 19-24 are from embryos of Azlactinia stelloides.

Fic. 19. Transverse section of larva, showing the formation of the fifth and sixth
pairs of mesenteries. Camera. Zeiss, A, 2.

Fic, 20. Transverse section of mesenterial filament of the third pair of mesen-
teries of larva with only four pairs of perfect mesenteries. Camera. Zeiss, D, 2.

Fic, 21. Transverse section through a mesentery of the fifth pair, just below the
point where it separates from the stomatodzeum. sf=stomatodzeal ectoderm, en! =
endoderm of mesentery, ec = ectoderm of column wall. Camera. Zeiss, D, 2.

Fic, 22. Transverse section through same mesentery and the adjacent one of the
first pair taken 0.08 mm. lower down. Lettering as in preceding figure, except ms=
median streak of mesenterial filament. Camera. Zeiss, D, 2.

Fic. 23. Transverse section of mesenterial filament of the first pair, from larva
from which Fig. 20 wastaken. Camera. Zeiss, D, 2.

Fic. 24. Transverse section of mesenterial filament of mesentery of the first pair,
taken just below the bend of the mesentery, from larva with twelve perfect mesen-
teries. Lettering as in Fig. 22; except s = lateral streaks of mesenterial filament.

BMeisel, bik Foster

ON INTERCALATION OF VERTEBR.!
G. BAUR.

WueEN we have two nearly related animals, which have a
different number of segments, the question arises, What is the
origin of this difference? There are two possibilities in regard
to the two forms; one may be derived from the other, either
by increasing or decreasing the number of segments. In each
of these two cases we have different possibilities. In the case
of increase, the new segments may either be developed by inter-
calation, or by division of the original segments, or by addition
at the caudal end of the animal. In the case of decrease, we .
can think of excalation, union, or loss of segments at the
caudal end.

Most morphologists are inclined to the opinion of addition
or subtraction of segments at the distal end. But there are
others, like Jhering and Albrecht, for instance, who adopt
intercalation.

Let us now consider some cases. In most of the higher
vertebrates we have a sacrum which is united to the vertebral
column by sacral ribs: this sacrum establishes a more or less
fixed point in the vertebral axis. We distinguish presacral
and postsacral, or caudal vertebra. The increased number of
presacral vertebrae may be produced, either by intercalation of
new vertebre, or by movement of the sacrum backwards.
The decreased number may be the result of excalation, or
of the movement of the sacrum forwards.

Cases of the movement of the sacrum have very often
been described, and quite a number have come under my
own observation. Positive cases of intercalation, however,
have seldom been recorded.

Fiirbringer? discusses the question in the chapter, “Uber die
Verschiebung (Wanderung) der Extremitaeten,” of his great

1 Paper read before the American Morphological Society, Boston, Dec. 29, 1890.
2 Fiirbringer, Max: Untersuchungen zur Morphologie und Systematik der Voegel,
Amsterdam, 1888, Vol. II, pp. 972-991.

ee

332 BAUR. [Vou. IV.

work on the morphology of birds. He speaks also about
the case of intercalation published by Albrecht (in the Bull.
Mus. Roy. d’Hist. Nat. de Belgique, II, 1883). Page 975 he
says :—

“Fir die Verschiebung der Gliedmassen und die dadurch
beeinflusste Umbildung der Wirbel sind manche Argumente
bisher beigebracht worden, fiir die Inter- und Excalation habe
ich sie dagegen bis jetzt vermisst.

“ Albrecht statuirt Segmentvermehrungen durch Theilungen
der Ursegmente, entscheidet sich somit fiir die Annahme einer
Interpolation in embryonaler Zeit. Nach meinen bisherigen
Anschauungen hatte ich gegen die Moglichgeit einer Interpola-
tion nichts einzuwenden, aber bei dem volligen Mangel irgend
welches sie stiitzenden Argumentes konnte ich ihr nur eine
rein theoretische oder begriffliche Bedeutung, aber keine Wahr-
scheinlichkeit zuerkennen.”

Albrecht examined a skeleton of Python seb@ in the Museum
at Brussels. Between the 194th and 197th vertebrz, he found
a complex of anchylosed vertebrz: this contained on the right
side two vertebral elements, one foramen intervertebrale, and
two ribs; on the left side three vertebral elements, two foramina
intervertebralia, and three ribs. Albrecht considers the super-
numerary vertebra on the left side as intercalated. Fiirbringer,
on the other hand, is inclined to regard it as the result of some
pathological process, which reduced the right half of the verte-
bra. He says: “ Welcher Art und Veranslassung dieser patho-
logische Process gewesen und wann er stattgefunden, kann ich
natiirlich nicht entscheiden, halte auch diese Frage fiir eine
nebensachliche ; aber die namentlich auf der Dorsalansicht sehr
auffallende unregelmassige Schraegstellung des fraglichen Halb-
wirbels, der Umstand, dass er kein reiner Halbwirbel ist, sondern
im Bereiche des Korpers und im Bereiche der Neurapophysen
noch Rudimente der rechten Seite darbietet, endlich die anchy-
losirung der 2! Wirbel, wahrend die Wirbelsaule sonst sehr
ausgelildete Gelenke besitzt machen es mir unmoglich hier an
einen normalen Fall von Wirbelinterpolation zu denken.”

I think I shall be able to show that Albrecht was correct in
his interpretation. Already Koken, in writing a review of
Albrecht’s paper (in one of the numbers of the ‘Neues Jahrbuch
fiir Mineralogie,” which I have not at hand), states that he has

No. 3.] ON INTERCALATION OF VERTEBRZ. 333

observed similar cases as Albrecht’s in 7vopidonotus. R. Owen!
has also described a case of the same nature in the skeleton of
a Python tigris, of which he says: “ Anchylosis has occurred
between the 148th and 14oth vertebrae. The 166th and the
167th vertebrae have been more completely and abnormally
blended together, so as to seem but one vertebra on the left
side, where that half of the neural arch and spine have com-
pletely coalesced, whilst on the right side each vertebra supports
its own rib. A similar abnormality occurs between the 184th
and 185th vertebrz.”

If intercalation takes place at all, we ought to expect traces
of it in such forms as show a great increase in the number of
vertebree, for instance, snakes, different groups of lizards, and
Plesiosaurs. In looking over such material I have found some
additional cases.

In a specimen of Pelamis bicolor (No. 763, Yale University
Museum) I find the 212th vertebra simple on the left side,
double on the right side; it bears one rib on the left, two ribs
on the right side. Exactly the same condition I have observed
in a cervical of a Plesiosaur, Czmoliasaurus plicatus, No. 48,001 of
the British Museum, London. One side has one, the other two
ribs. Mr. R. Lydekker? makes the following remark about this
vertebra : —

“The centrum of a small and malformed cervical vertebra,
from the Oxford Clay near Oxford. This specimen is imma-
ture, and on one side is divided into two portions, each with its
distinct costal facet.”

I do not doubt at all that in all these cases we have examples
of incomplete intercalation; and I am convinced that this
intercalation is the result of the division of myotomes, as ex-
pressed by Albrecht. The possibility of such intercalation I
have expressed already in No. 306 of the “ Zoologischer An-
zeiger,’’ 1889, where I said: “Ich glaube, dass eine Verschmel-
zung oder Spaltung von Myomeren auch schon wahrend der
Anlage des Embryo moglich ist. Mein Freund A. Bohm in
Miinchen theilte mir mit, dass er verschiedene Anzeichen von
Spaltung von Myomeren beobachtet habe.”

1 Descriptive Catalogue of the Osteological Series contained in the Museum of the
Royal College of Surgeons of England, Vol. I, p. 123, London, 1853.

2 Lydekker, Richard: Catalogue of the Fossil Reptilia and Amphibia in the British
Museum, Part II, London, 1889, p. 238.

334 BAUR. [VOE: AV:

Of course the direct proof of splitting of myotomes could only
be given by the study of the living embryo, which, if possible
at all, is exceedingly difficult. If we have the complete seg-
ments in the embryo or in the adult animal, we cannot decide
whether they consisted originally of a single myotome, or
whether they are the result of division. For instance, in the
peculiar consolidation of vertebrze in snakes, mentioned by
Owen, and also observed by me at different times, we do not
know whether this consolidation is the result of real union
of two segments, or of partial division of one segment. At
least we may adopt one explanation just as well as the other.
I am inclined, however, to accept partial division in these
cases. A great number of observations is necessary to see
in what relations the frequency of such consolidation stands
to the increased number of segments; in other words, whether
such complexes are more frequent in animals with the number
of segments considerably increased, as it appears to-day, than
in such which have a relatively small number.

By very careful study and comparison of the structure of the
single vertebrae, however, it is sometimes possible to determine
whether we have a case of intercalation or not. It is well known
that the typical number of the presacral vertebrze in the living
Crocodilia is twenty-four ; there are two sacrals: the first caudal
is peculiar, by being biconvex. In a specimen of Gavialis
gangeticus 1 found twenty-five presacral vertebrz.! As in all
living Crocodiles the first caudal vertebra is biconvex; but in
this case it is the twenty-eighth, in the other the twenty-
seventh. Is it not evident, therefore, that at some place be-
tween the occipital condyle and the first caudal a new vertebra
has beeen inserted? By careful comparison I find that this
new vertebra has been intercalated between the ninth and
tenth.

A similar case I have observed in Heloderma. In this
lizard the first caudal vertebra has also a peculiar character.
The small rib connected with it is perforated; this perforation
is absent in the other vertebra. By this peculiarity the first
caudal vetebra is distinguished from the rest. Four specimens
of Heloderma show the following condition. In the first speci-

1G. Baur: Anzahl der praesacralen Wirbel der Crocodilia. Zool. Anz. No. 238,
1886.

No. 3.] ON INTERCALATION OF VERTEBRE. 335

men the first caudal is the thirty-sixth (Heloderma horridum) ;}
in the second, the thirty-seventh (Heloderma horridum, observed
by me); in the third, the thirty-eighth? (Heloderma suspectum) ;
in the fourth it is the thirty-ninth (Heloderma suspectum, Clark
University). We have, therefore, four variations in four speci-
mens. There seems to me very little doubt that this difference
in the number of vertebra is produced by true intercalation.3

By these few but characteristic examples I believe to have
given positive evidence that intercalation of segments takes
place in vertebrates. I do not doubt that further examination
of more material will bring out more cases. What is necessary
to do, is to examine a great number of specimens of the same
and allied species of such forms as show an unusual increase
of segments, like the Varanidee, Scincide, Anguidz, Amphis-
beenide, Snakes, and so on. The embryology of such forms
would probably give important evidence, because we may expect
to find indication of myomeric division.

My opinion ts that in the increase of the number of segments
not only in vertebrates, but also in invertebrates, intercalation has
played a much greater rile than is generally admitted. At the
same time I admit addition of segments at the distal end, as well
as occasional sézgh¢ migration of the shoulder girdle and pelvis
in both directions. The question is an important one, and
I hope that some embryologist may take up the subject for
further study.

This question of increase of the number of segments is a
very interesting one from the standpoint of the evolutionist.
It is evident that intercalation can only take place in the very
early life of the embryo, when the myotomes are forming, and
that it is absolutely impossible that new segments can be
intercalated through any effort and exercise of the animal

1 Troschel, F.H.: Uber Heloderma horridum. Wiegm. Arch. f. Naturg., Jahrgang
19, Vol. I, Berlin, 1853, pp. 294-315. I am indebted to Mr. S. Garman, Cambridge,
for looking up this reference for me, the Journal not being at hand.

2 Shufeldt, R. W.: Contributions to the Study of Heloderma suspectum. Proc. Zool.
Soc., London, 1890, p. 214 (the peculiar character of the first caudal is not mentioned
by Dr. Shufeldt).

8 Whether this intercalation is produced by division of myotomes, or by addition
of myotomes from the beginning, I do not know; both ways are possible. I am
convinced that in a great number of cases intercalation takes place by adding new
segments in the embryo without division,

330 BAUR.

during the later period. The disposition to increase the number
must be, therefore, in the germ itself. The question is then, Is
the increase of the number of segments “accidental,” and are
forms which show this “accidental” increase of segments pre-
served through natural selection; or is this tendency to increase
the number of segments common to all individuals, not appear-
ing accidentally, but rather as the result of a definite stimulus ?
I can only adopt the latter possibility.

But how is it in this case? Many animals increase the length,
that of the neck for instance, not by addition of new segments,
but simply by elongating the single segments present. For
instance, the Giraffe among mammals, Chelys, Chelodina, Diro-
chelys, Hydromedusa among the tortoises. How is it that the
Plesiosaur elongates its neck by adding new segments, and the
Tortoise by stretching the single segments? Here we are before
a difficult question. It is clear the long neck of the Giraffe
and of some of the Tortoises develops during the evolution of
the animal, but this tendency must be potentially in the germ.
It is again the tendency to elongate the neck which is impressed
on the germ, but in a different way. An interesting case of
addition of segments is offered by the Sirenians. The Sirenians
are the only mammals, the Cetaceans excepted, which show an
increase of phalanges (four instead of three); a fourth phalange
is added in some digits at the distal end, but this takes place
during the postembryonic life of the animal, the embryo having
only three phalanges.

I am not able at present to give any explanation for these
phenomena, but I thought it worth while to mention them in
this connection.

NEUROBLASTS IN THE ARTHROPOD EMERYO;

WILLIAM M. WHEELER.

As some months will probably elapse before I shall be able
to complete my study of AXwphidium ensiferum, I take advantage
of the opportunity kindly offered me by Professor Whitman to
publish a few observations bearing on the development of the
nervous system of Arthropods.

In A7vphidium the nervous system begins to make its appear-
ance before the elongate blastopore closes and the fold of the
amnion and serosa envelops the head —at a time, therefore,
when the embryo is still in what I have called the “slipper”
stage. There may then be seen, scattered over the surface of
both procephalic lobes a number of pale circular spots, each
surrounded by a ring of the small cells forming the ventral
plate. The embryo still consists of a single layer of elements
except beneath the blastopore, where the meso-entoderm is
differentiating. In sections the pale circular spots are seen
to be due to centres of proliferation, each consisting of a few
enlarged cells. Surface study is rendered difficult as soon as
the envelopes have enclosed the head, and it is not till the
embryo has grown and stretched the overlying amnion that
the surface again becomes clearly visible. In the meantime
the nervous system must be studied in sections.

Beginning with the ventral nerve-cord, I find that the
ganglia arise as paired thickenings of the ectoderm in the
manner so often described for Arthropods. The thickenings
are the lateral cords (“Seitenstrange’’)—the region between
them, marked on the surface by a groove, is the median cord
(“ Mittelstrang”’ of Hatschek). Carefully made transverse sec-
tions through either lateral cord are seen to consist in early
stages of two kinds of ectoderm elements: smaller cells with
rather deeply stainable elongate oval nuclei and four large suc-
culent cells with pale spherical nuclei. These four large cells,
the zeuroblasts, lie side by side just beneath the smaller ecto-

337

338 WHEELER. [Vot. IV.

derm elements in a plane parallel to the surface of the yolk and
the outer surtace of the body. As every transverse section
through the ventral nerve-chain in this stage contains approxi-
mately four of these huge cells, I conclude that the embryo
presents eight longitudinal rows of neuroblasts. The rows
extend from the mouth to the anus, and are most clearly differ-
entiated anteriorly. When sections of the head are examined
it is found that the sporadic clusters of cells forming the pale
surface spots of the younger embryo have arranged themselves
as neuroblasts in eight rows in either procephalic lobe. Four
of these rows—those on either side of the stomodzeal orifice
and directly continuous with the rows of the ventral cord —
give rise to the brain proper, while four shorter lateral rows
form the optic ganglion. The retinal ganglion is delaminated
very early from the outer edge of the procephalic ectoderm.
From the first its elements do not resemble the neuroblasts
and appear to multiply irregularly.

<>
ey) (<4)
eS Pi“ 4 <2

4 = 7, S ey
TD
HES x)
LE ae Sk a> “

I give a slightly diagrammatic figure of a cross-section
through the posterior portion of a basal abdominal ganglion.
It is taken from an embryo somewhat older than the one just
described. The two lateral cords have been made to bulge
out beyond the general surface of the ectoderm by the pro-
liferation of the neuroblasts (S'-~S*), each of which now sur-
mounts a pillar of smaller elements. These, the future ganglion
cells, are budded off from the inner ends of the neuroblasts
and become cuneiform from mutual pressure. There is a
marked contrast between the terminal and daughter cells,
not only in size and shape, but also in intensity of stain.

No. 3.] MEVROBLASTS IN THE ARTHROPOD EMBRYO. 339

The cytoplasm of the neuroblasts is very pale and finely
granular, and their nuclei are pale and refractive, while the
daughter cells stain much more deeply throughout and thus
resemble the elements of the integumentary ectoderm (Z£).
The polar axes of the neuroblast spindles seem always to
be directed at right angles to the surface of the body in
embryos of this stage. The ‘“Punktsubstanz”’ (P) makes its
appearance in the bases of the lateral cords, which in the
section figured are separated by a pyramidal mass of cells, the
median cord. Heading this mass of cells is another neuroblast
(J7) apparently pushed below the surface by the bulging of the
lateral cords. Owing to the shape of the space to which the
terminal cell is confined, its daughter cells cannot arrange
themselves in a straight column, but lie heaped up somewhat
irregularly. While the four lateral neuroblasts represent the
cross-section of four continuous longitudinal rows of cells, the
median cord neuroblasts are isolated elements which arise
intersegmentally, but soon move forward between the two
connectives, and finally come to lie just back of the posterior
commissure in each segment. At a still later stage each is
incorporated together with the mass of cells to which it has
given rise in the posterior part of a ganglion. Inasmuch as
each postoral ganglion appears to be provided with one of
the median cord cells, these elements constitute a ninth un-
paired and interrupted row of neuroblasts extending like the
lateral rows from the mouth to the anus. The interruptions
occur at the points where the commissures are formed and at
the intercommissural spaces. I have not yet been able to find
median cord neuroblasts in very young embryos, but I do not
doubt that they differentiate from the primitive ectoderm at
the same time as the neuroblasts of the lateral cords.

The brain and optic ganglia arise as strings of cells budded
off from the sixteen rows in the same manner as the ventral
ganglia originate from the eight rows of neuroblasts.

In a stage succeeding the one figured, the daughter cells (CG)
of the neuroblasts themselves divide, the axes of their spindles
lying at right angles to the axis of the mother spindle. Thus
each neuroblast becomes the end of a pillar consisting of from
two to four rows of ganglion cells which have arisen by division
of the elements in the original single row.

340 WHEELER. [Vou. IV.

Like all the cells which they produce, the neuroblasts are
finally enclosed by the outer neurilemma. By the time the
embryo has undergone revolution and has enveloped nearly the
whole of the yolk, the ganglia are so crowded with cells that
the neuroblasts have very little space in which to proliferate.
They continue, however, to bud off new cells, notwithstanding
the short rows thus formed are forced to lie parallel instead of
at right angles to the surface of the ganglion. During this
process the neuroblasts gradually grow smaller, till they finally
become indistinguishable from their progeny, the ganglion cells.

I would emphasize the definite number and arrangement of
the neuroblasts in X7phidium, because I believe the eight rows
of the lateral cords to be the homologues of the two rows of
cells derived from the neuro-teloblasts in Annelids. The fact
that there are only two rows in Annelids, whereas there are
eight in X7phidium, can constitute no very serious obstacle to
this homology, since we have only to suppose that in the anne-
lid-like forms from which the Tracheates are descended, the
pair of primitive neuro-teloblasts divided twice to form four
pairs of proliferating centres for the longitudinal rows of neuro-
blasts. It is not improbable that forms with more than a single
pair of neuro-teloblasts may yet be found among existing Anne-
lids. The homology here advocated is rendered more probable
by the fact that the embryo Xzphzdium does not conform to
Graber’s “law” of metameric segmentation, but, like Myrio-
pods and Annelids, grows by the intercalation of segments in
front of the anal plate. The neuroblasts in the proliferating
zone at the end of the body are probably true teloblasts, since
they bud off the neuroblasts of the different segments. They
may be called primary neuro-teloblasts to distinguish them
from the secondary neuro-teloblasts that produce the strings
of ganglion cells.

It is more difficult to account for the interrupted series of
median cord neuroblasts. Do they represent the remnants of
a single unpaired row or of a pair of rows? Are they seconda-
rily derived from one or both of the inmost rows of lateral
cord neuroblasts (S"), or have they had an independent origin ?
These are questions which I cannot yet answer. The interrup-
tions in the median series are probably due to the formation
of the commissures; primitive conditions prevailing only in

No. 3.] WEUROBLASTS IN THE ARTHROPOD EMBRYO. 341

the intersegmental region where the original ectodermic layer
has been less affected by neural differentiation. The absence
of median neuroblasts in front of the mouth is easily accounted
for by the high degree of concentration and the lack of inter-
segmental spaces in the three pairs of ganglia that constitute
the brain.

The development of the ganglia from neuroblasts is by ne
means peculiar to Viphidium among insects ; it is quite as dis-
tinct in other Orthoptera. In the common locust (Melanoplus
femur-rubrum) 1 can detect the same number of rows of prolif-
erating cells both in the ventral nerve-cord and in the optic
ganglia and brain. In A7phidium and Melanoplus all stages
in the proliferation of the neuroblasts may be observed in a
single embryo; in the brain the columns of cells attain a con-
siderable length before the neuroblasts in the terminal segments
have budded off more than one or two cells.

Blatta germanica presents essentially the same conditions as
Melanoplus.

The “ganglioblasts” of Doryphora decemlineata described in
a former paper are the same as the cells which I now call
“neuroblasts.” Re-examination of my preparations has con-
vinced me that in this insect also the number of rows is
approximately four for either lateral cord. I fail, however,
to detect a definite string of cells extending inward from each
terminal cell; still there can be no doubt that the irregularly
arranged ganglion cells are budded off from the neuroblasts.
Doryphora, a highly modified form even among Coleoptera,
would be expected to present in the development of its nervous
system characters less primitive than those of the Orthoptera.

Neuroblasts in insect embryos have been described and fig-
ured by other investigators, but no one, to my knowledge, has
called attention to their definite number and to their striking
similarity to Annelid neuroblasts. Korotneff, in his paper on
Gryllotalpa,! figures four neuroblasts in the lateral cord in
two cases (Pl. XXX, Fig. 60; Pl. XXXI, Fig)! On) SU Ete
also describes the manner in which they differentiate from the
primitive ectoderm and proliferate to form ganglion cells (p.
589). His figures are taken from embryos too old to show

1 Zeitschr. f. wiss. Zool., Bd. 41, 1885.

342 WHEELER. [Vor LV.

the single band of cells proceeding from each neuroblast; the
daughter cells having divided, so that each teloblast surmounts
three or four rows of small cells. Graber, in a recent paper,!
calls attention to the huge cells that give rise to the ganglionic
thickenings in the Dipter Zwacz/za and in the Coleoptera Lznxa
and Melolontha.

Of great interest in this connection are the figures given by
Dr. Patten in his last paper.2- The nervous system of the young
scorpion embryo is represented as covered with peculiar “ sense-
organs,” which, strange to say, are arranged in four irregular
rows in either lateral cord of the ventral nerve-chain. A sin-
gle large ‘“sense-organ”’ occurs in the middle of the median
cord portion of each segment, and hence nearly corresponds in
position to the median neuroblast of X7zphidium after it has
moved forward between the connectives. The “sense’’-spots of
the scorpion are very similar to the spots which in the proce-
phalic lobes of the Locustid mark the small areas where neuro-
blasts are differentiating from the primitive ectoderm. Patten
gives no description of these “sense-organs”’ in his text, and
the cut representing a transverse section through a ventral
ganglion is on too small a scale to show the surface cells dis-
tinctly. Laurie? figures a transverse section through the pro-
cephalic lobes of an embryo in a stage that I take to corre-
spond to one of the young embryos figured by Patten. In this
figure (Pl. XV, Fig. 25) the ectoderm shows in the arrange-
ment of its nuclei a peculiar unevenness which may account
for the circular spots figured by Patten, but which would seem
to have no connection with the differentiation of neuroblasts
and ganglion cells. Nevertheless, the striking agreement in the
number and arrangement of the neuroblasts of X7phzdium and
the ‘‘sense-organs”’ of the scorpion, a form in most respects so
far removed from the Insecta, warrants the suggestion that the
so-called sense-organs in the Arachnid are, like the circular
spots in the procephalic lobes of X7¢phidium, simply the expres-
sion of an early differentiation of the primitive ectoderm into

1 Vergleichende Studien, etc. Denkschrift. d. k. Akad. d. Wiss. Wien, Bd. LVI,
1889, p. 48.

2 Quart. Journ. Micr. Sci., XX XI, 1890.

3 Quart. Journ. Micr. Sci., Vol. XXXI, 18go.

No. 3.] MEUVROBLASTS IN THE ARTHROPOD EMBRYO. 343

integumentary cells (dermatoblasts) and nerve-cells (neuro
blasts).}

1 Since making the above observations, my attention has been called to a recent
article by Viallanes (‘‘ Sur quelques points de Vhistoire du développement embryonnaire
de la Mante religieuse” (Jantis religiosa), Revue Biolog. du Nord de la France,
Tome II, No. 12, September, 1890). The description of the J/anéis brain as given
by this investigator agrees very closely with the results obtained from a study of
Aiphidium, Reserving a consideration of the segmentation of the brain and optic
ganglia for future publication, I here quote Viallanes’ remarks on the development
of the ganglia from neuroblasts (“cellules gangliogénes””): “ Le premier lobe proto-
cérébral constitué primitivement par une seule assise de grosses cellules (cellules
gangliogénes), ne tarde pas a multiplier ses éléments. Les cellules gangliogénes
entrent en division et produisent 4 leur face profonde de nouveaux éléments; ces
derniers, se divisant eux-mémes trés rapidement donnent naissance a de petites
cellules, pauvres en protoplasma, 4 noyau remarquablement chromatique, qui se
multiplient elles-mémes, et que nous pouvons dés maintenant désigner sous le nom
de cellules nerveuses ou ganglionnaires. Grace a cette prolifération, le premier lobe
protocérébral, de simple assise cellulaire qu’il était au début, est devenu un massif
cellulaire fortement convexe du cété de la plaque optique et formé maintenant de
deux couches bien distinctes, l’une externe (limitant la face convexe du lobe), formée
d’une seule assise de grandes cellules gangliogénes, l’autre interne, formée d’un
puissant amas de petites cellules nerveuses.”

CLARK UNIVERSITY, WORCESTER, MASs.,
January 15, 1891.

THE PELVIS OF THE TESTUDINATA, WITH
NOLES ON THE EVOLUTION OF tHe
PELVIS IN GENERAL.

G. BAUR.

TuaT Sphenodon is the most generalized living reptile there
cannot be any doubt to-day. Let us take the pelvis of Spheno-
don as a type of reptilian pelvis, and see in what relations the
pelvis of the Testudinata and other vertebrates stands. The
pelvis of Sphenodon consists of three ossified elements on each
side, the ilium, pubis, and ischium, which all meet in the acetab-
ulum. The ilium is a simple bone; pubis and ischium are more
complicated, each one consisting of two branches. These con-
ditions are best seen in the figure. The inner branch of the
pubis may be called extopubis ; the outer branch, ectopubis (pec-
tineal process aut.); the inner branch of the ischium, e7to-
ischium ; the outer, ectoischium (metischial process or tuberosity
of the ischium, Huxley }).

Fic. 1.— Sphenodon punctatum, Gray.

E, Epigastroid.

M, Mesogastroid.

H, Hypogastroid.

P, Pubis.

J, Ischium.

f; Foramen pubo-ischiadicum.
o, Foramen obturatorium.

The entopubes do not touch each other, nor do the ento-
ischia; the entopubes are also separated from the entoischia.
This separation is produced by a continuous rod of cartilage
placed in the middle line. This cartilage may be called the
gastral cartilage, gastrale, or gastrovd. The part in front of the
entopubis I call epigastroid (epipubis, part) ; the middle portion,

1 Huxley, Professor: On the Characters of the Pelvis in the Mammalia, and the
Conclusions respecting the Origin of Mammals which may be based on them. Proc.
Roy. Soc., No. 194, 1879, p. 405.

345

346 BAUR. [VoL. IV.

mesogastrotd,; the portion behind the entoischia, the hypogas-
troid (hypoischium, os cloacze). For the correct understanding
of the pelvis of vertebrates it is necessary to introduce these
terms. The foramina on each side between pelvis and ischium
are called foramina pubo-ischiadica; the small foramen in the
pubis, obturator foramen.!

It is very easy to derive all conditions seen in the pelvis of
the Testudinata from the condition of Sphenodon just de-
scribed.

We start from the Chelydroids as a general type. In the
young Macrochelys and Chelydra we have the gastral cartilage
complete and well developed. The epigastroid forms a very

Fic. 2.— Macrochelys Temminckit
(Troost. MSS.).

£, Epigastroid.
MW, Mesogastroid.
f/, Hypogastroid.
J; For. pubo-isch.
a, Entopubis.

6, Ectopubis.

c, Entoischium.
@, Ectoischium.

massive, long, anterior process ; the hypogastroid is very slen-
der, ending in a point behind. In old specimens the entopubes
touch each other in the middle line, separating epigastroid and
mesogastroid ; the entoischia also meet, separating mesogastroid
and hypogastroid. Ossification may take place from different
centres in the epigastroid, mesogastroid, and hypogastroid. En-
topubes and entoischia never meet, but are always separated by
the mesogastroid.

From the Chelydridz we reach the conditions seen in the
Cinosternide through the Dermatemydidz and Staurotypide.?

1 The obturator foramen may be placed completely in the pubis, or on the border
of the pubis, or in the pubo-ischiadic foramen.

? I am now able —thanks to Professor Riitimeyer — to give additional characters
for the Chelydride, Dermatemydidz, Staurotypide, based on the shoulder girdle, the
pelvis, and the conditions of the ninth and tenth dorsal vertebra.

Chelydride. No anterior process on entoscapula near acetabulum; no posterior
process on coracoid near acetabulum; mesogastroid well developed, separating com-
pletely entopubes and entoischia; no anterior process on ilium; rib-head of eighth

No. 3.] PELVIS OF THE TESTUDINATA. 347

In the two families mentioned we have about the same arrange-
ment as in the Chelydridz. In adult Cinosternidze we find the
three gastroids ossified, but very small; the epigastroid never

Fic. 3.— Dermatemys Mawit, Gray.

Pelvis from below, from a sketch of Professor Riitimeyer. -X, peculiar ossified pro-
cess, developed from the gastroid portion between the entoischia, also present in old
specimens of Chelydridz and Staurotypide.

reaches the extension seen in the Chelydride and Stauroty-
pide ; entopubes and entoischia nearly touch each other, being
only separated by the small, diamond-shaped mesogastroid. It
may be that in very old specimens the mesogastroid becomes
absorbed by entopubes and entoischia.

pleurale well developed; an entoplastron; generally no rib on tenth dorsal; number
of peripherals, 11.

Dermatemydide. An anterior process on entoscapula near acetabulum; a pos-
terior process on coracoid near acetabulum; mesogastroid well developed, separating
completely entopubes and entoischia; no anterior process on ilium; rib-head of eighth
pleurale present; a rib on tenth dorsal which is free from the eighth pleurale; num-
ber of peripherals, 11; an entoplastron.

Staurotypide (Claudius, Staurotypus). An anterior process on entoscapula near
acetabulum; a posterior process on coracoid near acetabulum; mesogastroid well de-
veloped, separating completely entopubes and entoischia; an anterior process on
ilium; rib-head on eighth pleurale absent; no rib on tenth dorsal; number of peri-
pherals, 10; an entoplastron.

Cinosternide. Like Staurotypidz, but mesogastroid reduced, not separating ento-
pubes and entoischia; no entoplastron.

348 BAUR. [Vo. IV.

In another direction, possibly from the Platysternide, devel-
oped the form of pelvis seen in the Emydidz and Testudinidz.
Among the more generalized forms of Emydidz, like JM/alaco-

E
Fic. 4.— Cinosternum pennsylvanicum, var.
£, Epigastroid.
#7, Hypogastroid.
u

clemmys, we find that entopubes and entoischia just begin to
touch each other. In the young animals there is a continuous
gastroid cartilage, only in older animals entopubes meet, and so
do the entoischia. In this stage we find a cartilaginous epigas-
troid, mesogastroid, and hypogastroid. The same condition I
have observed in Chrysemys picta. In the next stage entopubes
and entoischia unite, but the cartilaginous mesogastroid is still
present (Trachemys, Pseudemys, Terrapene, Clemmys, Gece-
myda). In Emys the mesogastroid becomes ossified, and forms
a slender element, pointed behind, and placed on the ventral
side on the anterior portion of the united entoischia. Cyclemys
is very near this stage. The epigastroid is always present in

E

Fic. 5. — Malacoclemmys geographica,
Tes:

£, Epigastroid.
MM, Mesogastroid.
H, Hypogastroid.

middle-aged specimens. It may calcify or ossify, or may become
absorbed by the entopubes. The hypogastroid I never found
ossified ; it is either cartilaginous, but very small, or absorbed

No. 3.] PELVIS OF THE TESTUDINATA. 349

by the entoischia. In the Testudinidz, or true land tortoises,
I have never seen an ossified gastroid. The whole gastroid
cartilage may become absorbed by the pubes and ischia in adult
animals, and it may happen that all elements co-ossify. This
I have also seen in a very old
specimen of Clemmys guttata.
In half-grown Testudinidz the
gastroid cartilage is complete,
but entopubes and entoischia
are already united.

Fic. 6. Fic. 7. — Trachemys elegans, Wied.
Testudo europea, L. E, Epigastroid.
£, Epigastroid. M, Mesogastroid.
H, Hypogastroid. fH, Hypogastroid.

The pelvis of Platysternum, the only representative of the
Platysternidz, seems to be of the pattern of the Chelydridz.
I have no specimen at hand. Boulenger says: “The pelvis is
intermediate between that of typical Emydoids, in which the
pelvis and ischium are in contact on the median line, limiting
two obturator foramens, and that of Chelydra, in which the two
bones diverge, and are connected by ligament. In Platysternum
the symphyseal branches of the pubis and ischium are parallel,
but yet connected only by ligament. I must remark here that
the former type of pelvis, ze. with two obturator foramens
separated by the union on the median line of the symphysial
branches of the pubis and ischium, occurs in all Testudinidz
(land and fresh-water) which I have examined, with the single
exception of Dermatemys, which belongs in this respect to the
Chelydroid type; also in the Cinosternidz, but not in the
Staurotypide, which belong to the latter type.” }

1 Boulenger, G. A.: Votes on the Osteology of the Genus Platysternum. Ann.
Mag. Nat. Hist., June, 1887, pp. 461-463, Pls. XVI, XVII.

350 BAUR. [Vo. IV.

From what I have said above, some of these statements
have to be modified. I believe also that what is called ligament
by Boulenger is really cartilage, and we would have, therefore,
in Platysternum, a cartilaginous mesogastroid separating ento-
pubes and entoischia. Boulenger does not mention the epigas-
troid and hypogastroid, but I conclude that these elements are
also present.

The condition of the pelvis seen in the Pinnata can also be
derived from that in the Chelydridz. In the living Cheloniidze
ectopubes are far separated from the ectoischia, but are con-
nected by the mesogastroid cartilage, which perhaps in very old
specimens may become ligamentous. The epigastroid is pres-
ent, rounded in front, and may become calcified or ossified in
old specimens ; the hypoischium is reduced entirely. Even in

E

Vv

Fic. 8. — Chelonia mydas, L.

£, Epigastroid.
M, Mesogastroid.
fH, Hypogastroid.

Oa)

B

pretty large specimens (length of bony coracoid 255 mm.) the
gastroid cartilage is still continuous. Between the entopubes
it is visible from above, between the entoischia from below, be-
ing always placed on the sharp angle in which these elements
meet. In some fossil Cheloniide, like Allopleuron, entopubes
and entoischia are nearer together. This leads to the condition
seen in the Dermochelyidz, in which entopubes and entoischia
seem to meet each other. I could not examine a fresh speci-
men of Dermochelys, and have to rely on Wagler’s! and Hoff-
mann’s? figures, Gervais’? not being at hand. According to
these figures the entopubes seem to touch the entoischia, but
there is some uncertainty about it. In Hoffmann’s figure the
left entopubis reaches the ischium, but the right one does not.

1 Wagler, Joh.: Matirliches System der Amphibien, 1830, Pls. I, Figs. 21, 22.
2 Hoffmann, C: Reféilien, in Bronn’s Klassen und Ordnungen, Taf. XI, f. 3.
8 Gervais, P.: Ostéologie du Sphargis Luth., Nouv. Arch. Mus. VIII. Paris, 1872.

INO.35] PELVIS OF THE TESTUDINATA. 351

In Wagler’s figures the cartilages are not indicated. It is im-
portant to re-examine young and fresh specimens. The pubo-
ischiadic foramina are very small; the mesogastroid seems to
be greatly developed. The entopubes have a strong posterior
branch, as in the Emydidz and Chelydride, and also some of
the fossil Cheloniida, and meet in the middle line. The ischia
are of the type of the Pinnata, and so is the ilium. Hoffmann
figures a very strong epigastroid. It seems that Dermochelys
developed from one of the more generalized Pinnata, which
still possessed the posterior branch of the entopubis of the
Chelydridze.

Fic. 9. — Trionyx.

£, Epigastroid.
M, Mesogastroid ligament.

In the Trionychidz we have a stage still more advanced than
that seen in the Cheloniide. The mesogastroid is only repre-
sented by ligament, and entopubes and entoischia are far
separated ; there is a well-developed cartilaginous epigastroid ;
the hypogastroid is exceedingly small. This condition must
already have existed in the forms of the Laramie Cretaceous,
in which the pelvis is not different from the living forms. That
the Trionychian ancestors had a pelvis with a cartilaginous
mesogastroid, and a pubis with a posterior entopubic process,
there cannot be any doubt. I also believe that the young or
embryonic Trionychidze will show the mesogastroid ligament
represented by cartilage.

PLEURODIRA.

It was the peculiar condition seen in the pelvis of the
Chelyiidz, which induced me to examine the pelvis of the Tes-

2152 BAUR. [ Wor, IV:

tudinata in fresh specimens. In Chelys, Hydraspis, and espe-
cially in Emydura,! the epigastroid is enormously developed,
more than in any other form. As it is well known, the ectopubes
and the branch connecting the entoischia with the ectoischia,
are co-ossified with the plastron, the ilium with the carapace.
In all the Pleurodira examined the epigastroid is united to the
plastron by ligament: this ligament is attached on the posterior
part of the suture between the hypoplastra. The epigastroid in
Chelodina? is only about half as long as in the other Chelyiide.
In the Podocnemididz and Sternothzridz it is well developed,
but not so elongated as in the Chelyiidee.

Fic, 10.— Chelys fimbriata, Schn.
Pelvis from above.
£, Epigastroid.
MM, Mesogastroid.
fl, Hypogastroid.

H

In young animals the gastroid cartilage is complete, but in very
old animals entopubes are united, and also the entoischia. The
hypogastroid is very small, or quite reduced ; but the mesogas-
troid is always cartilaginous, forming a long element, separating
entopubes and entoischia. In none of the Pleurodira the ento-
pubes touch the entoischia. In all living Pleurodira, as far as I

1 This fact was first seen by Hoffmann, who figured the long epigastroid in
Chelymys victoriac, in his Reptilien, Pl. VII, Fig. 6.

2 I may mention here the peculiar fact, that in all the specimens of Chelodina
examined by me there was only a single frontal, without any trace of a middle suture.
This is the only exception among Tortoises, so far as I know.

No. 3.] PELVIS OF THE TESTUDINATA. 353

know, the suture between the entopubes is short — in other
words, the entopubes are slender; in the Cryptodira and
Trionychia the entopubes are always much expanded. This
slenderness of the entopubes, which is also present in the ento-
ischia, seems to be produced by the co-ossification of ectopubes
and ectoischia with the plastron, for which purpose material of
the other elements has been used. In Compsemys of the
Amphichelydia, and in Plesiochelys we have a similar condition
of the pubis. In the Amphichelydia entopubes are widely
separated from the entoischia. In
Baéna we find the condition of
Chelydra, but the tendency is for
co-ossification of ectopubes and ec-
toischia with the plastron.

Fic. 11.— Zmydura Krefftit, Gray.

£, Epigastroid.
M, Mesogastroid.

It is now very easy to understand the evolution of the pelvis
of the Testudinata. The oldest Testudinata possessed a pelvis
very much like that seen in Sphenodon, but with the obturator
foramen between pubes and ischium ; the gastroid cartilage was
continuous; epi- and hypogastroids were present, and the meso-
gastroid separated the entopubes and entoischia. This form
was present in the Amphichelydia, and is still preserved in the
Chelydridz, Dermatemydide, Staurotypidae and Platysternidz.
The entopubes and entoischia gradually approached and united,
a. Cinosternide, 46. Emydidz, Testudinide; or became more
separated from each other, until they were represented by liga-
ment, a. Cheloniidz,! 4. Trionychia.

1 As I stated above, the marine ancestors of the Cheloniidze must have had a
pelvis more or less like the Chelydridz: from such a form the Dermochelyiidze
developed.

354 BAUR. [VoL. IV.

The ectopubes and entoischia remained separate, the posterior
branch of the entopubes became reduced; ectopubes and ecto-
ischia co-ossified with plastron : Pleurodira.

The pelvis of Sphenodon not only explains the pelvis of the
Testudinata, but also that of the Squamata and Ichthyosauria,!
in which entopubes and entoischia become far separated.

The pelvis of the Aétosauria, Belodontia, Megalosauria,? Ceti-
osauria,” is also easily referable to that of Sphenodon.

Iguanodontia? and Birds have become modified; in these
forms the entopubes have taken a parallel direction to the
entoischia. The ectopubis (pectinal process, praepubis) is more
or less developed, being directed forwards and outwards, in
the Agathaumidze (Ceratopsidz) the entopubes have become
exceedingly reduced, the ectopubes greatly developed. In
Birds the ectopubes are in a very rudimentary condition, and
may be placed functionally on the ilia.

But Sphenodon does not offer the most original form of a
pelvis in the higher vertebrates. The Proganosauria show con-
ditions still more primitive. In Palzohatteria, for instance, we
seem to have the gastroid cartilage very much more developed.
The entire space between pubes and ischia seems to have been
occupied by the gastroid cartilage. In Mesosaurus, an aquatic
Proganosaurian, we have conditions resembling the Plesiosauria.
In these forms the gastroid cartilage was probably interrupted,
a distinct mesogastroid being present.

The pelvis of the Theromora can be explained by the condi-
tion seen in Palzohatteria. The ossification of pubes and ischia
extended more and more, until the whole median portion of the
gastroid cartilage was absorbed, only leaving a small obturator
foramen. In this group pubes and ischia form a single broad
plate, separated by a suture in which also the obturator foramen
is placed. Still here we have the pubis turned forwards.

In Mammals the entopubes are turned backwards and united

1] may mention here that I have no doubt that the Ichthyosauria possessed, like
the Khynchocephalia, a small cartilaginous sternum. The whole morphology of the
shoulder girdle strongly supports this opinion.

? I consider, with Seeley, the Dinosauria as an absolutely unnatural group, which is
to be split into three distinct orders, — the Megalosauria, Cetiosauria, Iguanodontia,
corresponding to the Dinosaurian orders Teropoda, Sauropoda, Orthopoda. The
Dinosauria are comparable to the Enaliosauria, which contained also two entirely
different groups, the Ichthyosauria and Plesiosauria.

No. 3.] PELVIS OF THE TESTUDINATA. 355

to the entoischia, leaving a pubo-ischiatic foramen on each side.
We have to take a form of pubis as seen in Eryops! or Propap-
pus,? as related to the mammalian pelvis. I think it probable
that a form of pelvis as seen in Eryops, but not so much ossified,
was very near the original form of pelvis seen in Mammals. The
pelvis of Sphenodon seems to be in the same relation to that
of Palzohatteria as the pelvis of Mammals to that of an Eryops-
like form, in which ossification was not so much advanced.

We come now to the Crocodilia and Pterosauria. The pelvis
of these groups has always been a puzzle to the morphologist.
As it is well known, we find here two elements in front, which
do not take part in the formation of the acetabulum. There are
two opinions: most of the authors declare these elements as
the true pubes; others say the pubes are united with the ischia,
and the elements in question correspond to the marsupial bones
in Mammals, or the ypsiloid cartilages in Batrachia. Huxley is
of the opinion that the bones are homologues of the marsupial
bones in Pterodactyls, but that they represent true pubes in the
Crocodiles. ,

Before discussing this question, it is necessary to consider
the peculiar elements called marsupial bones, ypsiloid cartilages,
epipubes. The question is, are these elements portions of the
epigastroid cartilage, or are they developed independently from
it? The oldest Batrachia, the Proteida, do not have these ele-
ments ; they are well developed in Menopoma, Megalobatrachus
and many of the Urodela. According to Wiedersheim, who has
made very extensive ontogenetic researches on the pelvis of
vertebrates, this portion develops very late: “Ganz zuletzt
entsteht die Cartilago epipubis, und zwar in directem Zusammen-
hang mit dem allmahlich auftretenden Symphysengewebe.
.Dieselbe stellt ein oralwarts gerichtetes, zapfenartiges und
anfaenglich noch ginzlich ungegabeltes Gebilde dar.” — Azat.
Anz., 1889, Nr. 14, p. 435.

In a former communication, Wiedersheim was in doubt
about the homology of this element: “Uber die morphol-

1 Cope, E. D., Amer. Nat., 1884, Pl. III.

2 Lydekker, R., Catal. of Foss. Rept. and Amph., Part IV, London, 1890, p. 120,
Fig. 26.

8 Wiedersheim, R.: Zur Urgeschichte des Beckens. Ber. Naturf. Gesellsch., Freiburg
i. B, Vol. IV.

356 BAUR. [Vov. IV.

ogische Bedeutung der sogenannten Cartilago ypsiloides oder
epipubis halte ich mit meinem Urtheil vorderhand noch
zuriick.”’

In his latest paper on the subject (Anat. Anz., 4. Jan., 1890),
the epipubis is compared with the episternum: ‘‘ Wie dem Schul-
tergiirtel vorn das Episternum, so sitzt dem Beckengiirtel der
Urodelen und weniger Anuren vorn die Cartilago epipubis auf.
Sternum, Episternum und Cartilago epipubis der Amphibien
sind homologe Bildungen, vorauf bereits Gotte hingewiesen hat.”

Wiedersheim is inclined —to conclude from his text-books —
to consider these elements as the same as the epigastroid. I
do not believe that this is the case. The epigastroid is always,
also, in Necturus,! in which I could study its evolution on ma-
terial kindly given me by Professor C. O. Whitman, the anterior
portion of the gastroid cartilage, from which it is not developed
independently. I believe that the ypsiloid cartilages are of
secondary origin, developing independently from the gastroid
cartilage. The long epigastroid of the Chelyiidz is homologue
to the short epigastroid in Testudinide; homologue to the
anterior portion of the gastroid cartilage in Necturus; homo-
logue to that portion of the gastroid cartilage in Salamanders
and Dactyletra, to which the ypsiloid cartilages are connected.
I consider these cartilages as a later acquisition; they may
develop in any group, — Batrachia, Pterosauria, Monotremata,
Marsupialia. The question now is, how to name these elements.
The name epipubis is not good, having been used, also, for an
element which is not homologue. Ypsiloid cartilages and mar-
supial bones are also inappropriate names. I think it best to
introduce the name cartilago pyramidalis, to express the relation
of these elements to the musculus pyramidalis.

We have to return now to the Crocodilia and Pterosauria.
The peculiar condition of the pelvis of the Crocodilia exists
already in the Lias. This structure, therefore, is a very old
one, and I have some doubt whether embryology will give us
any help in this question. Perhaps it is possible to get some
light by examining the pelvis of the Pterosauria. I was always
inclined to consider the ischium of Crocodilia and Pterosauria
as this element alone, and the anterior bones as the true pubes ;

1 I may state here that in Necturus a distinct but very small sternum is present. I
found it in all specimens examined.

INO: 3.) PELVIS OF THE TESTCODINATA. 357

but I think now that this opinion is not correct, and that
we have to adopt the view of Leydig, Fiirbringer, and Seeley,
that the ischium contains also the pubis, and that the free ele-
ments in front are not the pubes. If we study the ischium of
a Pterodactyl, we find that it forms a very broad plate, which is
firmly co-ossified with the ilium ; between these two bones the
acetabulum is placed. In the ischium we find, according to
Zittel (Handbuch der Palacontologie, Vol. III, p. 786), a small
foramen below the acetabulum in Rhamphorhynchus and Dimor-
phodon. This foramen is said to be very much larger in Ptero-
dactylus antiqguus. In other species the “ischium”’ is divided
by the extension of this foramen into an anterior and posterior
branch. The bones called pubes are entirely excluded from the
acetabulum, and are connected with the anterior portion of
the “ischium.” They are either separated from each other,
expanded distally, or united and slender, having on each side
a lateral process. I think that this condition can only be ex-
plained in this way: The foramen in the broad “ischium” rep-
resents the obturator foramen; the pubis must therefore be
united with the ischium, of which it forms the anterior portion ;
the elements excluded from the acetabulum and connected with
the distal anterior end of the pubic portion of the ischium rep-
resent the cartilagines pyramidales.

From this standpoint I think we have to look also at the
pelvis of the Crocodilia. It seems to be more probable to con-
sider the bone generally called ischium as the united pubis and
ischium ; the so-called pubis as the cartilago pyramidalis. Palz-
ontology has to decide this question. It is possible that the
triassic ancestors of the Crocodilia (the Aétosauria and Belo-
dontia do not belong here) will bring some light. In some of
the Dicynodonts we find the pubis greatly reduced, and it is not
unlikely that the pelvis of the ancestors of the Crocodilia will
show some resemblance to such a form. By this of course I do
not wish to express that the Dicynodonts are in any way related
to the Crocodiles.

After having gone over the different groups of higher verte-
brates, we may now examine the history of the pelvis in the
lower types. Here I have little to add to the ontogenetic
researches of Wiedersheim, with whom I entirely agree in his
general results about the origin of the pelvis, which I can con-

358 BAUR. [Von IV.

firm after the study of the evolution of the pelvis in the most
primitive Batrachian Necturus.

From a form of pelvis as seen in Palzohatteria it is only one
step to the Batrachian pelvis; for instance, Necturus. Here
the gastroid cartilage is greatly developed, pierced only by the
small obturator foramen ; only the ischia are ossified ; the pubes
are not distinct from the gastroid cartilage. One step lower,
and we have the pelvis of the Dipnoa or Chlamydoselachus, only
represented by the gastroid cartilage.

Fic. 12. — Paleohatteria, Credner. Fic. 13. — Wecturus maculatus, Raf.
£, Epigastroid. £, Epigastroid.
M, Mesogastroid. M, Mesogastroid.
ff, Hypogastroid. ff, Hypogastroid.
Z, Ischium. J, Ischium.

Wiedersheim has expressed the opinion that the single median
gastroid cartilage (‘“unpaare ventrale Beckenplatte’’) in the Dip-
noa takes its origin from two halves. The pelvis of Necturus
is nearest to the pelvis of the Dipnoa, and in this form the
gastroid cartilage develops from two halves. In a specimen
25 mm. long the gastroid cartilage is represented by two lateral
portions ; in a little older stage the two halves are united, re-
main united, and extend now forwards, forming the long epigas-
troid portion.!

There is one element in the pelvis which has to be mentioned

1] may mention here the fact that Necturus, like Proteus (Wiedersheim), develops
its limbs like all the other Urodela examined, by budding. There is never an indica-
tion of more than four digits. This seems to be another proof for my theory that the
limbs of the Stapedifera have developed by budding, and that the ancestors of the
Stapedifera were not, as it is generally believed, polydactyle forms. Baur, G.: Bei-
trage zur Morphologie des Carpus und Tarsus, Jena, 1888.

No. 3.] PELVIS OF THE TESTUDINATA. 359

— the acetabular bone of the Mammalia.! This element, I think,
originated during the evolution of Mammalia, and may appear
in any group.

Mehnert? has expressed the opinion that a three-rayed pelvis
is the original form for all Amniota. This seems to be correct,
with the exception, perhaps, of the oldest Amniota, which seem
to have had a continuous gastral cartilage, as in Necturus for
instance, in which pubis and ischium became ossified. The his-
tory of the vertebrate pelvis seems to be this : —

1. Continuous gastral cartilage, extending between the femora.
Dipnoa, Selachia part.

2. Continuous gastral cartilage, in which the ischium devel-
oped as a separate ossification. Proteida.

3. Continuous gastral cartilage, in which pubis and ischium
appeared as separate ossifications. Batrachia part, Progano-
sauria part.

4. a. Pubic and ischiadic ossifications, extending over whole
gastral cartilage, Theromora, permian Batrachia part. Croco-
dilia, Pterosauria (?). 6. Gastral cartilage between pubis and
ischium disappearing ; appearance of foramina pubo-ischiadica ;
all other Amniota.

1 In the sketch of the pelvis of Dermatemys sent me by Professor Riitmeyer a
small acetabular bone is present.

2Mehnert, E.: Untersuchungen iiber die Entwicklung des Beckengiistels bet
einigen Saiigethieren. Morph. Jahrb., XV, p. 111, also Morph., Jahrb. XIII. Meh-
nert’s latest publication treats about the development of the pelvis in Amys orbicu-
laris, L. (Morph. Jahrb., Vol. XVI, Part 4). The account of the phylogeny of the
pelvis of the Testudinata given in this paper will modify some of the opinions
expressed by Dr. Mehnert.

CLARK UNIVERSITY, WORCESTER, MaAss.
February 6, 1891.

SPERMATOPHORES AS A MEANS OF HYPO-
DERMIC IMPREGNATION.

C. O. WHITMAN.

ALTHOUGH it is well known that spermatophores are of very
general occurrence among the invertebrates, and even among
many vertebrates, the assertion that, as a perfectly regular and
normal affair, in animals as highly organized as the leeches,
they represent an injecting apparatus, by means of which the
spermatic elements of one individual are forced through the body-
wall of another, at any point whatsoever, may appear almost in-
credible, even when supported by direct observation many times
repeated on different species. That such is certainly the case,
however, is very easily demonstrated, and any one can verify it
as often as he likes on almost any species of Clepsine that hap-
pens to be accessible. The observations to be presented in this
paper will make this fact abundantly evident.

But what becomes of these spermatic injections? Do they
ever reach the eggs and fertilize them? and if so, is this the
normal method of bringing the sexual products together? Al-
though I cannot affirm this as a positive certainty, the evidence
seems to me to fall but little short of being conclusive. I have
studied closely the habits of these leeches in Europe, Japan, and
America, and with especial reference to settling the question of
when and how impregnation is effected. Long-continued obser-
vation under most favorable circumstances has never given me
so much as a single indication that the genital pores are ever
united in the act of copulation. On the other hand, the plant-
ing of spermatophores on the surface of the body at any point
that happens to come first, is a common occurrence, which one
may often see repeated several times in the course of a few
hours by the same individual. I have followed the track of
the spermatozoa from the point of penetration to the coelomic
cavity in which the ovaries lie, but I have not pursued the
subject far enough to determine when or how the spermatozoa

pass through the wall of the ovisacs. That spermatozoa get
361

362 WHITMAN. [VoL TV.

into the ovisacs, and that fertilization takes place before oviposi-
tion, can be demonstrated by facts that admit of no doubt. The
passage through the wall of the ovisac—the only link in the
chain of direct evidence yet to be supplied —seems to be an
inference justified by all the known facts. Such a passage, in
the absence of any definite openings in the ovarian walls, would
have to be a forced one, depending upon the action of the
spermatozoa themselves. As these walls are represented by a
thin membrane which becomes enormously distended as the
eggs enlarge to maturity, the difficulty of penetration could not
be great; and the case of Peripatus and the Turbellarians seems
to show that spermatozoa are capable of effecting automatically
such a passage.

The view here taken finds a very strong confirmation in the
fact that precisely the same mode of copulation occurs in many
Turbellarians, in the Rotifers, and in Dinophilus, as the cita-
tions from Lang, Plate, and Harmer, given farther on, fully
show. The indications are that it occurs also in many oligo-
chzetous annelids, as well as in several genera of leeches besides
Clepsine, — perhaps in all the Rhynchobdellidz. The occur-
rence of such a mode of copulation among so many of the lower
bilateral animals appears not only to render explicable the pluri-
penial condition of many Turbellarians and some of the higher
worms, but also to clear up many puzzling observations in regard
to fertilization in animals that have no intromittent organ. It
is no longer necessary to suppose that the spermatophores found
attached to different parts of the body of Peripatus must be
carried through the vagina and up the uteri in order to reach
the eggs; and the discovery of spermatozoa projecting through
the ovarian walls of this animal, as reported by Moseley and
Sedgwick, ceases to be so complete a mystery. The difficulties
in the way of understanding how spermatophores can be of any
use when attached at a considerable distance from any genital
pore, and completely closed externally, as described by Vej-
dovsky for many annelids, may not be so great as they have
hitherto appeared.

The facts and bibliographical notes to be presented in this
paper are sufficient, I think, to make it at least probable that
the original function of the spermatophore was precisely what
it now is in the Turbellarians, the Rotifers, Dinophilus, and

No. 3-] SPERMATOPHORES. 363

Clepsine, — the injection of spermatozoa through the body-wall,
or hypodermic impregnation, as we may call it.

This mode of impregnation represents an important economi-
cal step in advance of the more primitive mode of setting the
seminal elements free in the water. The deposition of sperm-
capsules at random on any point of the external surface that
happens to be accessible at the moment of meeting, is improved
upon by restricting the act to a definite region, as one or more
segments of the clitellum in certain annelids, or the surface
around the external openings of the oviducts, as in the crayfish;
and, still further, by limitation to the edge of genital pores or
seminal receptacles, as in the copepods. The seminal reservoir
of the lobster, discovered by Bumpus,! marks an advance on the
conditions obtaining in the crayfish. .

The habit of discharging spermatophores directly into the
vaginal orifice, presupposing direct union of the sexual pores,
brings us to relations where such copulatory organs as we find
in the Gnathobdellidze would become useful. The penis is here
only an eversible end-piece of the vasa deferentia —a simple
tubular elongation of what in its simplest form would be repre-
sented by a pore.

Lang, who was the first to discover this mode of impregna-
tion in the Turbellaria, has thrown out the interesting sugges-
tion, that in these animals the penes may have been primarily
organs of attack and defence, which assumed secondarily the
office of copulatory organs. The grounds for the suggestion
may be seen from a citation to be introduced farther on. Such
a mode of origin, though it may be true for the Turbellaria,
does not invalidate the suggestion I have made for the Gnathob-
dellidz, except on the supposition that the penes represent
homologous organs in the two groups. Such a supposition
appears to be forbidden by the absence of these organs in the
lower leeches. It seems to me, therefore, altogether more prob-
able that in the higher leeches they have been independently
acquired, and that their evolution began after, or simultaneously
with, the establishment of the habit of true copulation.

The structural and ontogenetic resemblances of the penes in
the two groups cannot be taken as decisive proof of genetic
identity. The resemblances between the penis and the pharynx

1 The Embryology of the American Lobster. Journ. Morph., Vol. V. [ In press. ]

364 WHITMAN. [VoL. IV.

of a Turbellarian are of the same nature and equally close; but
no one would in this case be likely to mistake such resem-
blances for homologies.!

In view of the fact that the spermatophores of Clepsine were
discovered nearly half a century ago by Friedrich Miiller, and
described as a “phaenomenon cujus neque analogon inter reliqua
animalia repertre,’ and that they have since been observed by
Max Schultze, Leuckart, Leydig, and Schneider, it may appear
a little remarkable that their function should have so long
escaped detection. But when we consider how totally unpre-
pared were the minds of investigators for such a mode of
impregnation, we find no difficulty in understanding how it
comes to pass that the old belief still prevails, that all the
Hirudinea, penis or no penis, copulate in essentially the same
manner, and fecundate by conveying the spermatic fluid of one
individual (or of both reciprocally) directly into the female geni-
tal orifice of the other, thus placing it where it can pass unob-
structed into the so-called uterus, or, in the absence of such a
specialized part, into the ovarian sacs.

While this has been, and still is, the opinion generally re-
ceived, the possibilities of the eggs being fertilized after deposit,
of self-fertilization, and of parthenogenetic development, have
not been overlooked. The following notes and extracts are
designed to give the history of the subject, and to show what
questions have been left unsettled.

HIsToRICAL NOTES AND EXTRACTS RELATING TO THE
HIRUDINEA.

1. Clepsine complanata.

FRIDERICUS MUELLER. De Hirudinibus circa Berolinum hucusque observatis.
Berolini, 1844. pp. 33, 34.

“Cleps. complanatas, quamvis plurimas amoris tempore continua
>)

9

attentione observaverim, coéuntes nunguam observavi;* sed eodem

1 We are continually reminded that parallel development has been a much more
important factor in evolution than has generally been supposed. Similar bases,
similar needs, similar variations (because predetermined by like causes), guided by
parallel selective influences, which would be sustained by like environments, have
unquestionably resulted in numberless analogies which are usually allowed to pass as
evidences of genetic affinity.

2 Braun (Systematische Beschreibung einiger Egelarten, Berlin, 1805, p. 60) makes
the same observation.

No. 3.] SPERMATOPHORES. 365

fere ante ovorum partum tempore, quo Cleps. tessulatae coire solent,
singulare mihi in Cleps. complanatis sese obtulit phaenomenon, cujus
neque analogon inter religua animalia reperire, neque explicationem
dare valeo. Ad utrumque nimirum faciei ventralis latus ovgana singu-
laria filiformia, tres usque quinque corporis annulos longitudine
aequantia modo simplicia, modo ad basin usque bipartita exseruntur,
modo singula modo plura, modo in anteriore modo in posteriore cor-
poris parte. Haec per plures dies propendent, dum animal, alioquin
segnissimum, multo alacrius in vitro suo circumvagatur. Simul sub-
stantiae floccosae albidae magna copia secernitur, totam mox vasis in
quo servantur aquam turbidam reddens.

“ Inter phaenomenon hoc et propagationem relationem quandam exis-
tere, nullus dubito; plurimas enim Cleps. complanatas per tria semestria
domu observavi, neque vero alio unquam tempore ovgana haec filiformia
eas exserere vidi, dum amoris tempore ne una quidem inter triginta
et plures non exserebat. Praeterea his organis exsertis, ut in Cleps.
tessulata post coitum, ovariorum motus peristalticus, quo ova a funi-
culis suis solvuntur, incipit. Qwo vero munere fungantur haec organa,
nescio; anatomica quogue disguisitione nihil de eo docente. Nam cor-
pora quidem in crura dua reflexa divisa in tertio quovis annulo utrin-
que sub tractus intestinalis appendicibus latentes reperi, quibus replicatis
organa illa filiformia fortassis formantur; num autem cum testiculis,
quibus interjacent, aliave apparatus sexualis parte cohaereant, videre
haud contigit.

“Corpora similia etiam in C. verrucata, marginata, tessulata, inveni,
quamvis in nulla praeter complanatam specie organa filiformia exseri
vidi.”

MAX SCHULTZE. Zoologische Skizzen. Zeit. f. w. Zool. IV. 1853. pp. 186,
187.

“Hochst auffallend ist, dass bei /lanaria orva der Same in festen,
retortenformigen Spermatophoren verpackt iibergefiihrt wird, welche
man ein oder zwei an der Zahl nach der Begattung in dem beschriebe-
nen Raume [recept. sem.] findet. Die aus einer braunen, chitinartigen
Hiille bestehenden Spermatophoren platzen spater, und fallen nach
Entleerung des Inhaltes ganz zusammen. In diesem Zustande kann
man sie im ersten Friihjahr bei fast jedem Individuum dieser Species
sehen. Jch erinnere hier an die Beobachtungen von Fr. Miiller (Zet-

1 Nisi forte appendiculae generatrices a Morrenio (De Lumdr. terrestr. p. 77)
sic dicta, quas in Lumbrico terrestr. auctor laudatus, in aliis pluribus Lumbricinis
Cel. Dr. Hoffmeister et ipse observavimus, Cleps. complanatae organis filiformibus
analogae.

366 WHITMAN. [Von IV.

tung fiir Zoologie etc. von D’ Alton und Burmeister, No. 25, Juli, 1849),
welche ich selbst bestitigen kann, dass bei Clepsine complanata und
wahrscheinlich bet vielen Regenwiirmern die Begattung durch Sperma-
tophoren vermittelt wird.”

A very important observation was made by Filippi, and con-
firmed by Grube; namely, that individuals isolated for some
days before oviposition produced fertile eggs. This fact and
the absence of a penis suggested self-fertilization (Filippi), or
possibly an early internal fertilization (Grube).

F. DE FILIPPI. Lettera sopra l’ Anatomia e lo Sviluppo delle Clepsine. 1839.
Deis.

“Tutti i zoologi si accordano nel dire che le sanguisughe sono er-
mafrodite ; il che é vero anche per riguardo alle Clepsine ; ma in queste
gli organi de’ due sessi sono totalmente diversi che negli altri generi
della famiglia. E anche ammesso da tutti che le sanguisughe non pos-
sono fecondarsi da se, ma hanno bisogno del reciproco congiungimento
di due individui ; e qui mi occorre di far rimarcare un’ eccezione che ci
presentano le Clepsine. Infatti per le condizioni speciali de’ loro organi
generativi non pud nemmeno aver luogo in esse una fecondazione inte-
riore ; al che si aggtunga aver iol essempio di un individuo, il quale
mantenuto tsolato in un vaso di cristallo nella mia camera partori uova
che in seguito si svilupparono.”

ADOLPH EDUARD GRUBE. Untersuchungen ueber die Entwicklung der Clep-
sinen. K6nigsberg, 1844. p. II.

“ Gegen die Vermuthung, dass sich die Clepsinen ausserlich selbst
befruchten, spricht der Umstand, dass ich an den frischgelegten Dotter-
kugeln oder in der Eifliissigkeit nie Spermatozoen gefunden, was doch,
wenn die Samenfliissigkeit mit den Dottern zugleich ausgeschiittet wiirde,
kaum anders sein kénnte, gegen die Annahme in’s Besondere, dass sie
sich ausserlich gegenseitig befruchten, der Beweis, dass wenn ein mehrere
Tage abgesperrtes Individuum Eier legte, diese sammtlich zur Entwick-
lung kamen. Vielleicht also erfolgt die Begattung doch innerlich, aber
zu einer friihern Zeit? In diesem Fall miisste sie wenigstens 11 Tage
vor dem Eierlegen eintreten, denn so lange hatte ich eine Clepsine
abgesondert, deren Eier sich vollstandig entwickelten, und die Enden
der Samenleiter miissten dann sich umstiilpen und als Ruthen dienen,
oder die Spermatozoen gelangen in besondere Behalter geschlossen in
die weiblichen Genitalien.”’

No.3] - SPERMATOPHORES. 367

RUDOLF LEUCKART. Die Menschlichen Parasiten. I. 1863. pp. 675-680.

The place and mode of origin, and the form of the spermato-
phore were long ago correctly described by Leuckart. Speaking
of the terminal double sac of the male organs of the Rhynchob-
dellidz, he says : —

“ Der kornige Inhalt derselben dient zur Umhiillung des Samens und
formt denselben im Innern des Begattungsapparates zu einer gleichfalls
swethornigen plumpen Spermatophore, die bet der Begattung in die weib-
liche Oeffnung eingeschoben wird.” (pp. 675, 676.]

Leuckart assumes that in such forms as Clepsine there is
direct copulation, —that is, by union of the sexual orifices, —
and further, that fertilization is reciprocal (p. 673). But Leuck-
art has also seen spermatophores attached to the external pore
of the female organs ; for he says :—

“ Wo eine eigentliche Scheide fehlt (Bet den Riisselegeln), da wird die
Spermatophore in der weiblichen Oeffnung festgeklebt. Man sieht sie
hier noch halbe Tage lang nach der Begattung ansitzen, bis die Sper-
matozoen tn den LEterstocksschlauch iibergetrieben sind. Auch bet den
Arten mit Scheide findet man die Samenfiden spiiter im Innern der
LEterstock.” [p. 680. ]

2. Clepsine tessulata.

F. MULLER. Uber die Geschlechtstheile von Clepsine und Nephelis. Miiller’s

Archiv. 1846. p. 145.

“Mit dem Fusse festsitzend saugt jedes der beiden Individuen mit
dem Kopf sich an der Bauchseite des andern fest, worauf ez konisches
Organ aus der vordern Geschlechtsiffnung sich ausstilpt und in die
hintere des anderen Thieres eintritt; so vereinigt sitzen die Thiere meist
mehrere Tage lang.”

C. tessulata, if Miiller’s observation be correct, takes an excep-
tional position, which is all the more difficult to explain, as
C. marginata, and a number of other closely allied species found
in America and Japan, certainly all agree in the habit of
attaching their spermatophores to the exterior.

368 WHITMAN. [Vou. IV.

3. Clepsine var. Porte-chaine.
Mogq.-Tand. 1846. Pl. xiv. Fig. 5.

EBRARD. Nouvelle Monographie des Sangsues Médicinales. Paris, 1857.

“Aux derniers jours du mois de mai, en 1854, j’ai trouvé deux glossi-
phonies (variété dite Porze-chaine) qui étaient accouplées. Elles étaient
fixées, téte 4 téte, 2 la face inférieure d’une pierre par leurs ventouses
qui étaient trés-rapprochées. Leur corps était contourné de telle sorte
qu’un de ses cétés touchait la pierre, et que l’autre était libre; ed/es
étaient accolées par la surface abdominale. Une seule de ces annélides
fécondait l’autre ; car, les ayant séparées, je n’apercus qu’une verge. Les
ovaires de l’une d’elles se gonflérent et se colorérent peu a peu en blanc,
et guarante-cing jours apres elle fit des wufs.” (pp. 60, 61.]

Ebrard had the question of reciprocal fecundation in mind,
and entirely overlooked the spermatophores.

4. Clepsine marginata.

C. O. WHITMAN. The Embryology of Clepsine. 1878. pp. 8, 9.

“T have found that eggs taken from the ovary at the time they are
about to be laid develop in the normal manner, and have taken advan-
tage of this to watch the earliest changes in the ripe egg. I have done
this many times, and always with success. I regard this as very strong
evidence that zmpregnation takes place while the eggs are in the ovaries.
This is in harmony with the fact that I have found spermatozoa in the
ovary two or three days before the time for depositing the eggs. It is
barely possible that these spermatozoa found their way into the ovary
accidentally during the dissecting. I can only say that no testicular sacs
were ruptured during the process; but the vasa deferentia may have
been severed, as they are so minute that one cannot easily see them.
The unchanged condition of the germinal vesicle at the time the eggs have
attained their full size renders it probable that fecundation does not take
place more than four or five days at the longest before the deposit; but
this does not prove that copulation may not have taken place at a much
earlier date. J isolated an individual which had just sucked itself full of
blood, and which showed no signs of eggs through the body-wall, and after
fifteen days obtained eggs that developed in the usual manner. Recalling
the fact that the growth of the egg from the primary egg-cell requires only
twelve to fifteen days, it appears that this specimen was isolated about,
or just before, the time when the egg-cell began to grow. Jn another
case eggs were obtained at the end of twelve days, which developed in the
normal way.

No. 3.] SPERMATOPHORES. 369

“These facts raise a suspicion that Clepsine is capable of self-fecun-
dation. The question as to whether copulation occurs will be most
satisfactorily settled by isolating young individuals and keeping them
until they produce eggs.

May 2, 1878. —“ Five individuals were isolated in the summer of
1877, at the time of hatching. Each has been kept in a separate vessel
from that time to the present. Eggs were laid by one April 24th (this
year), and hatched May rst ; by two others, April 29th. The latter are
now in the germ-band stage. The eggs had in each case passed the
pronuclear stage, at the time they were first noticed, so that I was unable
to demonstrate by section the existence of a male pronucleus. As the
eggs developed in the normal manner, it is very probable that they were
fecundated. Here is an unquestionable case of se//(/ructification, or of
parthenogenesis — more probably the former.”

The above statements not only confirm the observation of
Filippi and Grube, as to isolated individuals producing fertile
eggs, but they also make it probable that fertilization is inter-
nal. To the evidence given by eggs taken artificially from
the ovaries, I can now add another which seems to be perfectly
conclusive. I have succeeded in finding a perfectly distinct
and indubitable male pronucleus in the ripe ovarian egg One,
marginata.

What I formerly regarded as positive proof, either of self-
fructification or of parthenogenesis, in the light of what I now
know about the use of spermatophores, is open to some doubt.
At the time of my experiment of rearing individuals from the
egg in isolation, the possibility of hypodermic impregnation
never crossed my mind, and I can now see where my observa-
tion was not sufficiently guarded to remove all doubt. In order
to bring the five individuals to maturity, I had to feed them
some ten or twelve times. I allowed them to take their meals
from the same fish, only thinking it necessary to watch them
from beginning to end, in order to see that no copulation took
place. Whether I ever allowed them to come in contact long
enough to deposit spermatophores, my notes do not show, and
here is where the doubt comes in. The experiment ought to be
repeated under conditions that would exclude every possibility
of contact, and C. marginata would be one of the best species
for such a purpose, as it is so easily reared. It would be well
to isolate a large number of individuals, so as to have material

370 WHITMAN. [Vou. IV.

enough to allow of taking the ripe eggs from the ovaries ina
few cases. The demonstration of the male pronucleus in such
eggs would show that the leech is able to fertilize itself inter-
nally. If the male pronucleus were not found before oviposi-
tion, and should be found some time after it, one could infer
external self-fecundation. If the male pronucleus proved to be
wanting in both cases, we should have conclusive evidence of
parthenogenesis, provided a considerable number of tests all
gave like results, and provided, further, that the rest of the eggs
developed embryos.

The importance of the experiment will be readily seen; for
should self-fertilization be clearly proved under the conditions
named, I think that fact, in the light of what we know about
the breeding habits, would be sufficient to make it extremely
probable that self-fertilization is a normal affair. That view
would compel us to look upon the spermatophores attached to
the surface as having nothing to do with fertilization. While
self-fertilization certainly seems very improbable, we are not to
forget that it is a possibility. It is believed to take place in
Cestodes, and perhaps also in some Trematodes and Turbellaria.
V. Baer (Mil. Arch., 1835, p. 224) long ago reported a case in
Limneus auricularis, and Oken (/szs, 1817, p. 320) obtained
fertile eggs from an individual of the same species reared in
isolation.

5. Nephelts.

ISAO IIJIMA. Origin and Growth of the Eggs and Egg-strings in Nephelis.
Quart, Jour. Micr, Sci. N.S. LXXXVI. April, 1882. pp. 196-197.

“The anterior portions of two individuals, attached by their suckers
to the glass vessel near each other, are spirally entwined in such a
manner that the ventral surfaces of their genital bands are always
brought into apposition. They maintain this position for a considerable
time, now and then changing the direction of their winding, and relax-
ing or tightening their hold. Sometimes the act is of short duration,
and two or three times renewed at short intervals. At other times, and
when disturbed, the act ceases altogether, or else they combine with
other individuals. It is evident that there can be no reciprocal fecunda-
tion while the leeches are coupling in the position above described.

“ As there ts no tntromtittent organ, itis probable that the male orifice
zweth tts prominent muscular lips clasps the female orifice, while the sper-
matozoa are forced onward by the action of the ejaculatory organ.

No. 3.] SPERMATOPHORES. 371

“T am unable to say precisely at what time of the year copulation
begins ; but I found spermatozoa in the ovaries of one leech on the
2oth of February, for the first time in this year. The act of coupling,
so far as my experience goes, takes place almost always in the morning.

“ ABNORMAL COPULATION. — J have often found individuals with a
small, two-horned, whitish body adhering to some portion of the genital
band. The position of this cornuous body was always on or near the
genital band, sometimes on the dorsal surface, sometimes on the extreme
margin, but more frequently on the ventral surface than elsewhere. It
consisted of two thin-walled bottle-shaped tubes (ca. 5 mm. long), she
broader ends of which were inserted, close to each other, into a small
aisc-like portion. This portion, the margin of which presented a villiform
appearance, was partially embedded in the epidernus. Around the disc
was a discolored area, which proved to be, on examination of sections, a
macerated portion of the epidermis. The two bottle-shaped tubes were
jilled with spermatozoa, and opened by means of two distinct holes in the
disc. From each of these openings a stream of spermatozoa was found,
penetrating to a considerable depth into the underlying tissues. In sec-
tion the substance of the two-horned body appeared dotted and longi-~
tudinally striated, but I was unable to recognize any cellular structure.

“ For a long time I was much puzzled as to the meaning of all this, but
from further observation was led to regard it as @ case of abnormal or
unsuccessful copulation.

“ When disturbed during the act of copulation, the two leeches usually
separate immediately ; but 2 one instance they did not separate even
after putting them into chromic acid. On examination I found that the
JSemale orifice of each leech was not in contact with the male orifice of the
other, but that each individual was attached to the ventral surface of
the other by tts male orifice, the female orifice remaining free. On sepa-
rating them by force, each male orifice left the two-horned olject on the
body of the other.

“In another instance that came under my notice, only one individual
had already deposited the two-horned body, while the other had a mass of
spermatozoa hanging from its male orifice. The latter was dissected,
and the two-horned body found occupying the whole interior of the
ejaculatory organ, with the cavity of which it exactly corresponded in
shape. It came out without any resistance. It thus became evident
that the two-horned body belongs to the interior of the ejaculatory
organ. That it forms no permanent part of the male organ seems evi-
dent, from the fact that sections made in the winter show no trace of
such a body. J have not thus far been able to determine whether this
body forms in the case of normal copulation also. As to its mode of

372 WHITMAN. [VoEr IVE

formation, I have nothing to offer except the conjecture that it may be
the hardened secretion of some of the glands of the ejaculatory organ.

“On many leeches were found scars, which very likely may have been
the marks left by these peculiar bodies.

“Tt is hardly to be doubted that the normal mode of charging the
ovaries with spermatophores is through the female orifice. It would
certainly be impossible for the spermatozoa to find their way into the
ovaries in many of those cases which we have described as abnormal,
especially where the injection takes place far in front of the male
orifice.”

Professor Iijima’s observations were made under my direction,
and at the time I certainly concurred with him in his conclu-
sions. Professor Iijima’s incredulity was wholly due, as one
may readily see from his own words, to the conviction that
there was only one way provided by nature whereby the sper-
matozoa could reach the eggs within the ovaries; namely,
through the female genital pore. The whole description tal-
lies so closely with what I have seen in Clepsine, that I feel
confident that the spermatophores serve the same end in both
genera.

Schneider’s observations on Nephelis are much less complete
than Iijima’s, and spermatophores seem to have entirely es-
caped him.

ANTON SCHNEIDER. Das Ei und seine Befruchtung. 1883. pp. 22, 32, 65.

“Die Begattung beginnt damit, dass sich die Thiere umeinander
winden. Ihre Korper sind dabei gleich gerichtet, so dass immer nur
ein Thier mit Samen versehen wird. Eine Ausstiilpung des sehr kurzen
Penis habe ich nicht beobachtet. Bei der Begattung geht viel Samen
verloren. Moquin-Tandon, gestiitzt auf eine Angabe von Bojanus,
nimmt an, dass die Korper der A/irudineen bei der Begattung entge-
gengesetzt gerichtet sind. Indess hat schon Ebrard*) in seinem sehr
lesenswerthen Werke die Begattung von Aiivudo medicinalis mit gleicher
Richtung der Korper vor sich gehen sehen. Auch Iijima hat dies bei
Nephelis bestatigt. Man kann die Begattung leicht beobachten. Wenn
man zwei Thiere, welche 3-4 Tage isolirt waren, vereinigt, findet die
Begattung sofort statt. Bei der von Iijima beobachteten Species findet
ausser dieser Begattung eine andre Art statt, indem Spermatophoren
von dem einen Thiere auf die Haut des andern befestigt werden. Bei
unsern Wephelis habe ich diese Spermatophoren nie gesehen, obgleich
mir das Vorkommen dieser Korper bei Pontoddella und Piscicola wohl
bekannt ist.”

No. 3-] SPERMATOPHORES. 373

6. Piscicola.
J. LEO. Verhaltnisse der Piscicola geometra. Miiller’s Arch. f. Anat, etc. 1835.

Pp. 425.

“Die Begattung dieser Thiere geschieht auf folgende Weise. Die
Fussscheiben zweier Individuen sind in einiger Entfernung von einander
auf einer Ebene angeheftet und die Korper erhalten sich schwebend
an den ausseren Offnungen der Geschlechtstheile dergestalt Bauch an
Bauch mit einander verschlungen, dass sie die Form eines X bilden,
wobei aber das Kopfende jedes Thieres nach derselben Seite zuriickge-
bogen ist, an welcher seine Fussscheibe haftet. Hinter der Umschling-
ung sind beide K6rper bedeutend angeschwollen und dicht vor dieser
Anschwellung sieht man in der Nahe der weiblichen Geschlechtsoffnung
cine weisse Masse hervortreten, die sich nach und nach vermehrt, und
unter dem Microscope sich als ein Siickchen mit einer weissen, Seinkor-
nigen und schleimigen Substanz erfiillt darstellt. Ich glaube, dass dese
Masse ohnerachtet des hiiutigen Uebersuges dennoch nichts anders als
der aus den weiblichen Geschlechtstheilen iiberfliessende minnliche Same
ist, dessen Oberfliche aber wahrscheinlich durch den Einfluss des Me-
diums 2u einer Haut gerinnt. Dass die Ruthe in die weibliche
Geschlechtsffnung eindringt bemerkt man erst, wenn sich die Thiere
von einander durch bewirkte Stérung trennen, in welchem Falle dieselbe
dann eine Zeit lang steif hervorsteht, wie es abgebildet ist.”

Leo’s description of the spermatophore falls considerably
short of being a discovery, and his remarks about the penis are
wide of the mark. He seems to have mistaken the proboscis
for a penis, as Leydig has pointed out.

T. BRIGHTWELL. Ueber die Hirudo geometra, Linn., und einige andere Arten
von Susswasser-Egeln. Froriep’s Neue NVotizen. XXII, No. 467. 1842. p. 65.

Somewhat later than Leo, Brightwell again saw the sperma-
tophore of Piscicola (“weisse Substanz’’), but without under-
standing it. During copulation, as Brightwell puts it,

“Man bemerkte auf jeder Seite des Theils, wo die Korper ihre Verei-
nigung bewirkten, e7e weisse Substanz. So blieben die Thiere gewohnlich
mehrere Stunden, in einem Falle sogar den ganzen Tag ueber verbun-
den. Als sie sich von einander trennten, léste sich von den Stellen, mit
denen sie aneinandergehangen hatten, eime wetsse, spinnewebenartige
Substanz ab, welche sich in einem Falle wie ein Ei ausnahm, sich aber
bei fernern Beobachtungen als ein Theil des Haiitchens herausstellte,

374 WHITMAN. [VoL. 1V.

von welchem die Eier umbhiillt sind. Innerhalb 24 Stunden nach dem
Begattungsacte wurden Lier gelegt.”

FRANZ LEYDIG. Zur Anatomie von Piscicola geometrica mit theilweiser Ver-
gleichung anderer einheimischer Hirudineen. Zetéschr. f. w. Zool. 1. 1849. p. 124.

“ Bei der Begattung stiilpt sich aus der mannlichen Geschlechtsoffnung
eine Blase, welche die Ausmiindungsstelle der Ductus def., sowie einen
Theil der gelappten Driise enthalt. Dieser hervorgestiilpte Theil gibt an
die weibliche Geschlechtsdffnung die Samenmasse ab, welche als weiss-
licher Korper auch nach der Begattung an der weiblichen Genitalmiind-
ung sitzen bleibt. Die weissliche Masse, naher untersucht, erweist sich
als eine gedoppelte Blase mit doppeltem Stel, an welcher die Membran
und die Stiele als aus dem Secret der gelappten Driise bestehend,
erkannt werden. Im Innern sind die Spermatozoiden in schén gelock-
ter Weise geschichtet enthalten, so dass man wohl den ganzen weissen
Korper als Spermatophoren bezeichnen kann. Schon im Ductus def.
briinstiger Individuen lagern sich die Spermatozoiden zu solchen
gelockten Biindeln zusammen, wie man sie nacher in den Spermato-
phoren findet. Es braucht also nur das Secret der gelappten Driisen
die Spermatozoiden bei der Ejaculation zu umhiillen, um die treffenden
weissen Korper zu bilden. Aus den Spermatophoren, welche halbe Tage
lang nach der Begattung an der weiblichen Geschlechtsiffnung sitsen
bleiben, bewegen sich die Spermatozoiden in den Eterstocksschlauch und
adringen bis zu dessen blindem Ende vor. Ein solcher Eterstock mit
eingewanderten Spermatozotden hat ein schon dem blossem Auge wahr-
nehmbar veriindertes, weissliches Aussehen.”

Schneider confirms Leydig’s account, and adds Pontobdella to
the list of leeches which attach their spermatophores to the
exterior. The definite location of the spermatophores in P2s-
cicola is noteworthy, in comparison with Nephelis and Clepsine.
Leydig assumes that the spermatozoa pass through the female
genital pore into the ovaries ; but he gives no proof of this. Per-
haps his persuasion that it must be so, was the only reason he
had for concluding that it was so. Schneider expressly states
that the spermatophores of Pontobdella are attached to any
point of the exterior except at the genital pore.

ANTON SCHNEIDER. Das Ei u. seine Befruchtung. 1883. pp. 32, 65.

PIscICOLA +GEOMETRICA. — “ Zwei Exemplare heften sich mit dem
hintern Saugnapf fest, in entgegengesetzter Richtung und in der Ent-
fernung, dass sie sich mit dem Vorderende erreichen kénnen. Sie

No. 3-] SPERMATOPHORES. 375

kriimmen das Vorderende und haken sich in einander. Solange die
Reife der Eier nicht eingetreten, ist dies nur ein Vorspiel der Begattung,
denn man findet in den Eileitern keinen Samen. Dagegen werden bei
dieser Gelegenhett Spermatophoren adbgesetst, welche man hiufig auf dem
Boden der Gefisse findet.”

7. Pontobdella.
SCHNEIDER, Zc.

“Die Spermatophoren von /%scicola und Pontobdella sind kurze keu-
lenformige Rohren, deren Wand aus agg/utinirten Spermatozoen besteht,
wahrend das Innere mit freien Spermatozoen erfiillt ist. Leydig hat
dieselben bei Piscicola entdeckt und auch nachgewtesen, dass sie bet der
Begattung an die weibliche Geschlechtsiffnung befestigt werden. Wie
schon oben bemerkt, werden diese Spermatophoren schon gebildet und
abgelegt zu einer Zeit, wo die Thiere noch nicht geschlechtsreif sind
und kein Samen in die Eileiter eintritt. Man findet die Spermatophoren
dann auf dem Boden der Gefasse, worin die Thiere leben.

“ Aehnlich wird die Begattung bei Ponfobdella vor sich gehen. Zur
Zeit als ich Pontobdella lebend beobachiete | April in Triest], fand keine
Begattung, sondern nur das Absetzen von Spermatophoren statt. Die
Thiere befestigten sich dieselben gegenseitig an beliebige Kéirperstellen,
nur nicht an die Geschlechtsiffnung.”

8. Hirudo.

The ten-eyed leeches (Hirudo, Aulostoma, etc.) all have a
well-developed intromittent organ, and observers now agree
that in copulation the male organ of one individual is inserted
in the female orifice of the other, and that fertilization is one-
sided, not reciprocal. No one, so far as I know, has ever
reported external spermatophores, and there is no reason to
suppose that hypodermic impregnation ever occurs in the Gnath-
obdellide. Some of the older authorities maintained that
fecundation was reciprocal, and Moquin-Tandon, as late as 1846,
declared this to be a settled fact. Ebrard, however, disputes
this point, and brings many facts to show that only one indi-
vidual is fertilized at a time. Perhaps the conflicting testimony
warrants the suggestion that fecundation may sometimes be
reciprocal, at other times not. If copulation happened between
two individuals equally ready to fecundate reciprocally, there

1 An error corrected by Vejdovsky.

376 WHITMAN. [VoL. IV.

would be no difficulty in their doing so, provided the position
taken were such as to admit of it. On the other hand, if one
individual only were ready to discharge the male function, only
one individual would be fecundated. I incline to take this view,
as it offers a complete reconciliation of otherwise contradictory
observations.

Ebrard’s experiments in isolating leeches are not only inter-
esting per se, but also in connection with the results before
given of isolation of Clepsine.

A. MOQUIN-TANDON. Monographie des Hirudinées. 1846. pp. 166-68.

“Bibiéna, Thomas, Vitet, Mérat, Derheims et Fée, ont pensé que les
Hirudinées se reproduisaient sans accouplement réciproque. Suivant
Filippi, les Glossiphonies seulement sont capables de se féconder toutes
seules.

“Weser, Cuvier, Carena, Virey et Blainville, ont admis, d’apres la
structure des organes sexuels, que chaque individu était incapable de se
reproduire sans s’accoupler avec un autre. Leur opinion a été trouvée
conforme 4a la nature, apres les observations de Hebb et de Evans de
Worcester (cités par Johnson), qui ont fait connaitre que l’acte du coit
se passait, chez ces Annelides, de la méme maniére que dans les Arions
et les Hélices. Depuis cette époque, Kuntzmann, Bojanus, Odier et
plusieurs autres naturalistes, ont eu l’occasion de voir des Hirudinées au
moment de l’accouplement, et ont confirmé les observations des deux
savants anglais.

“Dans l’accouplement des Sangswes médicinales, deux individus se
rapprochent, ventre contre ventre et en sens inverse, de telle sorte que
la ventouse orale de chacun est tournée, ou 4 peu prés tournée, vers la
ventouse anale de l’autre. On concoit que, dans cette position respec-
tive, les organes génitaux se trouvant également situés en sens inverse,
de maniére que chaque verge doit se rencontrer en face d’une vulve.
Les deux individus s’enlacent, et l’accouplement a lieu (Bojanus).
Quelquefois les Sangswes s’attachent ensemble par leurs ventouses anales
et laissent pendre librement leur partie antérieure (Burdach). Kuntz-
mann a cru reconnaitre que, dans l’union sexuelle, les deux verges sont
entortillées en spirale comme celle des Hélices; cette disposition est
sans doute accidentelle (Bojanus).

“Le docteur Gaspard a prétendu que, dans chaque accouplement,
un seul individu fécondait l’autre, lequel, aprés vingt-cing ou trente jours,
fécondait le premier dans un autre accouplement. II est bien démontré
aujourd’hui, par l’observation, gue le coit est réciprogue comme celui des
Escargots.

No. 3-] SPERMATOPHORES. 377

“Johnson a observé |l’accouplement de la Véphelis, et a reconnu qu’ il
était entiérement semblable 4 celui de la Sangswe médicinale.”

EBRARD. Nouvelle Monographie des Sangsues Médicinales. Paris, 1857. pp.
104, 105.

“Comment se fait-il donc que Bibiéna, Thomas, Vitet, Mérat leur
aient accordé la faculté de se reproduire sans accouplement préalable ?
Lis ont probablement été trompés par cette circonstance qwune sangsue
produit des cocons féconds trots, six, huit, et méme dix mots apres avoir
été tenue tsolée. C’est la un fait dont il ne m’est pas permis de douter,
quoique l’intervalle séparant l’accouplement des sangsues de la pose ou
production des cocons ait été évaluée par divers auteurs a trente ou
quarante jours.

“J'ai déja publié, en 1851, l’histoire d’une sangsue verte de Hongrie
qui, renfermée seule vers le 15 mai 1850, produisit le 15 et le 27 aout
des cocons féconds.

“En 1852, soupconnant que les cocons de sangsues ne donnant pas
le jour 4 des filets proviennent (de méme que cela arrive pour les ceufs
stériles des femelles d’oiseaux tenues en cage) de sangsues ne s’étant
pas accouplées, et, voulant m’en assurer, je placai, le 15 septembre, sept
sangsues vaches dans autant de bocaux contenant de la terre et de la
mousse, une sangsue dans chaque bocal (je les gorgeai et je les changeai
de terre en mars (1853). Six de ces sangsues postrent des cocons aux
mois de juillet et d’aofit suivant, c’est-a-dire apres neuf a dix mots
a’isolement, et, @ mon grand étonnement, des filets sortirent de ces cocons.

“En face de ces résultats si inattendus, je me demandai, je l’avouerai
franchement, si les sangsues, tout en s’accouplant, ne pouvaient pas
produire des cocons sans un accouplement préalable, ou plutot si un
rapprochement ne suffisait pas pour plusieurs années. Je me livrai, en
conséquence a de nouvelles experiences :—

“1° Je continuai 4 tenir séparés quatre des sangsues précédentes
qui étaient isolées depuis un an, et deux autres sangsues qui |’étaient
depuis deux mois, depuis le mois de juin, et avaient recemment pose
des cocons ; toutes furent stériles l'année suivante, quoique j’eusse pris
soin de les gorger Iégérement aux premiers jours du printemps.

“9° Je réunis, le 17 septembre, trois des sangsues ayant été tenues
isolées depuis un an, puis je les séparai le 20 octobre. Je séparai le 17
septembre quatre autres sangsues qui étaient renfermées depuis quelque
temps dans le méme bocal. Une de ces sangsues périt, cing des autres
posérent des cocons féconds pendant les mois d’aofit et de juillet de
l’année suivante.

“Tl est dés lors évident, ce me semble, que /es sangsues ont besoin de

378 WHITMAN. [Vor. IV.

saccoupler chaque année pour étre féecondées, mais que, semblables en
cela a plusieurs insectes, elles peuvent ne se reproduire que huit ou neuf
mois aprés avoir été féecondées. Ce fait de la reproduction ayant leu
dans une année autre que celle de l’accouplement a d’ailleurs son ana-
logue chez une hirudinée non médicinale ; car les glossiphonies [Clepsine ]
gue, aux premicres chaleurs du printemps et peu de jours apres leur
sortie de leur retraite hivernale, portent souvent déja des eufs sous
l’abdomen, ont certainement été fecondées dans Vannée précédente.”

9. Aulostoma.
EBRARD, Zc. p. 67.

“ Deux aulastomes que j’ai trouvées accouplées étaient rapprochées
téte a téte. Une seule fécondait l’autre ; car je n’apercus qu’une seule
verge lorsque je les séparai. L’une d’elles posa son premier cocon,
ceuf polysperme, quinze jours avant l’autre annélide.”’

10. Macrobdella.

Copulation has not, so far as I know, been observed in our
common Macrobdella. That copulation occurs there can be
little doubt. These leeches are remarkable for having so-called
“copulatory glands,” opening on the ventral side, a few rings
behind the @ pore. It was Leidy who suggested that these
glands are “ provided for the adherence of individuals in sexual
intercourse,” and their position supports this view. This pecu-
liarity makes it all the more desirable to witness the process of
copulation. From what we know of other leeches, it is proba-
ble that individuals captured in early spring, and kept some
days in isolation, would, on being brought together, very soon
copulate.

OBSERVATIONS ON CLEPSINE PLANA (7. Sp.).

The specimens on which my observations were first made
were two large species obtained from Charles River, at Water-
town, near Cambridge, in September, 1884. I am unable to
identify them with any species hitherto described. Five of the
nine individuals captured were dark brown, variously marked
with yellow above, and with twelve or thirteen longitudinal lines
below; the remaining four were yellowish brown both above
and below.

No. 3.] SPERMATOPHORES. 379

Clepsine parasitica, as described by Say! and Verrill,? agrees
in certain features closely with the dark species, and Verrill’s
C. papillifera var. carinata may be identical with the light
species. The descriptions, however, do not point out any char-
acters which can be relied upon for identification.

The dark specimens, to be described in the following paper
under the name C. plana, were ready to copulate whenever they
met ; but they always avoided the light species, which may be
provisionally designated as C. carinata. The latter showed no
disposition to copulate until early the following spring. They
remained quiet during the winter; but on being started up in
May, they began to deposit sperm-cases.

The observations which follow are confined mainly to C.
plana. Two of this species bore young still stuffed with yolk,
and another had eggs in its ovaries that were nearly mature.

When I first placed these Clepsines in a dish together, I
noticed several long white bodies attached by one end to the
dorsal surface of one or two individuals. I pulled them off for
examination, thinking that they were parasites of some kind.
Putting them under the microscope, I saw, to my great surprise,
a stream of spermatozoa slowly issuing from the end that had
been detached. At first I could hardly believe that these sperm-
cases belonged to the leech, never having detected the animal
in the act of depositing them, and not suspecting that they
could discharge their contents through the skin. My curiosity
having been thus aroused, 1 watched the leeches more closely,
and soon had an opportunity to see the whole operation. The
leeches were moving about as they usually do when first cap-
tured, before becoming wonted to new quarters. One indi-
vidual, coming in contact with another, fixed itself by its oral
sucker to some convenient point, and then, while pressing its
protruded male pore against the back of its fellow, planted a
fresh sperm-case. During the operation, which lasted only a
few seconds, the body in the region of the genital pores was
more or less constricted, somewhat as it is in the act of forming
an egg-cocoon. The constriction seemed to be the expression

1Thomas Say: Major Long’s Expedition to the Source of St. Peter's River, etc.,
in 1823. Vol. II, Appendix, p. 14. Keating’s Compilation, London, 1825.

2A. E. Verrill: Synopsis of North-American Fresh-water Leeches. Professor
Baird’s Report for 1872-73.

380 WHITMAN. [Vot. IV.

of an effort to press the sperm-case firmly to the surface of
attachment, and very likely the case was filled with spermatozoa
by the same act. After a few moments of steady pressure, —
just long enough to allow the sticky secretion to “set,’’ — the
leech released its head and slowly drew back, allowing the
spermatophore to be gradually pulled out of the two sac-like
ends of the vasa deferentia. I saw this operation repeated
several times by the same individual at intervals of about thirty
minutes.

Among twenty or thirty spermatophores, I found only one on
the ventral surface, and this was near the margin of the body;
the rest were attached to the dorsal side, sometimes between
two rings, sometimes in the middle of a ring, without any dis-
crimination of place, so far as I could see.

Although the sperm-case is formed in two distinct sacs, unit-
ing in a common pore, its two halves are firmly glued together,
as the result of being pulled out through the single pore, while
they are still in an adhesive condition. The moment they are
set free, they are hardened by the action of the water, and only
the small free ends sometimes remain distinct and separate.

One of the spermatophores first deposited measured 8 mm. in
length and 1 mm. in width. Some of the last obtained meas-
ured only 3 mm. or even less. Repetition of the act seemed
to exhaust the individual’s power of forming spermatophores.
Widely as they varied in size, they always showed essentially
the same form as that shown in Fig. 4, a and 0.

In the spermatophore we may distinguish (1) a short, con-
stricted, basal portion with a single tubular lumen, formed in the
median unpaired portion of the male organs; (2) an elongated
body with a double saccular lumen, formed in the enlarged
end-portions of the vasa deferentia communia; and (3) a free
end, consisting of two distinct parts, adherent or separate, with
lumen closed, or reduced to a narrow line, formed in the ends
of the ejaculatory ducts (d) at the point marked w in Fig. 5.
The wall of the spermatophore, which is thickest at the base
and thinnest in the saccular body, is composed of two well-
defined layers: an outer, thin, transparent, finely striated, non-
stainable, cuticular-like layer (Fig. 2, 0), which appears to fill
the angles between the two halves of the case (Fig. 4 6), and
to serve as a medium whereby the case is firmly glued to the

No. 3.] SPERMATOPHORES. 381

surface ; and an inner, denser, thicker, stainable layer (Fig. 2, 2).
The outer layer is so extremely thin over the saccular portion
of the fresh spermatophore that it is difficult to recognize it ;!
but it is easily demonstrated on sections. In the basal portion,
this layer thickens, and then expands to form a broad base, so
closely applied to the underlying cuticula as to form almost a
continuum with it (Fig. 2). The striations of this layer may be
due to the pull given to the sac as it is liberated from the
genital pore, or more probably, as I think, to its mode of forma-
tion by numerous gland-cells.

When first placed, the spermatophore usually stands nearly
perpendicular to the surface. It is tough and elastic, and con-
siderable force is required to detach it. The skin of the leech
around the place of attachment is at first strongly corrugated,
as if by contraction; but this appearance gradually passes away
after the sac is emptied, although the sacs often remain for
several days, or even weeks. Whether they are ultimately dis-
solved, or shed with the cuticula, or drop off as the result either
of vital processes in the underlying skin, or of the solvent action
of water, I am unable to say.

The mouth of the fresh spermatophore is completely plugged
with a peculiar secretion (Fig. 4, a, gs), made up of elongated ellip-
tical or spherical corpuscles (Fig. 4, c), varying from 0.02 mm. in
diameter to much smaller dimensions. These bodies dissolve
in water in the course of a few minutes. At first appearance,
they are coarsely granular, but rapidly become perfectly homo-
geneous and transparent, and, growing paler and paler, fade
away by insensible degrees. At first I took these bodies to be
cells, as some of them appeared to be nucleated; but having
traced them to their origin in glands of a definite region of the
vasa deferentia, | now think that the nucleus-like centre (Fig.
4, ¢) merely marks the depth to which the water had penetrated
at the moment of examination. Their great variation in size
is also in harmony with their origin as globular secretions.

This granular secretion probably serves a double purpose:
first, to protect the spermatozoa inclosed in the saccular portion
against contact with water; and secondly, as a means of open-
ing and clearing the way for the safer penetration of the sper-

1 Exposed to acetic acid, it swells, and is thus made evident without the aid of
sections.

382 WHITMAN. VoEsVE
matozoa. This mass is expelled through the skin in advance of
the spermatic elements; and the disappearance of the pigment
and the clarification of the tissues at the point of penetration,
all of which is noticeable in sections (Fig. 2), suggest that it
may have a softening effect on the tissues! This, however, is
pure conjecture. With plenty of material, the action of this
secretion on fresh pigmented tissue might possibly be deter-
mined experimentally ; but thus far I have not tried this.

If the leech is placed under a magnifying power of twenty or
forty diameters, immediately after receiving one of the sperma-
tophores, one may see the spermatozoa slowly flowing from the
narrow mouth of the case through the skin. In the course of an
hour the greater part of the contents has escaped, and the case
itself is reduced to less than half of its original diameter. As
soon as the case is planted, it begins to shrink; and this con-
traction, induced by the action of the water, is probably what
forces the spermatic fluid through the skin. When the sac is
first placed, the spermatozoa may be seen through the wall united
in close bundles. Soon after deposit, as one may see towards the
free end of the sac, these bundles begin to swell up, and the indi-
vidual spermatozoa begin to show themselves. The appearance
might raise a suspicion that a part of the spermatozoa undergo
histolytic changes, serving by expansion as a means of expelling
the rest, somewhat as described by Gruber in the Copepoda
(v. extract). I think, however, that Leuckart’s suggestion in
regard to the spermatophore of Astaczs is the explanation to be
adopted here, as it is perfectly certain that the sperm-case
gradually contracts as its contents escape. I find that a few
spermatozoa are always left in the case after it has reached the
limit of contraction, showing that the expelling force ceases to
act after this. As distinct spermatozoa were found in a sperm-
sac two days old, I infer that the sac is water-proof. If a
fresh sac be detached and exposed to the pressure of a cover-
slip, the sperm is rapidly driven out in the form of a white flaky
string, consisting of a viscid fluid, with numerous bundles of
spermatozoa.

In order to learn precisely where the spermatophore is formed,
as well as the origin and relative positions of the various ele-
ments with which it is to be charged, it will be necessary to

1 Tsjima’s observations on Nephelis favor this view.

No. 3:1] SPERMATOPHORES. 383

examine briefly the form, structure, and contents of the male
efferent ducts. A glance at Fig. 5 will show that these ducts
are differentiated into a number of different regions, each of
which seems to have a special function. Beginning with the pore,
which lies between the tenth and the eleventh ganglia, we finda
very short median tube, which bifurcates beneath the ventral
cord, giving rise to two diverging horns (s), which are continued
into a convoluted tube (zw, g, @) of nearly uniform, but much
smaller, diameter ; then follows an enlarged sigmoid coil (vs), and
finally the long narrow tube (vd@c), which receives the six short
testicular ducts (vd). The sigmoid portion is a thin-walled
reservoir completely filled with sperm-bundles, fulfilling the

Fic. 1.— Section from the posterior half of the ductus ejaculatorius, showing
bundles of spermatozoa (sf) massed together, and a few granular corpuscles (gc),
probably secretions from the anterior, glandular half of the duct. The muscular
layer (m) is strongly developed, and the large lumen is lined with a thin epithe-

lium (ép).

office of a vesicula seminalis. The convoluted portion con-
necting the vesicula seminalis with the terminal horn-like
enlargement (s) appears externally to be a nearly uniform tube,
and js usually called the ductus cjaculatorius (d). But an exam-
ination of the structure and contents of this portion reveals the
fact that it is really differentiated into two parts which fulfil
different functions. The posterior half (¢) has a thicker mus-
cular wall, and a much larger lumen than the anterior half
(w and g); it is lined with a thin epithelium, and is filled with
spermatozoa. In the anterior half this epithelial lining takes
the form of long columnar gland-cells, radially disposed, with the

384 WHITMAN. [Vot. IV.

nucleated ends next to the muscular wall. The reduction of the
lumen in this part is due to the development of this glandular
epithelium. It is in this glandular portion that the granular cor-
puscles (gc) which plug the spermatophore are produced.

I have a series of sections of this region, showing the gland-
cells fixed in the very act of secreting these corpuscles. The
corpuscles are somewhat pyriform in shape, with the smaller
end tapering to a fine thread, which is connected with the cen-
tral end of the producing cell (Fig. 2, gc). As soon as the cor-
puscle is fully liberated, it assumes a more or less elliptical form.
These corpuscles sometimes nearly fill the whole lumen of the
duct ; sometimes they lie in masses that resemble clusters of

Fic. 2.— Section from the glandular anterior half of the duct, in which the lining
epithelium (ef) is transformed into radial, pyramidal gland-cells, with nuclei at the
external bases. Granular corpuscles (gc) in process of formation. Muscular layer
thinner than in Fig. 1. The lumen of the canal is very much reduced by the thick-
ening of the epithelial lining.

blood-corpuscles. I find a few such clusters in the posterior
non-glandular region (¢), but I doubt if they move backward to
this part as a regular thing. They were more probably thrown
backward from the place of origin by irregular, peristaltic con-
tractions during the process of killing. At the anterior end (w)
of the corpuscle-secreting region, the glandular lining becomes
thicker, and passes imperceptibly into the still thicker lining of
the terminal horns. I find no corpuscles at this level (zw), and
it is here that the free end of the spermatophore is evidently
secreted. The saccular part of the spermatophore, as before
noticed, is secreted in the horns (s), while the narrower base is

No. 3.] SPERMATOPHORES 385

formed in the median unpaired tube lying beneath the nerve-
cord.

The foregoing facts enable us to form some idea of what
probably goes on each time a spermatophore is produced. My
conjecture is as follows: As soon as the sperm-case is ready for
the reception of its burden, the corpuscular secretion and the
spermatic fluid contained in the convoluted tube (w, g, and @)
are driven forward into it. The contents of this whole tube
between the horn (s) and the vesicle (vs) is probably required

Fic. 3.— Section of the terminal horn of the efferent duct, just below the point
of union with the ejaculatory coil. The epithelial layer is still more strongly devel-
oped. The cells are fixed in the act of secreting a spermatophore; the secretion
still shows its origin from numerous single cells.

for a single charge. The spermatic fluid would sweep the cor-
puscular mass before it, and leave it in the basal portion of the
sperm-case. Whether any portion of the contents of the veszcula
seminalis is needed in addition to that of the ductus ejaculatorius
to complete a single charge, is a matter of doubt. I am inclined
to think that the charge is measured off each time in the ejacu-
latory ducts, and that the contents of the vesecule seminales is
brought forward only to replace what has been ejected.

386 WHITMAN. [Vot. IV.

In this connection I desire to call attention to a peculiar
feature of the ovaries of this species. A little behind the point
of union of the two ovaries (Fig. 5) is seen a large cecal diver-
ticulum (ca) directed obliquely forward and outward, and termi-
nated by a fibrous prolongation. I have not examined the
minute structure of this part. I wish here merely to suggest
that the ovarian sac of Clepsine may have been derived from a
looped form like that of Nephelis. The free end of the loop in
Nephelis (according to Iijima, Zc.) terminates with a fibrous
prolongation similar to that of the cul-de-sac of C. plana. A
drawing of the same organs in C. marginata (Leipzig), made
when I was in Leuckart’s laboratory, shows this same fibrous
prolongation, arising, not from a cecal appendage, but from the
anterior angle of the simple ovary.

Notes AND EXTRACTS RELATING TO THE PHENOMENA OF
IMPREGNATION BY MEANS OF SPERMATOPHORES IN THE
TURBELLARIA, THE ROTATORIA, DINOPHILUS, THE CH#TO-
PODS, PERIPATUS, ETC.}

1. The Turbellaria.

ARNOLD LANG. Der Bau von Gunda segmentata, etc. JZitt. a. d. Zool. St. 2.
Neapel. IlI,1tand 2. pp. 222-24. 1882.

“Die Endapparate der Geschlechtsorgane sind bei den Polycladen
ausserst mannigfaltig gebaut. Betrachten wir diese Thiere als kriech-
ende Coelenteraten, so darf uns diese Mannigfaltigkeit nicht wundern,
denn dann sind die Polycladen die ersten Thiere, bei denen eine wahre
Copulation vorkommt. Dann setzt uns auch ein gewisser Copulations-
modus, den ich bei mehreren Arten verschiedener Gattungen von Poly-
claden beobachtet habe, nicht mehr in zu grosses Erstaunen.

“Lange Zeit hatte ich namlich vergeblich nach einer Erklarung der
eigenthiimlichen Thatsache gesucht, dass bei Zhysanozoon 2 mannliche
Offnungen und 2 Penes vorkommen, daneben aber eine einzige weib-
liche Offnung existirt. Fiir die Erklarung war wenig gewonnen, als ich
bei andern Gattungen von Polycladen dieselbe Thatsache entdeckte und
sogar bel einer neuen Art und Gattung bei einem jungen Zhzere 9, bei

1 As the literature on the subject of hypodermic impregnation has not hitherto
been collected, I have given at length such reports as seemed to me to be of especial
interest. Doubtless I may have overlooked much that would be of value in this con-
nection. What I have given will suffice to show that the subject is worthy of further
investigation,

No. 3-] SPERMATOPHORES. 387

einem 15 Lenes in zwei seitlichen Liingsreihen angeordnet vorfand.
Daneben bestand immer nur eine einzige weibliche Offnung. Auf die
richtige Spur brachte mich endlich eine Beobachtung, die ich vor zwei
Jahren an einer in die Nahe der Gattung Zeptoplana gehorenden Art
wiederholt anzustellen Gelegenheit hatte. Die meisten Exemplare dieser
Art, die mir von den Fischern gebracht wurden, trugen niimlich eine
geringere oder grossere Anzahl weisser, ziemlich resistenter Faden, die
ohne irgend welche Regelmissigheit auf der Bauch- oder auf der Riick-
sette, oder auf beiden zugleich im Korper der Thiere befestigt waren.
Zuerst dachte ich, dass ich es hier mit Parasiten zu thun habe. Die
genaue Untersuchung ergab jedoch, das die erwiihnten Anhinge weiter
nichts sind, als Spermatophoren. Sie bestehen aus einer structurlosen,
resistenten Hiillmembran, welche in ihrem Innern eiien Haufen Sper-
matozoen enthalt. An Schnitten und an lebenden Thieren zeigte es
sich, dass diese Spermatophoren mit Gewalt und unter Zerreissen des
LE pithels der Basalmembran und der Musculatur in den Korper der
Thiere eingesteckt worden waren. Der Samen ergiesst sich von den
Spermatophoren in alle Hohlriiume des Korpers, so dass man ofter im
Lumen der Darmiste Sperma in reichlicher Quantitit antrifft. Zufallig
gelangt er auch in die Eileiter, die im ganzen Korper sich verasteln.
flier findet die Befruchtung der Fier state.

“Die Untersuchung des Penis ergab Resultate, die mit dieser eigen-
thiimlichen Art der Copulation vollstandig in Einklang stehen. Er
besteht aus einem hintern driisigen Theil, welcher offenbar die structur-
lose Hiille der Spermatophoren bildet und aus einem vordern sehr mus-
culdsen und langgezogenen Theil, dessen Hohlung spiralig gewunden
ist. In Folge dieser Beschaffenheit des Penis kann das Spermataphor
wie ein Korkzieher in den Korper des die Misshandlung erleidenden
Individuums eingebohrt werden. Die weibliche Offnung dient bei dieser
Species ausschhesslich sur Eiablage. .. .

“Diese eigenthiimliche Copulationsart, neben der bei vielen Arten
auch eine richtige Begattung vorkommt, erklart nach meiner Ansicht so
vollstandig das Vorkommen mehrerer Penes' neben einer einzigen weib-
lichen Geschlechtséffnung, dass ich weitere Commentare fiir unnothig
halte. Jn wie weit sie als eine urspriingliche zu bezeichnen ist, wage ich
nicht zu entscheiden. Jedenfalls scheint es mir charakteristisch, dass
sie ausschliesslich bet denjenigen Thieren vorkommt, die ich fiir die altes-
ten Bilaterien halten muss und bei denen wahrscheinlich zum ersten Male
die Befruchtung durch eine Copulation eingeleitet wurde.”

lIn Histriodrilus Benedeni, Foet. (Histriobdella homari, P. J. v. Ben.), Alexandre
Foettinger finds three penes. It is possible that this “ archiannelid” accomplishes the

act of fertilization in the same manner as the pluri-penial Polyclads. Arch, de Biol.,
Vol. III, pp. 482-83 and 510. 1884.

388 WHITMAN. [Vou. IV.

ARNOLD LANG. Die Polycladen. Fauna und Flora des Golfes von Neapel.
XI. Monographie. 2d Halfte. pp. 636-38. 1884.

“Tch habe nur bei einer einzigen Art, namlich bei Stylochus neapol-
zanus, den Vorgang der Copulation im gewohnlichen Sinne des Wortes,
d. h. die Einfiihrung von Sperma in den weiblichen Begattungsapparat
durch den mannlichen beobachtet. . . .

“F's ist sehr wahrscheinlich, dass noch bei vielen anderen Polycladen
sich eine normale Begattung vollzieht. Bei einer Reihe von Formen
aber geschieht die Copulation in einer bis jetzt tm Thierreiche ganz allein
dastehenden, hichst merkwiirdigen Art and Weise. Der Umstand,
dass Zhysanozoon Brocchit zwei Penes und zwei getrennte mannliche
Geschlechtsapparat besitzt, hatte meine Neugierde, zu sehen, wie sich
bei diesen Organisations-verhaltnissen die Begattung bei dieser Art voll-
ziehe, schon lange wach gerufen. Diese wurde noch gesteigert, als ich
noch andere Pseudoceriden mit doppeltem mannlichen Begattungs-
apparat entdeckte, und gar erst, als ich den merkwiirdigen Anonymus
virilts mit seinen zahlreichen Penes, aber nur einer weiblichen Oeffnung
auffand. Die genauere Untersuchung der Einrichtung und des Baues
der Begattungsapparate dieser Formen brachte keine Aufklarung darii-
ber, in welcher Weise bei ihnen die Copulation sich vollziehen konne,
sie zeigte vielmehr, dass die ganze Organisation und Anordnung der in
Frage stehenden Apparate fiir eine richtige Begattung so unpassend wie
moglich ist. Den ersten Schritt auf dem Wege zur Aufklarung der
Verhaltnisse machte ich eines Tages, als man mir den prachtigen Prev-
doceros superbus brachte. Ich setzte ihn in ein Bassin, in welchem sich
mehrere schone Exemplare, von Yungia aurantiaca und Thysanozoon
Brocchit befanden. Das Thier kroch an den Wanden des Gefasses
umher, stiess zufallig auf eine Yumgia, wurde nun plotzlich sehr auf-
geregt, liess seine beiden Penes weit hervortreten, und glitt iiber das
Exempiar von Yungia hinweg. Bei seinem eiligen Umherkriechen traf
es noch ofter mit Exemplaren von Yungia und Zhysanozoon zusammen.
Jedesmal wenn dies geschah, wurden die Penes hervorgestreckt, so dass
ich mich veranlasst fiihlte, die Individuen, iiber die der Pseudoceros
superbus hinweg gekrochen war, aus dem Bassin heraus zu nehmen und
zuexaminiren. Da stellte sich heraus, dass alle diese Exemplare mehr
oder weniger zahlreiche Wunden hatten, und swar an allen moglichen
Korperstellen, und in den Wunden fanden sich ansehnliche weisse
Klumpen von Sperma (ich will hier noch beilaufig bemerken, dass
sowohl bei Pseudoceros superbus als bei den anderen Pseudoceriden die
Penes beim Schwimmen haufig weit hervor gestreckt werden). Diese
Beobachtung brachte mich zuerst auf den Gedanken, dass die mann-
lichen Begattungsapparate der Polycladen neben ihrer eigentlichen Func-

No. 3-] SPERMATOPHOKES. 389

tion auch noch die von Waffen zum Angriff oder zur Vertheidigung
haben konnten; sie rief mir zugleich eine alte Beobachtung die ich
gemacht hatte, in das Gedachtniss zuriick. Ich hatte namlich schon
mehrere Male in den Aquarien, in denen ich Zhysanozoon hielt, diese
Thiere mit vorgestrecktem Penis aufgeregt herum und _ iibereinander
hinweg kriechen sehen. Es bot sich mir bald die Gelegenheit, diese
Beobachtung wieder zu erneuern, und ich unterliess es diesmal nicht,
nach dem Ereignisse die Thiere zu untersuchen. Jfcin Erstaunen war
gross, als ich fand, dass sich auch die Exemplare von Thysanozoon
gegenseilig verletst und Haufchen von Sperma in die Wunden abgelegt
hatien. Dies brachte mich sum ersten Mal auf den Gedanken, dass
wenigstens bet den Polycladen mit doppeltem oder vielfachem minnlichen
Begatiungsapparatund einfacher weiblicher Geschlechtsiffnung die Begat-
tung sich so vollztehe, dass die Begattungsglieder eines Individuums an
irgend eines Korperstelle anstechen, Sperma in die Wunde entleeren, und
dass dann das Sperma szufillig in die im Korper reich verzweigten
Liileiter gelange. Ich fand sodann auf Schnitten in der That bei vielen
Pseudoceriden Sperma nicht nur in den Eileitern, sondern auch zx
Darmasten, im Parenchym, etc. Diese Art der Copulation erschien
mir aber doch so eigenthiimlich, so ganz verschieden von allem, was bis
dahin bekannt war, und hauptsachlich so unnatiirlich, dass sich immer
wieder Zweifel an der Richtigkeit meiner Auffassung der oben beschrie-
benen Vorgange in mir regten. Dvyese Zweifel verschwanden aber voll-
stindig, als ich die Entdeckung machte, dass bet Cryptocelis alba im
minniichen Begattungsapparat Spermatophoren erzeugt werden, die dazu
bestimmt sind, mit Gewalt in die Letbeswand anderer Individuen der-
selben Art eingepflanst zu werden. Als ich zuerst Individuen von Cryp-
tocelis alba bekam, die an den verschiedensten Kéorperstellen mit einer
wechselnden Anzahl dieser weissen, fadenformigen, zihen Spermatophoren
besetst waren, glaubte ich erst, dass es Parasiten seien, bis ich ein solches
Gebilde offnete und eine Unmasse von Spermatozoen von der Form der-
Jenigen von Cryptocelis alba heraustreten sah. Die Spermatophoren sind
unter Durchbrechung des Epithels, der Basalmembran und der Muscu-
latur so fest in den K6rper eingepflanzt, dass sie sich bis auf ihre
doppelte und dreifache Lange zu langen, diinnen Faden ausziehen
lassen, bevor sie sich loslésen. Die durch das Einpflanzen der Sper-
matophoren hervorgerufenen Wunden lassen deutliche Narben zuriick.
Man trifft sie bei zahlreichen Individuen an. Der grosse kraftige,
ausserst musculése Begattungsapparat von Crypéocedis erscheint seiner
Function sehr gut angepasst.

“Spermatophoren werden auch noch bei anderen Polycladen pro-
ducirt. Im K6rperparenchym von Prostheceraeus albocinctus fand ich
unzahlige Haufchen von Samenfaden, deren Kopfenden alle in einer

390 WHITMAN. [ VoL. IV.

Ebene lagen und deren Schwanze nach einer und derselben Seite
gerichtet waren. Ballen von Sperma fand ich auch sehr haufig vor
dem Eingang zum weiblichen Begattungsapparat von Lepfoplana fre-
mellaris. Diese Spermatophoren unterscheiden sich von denen der
Cryptocelis alba dadurch, dass sie nicht in eine Membran oder Kapsel
eingeschlossen sind.”

Td. ibid. ist Halfte. pp. 231-32.

“Wie im biologischen Theile auzeinandergesetzt werden wird, dienen
die mannlichen Copulationsorgane mehreren Polycladen nicht zu einer
eigentlichen Copulation im gewohnlichen Sinne des Wortes, d. h. sie
werden nicht in die weiblichen Copulationsorgane eingefiihrt. Sie
dienen vielmehr diesen Formen dazu, den Korper eines anderen Indi-
viduums derselben Art an irgend einer Stelle anzustechen und den
Samen in die so-erzeugte Wunde zu ergiessen, oder eigens bereitete
Spermatophoren mit Gewalt in den Korper des die Misshandlung erlei-
denden Individuums einzupflanzen. Diese eigenthiimliche, bis jetzt, so
viel ich weiss, bet allen Thieren ganz allein dastehende Art der Begat-
tung macht es verstindlich, dass bet Pseudoceriden und Anonymiden
neben einem einzigen weiblichen Begattungsapparat swet oder mehrere
minntliche vorkommen.

“Ich erblicke in thr aber ferner auch einen Vorgang, der auf die
phylogenetische Bedeutung der Copulationsorgane der Polycladen viel-
leicht etniges Licht wirft. Wenn, wie ich anzunehmen geneigt bin,
die Polycladen aus Coelenteraten durch Anpassung an die kriechende
Lebenweise hervorgegangen sind, so haben ihre Vorfahren keine Begat-
tungsapparate besessen. Es bleibt also die Schwierigkeit, die Entsteh-
ung dieser oft so complicirten Organe bei den Polycladen zu erklaren.
Ich weiss sehr wohl, dass diese Schwierigkeit gegenwartig noch nicht zu
beseitigen ist, doch diirften vielleicht die folgenden Bemerkungen einen
Fingerzeig abgeben, in welcher Richtung die Losung der Frage zu
suchen sein wird. Die minnlichen Begattungsapparate vieler Polycladen,
ganz besonders diejenigen, welche an thren Ende ein hartes Stilett tragen,
stehen nimlich sicher nicht ausschliesslich im Dienste geschlechtlicher
fFunctionen, sondern ste dienen auch als Waffen sum Angriff und
vielleicht auch zur Vertheidigung. Schon O. Schmidt, Hallez und v.
Graff haben fiir die harten Stilette der mannlichen Geschlectsapparate
gewisser Rhabdocoeliden diese Auffassung ausgesprochen, die ersten
beiden haben dieselbe sogar durch directe Beobachtung erhartet. Ich
habe bei Pseudoceriden ebenfalls direct beobachtet, dass die Penes als
Waffen gebraucht werden. Ein grosses und schénes Exemplar von
Pseudoceros superbus sah ich in einem meiner Aquarien, in denen sich

No. 3.-] SPERMATOPHORES. 391

mehrere Exemplare von Zhysanozo0on Diesingtt und Yungia aurantiaca
befanden, in grosser Aufregung umher kriechen und von Zeit zu Zeit
die beiden Penes weit vorstrecken. Es kroch Ofter iiber die anderen
erwahnten Polycladen hinweg und brachte ihnen durch Vorstossen der
Penes zahlreiche Wunden bei, in denen ich stets ein Haufchen Sperma
vorfand. Ganz ahnliches habe ich zu wiederholten Malen auch bei
Thysanozoon Diesingit beobachtet. Aber noch ein anderer Umstand
spricht zu Gunsten der gelegentlichen Verwendung der Copulationsor-
gane als Waffen. Das Lagerungsverhaltniss des mannlichen Begattungs-
apparates von S#ostomum zu Pharyngealtasche und Pharynx bringt es,
wie ich weiter unten nachweisen werde, mit sich, dass der Pharynx nicht
vorgestreckt werden kann, ohne dass nicht auch der mit einem harten
Stilett versehene Penis vorgestossen wird. Wenn wir uns nun ferner
daran erinnern, dass bei der Begattung mehrerer Polycladen-Arten eine
gewaltsame Verwundung der Individuen durch den Penis an den ver-
schiedensten Korperstellen erfolgt, so liegt der Gedanke doch gewiss
nahe, dass die Copulationsorgane der Polycladen urspriinglich Angriffs-
und Vertheidigungswaffen waren, die erst secundar in den Dienst ge-
schlectlicher Functionen traten. Von diesem Gesichtspunkte aus ist
das Vorhandenseir. einer grossen Anzahl von Begattungsorganen bei dem
urspriinglichen Genus Anonymus sehr leicht erklarlich, und die oben
erwahnte Begattungsweise erscheint uns viel weniger seltsam.

2. Rotatoria.

LUDWIG PLATE. Beitrige zur Naturgeschichte der Rotatorien. /en. Zeit. fiir
Nat. XIX. 1885. pp. 110-11.

“Dass die Spermatozoen bei den begatteten Weibchen frei in der
perienterischen Fliissigkeit sich umhertummeln, ist eine von vielen
Forschern wiederholt gemachte Beobachtung: aber wie sie hinein
gelangen, ist von denselben nicht erkannt worden. Cohn und Bright-
well konnten, da sie nur mit Lupen arbeiteten, weiter nichts bemerken,
als dass die Mannchen sich dicht an die Weibchen anhefteten, und
ersterer vermutete bei Hydatina und Conochilus einen besonderen, in
der Halsgegend befindlichen Genitalporus. Eyferth berichtet: ‘bei
Diglena catellina habe ich die Anheftung (der Mannchen) an die
Kloakenmiindung gesehen.’ Hudson! dagegen fand bei Asplanchna
Ebbesbornii ‘ein Mannchen, das mit der Spitze des Penis dem Weib-
chen anhing.’ Aber es war an der Aussenseite der Bauchflachenmitte
und nicht an der Oviductdffnung.’ Alle diese widersprechenden Anga-
ben erklaren sich leicht aus den Beobachtungen, die im speciellen Teile

1 Jour. Roy. Micr. Soc., Vol. III, Part 5, 1883, p. 622.

392 WHITMAN. [Vot. IV.

bei Hydatina senta geschildert wurden. Sie fiihrien zu dem merkwir-
digen und, soviel ich weiss,im ganzen Tierreich nur noch bet einigen
Planarien vorkommenden Ergebnis, dass der Penis die Korperwandung
des Weibchens bei der Copulation an irgend einer beltebigen Stelle durch-
bohrt, derselbe dagegen nicht, wie man erwarten solte, in die Kloake
gesteckt wird. Unter geeigneten Umstinden vermag daher auch dasselbe
Wetbchen gleichzeitig von mehreren Méannchen begatiet zu werden. Da
schon bei so vielen anderen Specien Sperma fret in der Leibeshohle flot-
werend gefunden worden ist, kann kaum beswetfelt werden, dass auch
bet diesen die Begattung in gleicher Weise vollzogen wird. Es fragt sich
nun, ob wir annehmen diirfen, dass auch zu jener Zeit, als die Mannchen
wie die Weibchen mit Mundoffnung und Darm versehen und in ihrer
ganzen Organisation noch nicht riickgebildet waren, der mannliche Same
auf dieselbe Weise in den weiblichen Korper gebracht wurde. Ehe bei
Seison, dem einzigen Radertier, dessen Mannchen noch nicht retrometa-
morphosiert ist, die Copulation nicht beobachtet worden ist, lasst sich
freilich die angeregte Frage nicht mit Sicherheit entscheiden. Da
jedoch Claus von dieser Gattung glaubt mit Sicherheit behaupten zu
konnen, dass die Samenfaden nicht frei in der Leibeshohle, sondern in
dem diinnhautigen Ovar sich befinden, scheint es mir das Wahrschein-
lichste cu sein, dass urspriinglich der Penis in die Kloake geschoben, und
auf diesem allein natiirlichen Wege das Sperma mit den Keimzellen
zusammengebracht wurde. Wir miissen dann annehmen, dass mit der
Riickbildung der Miannchen oder vielleicht bewirkt durch dieselbe eine
Anderung in der Art des Coitus eingetreten ist.”

PP- 37-39-

“Um den Akt der Begattung zu beobachten, thut man gut, gleich-
zeitig eine grossere Anzahl (6-10) Mannchen mit einem Weibchen in
einem kleinen Tropfen zu isolieren. Die ersteren besitzen namlich nicht
die Fahigkeit, die Nahe des anderen Geschlechts zu wittern, auch nicht
dann, wenn das siedende Gewimmel der Samenfaden, — welches manch-
mal erst am zweiten Tage nach dem Verlassen des Eis eintritt, —anzeigt,
dass sich die Tierchen in einem begattungsfahigen Zustande befinden.
Auch die Weibchen bekiimmern sich nicht um die Mannchen ; beide
werden lediglich durch den Zufall zusammengefiihrt, und bringt man
daher 1 oder 2 Mannchen mit einem oder wenigen Weibchen zusammen,
so muss man oft Stunden lang warten, bis eine Begattung wirklich eintritt.
Oft kommen beide Geschlechter vielfach mit einander in Beriihrung,
ohne zu copulieren, wie auch schon Cohn mit der Lupe beobachtet hat,
dass die Mannchen ‘ die Wiebchen umschwarmen, sich an diese anlegen,
meist aber von diesen . . . wieder zuriickgeschreckt werden.’ Bei der
Segattung wird merkwiirdiger Weise der Penis des Mannchens, nicht in

No. 3-] SPERMATOPHORES. 303

die Kloake oder in eine andere Offnung der Cuticula geschoben, sondern
durchbricht letztere an irgend einer beliebigen Stelle und befordert wahr-
scheinlich durch eine energische Contraction der Muskulatur des Hoders
die stibchenformigen Korper und das Sperma in die Leibeshihle. Wahr-
end der Begattung Kriimmt sich der Korper des Mannchens so, dass die
Bauchseite einen concaven Bogen darstellt. Die Zehen werden hierbei
nicht gebraucht, sondern auch der Ventralflache zugewendet, sodass, das
ganze Tier eine halbmondformige Gestalt annimmt und nur mit dem
ausgestreckten Penis sich festheftet. Da eine besondere Genitaloffnung
nicht vorhanden ist, kann ein Weibchen gleichzeitig von mehreren
Mannchen begattet werden, ein Vorgang, der in der Natur wegen der
Seltenheit der letzteren wohl kaum vorkommen diirfte, den ich aber
unter den oben angegeben Bedingungen wiederholt beobachtet habe ;
so sah ich 2, 3, 5, und einmal eine noch grossere Zahl von Mannchen
(6-8) gleichzeitig mit demselben Wetbchen copulieren. Es ist mir Ofters
aufgefallen, dass, wenn man Tiere beiderlei Geschlechts in einem kleinen
Tropfen isoliert, dieselben zunachst langere Zeit gleichgiiltig an einander
vorbeischwimmen ; umschwarmt jedoch erst ein Mannchen das Weibchen,
so sammeln sich bald mehrere der ersteren um dasselbe, gleichsam als
ob die Mannchen sich gegenseitig bemerkten. Bet der Begattung wird
der Penis nicht in seiner ganzen Linge durch die gebildete Offnung der
Cuticula in die Leibeshihle hereingeschoben, sondern klebt nur adusserlich
derselben an. Sie wird nur sehr klein sein, denn ich habe nie nach er-
folgter Begattung Spuren derselben in der Haut wahrnehmen konnen.
Leider kann ich nicht mit Sicherheit angeben, wie die Offnung zum
Ubertritt des Sperma ensteht, da die stete Beweglichkeit der copulie-
renden Tiere die Untersuchung sehr erschwert. Aus der Beobachtung
von Tieren, die gleichzeitig von mehreren Mannchen begattet und in
diesem Augenblicke durch ein Deckglas festgehalten wurden, glaube ich
jedoch schliessen zu diirfen, dass die grossen Borsten an der Pentsoff-
nung und die pfeilartigen Stibchen, die wegen threr Lage im Vas deferens
suerst herausgepresst werden, die Kérperwand des Weitbchens durch-
brechen. Alle diese zweifelhaften Punkte wiirden sich ohne Schwierig-
keit an den grossen Asplanchnaarten lésen lassen. An demselben Genus
wiirde man auch relativ leicht beobachten kd6nnen, welchen Einfluss das
Sperma auf die Bildung der Eier ausiibt, ob die Begattung auch eine
Befruchtung nach sich zieht oder ob,— wre ich glaube,— die Samen-
fiden in der Leibeshihle der Weibchen siimtlich nach etniger Zeit su
Grunde gehen, und der Begattungsakt durch das Auftreten der Parthen-
ogenese seine weiteren Folgen verloren hat. Es wire dies ein in der
Tierreithe nach unsern jetzigen Kenntnissen wohl einsig dastehender Fall,
den man als einen ‘ rudimentaren Vorgang’ in demselben Sinne besetch-
nen Kinnte, wie man Organe, ate ausser Function getreten sind, rudt-

394 WHITMAN. [Vot. IV.

mentir nennt. Leider wusste ich, als ich das Verhalten der Spermatozoen
in der Leibeshohle verfolgte, noch nicht, dass nur ein Teil der Geschlechts-
organe, ndmlich der Eierstock, bei Entscheidung jener Frage in Betracht
kommen kann und richtete meine Aufmerksamkeit daher nicht besonders
auf den Vorderrand des Dotterstockes. Was man an den begatteten
Tieren beobachtet, ist eigentlich nur sehr wenig. Das Sperma hauft sich
zuweilen in einem Klumpen innen um die gebildete Hautoffnung herum
an, flottiert jedoch in der Regel frei in der Leibeshohle, verteilt sich
zwischen allen Organen, zwischen den Faden des Gehirns so gut, wie in
der Nahe der Klebdriisen und sammelt sich weder in der Umgegend der
Geschlechtsorgane vorzugsweise an, noch habe ich je beobachten konnen,
dass die Samenfaden den Versuch gemacht hatten, in dieselben einzu-
dringen. Der Dotterstock eines gut genahrten Tieres bildet nachst dem
Darm das grosste Organ im Korper, und es ist natiirlich, dass sich an
siener Oberflache mehr Spermatozoen ansammeln als auf einem kleine-
ren, z. B. der contractilen Blase. Ubten die Fortpflanzungsorgane ferner
eine besondere Anziehungskraft auf das Sperma aus (wie Cohn es bei
Conochilus gesehen haben will), so miisste sich dasselbe vorn in der
Nahe des Keimstockes ansammeln, was mir sicherlich nicht entgangen
ware, auch ohne zu wissen, warum gerade dieser Teil von den Samen-
tierchen bevorzugt wiirde. Wahrend die eben in die Leibeshohle ge-
langten Spermatozoen sich zunachst noch sehr lebhaft bewegen und
daher, wenn sie der Genitaldriise dicht anliegen, durch das hin und her
Schlangeln ihres Schwanzes leicht den Eindruck hervorrufen konnen, als
ob sie sich in dieselbe einzubohren suchten, werden diese Bewegungen
nach einigen Stunden immer matter. Dabei schwellen sie am vorderen
Ende dick an, werden allmahlich kugelformig und im Innern vacuoli-
siert, kurz, es ist offenbar, dass sie einen lingeren Aufenthalt in der
pertenterischen Flissigheit nicht su vetragen vermigen, sondern darin
sterben und sich zersetzen. Untersucht man ein begattetes Tier nach
24 Stunden, so nimmt man nichts mehr von denselben wahr.

““Die ausgesprochene Ansicht, dass sich die Aydatina senta aus-
schitiesslich parthenogenetisch fortpflanst, stiitzt sich vornehmlich auf
den Mangel einer anziehenden Wirkung der Geschlechtsorgane auf
das Sperma, und darauf, dass nie das Eindringen des Samens in jene
beobachtet wurde.”

3. Dinophilus.

SIDNEY F. HARMER. Notes on the Anatomy of Dinophilus. Studies from
Morph. Lab. Univ. Cambridge. Vol. I, pp. 49, 50. 1890. [Reprint from
Jour. Mar. Biol. Assoc. N.S. Vol. 1.]

“So far as I am aware, copulation has not hitherto been actually proved
to take place in any species of Dinophilus. The proof that such a

No. 3.] SPERMATOPHORES. 395

process takes place in D. teniatus is very readily obtained by merely
placing a considerable number of individuals of both sexes in a small
quantity of sea-water, as in a watch-glass. Under these circumstances,
it is noticed, even a very short time after the animals have been placed
together, that here and there a male is attached, by means of its penis,
to the body of a female. In these cases, the terminal, conical portion of
the penis is protruded through the generative pore, and zs passed into the
skin of the female ; spermatozoa are then seen to have passed, from the vest-
cule seminales, through the skin of the female, and to be accumulating them-
selves into a mass immediately beneath the perforation made by the penis.

“There seems to be no localization of the spot at which spermatozoa
can be introduced into the female. The penis can obviously be inserted
into the skin at any point, as is shown by the fact that, in the cases actu-
ally observed, the point selected was sometimes in the region of the
neck, in other cases far back in the body of the female, and in other
cases near the middle of the body.

“The act of copulation has no relation to the maturity of the ova of
the female, nor is it prevented by the fact that the female has already
received an ample supply of spermatozoa by a preceding operation. It
was extremely difficult to discover any female, in which ovaries were
recognizably developed, which did not contain large numbers of sper-
matozoa in its body-cavity. These were observed in almost any part of
the body of the animal, their position being probably partly dependent
on the manner in which fertilization had been previously effected. The
spermatozoa show, however, a great tendency to accumulate into a large,
compact mass, situated in a space on the ventral side of the stomach.
In some cases tt was observed that the female was receiving spermatozoa
simultaneously from two males ; in others that while, for instance, ferti-
lization was being effected near the posterior end of the body, a great
mass of spermatozoa (obviously obtained on a previous occasion) was
visible at the anterior end of the body. In many cases the females were
enormously distended with spermatozoa, which could hardly have been
all received at one time.

“The common occurrence of great numbers of spermatozoa in the body
of the supposed female might suggest that D. entatus was hermaphro-
dite. Such a supposition is rendered sufficiently improbable by the
following considerations: (1) That no other species of Dénophilus is
known to be hermaphrodite ; (II) that the process of fertilization was
frequently observed in D. seniatus ; (III) that the spermatozoa so con-
stantly seen in the female of the same species were, without exception,
ripe and actively moving, no trace of spermmorulz or unripe sperma-
tozoa being discernible. Such stages in the development of the sper-
matozoa were never missed in any adult male individual.”

396 WHITMAN. [Vou. IV.

4. Chetopods.

a. THE TUBIFICIDH. — Spermatophores are of very general oc-
currence among the oligochzetous annelids. They were observed
in the seminal vesicles of the Tubificidz as long ago as 1828 by
Dugés, and in 1850 by Budge; but their significance escaped
these older authors, and even as late as 1861-62 Claparede
described them as opfalina-like parasites, under the name of
Pachydermon acuminatum and P. elongatum. It was not until
1869, in a joint work with Metschnikoff, that Claparede recog-
nized the real nature of his “ opalinoid parasites.”

In 1848 Kolliker saw the spermatophore of Spio attached to
the surface of the worm, and supposed it to be a Gvregarina.
The first to discover their meaning, according to Vejdovsky,
was Doyere (1854-55).

In some of these spermatophores the tails of the inclosed
spermatozoa project through the wall of the capsule, giving it
the appearance of being clothed with vibratile cilia. Such
spermatophores, in motion, resemble living organisms so closely
as to deceive the trained eye, as the Pachydermon of Claparede
well illustrates. Sometimes, as in Psammoryctes, these vibratile
portions adhere in bundles on the neck of the capsule, giving it
the appearance of being armed with recurved hooks like the
proboscis of an Echinorhynchus. Vejdovsky himself first de-
scribed these as “ Widerhaken,” but corrected himself in his
later monograph.

Among the earlier contributions to a more definite knowledge
of these structures are two papers by E. Ray Lankester,! and
one by Franz Vejdovsky.?

Lankester remarks on “The Structure and Origin of the
Spermatophores of Two Species of Tubifex”’ (pp. 180, 181, 187)
as follows :—

“The very curious structure of these built-up masses of spermato-
phores, the fact that they are an example of @ kind of organization
elsewhere without parallel, — a secondary aggregation, not due to growth
as ordinarily presented by organized beings, but to accumulation of free
independently developed elements, — gives them a claim on our atten-
tion, as well as the facts that they have been misunderstood by the

1 Quart. Journ. Mic. Sci., 1870 and 1871.
2 Zeitschr. f. w. Zool., XXVII, 1876.

No. 3.] SPERMATOPHORES. 397

ablest and latest writer (M. Claparéde) on the animals which present
them ; and that they exhibit marked variations in form in the various
genera and species of Oligochcet worms.”

‘«‘ Did we know of a number of free unicellular organisms after com-
plete development becoming fixed together by a cement to form a
secondary organism capable of locomotion and possibly of nutrition, we
should have a parallel to the spermatophores ; as it is, they are, I be-
lieve, the only examples of the building up of an organ or quasi-organ-
ism by agglomeration instead of histogenesis.”

“The sperm-ropes of Zudifex rivulorum I have found in the copula-
tory pouches both in summer and winter, but especially abundant and
well formed in the winter. They have a worm-like figure, with a curious
conical head, an average from =}, to ;; of an inch in length, and from
=1, to zi, of an inch in breadth, the narrowest part being that imme-
diately succeeding the conical head, which has a breadth of about zy'59
of an inch.”

“The general form of the sperm-rope is due to its being moulded in
the long neck of the copulatory pouch... .

“Tt appears that the material of which the sperm-ropes are formed,
namely, spermatozoa and a cementing matrix, must be introduced in a
viscid form from the male efferent duct, through the penis of one worm
into the copulatory reservoir of another, and in the neck of that reser-
voir a ‘setting’ occurs; for the sperm-ropes, when fully formed, are
very firm and compact bodies, of high light-breaking power. The wall
of the copulatory pouch is glandular, and undoubtedly furnishes a
secretion which occupies part of its cavity, and in all probability also
assists as a cementing material in the formation of the sperm-ropes.”

b. Tue Lumpricip®. — The account given by Vejdovsky ! of
the spermatophores of the Lumbricidae comes more closely in
several respects to what I have seen in the leeches. It is in
these worms that spermatophores have often been seen attached
to the surface of the body, usually on the first segments of the
sexual girdle. According to Vejdovsky, they were known to
the earlier naturalists of this century, and were usually described,
after Morren’s example, as “appendicule generatrices,” or as
“penes.” Friedrich Miiller (1849) first recognized them as sper-
matophores. Later (1857), however, they are referred to by
Ewald Hering as “unimportant formations.” Hering’s descrip-
tion (Zeitschr. f. w. Zool., IV, 1857), nevertheless, seems to be
of some value : —

1 System und Morphologie der Oligochaten, Prag., 1884.

398 WHITMAN. [VoL. IV.

“Nach der Begattung tragen die Wiirmer meist in der Gegend des
26 Segments, se/ten am Giirte/, jederseits einen kleinen plattkolbenfor-
migen, ungefahr 1!’ langen Anhang, den sogenannten Penis. ... Er
ist anfangs weich, wird aber allmahlig harter und besteht aus einer
hyalinen Substanz, in die am freien Ende ein Trépfchen Samenmasse
eingebettet ist. Er ist nachweisbar ein Product der Begattung und
besteht nach meiner Ansicht aus evhartetem Schleime.”

Leuckart (Bericht, 1854-55) remarks as follows : —

“Referent hat an der vorderen Bauchseite der Regenwiirmer zur
Brunstzeit nicht selten kleine spindelformige Gebilde angetroffen, die mit
Samenfaden gefiillt waren und wohl als Spermatophoren zu betrachten
sein diirften.”

Fraisse (Semper’s Arbeiten, V, 1879) devoted a special paper
to the spermatophores of the earthworms, giving figures illus-
trating their form. Fraisse came to the conclusion that the
outer homogeneous envelope of the capsule is not formed in
the male efferent ducts, nor in the seminal vesicles, but proba-
bly by the glands associated with the sexual sete. Vejdovsky
finds the spermatophores attached sometimes in the interseg-
mental furrows, and considers this evidence against the glands
(“tubercula”’) having any share in their formation. In opposi-
tion to Fraisse, Vejdovsky thinks it probable that the seminal
vesicles furnish the secretion which forms the envelope in
question.

Vejdovsky concludes his account with some remarks about
the difficulty of understanding how the spermatozoa inclosed in
spermatophores can reach the eggs. The female genital pore
is located in the fourteenth somite, and the spermatophores
are usually placed in the twenty-seventh to thirtieth somites.
Furthermore, no external opening could be found in the sper-
matophore, by which the contents could escape. These facts
naturally suggest to me the possibility of the spermatophores
emptying themselves as they doin Clepsine. Such a possibility,
however, is not mentioned by Vejdovsky, who suggests that the
spermatozoa may be freed during the formation of the cocoon.

The mode of attachment of the spermatophores appears to be
precisely the same as in Clepsine.

“ Thre Basts wiichst mit der Cuticula des Lethesschlauches susammen
und ist von der letsteren iiberhaupt nicht su unterscheiden.”

No. 3.] SPERMATOPHORES. 399

5. Lhe Capitellide.

Among the polychztous annelids and the Capitellide there
seems to be little that can be said to fall in the direct line of
our inquiry. In very many cases the sexual products are set
free in the water, and external fertilization takes place without
the intervention of spermatophores. In Polygordius, according
to Fraipont, the whole body undergoes a regressive metamor-
phosis at sexual maturity ; and the sexual products, as the result,
perhaps, of mechanical pressure upon the body-wall, weakened
by almost complete atrophy, burst through, and escape into the
water. The animal does not survive the evacuation of its sexual
cells.

In one of the Capitellidae (C/istomastus lineatus), according
to Eisig, the whole abdominal region, in which the sexual cells
are lodged, undergoes a histolytic metamorphosis at sexual
maturity ; and the sexual elements are set free by fragmentation
of this region. The thoracic region alone remains intact, and
this is supposed to be able to regenerate a new abdomen.

In Capitella we find spermatophores, but no evidence that
they are ever attached to the exterior. Dr. Eisig has made a
number of most interesting discoveries in regard to the mode
of copulation and fertilization in these worms; and although
they do not touch very closely the phenomena under considera-
tion in the leeches, I think they are not so remote as to be out
of place here.

Monog. XVI. Fauna und Flora von Neapel. 1887. pp. 284, 674-75, 790-93.

The organs of copulation in Capzte//a consist of (1) two pairs
of modified hzemal parapodia belonging to the eighth and ninth
somites ; (2) a copulation gland lying between the genital para-
podia of the ninth somite; and (3) a pair of genital sacs in the
eighth segment, which function as vesicle seminales, vasa effer-
entia, and penes. The female possesses a like pair of genital
sacs in the same segment, and these serve not only as organs
of copulation (vuz/v@), but also as receptacula seminis and ovt-
ducts. Dr. Eisig thinks the glandular porophore of the female,
like the clitellum of the oligochzeta, assists in copulation by its
sticky secretion.

From the position of the copulatory organs, Dr. Eisig con-

400 WHITMAN. [Vot. IV.

cludes that in copulation the two individuals lie back to back,
and that the terminal portions of the genital sacs of the male are
everted and inserted as penes in the corresponding non-everted
parts of the female.

The spermatozoa, originating like the ova in a so-called “ geni-
tal plate,” fall into the body-cavity in an imperfectly developed
state; after attaining maturity, they collect in the genital sacs,
and are there united into bundles or spermatophores [p. 793].
In this form they may pass, during the act of copulation, into
the genital sacs of the female, thence into her body-cavity,
where they meet and penetrate the ova. Fecundation is thus
accomplished before oviposition.

Although Dr. Eisig does not seem to have witnessed the act
of copulation, his statements rest on many concurrent evidences.

The most remarkable feature of this copulation remains to be
mentioned. Dr. Eisig reports [pp. 791-92] that the ripe males
copulate not only with adult females, but also with unripe fe-
males, and even with juvenes in which the sex has not yet
become manifest ; and what is still more astounding, wth young
individuals of their own sex. The proof of this lies in the fact
that the genital sacs of young males were found stuffed with
spermatophores before their genital plates had come into func-
tional activity. They must therefore have received their sper-
matozoa from without.

These facts prove that the male is capable of discharging his
function effectively without the aid of any co-operative act on
the part of the female, and in this respect the copulation bears
some resemblance to that of Clepsine. But there seems to be
no evidence of spermatophores attached to the exterior.

6. Arthropods.

The spermatophores of Eupagurus were seen by Swammerdam
(Bibel der Nature, p. 87), and described as “lauter regelmas-
sigen Theilchen.”” Whether they represented eggs or spermato-
zoa was left undecided. About a century later, in 1841-42, the
decapod spermatophores were rediscovered by Kolliker, and
their purpose and origin made known through his observations
and those of Von Siebold (cf Miil. Arch., 1842, pp. cxxxv-vi).
It seems probable, from Grobben’s observations (Claus’s Avbezten,
I, 1881, p. 67), that all decapods produce spermatophores. The

No. 3.] SPERMATOPHORES. 401

question as to how the spermatophores empty themselves, and
whether fecundation is internal or external, has received different
answers. Grobben (/.c., p. 75) does not attempt to decide by
what agent the spermatophore is made to burst and discharge
its contents. In the Brachyura they are supposed to be dis-
solved in the bursa copwlatrix of the female. Leuckart (Zeugung.,
p. 900) accounts for the escape of the contents of the sperm-
case thus: ‘‘ The walls gradually harden and compress the con-
tents until the capsule bursts, or (as in the case of Astacus and
insects) until it flows out of the open end.” Paul Meyer (Jen.
Zeitschr., 1877, p. 204) thinks it is not the water that causes
the spermatophore to burst (in Galathea and Pagura), and sug-
gests that the secretion with which the female fastens the eggs
to its legs may be the agent that accomplishes this.

It is in Peripatus and the Copepods that we find modes of
impregnation more or less closely analogous to what takes place
in the Rhynchobdellide.

ADAM SEDGWICK. The Development of Peripatus Capensis. Quart. Jour.
Micr, Sct. N.S. XCIX. July, 1885. pp. 453-54.

a. PERtpATUS. — “ The ovaries contain spermatozoa, some of which
project through the ovarian walls into the body-cavity. This condition
has been figured and described by Moseley."

“The ovaries always contain spermatozoa, but in smaller numbers
directly after the eggs have passed into the oviduct than at any other
time. ‘This is a very marked feature of an ovary, say, at the beginning
of April, when compared with an ovary from which the ova have just
passed into the oviducts, say, at the beginning of May, the former being
of an opaque white color to the naked eye, while the latter has a much
more transparent appearance.

“This fact would seem to imply that fresh spermatozoa pass each year
into the ovaries. This brings me to the question of the manner in which
the male discharges his function. The veszcwle seminales (testes of
Moseley and Balfour) are almost empty of spermatozoa in the months
of February, March, and April. At the end of April, however, they
begin to swell again and contain spermatozoa, which increase in number
as time goes on, until, in October, they are fully distended with sper-
matozoa in all stages of development. There seems to be no functional
intromittent organ, but the male deposits little oval spermatophores quite
casually on any part of the body of the female, and, for all that I know,

1 Phil. Trans., Vol. 164.

402 WHITMAN. [Vou. IV.

of the male also; e.g. 1 have often seen them on the head. How these
little packets of spermatozoa get into the vagina, and then up the utert,
which are always full of embryos, I cannot conceive. The spermatozoa
exhibit a certain amount of vibratory movement, and no doubt, once
within the vagina, they are set free from the spermatophore and make
their way up the female generative tube, between the embryos and the
uterine walls. Inasmuch as the deposition of spermatophores lasts from
June until January, each female probably has a large number of sperma-
tophores deposited on her, and some of these are probably near the gener-
ative opening, and are, somehow or another, transported through it into
the vagina.

“ Fertilization is apparently effected in the ovary. I have never seen
spermatozoa in any part of the female apparatus except in the ovaries,
and in small numbers in the upper end of the oviducts at the time when
the ova are entering the latter.”

AUGUST GRUBER. Beitrage zur Kenntniss der Generationsorgane der freileben-

den Copepoden. Zettschr. f. w. Zoology. 1879. p. 407.

6, CopEPoDA. — Fertilization is accomplished by means of
spermatophores in the Copepoda ; but here the sperm-cases are
always, so far as known, affixed to the body of the female in the
immediate neighborhood of the genital openings, and the con-
tents are never forced through the body-wall.

The whole history of the spermatophore has been carefully
traced out by Gruber. Its formation in Heterocope is considered
to be typical for the entire group.

The vas deferens forms two secretions, one of which forms the
elongated, sausage-shaped spermatophore, while the other is
incased along with the spermatozoa. The spermatophore is at-
tached to the vulva of the female by one end, which is drawn
out into a tubular neck. As soon as this is accomplished, the
contents of the capsule begin to flow out, as the result of a sort
of histolytic metamorphosis which overtakes the larger part of
the inclosed spermatozoa. These swell up to pale spherules,
which grow larger and larger, fuse, and finally give rise to a net-
work of polygonal spaces. Meanwhile the inclosed secretion '
has been gradually expelled, and now forms a slimy mass in the
vulva, into which, as a sort of secondary capsule, the remnant
of spermatozoa is driven. Oviposition follows. The eggs on
their way out of course meet the spermatozoa, and both are car-
ried out together.

No. 3.] SPERMATOPHORES. 403

In those species of the Calanidz which have one or a pair of
veceptacula seminis, the phenomena are the same, except that the
spermatophores are attached to the pores of these receptacles,
and their contents pass into the receptacles, instead of directly
into the valve. The spermatozoa find their way into the vulva
through connecting ducts, when the eggs appear.

In the Calanidze, the male catches the spermatophore, as it
is extruded, with his fifth pair of limbs, and then places it on
the body of the female. The secretion which begins at once to
escape from the neck of the capsule serves to fix the latter first
to the limb and then to the genital pore of the female [p. 424].

In other families, the male and female lie with their ventral
surfaces together; the male seizes with his antennz the last
pair of swimming-legs of the female, then bends his abdomen
forward, and fixes the spermatophore directly on the opening of
the receptaculum seminis by means of a special secretion [pp.
424-25].

c. GAMMARUS. — Della Valle! states that the oviducts end
blindly, and that what should be the opening at the base of the
fifth pair of legs is completely closed, except at the moment of
oviposition.

Copulation occurs while the female is still bearing young in
her pouch. The young leave the pouch; and the female moults,
the male assisting in the operation. The ventral surfaces are
together, so that the papillae of the male are in apposition with
the region of the oviducal extremities of the female. As soon
as the moulting is finished, ejaculation of spermatozoa occurs :
the ova appear half an hour later. The spermatozoa are not
inclosed in sperm-cases, but they adhere to the ventral surface
of the fifth segment and on the plates of the pouch. The ovi-
ducts are forced open by pressure from within, and the ova are
covered with a viscid gelatinous mass which binds them and the
spermatozoa together. For this abstract I am indebted to Dr.
McMurrich.

C. SPENCE BATE. Report on the Present State of our Knowledge of the Crus-

tacea. Report British Assoc. f. Adv. of Sc. 1880. pp. 230-32.

d. Astacus. — “Copulation of the crayfish takes place, according to
the observations of M. Chantran,’? during a period which includes the

1 Atti. Soc. Nat. Modena (3), WIII, 1889.

2 Comptes Rendus, July 4, 1870, LXXI, pp. 42-45; Ann. Nat. Hist., 4th Ser.,
Vol. VI, p. 265.

404. WHITMAN. [Vou. IV.

months of November, December, and January. The male seizes the
female with his large nippers, turns her over, and whilst he holds her
lying on her back, places himself in such a manner as to pour out the
fecundating material upon the two outer lamellz of the tail. After this
first operation, which lasts some minutes, he conveys her rapidly beneath
his pleon, in order to effect a second deposition of semen upon the
plastron round the external opening of the oviducts, by means of the
curious mechanism so accurately described by M. Coste, upon the plates
of the caudal fan. (Aipiswra.)

“ According to the degree of the maturity of the ova at the time of
the union of the sexes, oviposition takes place at a period varying from
ten to forty-five days after copulation. ... Immediately after ovi-
position (which usually takes place during the night, and is accompanied
with an emission of mucus for securing the eggs) we may detect in this
mucus and water the presence of spermatozoids, precisely similar to
those which are contained in the spermatophores attached to the
plastron, and derived from them.”

These spermatophores still remain attached to the plastron
long after oviposition.

“They consist of small white coriaceous filaments, either isolated or
mutually adherent ; they no longer show anything but a central cavity,
in which the microscope reveals only a few more or less withered sper-
matozoids. The wall of these spermatophores retains its thickness, and
remains, as before, composed of a concrete, striated, tenacious mucus.’’?

Fecundation [p. 230] is thus accomplished after oviposition. This
is concluded from the fact that spermatozoa are found in the mucus
surrounding the eggs, and from the fact that the spermatophores are
then empty.”

POSTSCRIFT.

Dr. C. T. Hudson, who has recently given us the results of
observations continued for upwards of thirty years on the Ro-
tifera, seems to doubt Plate’s statements regarding the injec-
tion of spermatozoa through the body-wall. In his presidential

1 Comptes Rendus, jan. 15, 1872, UXXIV, pp. 201-2; Ann. Nat. Hist., 4th Ser.,
IX, pp. 173-74.

2Cf. A. Lereboullet: Recherches d’Embryologie comparée sur le Développement
du Brochet, de la Perche et de l’Ecrevise. Paris, 1862. p. 652.

No. 3.] SPERMATOPHORES. 405

address! to the Royal Microscopical Society, referring to Plate’s
account, he says: “It is not necessary to comment further on
this strange theory than to say that Gosse has seen intercourse
take place at the cloaca in the case of Brachionus pala; M. E. F.
Weber, in that of Digtena catellina ; and Mr. J. Hood, not only
in floscularia ornata, Syncheta gyrina, Euchlanis triquetra, and
Melicerta tubicolaria, but also more than a score of times in
flydatina senta itself.”

“The frequent presence of spermatozoa in the perivisceral
cavity” is, however, one of “the doubtful points” for which
Dr. Hudson offers no explanation. No opening is known by
which they could reach this cavity. ‘Neither the oviduct, nor
the cloaca, is known to have an opening into the perivisceral
cavity, and yet the spermatozoa in several species have been
seen in that cavity, adhering to the outside of the ovary. How
did they get there?”

Plate’s observations answer this question, and it will not do
to dismiss them as “‘a strange theory,” since the same strange
thing is reported from so many different sources.

1 On Some Doubtful Points in the Natural History of the Rotifera. Journal
Royal Microscopical Society, February, 1891, p. 6.

406 WHITMAN.

EXPLANATION OF PLATE XIV.

LETTERS.
I-XXXIII= somites. mc. =ventral nerve-cord.
1-66. = number of rings. 72 — env:
al. =alimentary canal. nph. = nephridial pores.
alc. = gastric cecum. o. =outer layer of the spermatophore.
6, = brain. @, =cesophagus.
c. =ccelomic cavity leading up tothe wg. =cesophageal pair of glands.
ovaries. ov. =ovary.
ca. =cecal appendage of the ovary. p. = proboscis.
c.ep. =ccelomic epithelium. phgi. = first pair of pharyngeal glands.
cg. =caudal appendage. pAg*. = second pair of pharyngeal glands.
d. =ductus ejaculatorius. s. =enlarged end-portion of vas defe-
es. =egg-string. rens commune.
jf. =fibrous prolongation. sp. = spermatozoa.
g. =glandular part of the ejaculatory “4 =testes.
duct. vad, =vas deferens.
gs. =granular secretion. vac. =vas deferens commune.
7. =inner layer of thespermatophore. vs. =vesicula seminalis.
/m. =J\ongitudinal muscles. w. =end of ejaculatory duct.
Zu. =Jlumen of the base of the sper- x. =position of the spermatophore.
matophore. y. =course of the spermatozoa.

Fic. 1.— Transverse section of Clepsine plana, showing ccelomic cavities filled
with spermatozoa (red), which penetrated the epidermis at the level of the point
marked X. The ccelomic cavities communicate with the cavity in which the ovaries
(ov) lie at the point c. Only about one-half of the section is represented in the
figure. DGI25s

Fic. 2.— A part of one of the following sections, showing the base of the sper-
matophore and the spermatozoa issuing from it. The arrows show the direction of
penetration through the muscular layers. < 1120:

Fic. 3. — Section of ccelomic cavities with their peculiar epithelium. X 120.

Fic. 4a.— Spermatophore, just placed (3.4 mm. long), and full of spermatozoa.
The granular substance issuing from the mouth seems to serve the purpose of prepar-

ing the way for the penetration of the spermatic elements. x 55:
6=section of the case two days after the escape of the spermatozoa; ¢=cor-
puscles filling the mouth of the spermatophore. X 280.
Fic. 5.— The central nervous system and sexual organs, together with the pro-
boscis, cesophagus, and the so-called salivary glands. X 44.
Fic. 6.—The supra and sub-cesophageal ganglia, seen from the dorsal side.
X 50.

Fic. 7.— The same, seen from the side. X 50.

Journ. Morph. Vol

COWhitman, det

BMetsel ith Boston.

DESCRIPTION OF CZLEPSINE PIAA.
C. O. WHITMAN

THIS species, as already stated in the foregoing paper on
Hypodermic Impregnation, agrees in some features closely with
C. parasitica, as described by Say! and Verrill?; but the points
of agreement are not sufficient for identification; for I have four
quite distinct species, — and I have reason to think there are at
least several more, — each of which comes about equally near the
characters said to belong to C. parasitica. In view of the fact
that we have several large species of Clepsine which agree in
having a single pair of eyes, a median yellow vitta, and marginal
yellow spots, I am led to doubt the identity of Verrill’s C. para-
sitica with that described by Say. Say says, “This leech is
frequently found in the lakes of the Northwestern region, adher-
ing to the sternum, or inferior shell, of tortoises (Emys), particu-
larly to that of £. geographica of Lesueur.” Verrill’s specimens
were found in “ West River, near New Haven, Conn., on the lower
side of floating wood, and at Norway, Me.” I have found two
quite distinct species of these large Clepsines in the vicinity of
Milwaukee, either of which may, or may not, be Say’s species.
I have two eastern species, quite distinct from each other and
from the two Wisconsin species; one from Charles River,
Watertown, the other from a pond in Worcester. Whether
one, or the other, or neither of these is identical with Verrill’s
species, I am unable to say.

Say mentions as the constant characters: “A yellow vitta
before ; guadrate marginal spots each side; beneath with about
eleven longitudinal lines ; ocular points two.” Then comes the
following description: “Body dilated when at rest, narrowed
before; above varied with dull yellowish and blackish brown ;

1 Major Long’s Expedition to the Source of St. Peter's River, etc., in 1823. Keat-
ing’s compilation, Vol. II, Appendix, p. 14. London, 1825.

2 Synopsis of the North American Fresh-Water Leeches. Professor Baird’s Report
for 1872-73, p. 678. The same description was given in the American Jour. Sc. and
Arts, Vol. III, February, 1872, p. 128.

407

408 WHITMAN. [VoL. IV.

a yellow vitta commences at the anterior extremity, and is more
or less elongated, in some specimens less than one-fourth the
length of the body, and in others extending nearly, or quite, to
the posterior disc ; lateral margin with eighteen or twenty sym-
metrical, equal, and equidistant quadrate yellowish spots; pos-
terior disc above radiate with yellowish; ocular points two,
appropriate, sometimes apparently confluent ; beneath very flat,
whitish, with about eleven longitudinal lines; lateral edges very
acute.

“Length, in a state of repose, two inches; greatest breadth,
seven-tenths of an inch.”

According to Verrill: “This species is one of the largest
and most conspicuously colored of the genus.

““Body smooth, but distinctly annulated, much depressed,
broad, tapering anteriorly to the obtusely rounded head, broad
and emarginate posteriorly, with a broad, round, posterior sucker
or acetabulum, about half of which is exposed behind the end of
the body. Length, in extension, three inches; greatest breadth,
three to five tenths of an inch, according to the degree of exten-
sion. Ocelli usually united into one inconspicuous spot, placed
near the anterior margin of the head; two or three other minute
black spots, somewhat resembling ocelli, sometimes occur along
the margins of the head anteriorly.

“Upper surface variegated with green, yellow, and brown ;
the ground-color is usually dark greenish brown, with a broad
median vitta of pale greenish yellow, which at intervals expands
into several large, irregular spots; unequal, oval, and rounded
spots are also irregularly scattered over the back. The entire
margin is surrounded by a series of alternating square spots of
dark green and yellow. Lower surface longitudinally striped
with numerous purplish brown and black lines; the margin
spotted like that of the upper side.”

These descriptions give us the size, shape, color, number of
eyes, and locality. Size and shape do not help us much, since
they vary so much, and since so many species are so nearly
alike in these respects. The eyes are a very constant feature ;
but a number of distinct species agree in having only one pair
of eyes closely approximated near the anterior margin of the
head. The localities are so widely separated that they speak
against rather than for identity. Color, as every one knows

IND: 3:i] DESCRIPTION OF CLEPSINE PLANA. 409

who has had any experience in collecting and describing leeches,
is a very unsatisfactory guide to identification. We do not find
then in these descriptions either a single character or a combi-
nation of characters that may not belong to any one of several
different species.

Perhaps the claim might be made that “all these species are
apt to be quite variable in character in different localities, as
well as at different periods of growth” (Verrill, Zc. p. 677).
This is undoubtedly true of most of the characters above named.
But is it true that the variability is so great and so general that
distinctive characters are nowhere to be found, either in the
external or the internal organization? In other words, do the
“species ’’ and “varieties” grade into one another so closely
as to make it impossible to find really dzstinctive characters?
Must we be content with descriptions that only help us to bring
together more or less nearly related forms, and give up the
attempt to find constant morphological features which may
serve as a reliable means of identification? It must be admitted
that most authorities —if we may judge from their descriptions
—nhave taken this view. The result is that we still have no
uniform method of describing the Clepsinidz, and very few
descriptions that are not more a hindrance than an aid to
progress in their classification. This is a simple statement of
fact, and not a criticism reflecting upon any particular author’s
work. Early classifiers of the Hirudinea adopted what now
appears to be an altogether inadequate mode of description, but
which was perhaps all that the times seemed to require. The
example first set by European systematists has naturally enough
been followed by later authorities, in this country as well as in
others, and no one can be justly reproached for not having fore-
seen the necessity of a better system. If, then, I attempt to
point out defects of method, and to suggest how they may be
improved, I trust no one will find cause for thinking that I
am actuated by a spirit of captious criticism, or with any desire
to belittle the labors of others. The defects to be pointed out
are not the defects of any particular piece of work alone, but
rather those of a long-standing and generally received method
of describing the Hirudinea. The above descriptions of C/ep-
Sine parasitica are neither the best nor the worst of their kind ;
but they are fairly representative, and are here made the sub-

410 WHITMAN. [VoL. IV.

ject of special remark merely because they happen to touch the
case in hand.

Let us now consider briefly in what particulars these descrip-
tions fail to meet our needs. First of all they are not accom-
panied with any figures, and without such aids it is often
extremely difficult, if not impossible, to get at the author’s exact
meaning, however skilful he may be at depicting. The oral
sucker in these species presents a number of important diag-
nostic characters, not one of which is mentioned. The position
of the mouth varies for different species, but it is not alluded to.
Important specific distinctions are to be found in the number
and condition of the rings represented in the head, and espe-
cially in the buccal and post-buccal rings, all of which are
ignored. Nothing whatever is said about the number of rings
and segments in the animal, and the peculiarities of the abbre-
viated posterior somites pass unnoticed. The metameric sense-
organs were not then known, and although conspicuous enough,
escaped notice. The nephridiopores are neither numbered nor
otherwise defined, and the genital pores are not so much as
mentioned. All this for the external features. Not one of the
internal features received any notice, not even the diverticula
of the stomach.

Some time ago I attempted to show that a satisfactory basis
for the classification of the ten-eyed leeches (Hirudinida) was
to be found in the external metamerism. I shall now show that
the same method may be extended to the Clepsinide. The
determination of the number and homology of the rings is often
more difficult here than in the Hirudinidz ; but still it is always
possible, I believe, and it affords the only safe basis for distin-
guishing genera and species. It is the remarkable constancy of
these metameric characters that gives them such high value for
diagnostic purposes. With metamerism as a basis, all the external
features are readily defined topographically, and with a precision
and definiteness that cannot otherwise be attained. The first
thing to be done in describing a new form is, therefore, to
determine with all possible precision the number of the rings
and somites. This task is comparatively easy except at the
two extremities of the body, where, it must be confessed, it is
often quite difficult to decide upon the number of rings. This
is more especially true of the head region. Here it is often

No. 3.] DESCRIPTION OF CLEPSINE PLANA. AII

necessary to resort to various expedients. It is well to have
several individuals killed in weak chromic acid (4 to +%), in an
extended condition, If the leeches do not die extended, they
may easily be straightened by stretching a little. After lying
in the acid a short time, the rings usually become sufficiently
well defined to admit of study and comparison. It is advisable
always to supplement this study with that of the living leech.
Sometimes I have found it necessary to cut off the head and
study it in all positions while it is contracting and expand-
ing. For such study, a good dissecting microscope (e.g. that
of Zeiss) is indispensable. The metameric sense-organs enable
one to fix the limits of the somites, and are thus a most impor-
tant guide in the analysis of the head region. Having deter-
mined the composition of the first two or three somites, the
next important point to settle is the number of the ring which
forms the posterior boundary of the oral sucker. This buccal
ring is sometimes united either above or below with the preced-
ing or following ring, and hence both the buccal and the post-
buccals require careful examination from all sides and in all
states of contraction and extension. The ocular ring is fre-
quently a double ring, z.e. two rings more or less consolidated.
In order to describe accurately such rings, it is necessary to
note the relative width of the successive rings, particularly in
the head and the posterior extremity of the body. The method
of numbering rings and somites is shown in Figs. 1 and 3,
PL XV.

My chief reason for offering the following description at this
time is the fact that I have had to refer to this leech by name
in my paper on spermatophores, and it seems desirable to
show that the name stands for a reality. The description may,
however, be regarded as a preliminary one, inasmuch as I hope
to be able to describe this in connection with the more closely
allied species, and to furnish with the descriptions the much-
needed colored figures. The preparation of such figures, with
due attention to all the details which require to be accurately
reproduced, is already in progress.

CLEPSINE PLANA, 7. Sf.
The largest of the five specimens obtained measured as
follows : —

4l2 WHITMAN. [Vo. IV.

Length at rest, 5-6 cm.; width, 2.6 cm.

Length in extension, 8.5 cm. ; width, 1.8 cm.

Head, 4 mm. wide, scarcely marked off from the body, obtusely
pointed in extension, rounded or truncated at rest.

Body, ovate-elliptical in contraction, emarginate posteriorly,
very thin, showing two rows of very low, smoothly rounded, meta-
meric protuberances on the dorsal surface, and between these
similar, but scattered, non-metameric protuberances.

Disc, 9 mm. in diameter, circular, often largely covered by the
body.

Annult, 66 between the eyes and the anus, counting four
double rings (1, 2, 64, 65) as single rings.

Somites, XXVI in front of the anus, XX XIII in all.

Buccal annuli=s5th and 6th, united for about the middle
third of the ventral side, distinct towards the margins of this
side.

Post-buccals = 7th and 8th, completely united below.

Eyes, 2, rather obscure, and in contact at the anterior edge
of the first ring. In the young the eyes are conspicuous and
quite distinct, although nearly or quite contiguous.

Mouth, in front of the centre of the flat oral sucker.

Genital pores. — Male orifice in the 1oth somite, between
24th and 25th rings ; female orifice in the 11th somite, between
26th and 27th rings.

Testes = six pairs in 12th to 17th somites.

Anus, behind the 66th ring, between this and a postanal
rudiment, representing probably a remnant of one or two rings.

The annuli of the head. —In front of the eyes I was unable
to discover any distinct rings. In another species, C. chelydra,
from Wisconsin, there are three narrow rings in front of the
eyes; and the first is marked by the usual metameric sense-
organs. Although no metameric sense-organs were recognized
in front of the eyes in C. plana, the correspondence of other
metameric characters in the two species is sufficiently close to
enable me to identify the ocular rings as equivalents. The pre-
ocular part of the head is, therefore, probably equivalent to the
first somite of C. chelydre, and is so numbered in Fig. 1. The
ocular ring is double, representing the Ist and 2d rings of the
second somite, so incompletely divided that the evidence of
duplicity is seen mainly in the relative width of the ring.

No. 3.] DESCRIPTION OF CLEPSINE PLANA. 413

This ring, then, corresponds to the 4th and 5th rings of C.
chelydre.

The 2d ring (6th and 7th of C. chelydre) is also double,
as shown by its width and by a slight division, sometimes notice-
able at the margins. This ring represents the third ring of the
second somite, united with the first ring of the third somite, as
is plainly shown by the sense-organs being placed in the poste-
rior half of the ring.

The 3d and 4th rings are of equal width, and slightly nar-
rower than the double rings preceding them. These, together
with the posterior and sensory half of the 2d ring, constitute
the third somite.

The 5th (sensory) and 6th rings (buccals) are a trifle wider,
distinct above, but united below except at the margins. These
two rings form the posterior limit of the head, and together
form the first ring behind the suctorial surface of the ventral
side. When the leech is at rest, with the head attached, a
feeble constriction may usually be seen, which falls between
the 6th and 7th rings, and thus obscurely marks off the head.

The 7th and 8th rings, the post-buccals, are distinct above,
but consolidated below. From this point onward the rings are
regular and distinct, both above and below, until we reach the
twenty-third somite.

In the head we have found only two incomplete somites (Ist
and 2d), z.e. somites with less than three distinct rings. The
twenty somites, from III to XXII inclusive, are complete in
respect to the number of constituent rings. The body termi-
nates behind with four short and incomplete somites (X XIII-
XXVI). The twenty-third has two distinct rings (62d and
63d); the twenty-fourth, one plainly double ring (64th), consist-
ing of a sensory ring and a narrow, imperfectly defined rudi-
ment; the twenty-fifth, one ring (65th), giving evidence of its
double nature only at the margins; and the twenth-sixth, one
narrow and simple sensory ring (66th). Adding seven somites
for the disc to the twenty-six pre-anal somites, we have as the
whole number thirty-three, which agrees with the number of
ganglia in the ventral chain.

In regard to the abbreviated somites, it will be noticed that
we have here, as in all the other Hirudinea, the greatest reduc-
tion in the number of rings in the twoend somites. Reduction,

414 WHITMAN. [VOL. IV:

as I have before pointed out, seems to have begun at both
extremities, and to have advanced from these points towards
the middle region of the body. Its advance has been centrip-
etal, and the extent of its advance shows how far a form has
departed from the ancestral condition of uniform somites. It
is here that we discover a very important guide to the systematic
rank and relationship of different forms. This is most clearly
illustrated in Hirudo and Hzemadipsa.

It may be well here to call attention to a fact hitherto over-
looked ; namely, that metamerism among the leeches has under-
gone modification in two opposite directions. Variation by
centripetal reduction of the number of rings is universal ; varia-
tion by multiplication of rings characterizes, as a rule, only the
higher forms, Hirudo, Nephelis, etc. Clepsine rarely exhibits
the second mode of variation, and never to the extent that
Hirudo does. The difference between the Clepsinidz and the
Hirudinide in this respect has a physiological explanation.
Hirudo swims, and for this purpose a long flexible body is
required; Clepsine, with few exceptions, habitually creeps, and
for this mode of locomotion, supplementary rings have not been
essential. In variation by multiplication we have another means
of determining close systematic relations.! I would not be

1] am reminded of an error into which I fell in my paper on Japanese Leeches.
The error was the assumption that all somites having less than five rings were abbre-
viated. The assumption should have been, as I now feel convinced, that all somites
with less than ¢hvee rings are abbreviated, and all with more than three have been
increased by the division of one or two of the three primary rings. I have collected
considerable evidence, which cannot be given here, going to show that in the evolu-
tion of Hirudo, it was the second and third rings that underwent division, while the
first remained undivided. In the Hirudinide, then, we have supplemented somites
(five rings, rarely four), ‘yfe-somites (three rings), and abbreviated somites (0-2
rings). The type-somites I formerly regarded as abbreviated. The view here taken
helps to understand what before seemed unaccountable, that Hirudo and most of its
congeners present three successive somites (4-6) with only ¢2ree rings each. Allow-
ing these to be type-somites, we recognize in them a sort of meutral zone, standing
between the abbreviated and the supplemented somites. Usually one of these type-
somites only is preserved in the posterior region of the body; and sometimes we find
this somite already enlarged to four rings, by the division of its ¢hzrd ring, as is well
shown in the Japanese Leptosoma acranulatum.

Mr. Apathy —who, as I observe, seems to look upon “Some New Facts about
Leeches,” which I recently published, as worthy of being claimed as his own dis-
coveries — advances a different view, according to which the type-somite is supposed
to have had twelve rings. A review of his position must be postponed until I can
bring forward the evidences which seem to me fatal to such a view.

No. 3.] DESCRIPTION OF CLEPSINE PLANA. 415

understood as claiming that variation in these ways can always
be relied upon as an exact gauge of systematic relationship. I
am not unmindful that in such questions the eztzve organization
must be the final criterion in doubtful cases. Nevertheless, I
hold that we find in the variations above defined an important
guide.

Color. —Ground-color above a dull, dark brown, with a
slightly darker marginal border (Figs. 1 and 3) encircling body
and disc. Along the margin there are twenty-one dull yellow
spots, metamerically arranged, and having the same width as
the dark border. The first spot is very small and on the ocular
ring, the second on the second ring, the third on the two buccal
rings, the fourth mainly on the ninth and tenth rings. From
this point onward the spots mark the second and third ring of
each somite, except the last four (XXIII-X XVI), in which
they are absent. Anteriorly these spots have a triangular form
with the apex directed towards the median line. In the sixth
somite they begin to assume a V-shaped form, and this passes
into a U-shaped form along the middle and posterior regions.

The median portion of the tip of the head is whitish up to
the eyes. With the eyes begins the median yellow vitta, con-
stricted at irregular intervals. This vitta runs back to the
fourteenth ring, and then fades into an obscure patch of brown-
ish yellow reaching back to the twentieth ring. The extent of
the vitta is quite variable, as was pointed out by Say and Verrill.

There are two rows of yellow spots metamerically arranged,
marking the first ring of each somite (from VII onward), and
situated about midway between the margin and the median line.
These spots are at first small, circular, and placed just inside
the lateral row of sense-organs (/). From the twenty-sixth ring
backward, they become slightly elongated so as to reach and
encircle the corresponding sense-organs. All the sense-organs
of this row, anterior to the twenty-sixth ring, are encircled with
a narrow border of yellow, except the first two pairs (on 2d
and sth rings). The two median rows of sense-organs are
not thus marked, until we come to the last three pairs, which
have quite conspicuous borders. The outer lateral sense-organs
(of) are so marked only as far as the seventeenth ring.

Between the two rows of yellow spots there are other yellow
spots, scattered irregularly along the dorsal surface, varying in

416 WHITMAN. [ Vor. 1;

size from that of the larger metameric spots to much smaller
dimensions. All these spots, the metameric as well as the non-
metameric, mark low, rounded protuberances. The lateral sense-
organs (/) are not placed at the summit of these protuberances,
but close to the base, on the outer side.

On the disc we find yellow patches arranged in about six
radial lines, in each of which are seen from two to three sense-
organs. These radial rows of sense-organs correspond to the
median (wz), lateral (7), and outer lateral (0/) rows of the body.

The ventral surface is paler; the marginal dark border and
yellow v- or u-shaped spots are the same as above, only paler.
There are thirteen light longitudinal streaks alternating with
dark streaks (twelve in number) (Fig. 2).

On the ventral side I find six rows of sense-organs, differing
from those of the dorsal side only in being smaller and more
difficult to find. In all, then, we have fourteen rows of these
sense-organs, as in Hirudo.

The xephridial pores (nph.) lie in the first ring of the somite,
just in front of the line dividing the ring into an anterior and a
posterior half. They are ranged along the medial edge of a
broad, light streak, on the outer edge of which are seen the
lateral sense-organs.

The stomach has seven pairs of much-branched diverticula ;
the seventh diverticulum has five lateral czeca.

The serual organs have been figured and described in con-
nection with the subject of spermatophores (Pl. XIV, Fig. 5).

In the same place I have figured the entire central
nerve-system, and given two enlarged views of the supra-
and infra-cesophageal ganglia (Figs. 6 and 7).

The number of segments represented in the infra-
cesophageal ganglia is five, as shown by the number of
nerves, and by the presence of ten median ventral gan-
glia (1-5, Fig. 7). The arrangement of these ventral
ganglia, shown in the accompanying figure, may be of
some value in identifying the species.

S
Eo

Fic. 1.— Ventral ganglia of the infra-pharyngeal portion of the central nervous
system, showing that five metameres are represented in this region.

nr
etl
vr

418 WHITMAN.

EXPLANATION OF PLATE XV.

LETTERS.

I-XXVI = somites.

I-66 =rings.

(4-72) =corresponding rings of C. chelydre.
6.5-6 =buccal rings.

pb.7-8 =post-buccal rings.

if = lateral row of sense-organs.

m. = median row of sense-organs.

mg. = marginal row of sense-organs.
nph. = nephridiopores.

ol, = outer lateral row of sense-organs.

Fic. 1.—Diagram of the anterior portion of Clepsine plana. The first ring
bearing the single pair of eyes is double, and corresponds to the fourth and fifth
rings in C. chelydre, which appears to be the more typical form. The second ring
is also double, and equivalent to the sixth and seventh of C. chelydr@. The numbers
inclosed in parentheses in this and the two following figures refer to the same species.
Dotted areas show distribution of the yellow pigment. xX 5.

Fic, 2.— Ventral surface of four rings from the middle of the body, showing the
longitudinal pigment-lines and the relative position of the sense-organs, as well as the
nephridial pores. a5:

Fic. 3.— Diagram of the posterior portion of the leech, prepared with especial
reference to the topographical relations of the sense-organs and the annular compo-
sition of the last four somites of the body. § X5-

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